Antitumor antagonist consisting of mutated TGFβ1- rii extracellular domain and immunoglobulin scaffold

Abstract

To provide an antitumor antagonist comprising a first targeting domain comprising a mutated TGFβ1-R11 extracellular domain (mutated ECD); and an immunoglobulin scaffold having an amino terminus and a carboxy terminus.SOLUTION: The present disclosure relates to an antitumor antagonist comprising a first targeting domain comprising a mutated TGFβ1-R11 extracellular domain (mutated ECD); and an immunoglobulin scaffold having an amino terminus and a carboxy terminus, the one or more mutations reducing proteolytic cleavage of the antitumor antagonist. Also disclosed are methods for treating proliferative disorders, infections, and immunological disorders with the antitumor antagonists described herein.SELECTED DRAWING: Figure 20

Description

[Technical Field]

[0001] This invention claims priority to U.S. Provisional Application No. 62 / 869111, filed July 1, 2019, and PCT Application No. PCT / US19 / 39979, filed June 28, 2019, the contents of which are expressly incorporated herein by reference for all purposes.

[0002] The present invention relates generally to cancer therapy, and in particular to bispecific inhibitors that can modulate pathways associated with tumorigenesis, tumor immunity, and angiogenesis. [Background technology]

[0003] The inability of the host to eliminate cancer cells remains a major problem. Although an increasing number of therapeutic monoclonal antibodies have been approved for the treatment of various cancers, resistance to these antibodies is frequently observed due to different molecular pathways involved in cancer growth and metastasis. Although the immune system is a major mechanism for cancer prevention, cancer cells resist immunosurveillance. Natural control mechanisms have been identified that limit T cell activation to avoid secondary damage caused by excessive T cell activity. Tumor cells use this process to evade the immune response. Restoring the ability of immune effector cells, particularly T cells, to recognize and destroy cancer is a major goal of immunotherapy.

[0004] There is a need for binding antagonists or antibodies with enhanced therapeutic efficacy, and methods of using such types of reagents to treat cancer and chronic viral infections. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors of the present invention have discovered that antagonists having a TGFβ1 RII ECD region exhibit unacceptable levels of proteolytic degradation or clipping over time. One aspect of the present invention relates to an anti-tumor antagonist comprising a first targeting domain that specifically binds TGFβ1 and a second targeting domain that specifically binds PD-1 or PD-L1, wherein the first targeting domain comprises a mutated TGF-β1 RII extracellular domain (mutated ECD), which significantly reduces proteolytic cleavage of the anti-tumor antagonist. [Means for solving the problem]

[0006] Another aspect of the present invention relates to an anti-tumor antagonist comprising a first targeting domain comprising a TGFβ pathway inhibitor and a second targeting domain that specifically binds to VEGF.

[0007] Another aspect of the present invention relates to a method for treating a cell proliferative disorder in an individual, the method comprising administering to the individual in need of such treatment an effective amount of an anti-tumor antagonist of the present invention. [Brief explanation of the drawings]

[0008] [Figure 1] The complementarity-determining region (CDR) sequences of certain anti-PD-1 monoclonal antibodies (anti-PD-1 mabs) are shown, and the corresponding framework region (FR) sequences are shown in Figure 53, SEQ ID NOs: 176-205.

[0009] [Figure 2A] 1 shows several embodiments of anti-PD-1 antibody variable region sequences. [Figure 2B] 1 shows several embodiments of anti-PD-1 antibody variable region sequences.

[0010] [Figure 3]The CDR sequences of certain anti-PD-L1 monoclonal antibodies are shown, and the corresponding FR sequence series are shown in Figure 53, SEQ ID NOs:206-228.

[0011] [Figure 4A] 1 shows several embodiments of anti-PD-L1 antibody variable region sequences. [Figure 4B] 1 shows several embodiments of anti-PD-L1 antibody variable region sequences. [Figure 4C] 1 shows several embodiments of anti-PD-L1 antibody variable region sequences.

[0012] [Figure 5] The structures of four different bispecific antitumor antagonists, Bi-PB-1, Bi-PLB-1, Bi-PB-2, and Bi-PLB-2, each containing (1) an anti-PD-1 or anti-PD-L1 variable region and (2) a TGF-β RII ECD region, are shown.

[0013] [Figure 6] 6 shows exemplary functional region sequences corresponding to the bispecific antibody of FIG. 5.

[0014] [Figure 7] Exemplary heavy chain (HC) and light chain (LC) sequences corresponding to selected bispecific antibodies are shown in FIG.

[0015] [Figure 8]Three different bispecific antagonists, Bi-AB-1, Bi-A1B-1, and Bi-ZB-1, each containing the carboxy-terminal TGF-β1 RII extracellular domain (ECD) of a mutated IgG1(K447A) scaffold, are presented. Both Bi-AB-1 and Bi-A1B-1 contain anti-VEGF variable regions (VH1, VL1) from the amino terminus of Avastin (bevacizumab), and Bi-A1B-1 contains two amino acid substitutions (E6Q, L11V) in the VH region. Bi-ZB-1 contains the amino-terminal aflibercept region upstream of the IgG1 Fc(K447A) region.

[0016] [Figure 9A] The various functional domain sequences of the bispecific antagonists shown in FIG. 8 are shown. [Figure 9B] The various functional domain sequences of the bispecific antagonists shown in FIG. 8 are shown.

[0017] [Figure 10] The amino acid sequences of the heavy chain (HC) and light chain (LC) corresponding to the bispecific antagonists shown in FIG. 8 are shown.

[0018] [Figure 11] 1 summarizes the arrangement of functional regions of the bispecific antagonists shown in FIG. 8.

[0019] [Figure 12] Polyacrylamide gel stained with Coomassie brilliant blue showing non-reducing polyacrylamide gel electrophoresis (PAGE) determining increased expression levels of bispecific antibody antagonists Bi-AB-1 and Bi-A1B-1 containing E6Q and L11V mutations when transiently transfected into HEK293 cells.

[0020] [Figure 13](Figure 13A) Size-exclusion chromatography (SEC) graphs of Bi-AB-1 and Bi-A1B-1 are shown. (Figure 13B) Compared to the dimers (98.6% and 98.7%, respectively), Protein A-purified Bi-AB-1 and Bi-A1B-1 show low levels of high-molecular-weight (1% and 1%, respectively) and low-molecular-weight (0.4% and 0.4%, respectively) species.

[0021] [Figure 14] A PAGE gel of transiently expressed Bi-ZB-1 under non-reducing (FIG. 14A) and reducing (FIG. 14B) conditions is shown.

[0022] [Figure 15] A size exclusion chromatography (SEC) graph is shown, which shows that compared to the dimer (96.1%), Bi-ZB-1 purified by Protein A has lower levels of high molecular weight (HMW, 3.4%) and low molecular weight (LMW, 0.5%) species.

[0023] [Figure 16] Results are shown from a cell-based assay in which huVEGF165 is used to stimulate recombinant HEK-293 cells expressing huVEGFR2 and firefly luciferase under the control of an NFAT response element in the presence of serial dilutions of two anti-VEGF antagonists (Bi-A1B-1 and Antibody A). Biological activity is determined by measuring the decrease in luciferase-mediated fluorescence.

[0024] [Figure 17]An ELISA assay is shown in which Bi-A1B-1 simultaneously binds to TGF-β1 and VEGF165, where a 96-well plate coated with huTGF-β1 was incubated with serially diluted samples of Bi-A1B-1 followed by biotinylated huVEGF165, and the bound molecules were detected by streptavidin-HRP using TMB substrate.

[0025] [Figure 18] Results are shown for a cell-based assay in which recombinant HEK-293 cells expressing the human TGF-β1 RII receptor and firefly luciferase under the control of a SMAD response element were stimulated with huTGF-β1 in the presence of serial dilutions of Bi-A1B-1, Bi-ZB-1, and a control, each containing a TGF-β1 RII extracellular domain (ECD) fusion. Biological activity was determined by the decrease in luciferase-mediated fluorescence.

[0026] [Figure 19] Pharmacokinetics graph showing the in vivo half-life (T) of the bispecific antagonist Bi-A1B-1 after intravenous tail vein injection into 6-10 week old female CD1 mice. Serum of Bi-A1B-1 antagonist was collected at several time points post-injection and analyzed by ELISA.

[0027] [Figure 20] Two bispecific anti-tumor antagonists, Bi-PB-1.2 (Figure 20A) and Bi-PLB-1.2 (Figure 20B), are shown, each of which contains an antibody backbone from the variable region of PD-1 and PD-L1, respectively (IgG4 K447A or IgG1 K447A), and further contains the TGF-β-RII ECD fused to the carboxy terminus of each heavy chain CH3 region.

[0028] [Figure 21] The functional region sequences of the bispecific antibodies present in Figures 20A and 20B are shown.

[0029] [Figure 22] 2 shows the amino acid sequences of the heavy chain (HC) and light chain (LC) of the bispecific antagonists shown in FIGS. 20A and 20B.

[0030] [Figure 23] The locations of functional regions in the bispecific antagonists shown in Figures 20A and 20B are summarized.

[0031] [Figure 24] (Figure 24A) Native polyacrylamide gel (PAGE) analysis showing the expression of Bi-PB-1.2 and Bi-PLB-1.2 in a transient expression system compared to 1 μg of the parental control antibody (2P17). (Figure 24B) Size-exclusion chromatography (SEC) graph showing Protein A-purified Bi-PB-1.2, Bi-PLB-1.2, and the anti-PDL1-TGF-β1 RII ECD benchmark molecule. (Figure 24C) Bi-PB-1.2, Bi-PLB-1.2, and the anti-PDL1-TGF-β1 RII ECD benchmark molecule have low levels of high-molecular-weight (HMW) and low-molecular-weight (LMW) species compared to dimers.

[0032] [Figure 25] The results show that the dimeric, HMW, and LMW forms of Bi-PB-1.2 (FIG. 25A) and Bi-PLB-1.2 (FIG. 25B) exhibited good stability at 4° C. for at least 4 weeks.

[0033] [Figure 26] The results of PD-1 and TGF-β1 binding to Bi-PB-1.2 (26A and 26B, respectively) and the corresponding binding to anti-PD-1 and anti-PDL1-TGF-β1 RII ECD benchmark molecules (26C and 26D, respectively) are shown, along with the parent binding constants generated by them (26E).

[0034] [Figure 27] The results for binding to PD-L1 and TGF-β1 Bi-PLB-1.2 (27A, 27B, respectively) and the corresponding binding to anti-PDL1 and anti-TGF-β1 RII ECD benchmark molecules (27C, 27D) are shown, along with the parent binding constants generated by them (27E).

[0035] [Figure 28] (Figure 28) ELISA analysis showing Bi-PB-1.2 simultaneously binding to TGF-β1 and PD-1, where a 96-well plate coated with TGF-β1 is incubated with serially diluted samples of Bi-PB-1.2 followed by biotinylated huPD-1, and bound molecules are detected by streptavidin-HRP using TMB substrate. (Figure 28B) ELISA analysis showing Bi-PB-1.2 simultaneously binding to TGF-β1 and PD-L1, where a 96-well plate coated with TGF-β1 is incubated with serially diluted samples of Bi-PB-1.2 followed by biotinylated huPD-1, and bound molecules are detected by streptavidin-HRP using TMB substrate.

[0036] [Figure 29] (Figure 29A) Shows the ability of Bi-PB-1.2 and anti-PD-1 benchmark antibodies to block PD-1 binding to PD-L1. (Figure 29B) Shows the results of a cell-based assay demonstrating the ability of serially diluted Bi-PB-1.2 and anti-PDL1-TGF-β1 RII ECD benchmark molecules to block TGF-β1-activated luciferase expression under the control of SMAD response elements. Biological activity is determined by the decrease in luciferase-mediated fluorescence.

[0037] [Figure 30]Figure 30 shows the binding of Bi-PB-1.2 and benchmark antibodies to both anti-human PD-1 and anti-crab-eating monkey PD-1 to human PD-1 (Figure 30A) and crab-eating monkey PD-1 (Figure 30B), and the corresponding EC50 values, which reflect the half-maximal effective concentration (EC50) that produces a half-response between the baseline and maximum response for binding to human PD-1 and crab-eating monkey PD-1, respectively.

[0038] [Figure 31] (Figure 31A) Shows the ability of Bi-PLB-1.2 and anti-PD-L1 benchmark antibodies to block the binding of PD-L1 to PD-1. (Figure 31B) Shows the results of a cell-based assay demonstrating the ability of serial dilutions of Bi-PLB-1.2 and anti-PDL1-TGF-β1 RII ECD benchmark molecules to block TGF-β1-activated luciferase expression under the control of SMAD response elements. Biological activity is determined by the decrease in luciferase-mediated fluorescence.

[0039] [Figure 32] Figure 32A shows the binding of Bi-PLB-1.2 and benchmark antibodies to human PD-L1 (Figure 32A) and crab-eating monkey PD-L1 (Figure 32B) for both anti-human PD-L1 and anti-crab-eating monkey PD-L1, and the corresponding EC50 values, which reflect the half-maximal effective concentration (EC50) that produces a response between the baseline and maximum response for binding to human PD-L1 and crab-eating monkey PD-L1, respectively.

[0040] [Figure 33] Figures 33A-33B show that Bi-PB-1.2 increased IFN-γ secretion in human PBMCs (donor 1, Figure 33A; donor 2, Figure 33B) compared to negative control treatment. Figures 33C-33D show that Bi-PB-1.2 increased IL-2 secretion in human PBMCs (donor 1, Figure 33C; donor 2, Figure 33D) compared to negative control treatment.

[0041] [Figure 34] Figures 34A-34B show that Bi-PLB-1.2 increased IFN-γ secretion in human PBMCs (donor 8, Figure 34A; donor 9, Figure 34B) compared to parental anti-PD-L1 antibody and negative control treatment. Figures 34C-34D show that Bi-PLB-1.2 increased IL-2 secretion in human PBMCs (donor 8, Figure 34C; donor 9, Figure 34D) compared to parental anti-PD-L1 antibody and negative control treatment.

[0042] [Figure 35] Following intravenous tail vein injection into 6-10 week-old female CD1 mice, Bi-PB-1.2 and Bi-PLB-1.2 demonstrated improved pharmacokinetic profiles compared to the benchmark antibody. Serum concentrations of Bi-PB-1.2, Bi-PLB-1.2, and the benchmark antibody antagonist were collected at several time points after injection and analyzed by ELISA (Figures 35A, 35C, and 35E, respectively) and Western blotting (Figures 35B, 35D, and 35F, respectively).

[0043] [Figure 36] Bi-PB-1.2 produced by stably transfected CHO cells was stored at 4°C, and the percentage of low molecular weight (LMW) species increased over time, and the percentage of dimeric species decreased over time, consistent with increased clipping measured by size-exclusion ultra-high performance liquid chromatography (SE-UHPLC).

[0044] [Figure 37] Figure 1 shows a size exclusion chromatography (SEC) graph of the bispecific antagonist (Bi-PB-1.2 (PD1-TGF-β1 RII ECD) and PLB-1 benchmark (BM) antibodies expressed in stably transfected CHO cells. Bi-PB-1.2 produced by transiently transfected HEK293 cells shows a shoulder around the main peak consistent with increased clipping.

[0045] [Figure 38] 1 shows a SEC graph of Bi-PB-1.2 fraction obtained by cation exchange chromatography (CEX).

[0046] [Figure 39] The fragments and clipped fragments determined by mass spectrometry were confirmed, which indicates that Bi-A1B-1 and Bi-PB-1.2 have similar clipping sites.

[0047] [Figure 40] Based on mass spectrometry, clipping sites in the heavy chain amino acid sequences of Bi-A1B-1 and Bi-PB-1.2 are shown.

[0048] [Figure 41A] 1 shows various mutant TGF-β1 RII ECD sequences used to study wild-type TGF-β1 RII ECD clipping. [Figure 41B] 1 shows various mutant TGF-β1 RII ECD sequences used to study wild-type TGF-β1 RII ECD clipping. [Figure 41C] 1 shows various mutant TGF-β1 RII ECD sequences used to study wild-type TGF-β1 RII ECD clipping.

[0049] [Figure 42A] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42B] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42C]Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42D] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42E] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42F] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42G] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52. [Figure 42H] Figures 41A-41C show Bi-PB-1.2 heavy chain sequences containing mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown below in Figures 46-52.

[0050] [Figure 43] Figures 41A and 41C show Bi-PLB-1.2 heavy chain sequences containing selected mutant TGF-β1 RII ECD sequences. Analysis of these mutations is shown in Figure 52 below.

[0051] [Figure 44A] Figures 49, 50 and 52 below show Bi-A1B-1 heavy chain sequences containing selected mutant TGF-β1 RII ECD sequences. [Figure 44B] Figures 49, 50 and 52 below show Bi-A1B-1 heavy chain sequences containing selected mutant TGF-β1 RII ECD sequences. [Figure 44C]Figures 49, 50 and 52 below show Bi-A1B-1 heavy chain sequences containing selected mutant TGF-β1 RII ECD sequences. [Figure 44D] Figures 49, 50 and 52 below show Bi-A1B-1 heavy chain sequences containing selected mutant TGF-β1 RII ECD sequences.

[0052] [Figure 45] The amino acids targeted by the mutant TGF-β1 RII ECD sequences shown in Figures 41A-41C are indicated.

[0053] [Figure 46A] Reducing polyacrylamide gel (PAGE) analysis is shown, which indicates that after protein A-parental purification of the molecule from stable Chinese hamster ovary cell (CHO) transformants, the Bi-PB-1.2 TGFβ1 ECD mutant has similar expression levels and yields as the parental wild-type Bi-PB-1.2. [Figure 46B] Reducing polyacrylamide gel (PAGE) analysis is shown, which indicates that after protein A-parental purification of the molecule from stable Chinese hamster ovary cell (CHO) transformants, the Bi-PB-1.2 TGFβ1 ECD mutant has similar expression levels and yields as the parental wild-type Bi-PB-1.2. [Figure 46C] Reducing polyacrylamide gel (PAGE) analysis is shown, which indicates that after protein A-parental purification of the molecule from stable Chinese hamster ovary cell (CHO) transformants, the Bi-PB-1.2 TGFβ1 ECD mutant has similar expression levels and yields as the parental wild-type Bi-PB-1.2.

[0054] [Figure 47A] SEC graphs corresponding to Bi-PB-1.2 and mutations (Bi-PB-1.2-Δ7, Bi-PB-1.2B to Bi-PB-1.2H) are shown, which show reduced clipping (no shoulder) for these mutations compared to Bi-PB-1.2. [Figure 47B]SEC graphs corresponding to Bi-PB-1.2 and the mutations (Bi-PB-1.2-Δ15, and Bi-PB-1.2-Δ20) are shown, which show reduced clipping (no shoulder) for these mutations compared to Bi-PB-1.2.

[0055] [Figure 48] The percentages of high molecular weight (HMW) species, dimers, and low molecular weight (LMW) species reflected in the SEC graphs of Figures 47A-47B are shown.

[0056] [Figure 49] We present results from cell-based assays demonstrating the ability of serially diluted variants of Bi-PB-1.2 and Bi-A1B-1 generated by transient transfection of HEK293 cells and controls (i.e., parental Bi-PB-1.2, Bi-A1B-1, and PLB-BM (benchmark) and negative control antibodies) to block TGF-β1-activated luciferase expression under the control of SMAD-responsive elements. Biological activity is determined by the decrease in luciferase-mediated fluorescence.

[0057] [Figure 50] The IC50 values ​​of the Bi-PB-1.2 and Bi-A1B-1 variants versus the parental control are shown, reflecting their ability to block TGF-β1-activated luciferase expression under the control of SMAD-responsive elements. Biological activity is determined by the decrease in luciferase-mediated fluorescence.

[0058] [Figure 51] Bi-PB-1.2 (Figure 51A), Bi-PB-1.2C (Figure 51B), and Bi-PB-1.2D (Figure 51C) showed similar pharmacokinetics in paired mice, characterized by a T1 / 2 of approximately 5 to 8 days.

[0059] [Figure 52A]Reducing polyacrylamide gel (PAGE) analysis is shown, which shows reduced levels of clipped fragments from the C mutant compared to the parental wild-type Bi-PB-1.2, Bi-PLB-1.2, and Bi-A1B-1. [Figure 52B] Reducing polyacrylamide gel (PAGE) analysis is shown, which shows reduced levels of clipped fragments from the C mutant compared to the parental wild-type Bi-PB-1.2, Bi-PLB-1.2, and Bi-A1B-1.

[0060] [Figure 53] The framework regions (FRs) corresponding to the anti-PD-1 CDRs (SEQ ID NOs: 176-205) and anti-PD-L1 CDRs (SEQ ID NOs: 206-228) in Figures 1 and 3 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0061] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be understood by one of ordinary skill in the art of the disclosed methods and compositions. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly indicates otherwise. Thus, by way of example, "a peptide" includes "one or more" peptides or "plurality" of such peptides. With respect to the teachings of this specification, any issued patents or published patent applications cited herein are expressly incorporated herein by reference.

[0062] As used herein, the term "PD-1" refers to any form of PD-1 and variants thereof, where the variants retain at least some PD-1 activity. Unless otherwise specified (e.g., when human PD-1 is specified), PD-1 includes native-sequence PD-1 from all mammals (e.g., humans, dogs, cats, horses, and cows).

[0063] As used herein, the term "PD-L1" refers to any form of PD-L1 and variants thereof, where the variants retain at least some PD-L1 activity. Unless otherwise specified (e.g., by specific reference to human PD-L1), PD-L1 includes native-sequence PD-L1 in all mammals (e.g., humans, dogs, cats, horses, and cattle).

[0064] The term "agonist" refers to a substance that stimulates (i.e., induces, induces, enhances, or increases) the biological activity or effect of another molecule. The term agonist includes substances that bind to receptors (e.g., antibodies) and substances that stimulate receptor function (e.g., by activating associated proteins) in the absence of receptor binding.

[0065] The term "antagonist" or "inhibitor" refers to a substance that avoids, blocks, inhibits, neutralizes, or reduces the biological activity or effect of another molecule, such as a receptor or ligand.

[0066] As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen via one or more immunoglobulin variable regions. An antibody can be a whole antibody, an antigen-binding fragment, or a single chain thereof. The term "antibody" encompasses a wide variety of biochemically recognizable polypeptide species. Those skilled in the art understand that heavy chains are classified as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), as well as several subclasses thereof (e.g., γ1-γ4). The nature of this chain determines the "class" of an antibody, as IgG, IgM, IgA, IgD, or IgE, respectively. These immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, are well characterized and known to have specialized functions. In light of this specification, modified forms of each of these classes and isotypes are readily recognizable to those skilled in the art and are within the scope of this specification. All classes of immunoglobulins are within the scope of this specification, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules.

[0067] The antibodies or antibody antagonists of the present invention can include, but are not limited to, polyclonal, monoclonal, multispecific, bispecific, trispecific, human, humanized, primatized, chimeric, and single-chain antibodies. The antibodies disclosed herein can be from any animal origin, including birds and mammals. Preferably, the antibodies can be human, mouse, rat, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable regions can be of chondricthoid origin (e.g., from sharks).

[0068] The term "antibody fragment" or "antigen-binding fragment" refers to portions of antibodies, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing the VL or VH regions, fragments produced by Fab expression libraries, and anti-idiopathic (anti-Id) antibodies. Regardless of structure, antibody fragments bind to the same antigen recognized by the whole antibody. The term "antibody fragment" includes DARTs and diabodies. The term "antibody fragment" includes any synthetic or genetically engineered protein containing an immunoglobulin variable region that functions like an antibody by binding to a specific antigen to form a complex. A "single-chain fragment variable (scFv)" refers to a fusion protein of the heavy chain variable region (VH) or light chain variable region (VL) of an immunoglobulin. In certain embodiments, the variable regions are linked to a short connecting peptide of 10 to about 25 amino acids. The connecting peptide can be rich in glycine for flexibility, serine or threonine for solubility, and can link the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant regions and the introduction of the connecting peptide, the protein still retains the specificity of the original immunoglobulin. For IgG, a typical immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 to 70,000 Daltons. The four polypeptide chains are usually joined by disulfide bonds in a "Y" configuration, where the light chain brackets the heavy chain and the heavy chain begins at the opening of the "Y" and extends to the variable region.

[0069] Both light and heavy chains are divided into regions of structural and functional homology. Terms such as "constant" and "variable" are used functionally. Here, the two portions, the light chain variable region (VL) and the heavy chain variable region (VH), determine antigen recognition and specificity. Conversely, the light chain constant region (CL) and the heavy chain constant region (CH1, CH2, or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, in a typical antibody, the further the constant region domain is from the antigen-binding site or the amino terminus of the antibody, the greater the number of the constant region domain. In a typical antibody, the N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0070] As described above, the variable region allows an antibody to selectively recognize and specifically bind to an antigen epitope. That is, the VL and VH regions of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of each of the VH and VL. In some cases, for example, a particular immunoglobulin is derived from a Camelidae species or is designed based on Camelidae immunoglobulins. Alternatively, the immunoglobulin molecule consists of only heavy chains without light chains, or only light chains without heavy chains.

[0071] In naturally occurring antibodies, the six CDRs present in each antigen-binding region are short, non-contiguous amino acids that form the antigen-binding region in specific locations when the antibody assumes its three-dimensional structure in an aqueous environment. The remaining amino acids in the antigen-binding region, called the "framework" region, exhibit less intermolecular variability. The framework regions mostly adopt a β-sheet structure, and the CDRs form rings that connect the β-sheet structure, and the rings also form part of the β-sheet structure under some circumstances. Thus, the framework regions function as a scaffold to position the CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding region formed by CDR positioning defines a surface complementary to the immunoreactive antigen epitope. This complementary surface promotes non-covalent binding of the antibody to the homologous epitope. For any given heavy or light chain variable region, because they are precisely defined, one skilled in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively.

[0072] As used herein, the terms "VH1" and "VH2" refer to immunoglobulin heavy chain variable regions corresponding to two different binding specificities. Similarly, "VL1" and "VL2" refer to immunoglobulin light chain variable regions corresponding to two different binding specificities. When used together, it is understood that the VH1 and VL1 regions define a common binding specificity, and the VH2 and VL2 regions define a second binding specificity.

[0073] The term "framework region (FR)" as used herein refers to variable region residues other than CDR residues. Each variable region typically has four FRs flanking the corresponding CDRs. For example, a VH region typically has four HFRs flanking three HCDRs, i.e., HFR1, HFR2, HFR3, and HFR4, in the following configuration: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4. Similarly, an LH region typically has four LFRs flanking three LCDRs, in the following configuration: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. Exemplary FRs of the antagonists described herein are summarized in Figure 53.

[0074] Light chains are classified as K (kappa) or λ (lambda). Various heavy chains can be combined with K or λ light chains. Generally, when fusion tumor cells, B cells, or genetically engineered host cells produce immunoglobulins, the light and heavy chains are covalently linked to each other, and the "Fc" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0075] As used herein, the terms "light chain constant region" or "CL" are used interchangeably herein and refer to the amino acid sequence derived from the light chain of an antibody. Preferably, the light chain constant region comprises at least one constant K region or constant λ region.

[0076] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least a CH1 region, a hinge region (e.g., an upper, middle, and / or lower hinge region), a CH2 region, a CH3 region, or a variant or fragment thereof. By way of example, an antigen-binding polypeptide as used herein may comprise a polypeptide chain comprising a CH1 region, a polypeptide chain comprising a CH1 region, at least a portion of a hinge region, and a CH2 region, a polypeptide chain comprising a CH1 region and a CH3 region, a polypeptide chain comprising a CH1 region, at least a portion of a hinge region, and a CH3 region, or a polypeptide chain comprising a CH1 region, at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, a polypeptide herein comprises a polypeptide chain comprising a CH3 region. Furthermore, an antibody used in the present invention can lack at least a portion of a CH2 region (e.g., all or part of a CH2 region). It should be understood that a heavy chain constant region can be modified such that its amino acid sequence differs from that of a naturally occurring immunoglobulin molecule.

[0077] For example, as reflected in the disclosure below, Applicant has discovered that the CH3 region can tolerate or accommodate large insertions (e.g., greater than 100 amino acids) of the CH3 region into the Fc ring (see, e.g., Bi-PB-2, Bi-PLB-2 in Figure 5B). Thus, in the present invention, any of the disclosed inhibitor regions can be inserted into the Fc ring in a manner similar to the insertion of the TGFβ1 RII ECD region into the Fc ring of the heavy chain sequences set forth in SEQ ID NOs:151-154.

[0078] The heavy chain constant regions of the antibodies disclosed herein can be derived from various immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can comprise a CH1 region derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain constant region can comprise a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0079] A "light chain-heavy chain pair" refers to a set of light and heavy chains that can dimerize via disulfide bonds between the CL region of the light chain and the CH1 region of the heavy chain.

[0080] The subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are known. As used herein, the term "VH region" comprises the amino-terminal variable region of an immunoglobulin heavy chain, and the term "CH1 region" comprises the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 region is adjacent to the VH region and amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0081] As used herein, the term "CH2 region" includes the portion of a heavy chain molecule, e.g., the portion extending from about residue 244 to residue 360 ​​of an antibody using conventional numbering schemes (residues 244-360, Kabat numbering system; residues 231-340, EU numbering system). A characteristic of the CH2 region is that it is not tightly paired with other regions. Two N-linked branched carbohydrate chains are inserted between the two CH2 regions of an intact native IgG molecule. The CH3 region extends from the CH2 region to the C-terminus of the IgG molecule and includes approximately 108 residues.

[0082] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 and CH2 regions. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct regions: the upper hinge region, the middle hinge region, and the lower hinge region.

[0083] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group, which can form a disulfide bond or crosslink to a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 in the Kabat numbering system (positions 226 and 229 in the EU numbering system).

[0084] As used herein, a "variant" of an antibody, antibody fragment, or antibody region refers to an antibody, antibody fragment, or antibody region that (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the original antibody, antibody fragment, or antibody region, and (2) specifically binds to the same target as the original antibody, antibody fragment, or antibody region. When a sequence identity value is expressed as "at least x% identical" or "at least x% identical," it is understood that the embodiment includes any and all integer percentages above the lower limit. Furthermore, it is understood that an amino acid sequence appearing herein should also be interpreted to disclose or include amino acid sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence.

[0085] When a sequence homology range appears herein (e.g., the phrase "about 80% to about 100%"), it is understood that the embodiment includes any and all subranges within that range, the lower limit of which can be any integer between 80 and 100.

[0086] As used herein, the phrase "humanized antibody" refers to an antibody derived from a non-human antibody, typically a murine monoclonal antibody. Alternatively, a humanized antibody can be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parent non-human antibody, but exhibits less immunogenicity compared to the parent antibody when administered to humans.

[0087] As used herein, the phrase "chimeric antibody" refers to an antibody in which the immunologically active regions or sites are obtained or derived from a first species and the constant regions (which may be complete, partial, or modified as provided herein) are obtained from a second species. In certain embodiments, the targeted binding regions or sites are derived from a non-human source (e.g., mouse or primate) and the constant regions are derived from a human.

[0088] The scope of the multispecific antibodies of the present invention includes asymmetric IgG-like antibodies (e.g., triomab / quadroma, Trion Pharma / Fresenius Biotech), knobs-into-holes antibodies (Genentech), Cross MAbs (Roche), electrostatic pair antibodies (AMGEN), LUZ-Y (Genentech), strand exchange engineered domain (SEED) antibodies (EMD Serono, Biolonic, Merus), Fab-exchange antibodies (Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig (GSK / Domantis), 2-in-1 antibodies (Genentech), cross-linking MAbs (Karmanos Cancer Center), mAb2 (F-star), Cov X-body (Cov X / Pfizer), double variation domain (DVD)-Ig fusions (Abbott), IgG-like bispecific antibodies (Eli Lilly), Ts2Ab (Medimmune / AZ), BsAb (ZymoGenetics), HERCULES (Biogen Idec, TvAb, Roche), scFv / Fc fusion, SCORPION (Emergent BioSolutions / Trubion, ZymoGenetics / BMS), dual affinity retargeting technology technology, Fc-DART), MacroGenics, Bis(scFv)2-Fabs (National Center for Antibody Drug Research), F(ab)2 fusions (Medarex / AMGEN), Dual Action or Bis-Fab (Genentech), Dock-and-Lock (DNL, ImmunoMedics), Fab-Fv (UCB-Celltech), scFv- and diabody-based antibodies (e.g., bispecific T cell engaging antibodies (BiTEs, Micromet), tandem diabodies (Tandab, Affimed), DARTs (MacroGenics), single-chain diabodies, TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scFv fusions (Merrimack), COMBODIES (Epigen Biotech), and IgG / non-IgG fusions (e.g., immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

[0089] "Specifically binds by" or "has specificity for" generally refers to an antibody binding to an epitope via its antigen-binding region, and such binding requires a certain degree of complementarity between the antibody-binding region and the epitope. Based on this definition, if an antibody binds to an epitope more readily via its antigen-binding region than to a random, unrelated epitope, it is said to "specifically bind" to that epitope. The term "specificity" is used herein to define the relative affinity with which a particular antibody binds to a particular epitope. As an example, antibody "A" can be considered to have higher specificity for a particular epitope than antibody "B," or antibody "A" can be described as binding to epitope "C" with higher binding to related epitope "D." In some embodiments, an antibody or antibody fragment binds to an antigen more readily than an antibody. -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 An antibody or antibody fragment "has specificity for an antigen" if it forms a complex with a dissociation constant (Kd) less than or equal to M.

[0090] The phrase "immune checkpoint regulator" refers to a functional class of agents that inhibit or stimulate signaling through immune checkpoints. "Immune checkpoint regulators" include cell surface receptors and their associated ligands, which together provide a means of inhibiting or stimulating T cell activation-related signaling pathways. Cell surface receptor immune checkpoint regulators are generally TNF receptors or B7 superfamily members that induce checkpoint signaling pathways that inhibit immune responses, and include agents that bind to negative costimulatory molecules, including, but not limited to, PD-1, TIGIT, LAG-3, TIM-3, BTLA, VISTA, CTLA-4, and their respective ligands.

[0091] The phrases "immune checkpoint modulator antagonist," "immune checkpoint binding antagonist," and "immune checkpoint antagonist" are used interchangeably herein and refer to a class of agents that interfere with (or inhibit) the activity of immune checkpoint modulators, such that they bind to the checkpoint modulator or its ligand, thereby blocking or inhibiting signaling through the checkpoint modulator receptor. Inhibiting this signaling can reverse immune suppression and re-establish or enhance T cell immune efficacy against anti-tumor cells. Exemplary immune checkpoint antagonists include, but are not limited to, PD-1 and its ligands, PD-L1 and PD-L2, TIGIT and its CD155 ligand, PVR, and liver sinusoidal endothelial cell lectin (LSECtin) and galectin-3, CTLA-4 and its ligands, B7-1 and B7-2, TIM-3 and its ligands, galectin-9, CD122 and its CD122R ligand, CD70, B7H3, B and T lymphocyte attenuator (BTLA), and LAG-3 and its ligands, including VISTA. Immune checkpoint modulator antagonists can include antibody fragments, peptide inhibitors, dominant-negative peptides, and small molecule drugs, in isolated form or as part of a fusion protein or conjugate.

[0092] "Immune checkpoint binding agonist" and "immune checkpoint agonist" are used interchangeably herein and refer to a class of agents that stimulate immune checkpoint modulators, such that binding to a checkpoint modulator or its ligand results in stimulation of a signal via a checkpoint modulator receptor. Stimulating this signal can re-establish or enhance T cell immune responses against tumor cells. Exemplary immune checkpoint modulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, OX40 (CD134), glucocorticoid TNFR family-related protein (GITR), and 4-1BB (CD137), and their ligands. Other checkpoint modulator agonists belong to the B7-CD28 superfamily, including CD28 and ICOS. Immune checkpoint modulators can include antibody fragments, peptide inhibitors, dominant-negative peptides, and small molecule drugs, either isolated or as part of a fusion protein or complex.

[0093] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of that target. In certain embodiments, the term can refer to an antibody that prevents the biological function of a target (e.g., PD-1) to which it binds.

[0094] As used herein, an "anti-PD-1 antagonist antibody" refers to an antibody that can inhibit PD-1 biological activity and / or downstream events mediated by PD-1. Anti-PD-1 antagonist antibodies include those that block, antagonize, inhibit, or reduce (to any level, including significantly) PD-1 biological activity, including PD-1 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and downstream events mediated by PD-1, such as inhibition of anti-tumor immune responses. For purposes of the present invention, the term "anti-PD-1 antagonist antibody" (interchangeable with the terms "antagonist PD-1 antibody," "antagonist anti-PD-1 antibody," or "PD-1 antagonist antibody") should be clearly understood to include the above-identified terms, titles, and functional states and properties, as well as PD-1 itself, PD-1 biological activity, or any result that significantly substantially eliminates, reduces, or neutralizes such biological activity. In certain embodiments, the anti-PD-1 antagonist antibody binds to PD-1 and upregulates an anti-tumor immune response.

[0095] As used herein, an "anti-PD-L1 antagonist antibody" refers to an antibody that can inhibit PD-1 biological activity and / or downstream events mediated by PD-L1. Anti-PD-L1 antagonist antibodies include those that block, antagonize, inhibit, or reduce (to any level, including significantly) PD-L1 biological activity, including PD-L1 binding and downstream signaling, inhibition of T-cell proliferation, inhibition of T-cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and downstream events mediated by PD-L1. For purposes of the present invention, the term "anti-PD-L1 antagonist antibody" (which may be used interchangeably with the terms "antagonist PD-L1 antibody," "antagonist anti-PD-L1 antibody," or "PD-L1 antagonist antibody") should be clearly understood to include the above-identified terms, titles, and functional states and properties, as well as PD-L1 itself, PD-L1 biological activity, or any result that significantly substantially eliminates, reduces, or neutralizes such biological activity. Such anti-PD-L1 antagonist antibodies, by design, bind to PD-L1 and upregulate anti-tumor immune responses.

[0096] The phrase "dominant-negative protein" or "dominant-negative peptide" refers to a protein or peptide that is derived from a wild-type protein that has been modified, generally by mutation and / or deletion, such that the modified protein or peptide interferes with the function of the endogenous wild-type protein from which it is derived.

[0097] The phrase "VEGF binding antagonist" refers to a functional class of agents that bind to VEGF-A or its receptor, VEGFR-2, such that the result of binding blocks or inhibits VEGFR-2 activation by VEGF-A. As used herein, the term "VEGF binding antagonist" includes antibody fragments, peptide inhibitors, dominant negative peptides, and small molecule drugs, in isolated form or as part of a fusion protein or conjugate.

[0098] The phrase "Tie2 tyrosine kinase receptor binding antagonist" refers to a functional class of agents that bind to the Tie2 tyrosine kinase receptor or one of its ligands, and thereafter block or inhibit Tie2 tyrosine kinase receptor activation by one or more of its ligands (i.e., Ang1, Ang2, Ang3, and Ang4). As used herein, the term "Tie2 tyrosine kinase receptor binding antagonist" includes antibody fragments, peptide inhibitors, dominant-negative peptides, and small molecule drugs, in isolated form or as part of a fusion protein or complex.

[0099] The term "one or more mutations in which reduce proteolytic cleavage of the anti-tumor antagonist" should be interpreted in the context of an otherwise identical antagonist containing a wild-type ECD.

[0100] The phrase "small molecule drug" refers to a non-polymeric molecular entity, generally organic or organometallic, that has pharmaceutical activity and a molecular weight of less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da. While small peptide or nucleic acid analogs can be considered small molecule drugs, the phrase includes most pharmaceutical compounds known as "drugs" other than proteins or nucleic acids. Examples include chemotherapeutic antitumor drugs and enzyme inhibitors. Small molecule drugs can be synthetic, semi-synthetic (i.e., derived from naturally occurring precursors), or biologically derived.

[0101] When describing polypeptide regions with hyphens between each region (eg, CH2-CH3), it is understood that the order of the listed regions is from the amino terminus to the carboxy terminus.

[0102] The term "immunoconjugate" refers to an antibody covalently fused to an inhibitory peptide or small molecule drug, which can be linked to the C-terminus of the constant heavy chain or the N-terminus of the variable light and / or heavy chain.

[0103] A "linker" can be used to link a peptide or small molecule drug (e.g., a maytansinoid) to an anti-tumor antagonist in a stable, covalent manner. Under conditions under which the compound or antibody remains active, the linker may be susceptible to, or substantially immune to, acid-induced, photo-induced, peptidase-induced, esterase-induced, and disulfide bond cleavage. Suitable linking peptides are known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. As described herein and known in the art, linkers include GGGGS, charged linkers, and their parent water forms. The immunoconjugate may further include a flexible 3-15 amino acid peptide (or spacer) between the anti-tumor antagonist and the peptide and / or small molecule drug. In some embodiments, the connecting peptide comprises 3, 4, 5, 6, 7, or 8 overlapping GGGGS. In some embodiments, the connecting peptide consists of 3 or 4 overlapping GGGGS.

[0104] As used herein, the term "scaffold" refers to any amino acid polymer that exhibits the properties necessary to support antagonist function, including increasing antibody specificity, enhancing antibody function, or supporting antibody structure and stability. A scaffold can be grafted with the binding region of a donor polypeptide to confer the binding specificity of the donor polypeptide to the scaffold.

[0105] As used herein, the phrase "multispecific inhibitor" refers to a molecule comprising at least two targeting domains with different binding specificities. In some embodiments, the multispecific inhibitor comprises a scaffold and two or more polypeptides that target antigen-binding regions of immunoglobulins of different antigens or epitopes. In certain embodiments, the multispecific inhibitor is a bispecific antibody or antagonist. In other embodiments, the multispecific inhibitor is a trispecific antibody or antagonist.

[0106] As used herein, the phrase "bispecific" refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting the biological function of the target molecule after binding to the target molecule. In some embodiments, the bispecific checkpoint modulator antagonist is a polymeric molecule comprising two or more peptides. In some embodiments, the targeting domain comprises the antigen-binding region or CDR of an antibody. In some embodiments, the bispecific inhibitor is a bispecific antibody.

[0107] The terms "bispecific antibody" and "bispecific antagonist" are used interchangeably herein and refer to antibodies that specifically bind to two different antigens (or epitopes). In some embodiments, the bispecific antibody is a full-length antibody that binds to an antigen (or epitope) with one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) with its second binding arm (a different HC / LC pair). In these embodiments, the bispecific antibody has two antigen-binding arms that are different (both in specificity and CDR sequence) and are monovalent for each antigen to which they are linked.

[0108] In other embodiments, the bispecific antibody is a full-length antibody that binds to two different antigens (or epitopes) with each binding arm (two pairs of HC / LC). In these embodiments, the bispecific antibody has two identical antigen-binding arms (same specificity and identical CDR sequences), and each linked antigen is bivalent.

[0109] Exemplary bispecific antibodies include asymmetric IgG-like antibodies (e.g., triomab / quadroma, Trion Pharma / Fresenius Biotech), knob-into-hole antibodies (Genentech), cross-MAbs (Roche), electrostatic pair antibodies (AMGEN), LUZ-Y (Genentech), strand-exchange domain (SEED) antibodies (EMD Serono, biolonic, Merus), Fab-exchange antibodies (Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig (GSK / Domantis), 2-in-1 antibodies (Genentech), cross-linking MAbs (Karmanos Cancer Center), mAb2 (F-star), Cov X-body (Cov X / Pfizer), dual variable region (DVD)-Ig fusions (Abbott), IgG-like bispecific antibodies (Eli Lilly), Ts2Ab (Medimmune / AZ), BsAb (ZymoGenetics), HERCULES (Biogen Idec, TvAb, Roche), scFv / Fc fusions, SCORPION (Emergent BioSolutions / Trubion, ZymoGenetics / BMS), dual affinity retargeting technology (Fc-DART), MacroGenics, Bis(scFv)2-Fabs (National Center for Antibody Drug Research), F(ab)2 fusions (Medarex / AMGEN), dual action or Bis-Fab (Genentech), Dock-and-Lock (DNL, ImmunoMedics), Fab-Fv (UCB-Celltech), scFv- and diabody-substrate antibodies (e.g., bispecific T cell engaging antibodies (BiTEs, Micromet), tandem diabodies (Tandab, Affimed), DARTs (MacroGenics), single-chain antibodies, TCR-like antibodies (AIT, Receptor Logics), human serum albumin scFv fusions (Merrimack), COMBODIES (Epigen Biotech), as well as IgG / non-IgG fusions (e.g., immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics)

[0110] The terms "treat" and "treatment" refer to ameliorating one or more symptoms associated with a cell proliferative disorder, preventing or delaying the onset of one or more symptoms of a cell proliferative disorder, and / or reducing the severity or frequency of one or more symptoms of a cell proliferative disorder.

[0111] The phrases "to a patient in need thereof," "to a patient in need of treatment," or "a subject in need of treatment" include any individual (e.g., a mammalian individual) who would benefit from the administration of an anti-tumor antagonist herein for the treatment of a cell proliferative disorder.

[0112] The terms "therapeutically effective amount," "pharmacologically effective amount," and "physiologically effective amount" are used interchangeably and refer to the amount of anti-tumor antagonist required to provide a threshold level of antagonist activity in the bloodstream or target tissue. The exact amount will depend on many factors (e.g., the specific active agent, the components and physical properties of the composition, the expected patient population, patient considerations, etc.) and can be readily determined by one of ordinary skill in the art based on the information provided herein or other information available in the relevant literature.

[0113] As used herein, the terms "improvement," "increase," or "decrease" refer to a value or parameter measured relative to a benchmark, where the benchmark value is measured, for example, in the same individual prior to initiating a treatment described herein, or in a control individual (or control individuals) in the absence of a treatment described herein.

[0114] A "control individual" is an individual afflicted with the same cell proliferative disorder as the treated individual and is approximately the same age as the treated individual (ensuring that the stage of disease in the treated and control individuals is comparable). The treated individual (also referred to as a "patient" or "subject") may be a fetus, infant, child, adolescent, or adult with a cell proliferative disorder.

[0115] The term "cell proliferative disorder" refers to a disorder characterized by abnormal proliferation of cells. A proliferative disorder does not imply a limitation relative to the rate of cell proliferation, but merely a loss of normal controls affecting growth and cell division. Thus, in some embodiments, cells of a proliferative disorder may exhibit the same rate of cell division as normal cells, but do not respond to signals that limit such proliferation. Neoplasms, cancers, or tumors fall within the scope of "cell proliferative disorders."

[0116] The term "cancer" or "tumor" refers to any of a variety of malignant neoplasms characterized by cellular proliferation with the ability to invade surrounding tissues and / or metastasize to new colonization sites, including leukemia, lymphoma, carcinoma, melanoma, sarcoma, germ cell tumors, and blastoma. Exemplary cancers treated by the methods herein include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, prostate cancer, gastric cancer, and uterine cancer, leukemia, and medulloblastoma.

[0117] The term "leukemia" refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Exemplary leukemias include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukocytic leukemia, nonleukemic leukemia, basophilic cell leukemia, and stem cell leukemia (blast cell leukemia). leukemia), bovine leukemia, chronic myelocytic leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic cell leukemia, Gross' leukemia, hairy cell leukemia, hematocytic leukemia, hematocytic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia leukemia), lymphoblastic leukemia, lymphocytic leukemia leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyelocytic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Riedercell leukemia, Schilling's leukemia These include stem cell leukemia, subleukemic leukemia, and anaplastic cell leukemia.

[0118] The term "carcinoma" refers to a malignant growth of epithelial cells that tends to infiltrate surrounding tissues and metastasize. Exemplary malignant tumors include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, adrenocortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, intrahepatic cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedocarcinoma, and the like. carcinoma, corpus carcinoma, cribriform carcinoma, armored carcinoma, carcinoma encuirasse, skin cancer, cylindrical carcinoma, columnar cell carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epidermal carcinoma, epithelial adenocarcinoma, exophytic carcinoma, ulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, giant cell carcinomagigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, lipomatous carcinoma carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinomacarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous cell carcinoma, string carcinoma, telangiectasia carcinoma These include carcinomas of the rectum, telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0119] The term "sarcoma" refers to a tumor made up of a substance similar to embryonic connective tissue and generally consists of tightly packed cells embedded in a fibrous or homogeneous material. Exemplary sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastoid sarcoma, grape sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, and the like. sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulomatous sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma (e.g., non-Hodgkin's lymphoma), immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma These include parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectatic sarcoma.

[0120] The term "melanoma" refers to a tumor arising from the melanocytic system of the skin or other organs. Melanoma includes, for example, acra-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

[0121] Other cancers include, for example, Hodgkin's Disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung tumor, primary brain tumor, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenocortical carcinoma. I. Bispecific antagonists targeting the TGF-β pathway

[0122] TGF-β is considered a major cytokine mediating immunosuppression by inducing and maintaining T regulatory cells and directly inhibiting innate and adaptive immune cells (e.g., NK cells, DCs, and T cells). TGF-β also plays a role in angiogenesis and tumor vascular stabilization, directly affecting tumors by promoting epithelial-to-mesenchymal transformation, which leads to cell migration and invasion. As a potent inducer of angiogenesis, TGF-β1 provides an important support system for solid tumors and plays a key role in tumor cell dissemination. Therefore, TGF-β status is a potent predictor of anti-PD1 / PD-L1 resistance and overall survival in various cancers.

[0123] Many cells synthesize TGF-β, and nearly all cells have specific receptors for these peptides. TGF-β1, TGF-β2, and TGF-β3 all exert their functions through the same receptor signaling system. The active form of TGF-β is a dimer, which transmits signals by forming an additional heterotetramer, consisting of serine-threonine type 1 and type 2 receptors (TGF-β RI and TGF-β RII), respectively. Recently, inhibitors of the TGF-β pathway have been developed in the form of antibodies or binding fragments directly against TGF-β1 or TGF-β1 RII, such as dominant-negative fusion protein fragments containing the extracellular domain (ECD) of TGF-β1 RII.

[0124] However, in developing these binding reagents, the inventors of the present invention discovered that antagonists containing the TGF-β1 RII ECD region exhibited unacceptable levels of proteolysis or clipping over time. To address this issue, variants of the TGF-β1 RII ECD mutations that reduce clipping within this region were developed. As further described herein, TGF-β1 RII ECDs can be combined with other binding agents targeting other checkpoint regulator pathways or angiogenesis pathways to generate bispecific antagonists that exhibit significantly reduced levels of proteolysis or clipping over time.

[0125] In one aspect, the present invention provides a bispecific antagonist that inhibits the TGFβ pathway and the PD-1 / PD-L1 checkpoint regulator pathway.

[0126] In another embodiment, the present invention provides bispecific antagonists that inhibit the TGFβ pathway and the vascular endothelial growth factor (VEGF) pathway, vascular endothelial growth factor receptor (VEFGR), or both simultaneously. A. Bispecific antagonists targeting the TGF-β and PD-1 / PD-L1 pathways

[0127] In some embodiments, the bispecific antagonist comprises a first targeting domain that specifically inhibits the TGF-β pathway and a second targeting domain that specifically binds to PD-1 or PD-L1, hi some embodiments, the first targeting domain comprises a TGF-β pathway inhibitor. TGF-β pathway inhibitors

[0128] The TGF-β pathway contains a multifunctional group of peptides that regulate cell proliferation and differentiation, migration and adhesion, extracellular matrix modification (tumor matrix and immunosuppression), angiogenesis and connective tissue proliferation, apoptosis, and other functions in many cell classes. TGF-β is considered to be the primary cytokine mediating immunosuppression by inducing and maintaining T regulatory cells and directly inhibiting innate and adaptive immune cells (e.g., NK, DC, and T cells). TGF-β directly influences tumors through angiogenesis and tumor vasculature stabilization, as well as epithelial-mesenchymal transformation, which leads to cell migration and invasion. As a potent inducer of angiogenesis, TGF-β1 provides a critical support system for solid tumors and plays a key role in tumor cell dissemination.

[0129] TGF-β status is a strong predictor of anti-PD1 / PD-L1 resistance and overall survival in various cancers. For example, high TGF-β1 levels are associated with poor response to PD1 / PD-L1 blockade in patients with metastatic urothelial carcinoma (Nature (2018) 554(7693):544-548), and high TGFβ signatures are associated with poor prognosis in 33 different cancer types (Immunity (2018) 48:812-830). Inhibiting TGFβ simultaneously with anti-PD / PD-L1 can release additional CD8 effector cells to kill tumor cells and stimulate other cell classes to increase tumor-killing capabilities.

[0130] Many cells synthesize TGF-β, and nearly all cells have specific receptors for these peptides. TGF-β1, TGF-β2, and TGF-β3 all exert their functions through the same receptor signaling system. The active form of TGF-β is a dimer that transmits signals by forming an additional heterotetramer, consisting of serine-threonine type 1 and type 2 receptors (TGF-β RI and TGF-β RII), respectively.

[0131] As used herein, a TGF-β pathway inhibitor can be in the form of an antibody or binding fragment directed against TGF-β1 or TGF-β1 RII, such as, for example, a dominant-negative fusion protein fragment comprising the extracellular domain (ECD) of TGF-β1 RII. TGF-β1 RII ECD and mutants

[0132] In some embodiments, the TGF-β pathway inhibitor comprises a TGF-β1 RII ECD. An exemplary human TGF-β1 RII ECD (wild-type) has the amino acid sequence set forth in SEQ ID NO:89. The present inventors have discovered that antagonists comprising the TGF-β1 RII ECD region exhibit unacceptable levels of proteolysis or clipping over time upon storage, as shown in Example 9. Thus, in some embodiments, the TGF-β pathway inhibitor of the present invention comprises a mutant of the human TGF-β1 RII ECD (hereinafter referred to as a "TGFBR mutant") that contains one or more modifications that reduce proteolysis or clipping. In some embodiments, the modifications include amino acid residue substitutions, amino acid residue deletions, amino acid residue insertions, or combinations of the foregoing. In some embodiments, the TGFBR mutant contains one or more substitution mutations at positions 6, 7, 13, 16, 17, 20, 22, and / or 23 of SEQ ID NO:89. In some embodiments, the one or more substitution mutations is the substitution of a non-polar amino acid residue with a polar or hydrophobic amino acid residue.

[0133] In some embodiments, the TGFBR mutant comprises one or more amino acid deletions compared to the wild-type human TGF-β1 RII ECD sequence. In some embodiments, the one or more deletions refer to a region comprising amino acid residues 1-20 of SEQ ID NO:89, and can include any residue therein. In more specific embodiments, the one or more deletions comprise a deletion of amino acid residues 1-7, 1-12, 1-13, 1-15, 1-20, 7-12, 7-13, 7-15, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, and / or 17-20 of SEQ ID NO:89. In even more specific embodiments, the one or more deletions comprise amino acid residues 6, 7, 12, 13, 15, 16, 17, or 20 of SEQ ID NO:89.

[0134] In some embodiments, the TGFBR mutant comprises (1) a deletion of one or more amino acid residues in the region of amino acid residues 1-20 of SEQ ID NO:89 and a substitution of one or more amino acid residues 6, 7, 13, 16, 17, 20, 22, and / or 23 of SEQ ID NO:89. In some embodiments, the TGFBR mutant comprises (1) a deletion of amino acid residues 1-7 of SEQ ID NO:89 and (2) an amino acid substitution of amino acid residues 13, 16, 17, 20, 22, and 23 of SEQ ID NO:89, or two amino acid substitutions of amino acid residues 16 and 17 of SEQ ID NO:89. In some embodiments, one or more polar amino acid residues are used to substitute one or more of amino acid residues 6, 7, 13, 16, 17, 20, 22, and / or 23 of SEQ ID NO:89. In some embodiments, one or more hydrophobic amino acid residues are used to replace one or more of amino acid residues 6, 7, 13, 16, 17, 20, 22 and / or 23 of SEQ ID NO:89.

[0135] In some embodiments, the TGFBR mutant comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 114-123 and 251-270 (Figures 41A-41C).

[0136] In some embodiments, the TGFBR mutant comprises an immunoglobulin heavy chain, and the immunoglobulin heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-133, 148, 150, and 271-290 (Figures 42A-42H, 44A).

[0137] In other embodiments, the TGFBR mutant comprises an immunoglobulin heavy chain, and the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO:145, SEQ ID NO:147, or SEQ ID NO:303 (Figure 43).

[0138] In other embodiments, the TGFBR mutant comprises an immunoglobulin heavy chain, and the immunoglobulin heavy chain comprises the amino acid sequence of SEQ ID NO:148, SEQ ID NO:150, or SEQ ID NOs:291-302 (Figures 44A-44D).

[0139] It should be understood that regardless of whether the TGFBR variants described herein comprise an immunoglobulin heavy chain, and whether the immunoglobulin heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-133, 145, 147, 148, 150, and 271-303 (herein "specific amino acid sequences"), the embodiment should further be construed to mean other heavy chain embodiments in combination with all heavy chain sequences derived therefrom, wherein one or more existing anti-PD-1 amino acid sequence (comprising HCDR1, HCDR2, and HCDR3 as shown in Figure 1), or one or more existing anti-PD-L1 amino acid sequence (comprising HCDR1, HCDR2, and HCDR3 as shown in Figure 3) substitutions replace the existing anti-PD1 CDR sequence or anti-PD-L1 CDR sequence present in the "specific amino acid sequence."

[0140] Similarly, with respect to the preceding paragraph, any immunoglobulin light chain described as being paired with a heavy chain set forth in the foregoing SEQ ID NOs:124-133, 145, 147, 148, 150, and 271-303 should be construed to represent other light chain embodiments in combination with all light chain sequences derived therefrom, where one or more existing anti-PD-1 amino acid sequence (including LCDR1, LCDR2, and LCDR3 as shown in Figure 1) or one or more existing anti-PD-L1 amino acid sequence (including LCDR1, LCDR2, and LCDR3 as shown in Figure 3) substitutions replace the existing anti-PD1 CDR sequences or anti-PD-L1 CDR sequences present in the "particular amino acid sequence."

[0141] Similarly, it should be understood that regardless of whether the TGFBR variants described herein comprise an immunoglobulin heavy chain, and whether the immunoglobulin heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-133, 145, 147, 148, 150, and 271-303 (herein "specific amino acid sequences"), the embodiment should further be construed to mean other heavy chain embodiments in combination with all light chain sequences derived therefrom, wherein the "specific amino acid sequence" replaces a different anti-PD1 heavy chain variable region (HCVR) amino acid sequence with an existing anti-PD1 HCVR, wherein the anti-PD1 HCVR is selected from the group consisting of SEQ ID NOs: 29, 31, 33, 35, 37, and 39, as shown in Figures 2A-2B, or wherein the "specific amino acid sequence" replaces a different anti-PD-L1 HCVR with an existing anti-PD-L1 HCVR amino acid sequence, wherein the anti-PD-L1 The HCVR is selected from the group consisting of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84 and 86 as shown in Figures 4A-4C.

[0142] Similarly, with respect to the preceding paragraph, any immunoglobulin light chain described as being paired with the aforementioned heavy chain in a "particular amino acid sequence" should be construed to represent other light chain embodiments in combination with all light chain sequences derived therefrom, where in the "particular amino acid sequence" an existing anti-PD1 light chain variable region (LCVR) amino acid sequence replaces a different anti-PD1 LCVR, and the anti-PD1 LCVR is selected from the group consisting of SEQ ID NOs: 30, 32, 34, 36, 38, and 40 shown in Figures 2A-2B; or where in the "particular amino acid sequence" an existing anti-PD-L1 LCVR amino acid sequence replaces a different anti-PD-L1 LCVR, and the anti-PD-L1 LCVR is selected from the group consisting of SEQ ID NOs: 73, 75, 77, 79, 81, 83, 85, and 87 shown in Figures 4A-4C.

[0143] In some embodiments, the TGF-β pathway inhibitor is fused to the carboxy terminus of an IgG in a bispecific anti-tumor antagonist, for example, as shown in Figures 5A-5B, 8A-8C, and 20A-20B. Alternatively, the TGF-β1 RII ECD can be fused to the amino terminus of an IgG in a bispecific anti-tumor antagonist. In yet another embodiment, the TGF-β1 RII ECD is inserted within the IgG Fc receptor (i.e., the CH2 or CH3 region) of an IgG, for example, as shown in Figure 5B.

[0144] In some embodiments, anti-TGF-β1, anti-TGF-β1 RII, or variable region fragments thereof can be used in place of the TGF-β1 RII ECD. Exemplary anti-TGF-β1 antibodies are as described in U.S. Patent Nos. 7,067,637, 7,494,651, 7,527,791, and 7,619,069. Exemplary anti-TGF-β1 RII antibodies are as described in U.S. Patent No. 7,579,186. Alternatively, the TGF-β1 RII ECD can be replaced with one or more anti-TGF-β1 and / or anti-TGF-β1 RII peptide inhibitors. An exemplary TGF-β1 peptide inhibitor is KRIWFIPRSSWYERA (SEQ ID NO:155).

[0145] Anti-PD-1 antibodies and anti-PD-1 antibody fragments In some embodiments, the bispecific TGFβ pathway antagonist comprises an anti-PD-1 antibody or antibody fragment. In one embodiment, the PD-1 inhibitor used in the present invention is an antibody, or an antigen-binding portion thereof, comprising an immunoglobulin heavy chain complementarily determining region 1 (CDR1) sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 9, and 12; an immunoglobulin heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 10, and 13; an immunoglobulin heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 6, 8, 11, and 14; an immunoglobulin light chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21-23, and 26; an immunoglobulin light chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 16, 19, 24, and 27; or an immunoglobulin light chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 17, 20, 25, and 28.

[0146] In another embodiment, the PD-1 inhibitor (e.g., second targeting domain) used in the invention is an antibody, or antigen-binding portion thereof, comprising an immunoglobulin heavy chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 9, and 12; an immunoglobulin heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 2, 5, 10, and 13; an immunoglobulin heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 3, 6, 8, 11, and 14; an immunoglobulin light chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21-23, and 26; an immunoglobulin light chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 16, 19, 24, and 27; or an immunoglobulin light chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 17, 20, 25, and 28.

[0147] In another embodiment, the PD-1 inhibitor used in the present invention comprises an immunoglobulin heavy chain variable region (HCVR) having at least 80%, 85%, 90%, 95%, or 99% identity to an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, 35, 37, and 39, an immunoglobulin light chain variable region (LCVR) having at least 80%, 85%, 90%, 95%, or 99% identity to an LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 32, 34, 36, 38, and 40, or both of the foregoing.

[0148] In another embodiment, the PD-1 inhibitor used in the present invention comprises an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs:29, 31, 33, 35, 37, and 39, an LCVR amino acid sequence selected from the group consisting of SEQ ID NOs:30, 32, 34, 36, 38, and 40, or both of the foregoing.

[0149] In one embodiment, the PD-1 inhibitor comprises an HCVR having at least 80%, 85%, 90%, 95%, or 99% identity to the HCVR amino acid sequence of SEQ ID NO:39, an LCVR having at least 80%, 85%, 90%, 95%, or 99% identity to the LCVR amino acid sequence of SEQ ID NO:40, or both of the foregoing.

[0150] In another embodiment, the PD-1 inhibitor has an immunoglobulin heavy chain variable region comprising: (1) an HCDR1 of SEQ ID NO:12, an HCDR2 of SEQ ID NO:13, and an HCDR3 of SEQ ID NO:14; (2) an HFR1 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO:196, an HFR2 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO:190, an HFR3 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO:201, and an HFR4 having at least 80%, 85%, or 90% identity to the amino acid sequence of SEQ ID NO:187; and (1) an LCDR1 of SEQ ID NO:26, an LCDR2 of SEQ ID NO:27, and an LCDR3 of SEQ ID NO:28; The immunoglobulin light chain variable region includes an LFR1 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:202, an LFR2 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:203, an LFR3 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:204, and an LFR4 having at least 80%, 85% or 90% identity to the amino acid sequence of SEQ ID NO:205.

[0151] In another embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-1 inhibitor comprising an immunoglobulin heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:92 or SEQ ID NO:113, an immunoglobulin light chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:93, or both of the foregoing. In a more specific embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-1 inhibitor comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:92 or SEQ ID NO:113, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:93, or both of the foregoing.

[0152] In another embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-1 inhibitor comprising an immunoglobulin heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:94 or SEQ ID NO:172, an immunoglobulin light chain having at least 80%, 85%, 90%, 95%, or 99% identity to the LCVR amino acid sequence of SEQ ID NO:95, or both of the foregoing. In a more specific embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-1 inhibitor comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:94 or SEQ ID NO:172, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:95, or both of the foregoing. In another embodiment, the PD-1 / TGF-β1 anti-tumor antagonist comprises a TGF-β1 RII ECD inserted within the CH3 ring, as shown in Figure 5B.

[0153] In one embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-1 inhibitor comprising a heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:151, a light chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:152, or both of the foregoing. In a more specific embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-1 inhibitor comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:151, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:152, or both of the foregoing.

[0154] Other PD-1 targeted antagonists include, for example, nivolumab (BMS-936558, MDX-1106, OPDIVO 商標 ), human immunoglobulin G4 (IgG4) mAb (Bristol-Myers Squibb), pembrolizumab (MK-3475, lambrolizumab, Keytruda 商標 Anti-PD-1 antibodies include, for example, ABCAM (AB137132), BIOLEGEND (AB137133), humanized immunoglobulin G4 (IgG4) mAb (Merck), pidilizumab (CT-011) (Medivation), and AMP-224 (Merck). 商標 (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4). The anti-PD-1 targeting anti-tumor antagonist can comprise any of the HCVRs, LCVRs, and / or CDRs derived from any of the anti-PD-1 antibodies described herein, including those shown in Figures 1-2.

[0155] Anti-PD-L1 antibodies and anti-PD-L1 antibody fragments In some embodiments, the bispecific TGF-β1 pathway antagonist further comprises an anti-PD-L1 antibody or antibody fragment. In one embodiment, the PD-L1 inhibitor used in the invention is an antibody, or an antigen-binding portion thereof, comprising an immunoglobulin heavy chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 41, 44, 50, and 53; an immunoglobulin heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 42, 45, 47, 49, 51, and 54; an immunoglobulin heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 43, 46, 48, 52, and 55; an immunoglobulin light chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 56, 59, 63, 66, and 69; an immunoglobulin light chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 57, 60, 64, 67, and 70; or an immunoglobulin light chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 58, 61, 62, 65, 68, and 71.

[0156] In another embodiment, the PD-L1 inhibitor (e.g., second targeting domain) used in the invention comprises an immunoglobulin heavy chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 41, 44, 50, and 53; an immunoglobulin heavy chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 42, 45, 47, 49, 51, and 54; an immunoglobulin heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 43, 46, 48, 52, and 55; an immunoglobulin light chain CDR1 sequence selected from the group consisting of SEQ ID NOs: 56, 59, 63, 66, and 69; an immunoglobulin light chain CDR2 sequence selected from the group consisting of SEQ ID NOs: 57, 60, 64, 67, and 70; or an immunoglobulin heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 58, 59, 63, 66, and 69. NOs: An antibody, or antigen-binding portion thereof, comprising an immunoglobulin light chain CDR3 sequence selected from the group consisting of 58, 61, 62, 65, 68, and 71.

[0157] In another embodiment, the PD-L1 inhibitor used in the invention comprises an immunoglobulin HCVR having at least 80%, 85%, 90%, 95%, or 99% identity to an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84, and 86, an immunoglobulin LCVR having at least 80%, 85%, 90%, 95%, or 99% identity to an LCVR amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 75, 77, 79, 81, 83, 85, and 87, or both of the foregoing. In another embodiment, the PD-L1 inhibitor used in the invention comprises an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs:72, 74, 76, 78, 80, 82, 84, and 86; an LCVR amino acid sequence selected from the group consisting of SEQ ID NOs:73, 75, 77, 79, 81, 83, 85, and 87; or both of the foregoing.

[0158] In one embodiment, the PD-L1 inhibitor comprises an HCVR having at least 80%, 85%, 90%, 95%, or 99% identity to the HCVR amino acid sequence of SEQ ID NO: 86, an LCVR having at least 80%, 85%, 90%, 95%, or 99% identity to the LCVR amino acid sequence of SEQ ID NO: 87, or both of the foregoing. In a more specific embodiment, the PD-L1 inhibitor comprises an HCVR having the amino acid sequence of SEQ ID NO: 86, an LCVR having the amino acid sequence of SEQ ID NO: 87, or both of the foregoing.

[0159] In another embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-L1 inhibitor comprising an immunoglobulin heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:94 or SEQ ID NO:172, an immunoglobulin light chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:95, or both of the foregoing. In a more specific embodiment, the TGF-β1 anti-tumor antagonist comprises a PD-L1 inhibitor comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:94 or SEQ ID NO:172, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:95, or both of the foregoing.

[0160] In another embodiment, the PD-L1 / TGF-β1 anti-tumor antagonist comprises a TGF-β1 RII ECD inserted within the CH3 ring, as shown in Figure 5B.

[0161] In certain embodiments, the TGF-β1 anti-tumor antagonist comprises a PD-L1 inhibitor comprising an immunoglobulin heavy chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 153, an immunoglobulin light chain having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 154, or both of the foregoing. In more specific embodiments, the TGF-β1 anti-tumor antagonist comprises a PD-L1 inhibitor comprising an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 153, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO: 154, or both of the foregoing.

[0162] Other anti-PD-L1 targeted antagonists include anti-PD-L1 antibodies such as atezolizumab (MPDL3280A, RG7446, Tecentriq), a fully humanized IgG1 mAb (Genentech / Roche), BMS-936559 (MDX-1105), a fully human IgG4 mAb (Bristol-Myers Squibb), durvalumab (MEDI4736, Imfinzi), a human IgG1 antibody (Medimmune / AstraZeneca), and avelumab (MSB0010718C, Bavencio), a fully human IgG4 monoclonal antibody (Merck, EMD Serono). Anti-PD-L1 targeted anti-tumor antagonists may comprise any of the HCVRs, LCVRs, and / or CDRs derived from any of the anti-PD-L1 antibodies described herein, including those shown in Figures 3-4.

[0163] B. Bispecific Antagonists Targeting TGF-β1 and Angiogenesis Pathways In another aspect, the bispecific anti-tumor antagonist of the invention comprises a first targeting domain that specifically binds TGFβ1 or TGFβ1 RII and a second targeting domain that specifically binds VEGF-A, VEGFR, Ang1, Ang2, Tie2R, or a combination of the foregoing. In these embodiments, any of the TGFβ1- or TGFβ1 RII-binding fragments described above can be used in conjunction with the second targeting domain.

[0164] Angiogenesis pathway Angiogenesis (the development of new blood vessels from pre-existing vessels) is necessary for tumor growth and migration. Inhibition of angiogenesis is a potentially valuable strategy for the treatment of diseases (e.g., cancer), where the course of the disease (e.g., metastasis) depends on angiogenesis. Inhibition of angiogenesis causes tumor cell death, which can deliver tumor antigens to the host's antigen presentation pathway. Angiogenesis pathway inhibitors can be in the form of, for example, antibodies, variable region fragments, or dominant-negative fusion protein fragments.

[0165] 1. VEGF / VEGFR pathway The major VEGF pathway is mediated by the transmembrane tyrosine kinase VEGF R2. Various isoforms of VEGF (particularly VEGF-A) can bind to VEGF-R2 and be dimerized and activated by phosphorylation by various downstream tyrosine kinases.

[0166] In some embodiments, antagonists of the present invention include VEGF pathway antagonists that bind to VEGF-A or its receptor VEGFR-2, and thus, after binding, block or inhibit VEGFR-2 activation by VEGF-A.

[0167] In one embodiment, the TGFβ pathway antagonist further comprises a VEGF pathway antagonist in the form of a dominant-negative VEGFR antagonist directed against the extracellular domain (ECD) of human VEGF receptor 1 or 2. In a specific embodiment, the TGFβ pathway antagonist comprises aflibercept (also known as Zaltrap), which comprises a recombinant fusion protein of a VEGF-A binding region, the recombinant fusion protein being a fusion of the extracellular domains of human VEGF receptors 1 and 2 with a human IgG1 Fc portion. VEGFR ECDs (e.g., aflibercept) function as soluble receptor decoys for VEGF-A.

[0168] In one embodiment, the bispecific anti-tumor antagonist comprises a first targeting domain comprising a TGFβ pathway inhibitor and a second targeting domain that specifically binds to VEGF-A, wherein the second targeting domain comprises aflibercept. A suitable source of aflibercept comprises the amino acid sequence as shown in SEQ ID NO:88. In some embodiments, the bispecific anti-tumor antagonist comprises a modified immunoglobulin heavy chain, the modified immunoglobulin heavy chain comprising an amino-terminal aflibercept region as shown in FIG. 8C, the carboxy terminus of which binds to TGF-β1 RII ECD or a TGF-β1 RII ECD variant via the IgG1 or IgG4 Fc receptor (Bi-ZB-1) chain. In one embodiment, the modified immunoglobulin heavy chain has the amino acid sequence of SEQ ID NO:105. Alternatively, the TGF-β1 RII ECD or TGF-β1 RII ECD variant can be positioned at the amino terminus and the aflibercept domain can be positioned at the carboxy terminus.

[0169] In another embodiment, the TGFβ pathway antagonist further comprises a VEGF pathway antagonist, wherein the VEGF pathway antagonist comprises an anti-VEGF A or anti-VEGFR2 variable region sequence, such as those derived from bevacizumab. 商標Bevacizumab A.4.6.1 is a humanized antibody containing human IgG1 framework regions (FRs) and antigen-binding complementarity-determining regions derived from the murine anti-hVEGF monoclonal antibody A.4.6.1, which blocks the binding of human VEGF-A to VEGFR1 and VEGFR-2. Approximately 93% of the bevacizumab amino acid sequence (including most of the framework regions) is derived from human IgG1, and approximately 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated. In one embodiment, the bevacizumab HCVR used in the present invention has the amino acid sequence set forth in SEQ ID NO:90, and the bevacizumab LCVR has the amino acid sequence set forth in SEQ ID NO:91.

[0170] In some embodiments, the bevacizumab / avastin antibody or fragment thereof as described in U.S. Patent No. 7,575,893 may contain amino acid substitutions. Exemplary amino acid substitutions include, but are not limited to, E1Q, E6Q, L11V, Q13K, L18V, R19K, A23K, or combinations thereof. In one embodiment, the mutated HCVR of bevacizumab used in the present invention has the amino acid sequence shown in SEQ ID NO:96, which can couple with the LCVR of SEQ ID NO:91.

[0171] In one embodiment, the bispecific anti-tumor antagonist comprises a first targeting domain that specifically binds TGFβ1, such as a TGFβ1 RII ECD (or comprising an anti-TGFβ1 or anti-TGFβ1 RII variable region), and a second targeting domain that specifically binds VEGFA or VEGFR2, such as bevacizumab or any other anti-VEGF or anti-VEGFR2 antibody, variable region fragment thereof, or functionally active mutant fragment thereof.

[0172] In certain embodiments, the bispecific anti-tumor TGF-β / VEGF-VEGFR2 antagonist comprises an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:102, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:103, or both of the foregoing, e.g., Bi-AB-1 (FIG. 8A), which comprises wild-type bevacizumab variable region sequences.

[0173] In another embodiment, the bispecific anti-tumor TGF-β antagonist comprises an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO:104, an immunoglobulin light chain having the amino acid sequence of SEQ ID NO:103, or both of the foregoing, e.g., Bi-A1B-1 (Figure 8B), which comprises a mutated bevacizumab variable region sequence.

[0174] Other anti-VEGF or anti-VEGFR antibodies or fragments thereof include ranibizumab (brand name Lucentis) 商標 bevacizumab), monoclonal antibody fragments derived from the same parent murine antibody as bevacizumab, the G6 or B20 series antibodies (e.g., G6-23, G6-31, B20-4.1) described in U.S. Patent Publication Nos. 2006 / 0280747, 2007 / 0141065, and / or 2007 / 0020267, antibodies described in U.S. Patent Nos. 7,297,334, 7,060269, 6,884,879, 6,582,959, 6,703,020, 6,054,297, and antibodies described in U.S. Patent Publication Nos. 2007 / 059312, 2006 / 009360, 2005 / 0186208, 2003 / 0206899, 2003 / 0190317, and 2003 / 0203409.

[0175] An exemplary anti-VEGFR-2 antibody antagonist is the humanized IgG1 monoclonal antibody ramucirumab, which binds to the extracellular domain of VEGFR-2, thereby blocking its interaction with VEGF-A. Other anti-VEGFR-2 antibodies are described in U.S. Patent Nos. 7,498,414, 6,448,077, and 6,365,157.

[0176] In some embodiments, the anti-tumor antagonist can further comprise one or more small molecule antagonists of the VEGF pathway, such as multikinase inhibitors of VEGFR-2, including, for example, sunitinib, sorafenib, cediranib, pazonpanib, and nintedanib. 2. Ang-Tie2R pathway

[0177] In some embodiments, the bispecific antagonist comprises a targeting domain comprising at least one Tie2 receptor binding antagonist. The Tie2 tyrosine kinase receptor binding antagonist binds to the Tie2 tyrosine kinase receptor or one of its ligands (i.e., Ang1, Ang2, Ang3, and Ang4), and thus, after binding, blocks or inhibits activation of the Tie2 tyrosine kinase receptor via one or more ligands. Like VEGF, angiopoietin-2 (Ang2) plays an important role in tumor angiogenesis. ANG2 and VEGF act together to prevent antigen presentation by dendritic cells and macrophages, promoting T cell proliferation and proliferation. reg Enhances aggregation and T eff Blockade of VEGF and Ang2 has previously been shown to improve survival in the mouse breast cancer model Tg MMTV-PyMT, and the addition of anti-PD-1 can further improve the response.

[0178] Tie2 tyrosine kinase receptor-binding antagonists used in the present invention can include antibody fragments, peptide inhibitors, dominant-negative peptides, and small molecule drugs in isolated form or as part of a fusion protein or conjugate. In one embodiment, the Tie2 receptor-binding antagonist is trebananib, an inhibitory peptide derived from TBN-P. In a specific embodiment, the inhibitory peptide comprises the amino acid sequence AQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 157). In another embodiment, the Tie2 receptor-binding antagonist comprises SEQ ID NO: 158 (TBN-P-IgG).

[0179] Other peptide inhibitors of Tie2 activation (including Ang-2 inhibitors) that can be used in the present invention include A-11 (Compugen), which contains the amino acid sequence: ETFLSTNKLENQ (SEQ ID NO:167), CVX-060 peptide: QK(Ac)YQPLDEK(Ac)DK(0P)TLYDQFMLQQG (SEQ ID NO:168, Pfizer), CVX-037 peptide: (DFB)TNFMPMDDLEK(0P)RLYEQFILQQG (SEQ ID NO:169, Pfizer), and CGEN-25017 (Compugen). Other peptide inhibitors of Tie2 activation are described in U.S. Patent No. 7,138,370.

[0180] Antibody inhibitors that inhibit Tie2 activation (and / or angiopoietin-2) for use in the present invention include AMG-780 (Amgen), MEDI-3617 (MedImmune / AstraZeneca), DX-2240 (Dyax / Sanofi-Aventis), REGN-910 (Sanofi / Regeneron), RG7594 (Roche), LC06 (Roche), TAvi6 (Roche), AT-006 (Roche / Affitech). Other Tie2 receptor-binding antibody antagonists and their antibody binding sequences are described in U.S. Patent Nos. 7,521,053, 7,658,924, and 8,030,025, and U.S. Patent Publication Nos. 2013 / 0078248, 2013 / 0259859, and 2015 / 0197578.

[0181] Tie2 binding antagonists for use in the present invention can further include small molecule inhibitors of CGI-1842 (CGI Pharmaceuticals), LP-590 (Locus Pharmaceuticals), ACTB-1003 (Act Biotech / Bayer AG), CEP-11981 (Cephalon / Teva), MGCD265 (Methylgene), Regorafenib (Bayer), Cabozantinib / XL-184 / BMS-907351 (Exelixis), Foretnib (Exelixis), MGCD-265 (MethylGene Inc.).

[0182] In some specific embodiments, the bispecific checkpoint modulator antagonist is a full-length antibody that binds to human PD-1 or PD-L1 with one of its two binding arms (an HC / LC pair) and to a different antigen (or epitope) with its second binding arm (a different HC / LC pair). In these embodiments, the bispecific antibody has two different antigen-binding arms (both with specificity and CDR sequences), and each of the antigens to which it is linked is monovalent.

[0183] In some embodiments, the bispecific checkpoint modulator antagonist is a full-length antibody that binds to human PD-1 and / or PD-L1 with two binding arms (HC / LC pair), respectively. In these embodiments, the bispecific checkpoint modulator antagonist has two identical antigen-binding arms (with the same specificity and identical CDR sequences), and each of the antigens to which it is linked is bivalent.

[0184] Immunoglobulin and non-immunoglobulin scaffolds The bispecific anti-tumor antagonists of the present invention can be constructed using immunoglobulin or non-immunoglobulin scaffolds as described herein. In some embodiments, the bispecific antagonists of the present invention are constructed using an immunoglobulin scaffold of IgG1, IgG2, or IgG4, comprising CH1, CH2, and / or CH3 regions. The use of an IgG1 scaffold is preferred for cancer treatment when the target is an antigen-presenting cell capable of antibody-dependent cell-mediated cytotoxicity (ADCC). The use of an IgG4 scaffold allows for antigen targeting when antigen binding alone is sufficient to produce the desired therapeutic effect. IgG4 antagonists prevent the defective effector functions associated with IgG1 antibodies, including FcγR binding and complement activation.

[0185] Preferably, the first and second targeting domains are present in a humanized IgG1 or IgG4 scaffold. The second targeting domain can be fused to the carboxy terminus of the IgG1 or IgG4 scaffold. Furthermore, the IgG1 or IgG4 scaffold can have an N297A or K447A amino acid substitution. In some embodiments, the first targeting domain can comprise one or more framework regions, wherein the framework regions comprise one or more amino acid substitutions selected from the group consisting of E1Q, E6Q, L11V, Q13K, L18V, R19K, A23K, or any combination thereof. In other embodiments, one or more amino acid residues in the IgG1 or IgG4 scaffold are deglycosylated or mutated to generate aglycosylated variants. Exemplary immunoglobulin scaffolds for the bispecific molecules described herein can be selected from the group consisting of SEQ ID NOs: 159-166 and 173-175.

[0186] Any of the antibodies or antagonists may be constructed in the form of a monoclonal antibody, chimeric antibody, humanized antibody, scFv, or multispecific antibody. Furthermore, any of the antibody antagonists described herein may comprise multiple binding specificities targeting PD-1, PD-L1, VEGF, VEGFR, angiopoietin, and / or Tie2R. Any of the antibody antagonists may also be designed to target multiple epitopes on a particular target. And, in some embodiments, the checkpoint antagonist and / or angiogenesis specificity may be comprised in the form of a dominant-negative fusion protein, e.g., corresponding to the extracellular domain (ECD) of a receptor.

[0187] The HCVRs and LCVRs described herein may be linked to naturally occurring or non-naturally occurring or mutated Fc regions, such as a non-effector (IgG1 N297A / G) or most non-effector Fc (e.g., human IgG2 or IgG4), or alternatively, to enhance Fc binding to one or more activating Fc receptors (FcγRI, FcγRIIa, or FcγRIIIa) to activate T cells in the tumor environment. reg Thus, in certain embodiments, the HCVRs and LCVRs of anti-PD-1, anti-PD-L1, and / or anti-VEGF antibodies described herein can be linked to an Fc region that includes one or more modifications, generally to alter one or more functional properties of the antibody (e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity). The antibodies described herein can also be chemically modified (e.g., the antibody can be heterologously linked to one or more chemical moieties) or their glycosylation can be altered to alter one or more functional properties of the antibody. More specifically, in certain embodiments, antibodies of the invention can include modifications to the Fc region to generate Fc variants with (a) improved or decreased antibody-dependent cellular cytotoxicity (ADCC), (b) improved or decreased complement-mediated cytotoxicity (CDC), (c) improved or decreased C1q affinity, and / or (d) improved or decreased Fc affinity compared to the parent Fc. These Fc region variants generally include at least one amino acid modification in the Fc region. Combination amino acid modifications are particularly desirable. By way of example, the variant Fc region can include, for example, two, three, four, five, etc. substitutions at particular Fc region sites as determined herein.

[0188] For applications requiring complete avoidance of effector function (e.g., where antigen binding alone is sufficient to produce the desired therapeutic effect and effector function only results in undesirable side effects (or increases the risk of such side effects)), IgG4 antibodies can be used, or antibodies or fragments or substantial portions thereof can be engineered lacking the Fc region, or the Fc can be mutated to completely eliminate glycosylation (e.g., N297A). Alternatively, composite constructs of human IgG2 (CH1 and hinge regions) and human IgG4 (CH2 and CH3 regions) can be generated that lack effector function and the ability to bind to FcγRs (e.g., IgG2) and activated complement (e.g., IgG4). When an IgG4 constant region is used, it is generally preferred to include the S228P substitution, which mimics the hinge sequence of IgG1, thereby stabilizing the IgG4 molecule and reducing Fab arm exchange between the therapeutic antibody and endogenous IgG4 in treated patients.

[0189] In certain embodiments, anti-PD-1, anti-PD-L1, anti-VEGF, anti-angiopoietin, and / or anti-Tie2R antibodies or fragments thereof can be modified to improve their biological half-life. A variety of different methods can be used, including, for example, increasing the binding affinity of the Fc region to FcRn. In one embodiment, the CH1 or CL region of the antibody is modified to contain a salvage receptor binding epitope taken from two loops in the CH2 region of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022. Residues in the Fc region are coded as in the EU index numbering system. The sequence variants disclosed herein are provided by reference to residue numbering, with the amino acid following the number in place of the naturally occurring amino acid, and optionally the naturally occurring residue preceding the position. When multiple amino acids can be present at a particular position, e.g., when the sequence differs between naturally occurring isotypes, or when the position can be replaced by multiple mutations, they are separated by slashes (e.g., "X / Y / Z").

[0190] Exemplary Fc variants that enhance binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that enhance Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8):6213-6216; Hinton et al., 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al., (20 01) J.Biol.Chem., 276(9):6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 4 These include 34F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H (Dall'Acqua et al., (2002) J. Immunol., 169:5171-5180; Dall'Acqua et al., (2006) J. Biol. Chem., 281:23514-23524; and U.S. Pat. No. 8,367,805).

[0191] For example, modifications of specific conserved residues in IgG Fc (1253, H310, Q311, H433, N434), such as the N434A mutant (Yeung et al., (2009) J. Immunol. 182:7663), have been proposed (WO98 / 023289) as a way to improve FcRn affinity to increase antibody half-life in circulation. A combined Fc mutant containing M428L and N434S has been shown to improve FcRn binding and increase serum half-life by up to 5-fold (Zalevsky et al., (2010) Nat. Biotechnol. 28:157). A combined Fc mutant containing T307A, E380A, and N434A modifications also extends the half-life of an IgG1 antibody (Petkova et al., (2006) Int. Immunol. 18:1759). Furthermore, combined Fc variants containing the mutations M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M, and M428L-N434S have also been shown to extend half-life (U.S. Patent Publication No. 2006 / 173170). Also, a combined Fc variant containing M252Y, S254T, and T256E has been reported to improve half-life by nearly four-fold (Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514). Homodimers and heterodimers

[0192] One of the challenges in efficiently producing bispecific antibody preparations is mismatching between light and heavy chains when chains of different binding specificities are co-expressed. Table 1 shows several amino acid substitution options for overcoming heavy chain mismatching of different binding specificities, which "enforce" or preferentially promote correct binding between the desired heavy chains. Any method that prevents or reduces mismatching between heavy chains can be used in producing bispecific anti-tumor antagonists according to the present invention.

[0193] The "knobs-into-hole" (KiH) approach relies on modifications at the interface of the two CH3 regions, where most interactions occur. This generally acts as a key to introduce bulky residues into the CH3 region of one antibody heavy chain. A "hole" is created in the other heavy chain, which, like the key, can accommodate the bulky residue. The resulting heterodimeric Fc portion can be further stabilized by artificial disulfide bonds.

[0194] An alternative approach is based on charged residues with ionic exchange or spatial complementarity. This involves altering the charge polarity of the CH3 interface, thereby supporting favorable attractive interactions and heterodimer formation while retaining the hydrophobic core, while simultaneously inhibiting unfavorable repulsive charge interactions for homodimerization. See Table 1. The amino acid codes in Table 1 can be applied to the heavy chain amino acid sequences of the antibodies described herein according to the Kabat numbering scheme.

[0195] Alternatively, bispecific molecules of the present invention can be constructed using non-immunoglobulins containing a leucine zipper (LZ) region. Leucine zippers are a common three-dimensional structural motif in proteins, commonly found as part of the DNA-binding domains of various transcription factors. A single LZ contains 4-5 leucine residues approximately every 7 residues, forming an amphipathic α-helix with a hydrophobic region along one side. In certain embodiments, a heterodimeric protein scaffold comprises an LZ derived from the c-jun transcription factor and an LZ derived from the c-fos transcription factor. Although c-jun can form jun-jun homodimers and c-fos cannot, the formation of jun-fos heterodimers is more favorable than the formation of jun-jun homodimers.

[0196] The leucine zipper region can replace the CH2-CH3 sequence of the protein scaffold or can be placed at the carboxy terminus of the two heavy chains of the bispecific anti-tumor antagonist. In the latter case, a furin cleavage site can be introduced between the CH3 carboxy terminus and the amino terminus of the leucine zipper. When the heavy and light chains of the bispecific anti-tumor antagonist are coexpressed in an appropriate mammalian cell expression system, this can facilitate furin-mediated cleavage of the leucine zipper following the heterodimerization step (see Wranik et al., J. Biol. Chem., 287(5):43331-43339, 2012). [Table 1]

[0197] The amino acid codes in Table 1 follow the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein. The mutations described in Table 1 can be applied to the sequences of any immunoglobulin IgG1 heavy chain and other immunoglobulin classes and subclasses (or isotypes) thereof, whether published or not.

[0198] When the heavy and light chains of a bispecific antibody are co-expressed, a light chain with a particular binding specificity can also mismatch with a heavy chain with a different binding specificity. Thus, in certain embodiments, the heavy chain portion, light chain portion, or bipartite portion can be modified relative to the "wild-type" antibody chain from which it is derived to prevent or reduce mismatches between the heavy chain constant regions and between the light chain constant regions and their corresponding heavy chains.

[0199] The problem of light chain mismatching can be solved in several ways. In some embodiments, spatially complementary mutations and / or disulfide bonds can be introduced at the two VL / VH junctions. In other embodiments, mutations can be introduced by ionic or electrostatic interactions. In some embodiments, light chain mismatching can be prevented or reduced by using a first arm with a S183E mutation in the heavy chain CH1 region and a S176K mutation in the light chain CL region. A second arm can contain a S183K mutation in the heavy chain CH1 region and a S176E mutation in the light chain CL region. In other embodiments, a "cross-Mab" approach is used, in which one arm of the bispecific anti-tumor antagonist (e.g., Fab) remains unchanged, while one or more regions of the light chain located at the heavy chain:light chain junction are exchanged for one or more regions of the heavy chain in another arm containing a different binding specificity.

[0200] Methods for preventing heavy and light chain mismatches in immunoglobulin sequences (including specific mutations) disclosed above are described in U.S. Patent Publication Nos. 2014 / 0243505 and 2013 / 0022601. Conjugates

[0201] In certain embodiments, the anti-tumor antagonist of the present invention is chemically coupled to one or more peptides and / or small molecule drugs. The peptides or small molecule drugs can be the same or different. The peptides or small molecule drugs can be linked, for example, to reduced SH groups and / or carbohydrate side chains. Methods for preparing covalent or non-covalent conjugates of peptides or small molecule drugs and antibodies are known in the art, and any of these known methods can be used.

[0202] In some embodiments, peptide or small molecule drugs are linked to the hinge region of a reduced antibody component via disulfide bond formation. Alternatively, these drugs can be linked using heterobifunctional crosslinkers such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). General techniques for such coupling are known in the art. In some embodiments, peptide or small molecule drugs are coupled via carbohydrate moieties in the antibody Fc region. The carbohydrate groups can be used to enhance loading of the same reagents coupled to thiol groups, or the carbohydrate groups can be used to attach different therapeutic or diagnostic agents. Methods for coupling peptide inhibitors or small molecule drugs to antibodies via their carbohydrate moieties are known to those skilled in the art. For example, in one embodiment, the method involves reacting an antibody component bearing an oxidized carbohydrate moiety with a vector polymer bearing at least one free amine function. This reaction results in an initial Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate. Exemplary methods for coupling small molecule drugs and peptides to antibodies are described in U.S. Patent Publication No. 2014 / 0356385.

[0203] Preferably, the anti-tumor antagonists herein retain some of the specific desirable properties and pharmacokinetic characteristics of antibodies, including desirable in vitro and in vivo stability (e.g., long half-life and shelf-life stability), efficient delivery to desired target cells, improved affinity for the conjugate, desirable improved antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, and reduced renal clearance or elimination rates. Thus, the design of anti-tumor antagonists can take into account attention to size and the need for specific constant region effector functions.

[0204] The bispecific anti-tumor antagonists described herein may be any of the following: 50kD to 300kD, 50kD to 250kD, 60kD to 250kD, 80kD to 250kD, 100kD to 250kD, 125kD to 250kD, 150kD to 250kD, 60kD to 225kD, 75kD to 225kD, 100kD to 225kD, 125kD to 225kD, 150kD to 225kD, 60kD to 200kD, 75kD to 20 ... The range may have a size in the ranges of 125 kD to 200 kD, 150 kD to 200 kD, 60 kD to 150 kD, 75 kD to 150 kD, 100 kD to 150 kD, 60 kD to 125 kD, 75 kD to 125 kD, 75 kD to 100 kD, or any range including any combination of the integers listed in the ranges described above, or any range specifically specified in any combination of the integers in the ranges described above. kit

[0205] The present invention provides kits comprising checkpoint modulator antagonists or anti-tumor antagonists of the invention. In some embodiments, the kits include one or more bispecific immune checkpoint modulators, where the bispecific immune checkpoint modulators include at least one TGFβ pathway inhibitory domain. In some embodiments, the kits include other reagents, including secondary antibodies used for detection and other human antibodies described herein (e.g., human antibodies with complement activity that bind to different epitopes of the same antigen). The kits generally include a label with instructions indicating the intended use of the contents of the kit. This so-called label includes any written or recorded material provided with or accompanying the kit.

[0206] II. Methods of Use of Antitumor Antagonists The anti-tumor antagonists of the present invention have many in vitro and in vivo utilities, including, for example, enhancing immune responses and treating cancer, infectious diseases, or autoimmune diseases.

[0207] The anti-tumor antagonists of the present invention can be administered to cells in culture in vitro or ex vivo, or to human individuals, e.g., in vivo, to enhance immune responses in various diseases. Accordingly, the present specification provides a method for modifying an individual's immune response by administering an antibody or antigen-binding fragment described herein to an individual to enhance, stimulate, or upregulate the immune response in the individual. Preferably, the individual includes a human patient to whom an immune response is to be enhanced. The method is particularly suitable for treating patients with diseases that can be treated by enhancing the immune response (e.g., a T cell-mediated immune response). The method is particularly suitable for treating cancer or chronic infectious diseases in vivo. For example, to enhance antibody-specific immune responses, the anti-tumor antagonist can be administered together with a desired antigen or an antigen already present in the individual to be treated (e.g., an individual with a tumor or virus). When the anti-tumor antagonist is administered together with another agent, the two can be administered separately or simultaneously.

[0208] In some embodiments, the checkpoint regulator antagonist in the above methods is an anti-PD-1 antibody, an anti-PD-L1 antibody, a VEGF antibody, a VEGFR antibody, or any fragment thereof having a TGFβRII ECD, a VEGFR ECD, or both of the foregoing.

[0209] In any of the antibody embodiments described herein, the antibody is preferably a human or humanized antibody.

[0210] The scope of the present invention includes methods for detecting and / or measuring the presence of a targeting molecule in a sample, comprising contacting the sample and a control sample with an antibody, antibody fragment or bispecific antagonist of the invention, which specifically binds to the targeting molecule under conditions that allow the formation of a complex between the antagonist and the targeting molecule, and then detecting the formation of the complex, wherein a difference between the formation of the complex with the sample and the control sample indicates the presence of the targeting molecule in the sample.

[0211] Given the ability of the anti-tumor antagonists of the present invention to block inhibitory or co-inhibitory T cell responses (e.g., antigen-specific T cell responses), the present specification provides in vitro and in vivo methods for stimulating, enhancing, or upregulating antigen-specific T cell responses (e.g., anti-tumor T cell responses) using the antibodies described herein. In certain embodiments, CD3 stimulation (e.g., by co-culture with cells expressing membrane CD3) is also provided, which can be provided simultaneously with, before, or after checkpoint modulator antagonist treatment. By way of example, the present specification provides a method for enhancing antigen-specific T cell responses, comprising contacting the T cells with a checkpoint modulator antagonist described herein (and optionally with CD3) to enhance the antigen-specific T cell response (e.g., by eliminating the inhibitory response mediated by the checkpoint modulator). Any suitable indicator of an antigen-specific T cell response can be used to measure the antigen-specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of the antibody and / or increased cytokine production in the presence of the antibody. In a preferred embodiment, interleukin-2 and / or interferon-γ produced by antigen-specific T cells is increased.

[0212] The present invention encompasses methods of enhancing an immune response (e.g., an antigen-specific T cell response) in an individual, comprising administering to the individual a bispecific anti-tumor antagonist described herein to enhance the immune response (e.g., an antigen-specific T cell response). In a preferred embodiment, the individual has a tumor, and the immune response against the tumor is enhanced. The tumor may be a solid tumor or a liquid tumor (e.g., a hematological malignancy). In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is a non-immunogenic tumor. In some embodiments, the tumor is PD-L1 positive. In other embodiments, the tumor is PD-L1 negative. The individual may also have a virus, and enhance the immune response against the virus after administration of a bispecific anti-tumor antagonist as described herein.

[0213] In one embodiment, a method of inhibiting tumor cell growth in an individual comprises administering to the individual a bispecific anti-tumor antagonist as described herein to inhibit tumor growth in the individual. Also provided is a method of treating a chronic viral infection in an individual comprising administering to the individual a bispecific anti-tumor antagonist as described herein to treat the chronic viral infection in the individual.

[0214] Also included herein are methods for depleting Treg cells from the tumor microenvironment of an individual having a tumor (e.g., a cancerous tumor), comprising administering to the individual a therapeutically effective amount of a bispecific anti-tumor antagonist described herein, wherein the bispecific anti-tumor antagonist depletes Treg cells in the tumor microenvironment. reg The Fc can be, for example, an Fc that has an effector function or that enhances an effector function (e.g., binding or enhanced binding to one or more activating Fc receptors).

[0215] In a preferred embodiment, T reg Depletion is the depletion of T cells in the tumor microenvironment. eff without significant depletion or inhibition of T and outside the tumor microenvironment effCells and T reg In certain embodiments, the individual has, for example, a tumor microenvironment that is free of T cells. eff In certain embodiments, the bispecific antagonists can deplete Tregs in tumors and / or tumor-infiltrating lymphocytes (TILs). For example, in the CT26 tumor model, anti-mouse TIGIT antibodies, such as mouse IgG2a (which displays effector function), can deplete Tregs. reg It has been shown to partially deplete CD8+ T cells but not CD4+ T cells. Non-effector antibodies or antagonists, such as the murine IgG1 D265A standard, do not deplete T cells.

[0216] In certain embodiments, the bispecific anti-tumor antagonists described herein are administered to an individual as adjuvant therapy. Treatment of cancer patients with bispecific anti-tumor antagonists according to the present invention results in long-term, durable responses, such as long-term survival rates of at least 1, 2, 3, 4, 5, 10, or more, or recurrence-free survival rates of at least 1, 2, 3, 4, 5, 10, or more, compared to current standard therapies. In certain embodiments, treatment of cancer patients with bispecific anti-tumor antagonists prevents cancer recurrence or delays cancer recurrence by at least 1, 2, 3, 4, 5, 10, or more. Thus, treatment with these antagonists can be used as primary or secondary therapy.

[0217] In certain preferred embodiments, the individual has a cell proliferative disorder or cancer. Provided herein are methods for treating an individual with cancer, comprising administering to the individual a bispecific anti-tumor antagonist described herein to treat the individual (e.g., inhibiting or reducing cancerous tumor growth and / or causing tumor regression). The bispecific anti-tumor antagonists described herein can be used alone to inhibit cancerous tumor growth. Alternatively, any of these anti-tumor antagonists can be used in conjunction with other agents, such as, for example, other anti-tumor targeting agents, immunogenic agents, standard cancer therapies, or other antibodies, as described below.

[0218] Accordingly, provided herein are methods of treating cancer (e.g., inhibiting tumor cell growth) in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific anti-tumor antagonist described herein, wherein the antibody preferably comprises human or humanized immunoglobulin sequences.

[0219] Cancers whose growth can be inhibited using the antibodies of the present invention include cancers that respond to immunotherapy. Non-limiting examples of cancers that can be used for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-NSCLC, glioma, intestinal and gastric cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer. Neck cancer (or carcinoma), stomach cancer, germ cell tumors, childhood sarcomas, natural killer of the paranasal sinuses, melanoma (e.g., metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, urethral cancer, renal pelvic cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, myeloid axis tumors, brain stem glioma, pituitary adenoma, cystic ulcer Posi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), virus-associated cancers (e.g., human papillomavirus (HPV)-associated tumors) and hematological malignancies derived from one of the two major blood cell repertoires (i.e., myeloid cell lines that produce granuloma cells, erythrocytes, blood clotting cells, macrophages and mast cells, or lymphoid cell lines that produce B, T, NK and lymphocytes), such as acute, chronic, lymphocytic and / or myeloid leukemias (e.g., acute leukemia, chronic myeloid leukemia, acute leukemia, chronic myeloid leukemia, chronic lymphocytic and / or myeloid leukemia, acute leukemia, chronic myeloid leukemia, acute ... Hematologic malignancies (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2, mature cell), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with increased eosinophils [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulosa sarcoma and green tumors, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL),B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, pleomorphic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic spectrum of lymphoid tumors Leukemias, lymphomas and myeloid neoplasms of all classes, including organ neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome), lymphoplasmacytic lymphoma with Waldenstrom macroglobulinemia myeloid tumors such as IgG myeloid tumors, light chain myeloid tumors, non-secretory myeloid tumors, smoldering myeloid tumors (also known as indolent myeloid tumors), solitary plasma cell neoplasms and multiple myeloid tumors, chronic lymphocytic leukemia (CLL), hairy cell lymphoma, myeloid hematopoietic tumors, tumors of stromal origin (including fibrosarcoma and rhabdomyosarcoma), seminoma, teratoma, central and peripheral nerve tumors (including astrocytoma), schwannomas, tumors of stromal origin including fibrosarcoma, rhabdomyosarcoma and osteosarcoma, and melanoma, xeroderma pigmentosum, Other tumors including lymphoid hematopoietic tumors such as T-cell and B-cell tumors, including but not limited to T-cell cytotoxics such as keratoacanthoma, seminoma, thyroid follicular carcinoma and teratomas, T-cell cytotoxics such as T-prolymphocytic leukemia (T-PLL) including the small cell and encephalomyeloid class, large granular lymphocyte leukemia (LGL), preferably the T-cell class, and T-NHL hepatosplenic lymphoma, peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes), angiocentric (intranasal) T-cell lymphoma, head or neck cancer, kidney cancer, rectal cancer,The methods described herein include thyroid cancer, acute myeloid lymphoma, and any combination of the foregoing cancers. The methods described herein are used to treat metastatic cancer, refractory cancer (e.g., cancers that have previously been treated with immunotherapy, e.g., blocking CTLA-4 or PD-1 antibodies), and recurrent cancer.

[0220] The bispecific anti-tumor antagonists of the present invention can be administered alone, in combination with, or simultaneously with other anti-tumor antagonists. Alternatively, in cancer vaccine strategies, the bispecific anti-tumor antagonists can also be administered in combination with or simultaneously with immunogenic agents, such as cancerous cells, tumor vaccines, purified tumor antigens (including recombinant proteins, peptides, and saccharide molecules), cells transfected with genes encoding immune-stimulating cytokines (He et al., (2004) J. Immunol. 173:4919-28), or oncolytic viruses.

[0221] Many experimental antitumor vaccine strategies have been devised. In one of these strategies, the vaccine uses autologous or allogeneic tumor cells. Some of these cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation, useful for cancer vaccination (Dranoff et al., (1993) Proc. Natl. Acad. Sci. USA 90:3539-43). Cancer vaccines have been shown to increase effector T cell infiltration into tumors in preclinical models. The major forms of cancer vaccines include peptide vaccines, vector-based antigen-specific vaccines, precellular vaccines, and dendritic cell vaccines. All vaccine-based therapies are designed to deliver single or multiple antigen epitopes or whole-cell antigens to patients and induce tumor-specific effector T cells. Therefore, vaccine-based therapies may be the most efficient method for inducing T cell infiltration into tumors.

[0222] Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10:281-7). Often, these tumor-specific antigens are differentiation antigens expressed in tumors and tumor-producing cells, such as the melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be targets for tumor-specific T cells found in the host.

[0223] Inhibition of checkpoint regulator pathways, TGF-β pathways, and / or angiogenesis pathways can be used in conjunction with a set of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are considered self-antigens by the immune system and are therefore tolerated. Tumor antigens include the telomerase protein, which is required for chromosomal telomere synthesis and is expressed only in a limited number of somatic cells in over 85% of human cancers (Kim et al., (1994) Science 266:2011-2013). Somatic mutations can result in protein sequence exchange or fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome) or idiotypes derived from B-cell tumors, resulting in tumor antigens being expressed in cancer cells as "neo-antigens."

[0224] Non-limiting examples of tumor vaccines include sipuleucel-T (Provenge®), an FDA-approved tumor vaccine for metastatic prostate cancer; tumor cells transfected to express the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), such as the whole-cell GM-CSF-secreting irradiated xenogeneic pancreatic cancer vaccine (GVAX, Johns Hopkins); breast cancer antigens, neu, legumain, and, when administered in combination with anti-PD-1 antibodies, can prolong vaccine-induced disease-free survival in mice bearing breast cancer (Karyampudi L. et al., (2014) Cancer Res 74:2974-2985); polypeptide vaccines consisting of immunogenic peptides derived from β-catenin, such as melanoma antigen peptides such as gp100, MAGE antigens, Trp-2, MARTI, and / or tyrosine. Other tumor vaccines include proteins derived from viruses associated with human cancer, such as human papillomavirus (HPV) (e.g., Gardasil®, Gardasil 9R, and Cervarix®), hepatitis B virus (e.g., Engerix-B and Recombivax HB), hepatitis C virus (HCV), and Kaposi's sarcoma-associated herpesvirus (KSHV). Another form of tumor-specific antibody that can be used in TIGIT inhibition is heat shock proteins (HSPs) isolated and generated from tumor tissue itself. These heat shock proteins contain protein fragments derived from tumor cells, and these HSPs are highly efficient at inducing tumor immunity in delivery to antigen-presenting cells. Talimogene laherparepvec (T-VEC or Imlygic®) is an FDA-approved oncolytic virus for some patients with metastatic melanoma that cannot be surgically removed.

[0225] Dendritic cells (DCs) are effective antigen-presenting cells that can be used to induce antigen-specific responses. DCs can be produced ex vivo and loaded with various proteins, peptides, and tumor cell extracts (Nestle et al., (1998) Nature Medicine 4:328-332). DCs can also be transduced by genetic means to express these tumor antigens. For immunization purposes, DCs can also be directly fused to tumor cells (Kugler et al., (2000) Nature Medicine 6:332-336). As a method of vaccination, DC immunization can be effectively coupled with checkpoint regulator blockade to activate (release) more effective antitumor responses.

[0226] Inhibition of checkpoint regulator pathways, TGF-β pathways, and / or angiogenesis pathways can also be combined with standard cancer treatments (e.g., surgery, radiation therapy, and chemotherapy). In particular, checkpoint regulator inhibitors can be effectively combined with chemotherapy regimens. In this case, it may be possible to reduce the dose of the administered chemotherapy agent (Mokyr et al., (1998) Cancer Research 58:5301-5304). An example of such a combination is the combination of an antitumor antagonist with decarbazine for the treatment of melanoma. Another example of a combination is the combination of a checkpoint regulator antagonist or an antitumor antagonist with interleukin-2 (IL-2) for the treatment of melanoma. For example, the scientific rationale for using checkpoint regulator inhibition, TGF-β1 / TGF-β1 RII inhibition, and / or angiogenesis inhibition in combination with chemotherapy is to enhance tumor antigen levels in the antigen presentation pathway by promoting cell death induced by the cytotoxic effect of most chemotherapy compounds. Other combination therapies that may synergize with checkpoint regulator inhibition, TGF-β1 / TGF-β1 RII inhibition, and / or angiogenesis inhibition through cell death are radiation therapy, surgery, and androgen deprivation, each of which creates a source of tumor antigens in the host.

[0227] The bispecific anti-tumor antagonists described herein are also constructed to target Fcα or Fcγ receptor-expressing effector cells to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). By way of example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies are used to target macrophages to tumor sites. This type of targeting class can be adapted to embodiments of the present invention to more effectively activate tumor-specific responses. The T cell arm of these responses can be enhanced by inhibiting one or more of the checkpoint regulator antagonists described herein. Alternatively, bispecific antibodies can be used to deliver antigens directly to DCs, where the bispecific antibodies bind to tumor antigens and specific cell surface markers of dendritic cells.

[0228] Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed in tumors. Immunosuppressive proteins include TGF-β, IL-10, and Fas ligand. Antibodies against each of these entities can be used in combination with the anti-tumor antagonists described herein to counteract the effects of immunosuppressive drugs and enhance the host's tumor immune response.

[0229] Other antibodies that activate the host immune response can be used in combination with the anti-tumor antagonists described herein. This includes molecules that activate DC function and antigen presentation on the surface of dendritic cells. Anti-CD40 antibodies can effectively replace auxiliary T cell activity (Ridge et al., (1998) Nature 393:474-478) and can be used in combination with the bispecific antagonists described herein. For example, activating antibodies against OX-40 (Weinberg et al., (2000) Immunol 164:2160-2169), CD137 / 4-1BB (Melero et al., (1997) Nature Medicine 3:682-685 (1997), and ICOS (Hutloff et al., (1999) Nature 397:262-266) T cell costimulatory molecules can also enhance T cell activation levels. Additionally, inhibitors of other immune checkpoint modulators can be used in combination with other anti-tumor antagonists described herein, as further described below.

[0230] Bone marrow transplantation is currently used to treat a variety of tumors of hematopoietic origin. This treatment can cause graft-versus-host disease, but checkpoint modulator blockade can be used to reduce the graft-versus-tumor response, thereby improving the efficacy of donor-transplanted tumor-specific T cells.

[0231] In certain embodiments, the anti-tumor antagonists described herein can be administered to patients suffering from infectious diseases, particularly chronic infections. In this case, similar to its application to cancer, antibody-mediated checkpoint regulator inhibition can be used alone or in combination with vaccines as an adjuvant to enhance immune responses against pathogens, toxins, and self-antigens. Exemplary pathogens that can be used in this therapy include, but are not limited to, HIV, hepatitis viruses (types A, B, and C), influenza viruses, herpes viruses, dinoflagellates, malaria, leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa. Checkpoint regulator inhibition, TGF-β1 / TGF-β1 RII inhibition, and / or angiogenesis inhibition are particularly effective against infectious diseases caused by pathogens (e.g., HIV with new or altered antigens present during the course of infection). Administration of the bispecific anti-tumor antagonist allows these antigens to be considered foreign in order to elicit appropriate T cell responses.

[0232] Other pathogenic viruses causing infections that can be treated by the methods described herein include HIV, hepatitis viruses (types A, B, and C), herpes virus infections (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, human herpesvirus type IV, etc.), and infections caused by adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, German measles virus, parvovirus, pox virus, HTLV virus, dengue virus, papilloma virus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, or combinations thereof.

[0233] Exemplary pathogenic bacteria or diseases that can be treated by the methods described herein include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Meningococcus and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, cholera, leptospirosis, tetanus, botulism, anthrax, as well as plague and Lyme disease.

[0234] Exemplary pathogenic fungi of infections that can be treated by the methods described herein include Candida (e.g., Candida albicans, Candida cruzi, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g., Aspergillus fumigatus, Aspergillus niger, etc.), Mucormycosis (e.g., Mucormycosis, Apsidia, Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0235] Exemplary pathogenic parasites that can be treated by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippostrongylus brasiliensis.

[0236] In all of the above methods, checkpoint regulator inhibition, TGF-β1 / TGF-β1 RII inhibition, and / or angiogenesis inhibition can be combined with other forms of immunotherapy (e.g., cytokine (e.g., interferon, GM-CSF, G-CSF, IL-2) treatment) or used in combination with bispecific antibody therapy using two different binding specificities to provide enhanced tumor antigen presentation.

[0237] The bispecific anti-tumor antagonists described herein can be co-administered with any antibody and one or more antigens of interest (e.g., vaccines) to enhance antigen-specific immune responses. Accordingly, the present specification provides methods for enhancing an immune response to an antigen in an individual, comprising administering to the individual (i) the antigen and (ii) a bispecific anti-tumor antagonist, thereby enhancing the immune response to the antigen in the individual. The antigen can be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those described in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens derived from viruses, bacteria, or other pathogens described above.

[0238] In certain embodiments, peptides or fusion proteins comprising the binding epitopes of the bispecific anti-tumor antagonists can replace the anti-tumor antagonists or be used as vaccines other than the anti-tumor antagonists.

[0239] Suitable routes for in vivo and in vitro administration of the antibody compositions (e.g., human monoclonal antibodies, multispecific antibodies, or immunoconjugates) described herein are well known to, and can be selected by, those skilled in the art. By way of example, the antibody compositions can be administered by injection (intravenous or subcutaneous). The appropriate dose of molecule used depends on the age and weight of the individual, as well as the concentration and / or formulation of the antibody composition.

[0240] Combination therapies In another aspect, the present invention provides a combination therapy for enhancing antigen-specific T cell responses in an individual. In one embodiment, the method comprises contacting T cells in combination with a bispecific anti-tumor antagonist and a second antibody, antibody fragment, antagonist, or drug to enhance the antigen-specific T cell response or apoptosis pathway. By way of example, in some embodiments, a first bispecific anti-tumor antagonist specifically binds to a first checkpoint modulator, e.g., PD-1 or PD-L1, and a second bispecific anti-tumor antagonist specifically binds to a different checkpoint modulator or a different epitope. In some embodiments, the second antibody or antibody fragment comprises different HCVRs, LCVRs, or CDR(s) from PD-1 and / or PD-L1.

[0241] In a related embodiment, a method for reducing or depleting regulatory T cells in an individual in need of a tumor comprises administering an effective amount of an antibody or antibody fragment in combination with a second antibody, antibody fragment, antagonist, or drug to reduce the number of regulatory T cells in the individual.

[0242] In some embodiments, the individual has a cell proliferative disorder or cancer as described herein.

[0243] In other embodiments, the individual has a chronic viral infection, an inflammatory disease, or an autoimmune disease as described herein.

[0244] Providing two distinct signals to T cells is a widely accepted model of lymphocyte activation in resting T lymphocytes. This model further provides self- and non-self-discrimination and immune tolerance. The initial or antigen-specific signal is transduced by the T cell receptor (TCR) after recognition of a foreign antigenic peptide in the presence of a major histocompatibility complex (MHC) background. The secondary or costimulatory signal is delivered to T cells by costimulatory molecules expressed on antigen-presenting cells (APCs). This promotes T cell clonal expansion, cytokine secretion, and effector function. Poor costimulation can cause T cells to become resistant to antigen stimulation, potentially leading to tolerance responses to exogenous or endogenous antigens.

[0245] In the two-signal model, T cells receive positive costimulatory signals and negative co-inhibitory signals. Regulation of these positive and negative signals is important for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative signals appear to be necessary for inducing T cell tolerance, while positive signals promote T cell activation. A combination of costimulatory and co-inhibitory signals is provided to T cells upon exposure to antigen, and the interplay between these signals is essential for controlling the breadth of the immune response. Furthermore, as infection or immune challenge resolves, worsens, or persists, the signals provided to T cells change, and these changes profoundly influence responding T cells and regenerate the immune response.

[0246] The mechanism of costimulation has therapeutic implications, as it suggests that manipulation of costimulatory signals provides a means to enhance or terminate cell-based immune responses. Recently, it has been discovered that T cell dysfunction or anergy can occur concomitantly with the induced and persistent expression of immune checkpoint modulators, such as programmed cell death polypeptide (PD-1) and its ligands PD-L1 and PD-L2. PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Thompson RH et al., Cancer Res 2006, 66(7):3381). Furthermore, compared with T lymphocytes from normal tissues and peripheral blood, most tumor-infiltrating T lymphocytes predominantly express PD-1, suggesting that upregulation of PD-1 on tumor-reactive T cells may lead to impaired antitumor immune responses (Blood 2009, 114(8):1537). This is due to exploitation of PD-L1 signaling, mediated by the interaction between PD-L1-expressing tumor cells and PD-1-expressing T cells, resulting in attenuation of T cell activation and evasion of immune surveillance. Blockade of PD-L1 / PD-1 interaction provides a means to enhance T cell immunity, including CD8+ T cell-mediated killing of cancer cells and tumors. Similar enhancement of T cell immunity is observed by inhibiting the binding of PD-1 to its binding partner, B7-1. Thus, therapeutic targeting of PD-1 and other immune checkpoint modulators is an area of ​​interest.

[0247] Combined inhibition of TGF-β1 signaling, checkpoint modulator signaling, and / or angiogenic signaling with other signaling pathways downregulated in tumor cells may provide a means to enhance therapeutic efficacy. In recent years, several immune checkpoint modulators in the form of receptors and their ligands have been identified. An important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, which includes CTLA-4 and its ligands, B7-1 and B7-2, PD-1 and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Other immune checkpoint modulator antagonists include, but are not limited to, TIM-3 and its ligand, galectin-9, LAG-3 and its ligand, including liver sinusoidal endothelial cell lectin (LSECtin) and galectin-3, CD122 and its CD122R ligand, as well as CD70, B7H3, B and T lymphocyte attenuator (BTLA), and VISTA (Le Mercier et al., (2015) Front. Immunol., (6), Article 418). Additionally, several checkpoint modulator antagonists have been identified and are being tested in various clinical and preclinical models and / or approved by the FDA (Kyi et al., FEBS Letters, 588:368-376 (2014)). The concept of inhibitory receptor blockade, also known as immune checkpoint blockade, has been demonstrated, for example, by the efficacy of the FDA-approved PD-1 inhibitors, nivolumab and pembrolizumab, and the anti-CTLA-4 antibody, ipilimumab, in metastatic melanoma.

[0248] Immune checkpoint antagonists block or inhibit signaling through checkpoint modulator receptors by binding to checkpoint modulators or their ligands, thereby modulating or interfering with the activity of immune checkpoint modulators. Inhibiting this signaling can reverse immunosuppression, thereby reconstituting or enhancing T cell immunity against anti-tumor cells. Conversely, immune checkpoint agonists (e.g., costimulatory molecules) stimulate signaling through checkpoint modulator receptors by binding to checkpoint modulators or their ligands, thereby stimulating the activity of immune checkpoint modulators. Stimulating such signaling can reconstitute or enhance T cell immunity against anti-tumor cells.

[0249] Thus, in one embodiment, a method of stimulating an immune response in an individual comprises administering to the individual a bispecific anti-tumor antagonist as described herein in combination with other immune checkpoint modulators as described above, thereby stimulating an immune response in the individual, e.g., inhibiting tumor growth or stimulating an antiviral response, etc. In particular, the bispecific anti-tumor antagonists can be administered as separate antagonists or as a heterogeneous multispecific antagonist comprising multiple products.

[0250] In some embodiments, to stimulate an immune response, the bispecific anti-tumor antagonist of the present invention can be conjugated to (i) an antagonist of the IgSF family protein, B7 family, or TNF family that inhibits T cell activation, or an antagonist of a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, or other immunosuppressive cytokines), and / or (ii) an agonist of a stimulatory receptor of the IgSF family, B7 family, or TNF family, or an agonist of a cytokine that stimulates T cell activation, to stimulate an immune response. In other embodiments, the bispecific anti-tumor antagonist is administered to the individual in combination with an anti-CTLA-4 antibody or CTLA-4 antagonist. Exemplary CTLA-4 antibodies for use in accordance with the present invention include ipilimumab, trevilizumab, and tremelimumab.

[0251] In some embodiments, bispecific antagonists targeting different checkpoint modulator antagonists can be administered to individuals with cancers that highly express immune checkpoint modulator ligands. For example, in one embodiment, cancer individuals with high expression of PVR (CD155) and / or Nectin-2 (CD112) and / or low expression of PD-1 can be selected. Monotherapy can be performed using only anti-TIGIT or anti-LAG-3 antibodies or fragments thereof, or combination therapy can be performed using PD-1 antagonists or other immune checkpoint modulator antagonists. The bispecific anti-tumor antagonists of the present invention can be administered simultaneously with other agents (e.g., antibodies, antagonists, or drugs) in amounts effective to stimulate immune responses and / or apoptosis to further enhance, stimulate, or upregulate immune responses and / or apoptosis in individuals.

[0252] In some embodiments, the bispecific anti-tumor antagonist is administered after treatment with a different anti-tumor antagonist. By way of example, in some embodiments, the bispecific anti-tumor antagonist of the present invention can be administered only after a single specific anti-tumor antagonist has failed, caused an incomplete therapeutic response, or caused tumor recurrence or recurrence (e.g., "PD-1 failure"). In some embodiments, cancers that exhibit such failure, for example, with PVR and / or Nectin-2, can be screened, and only those high-level expressers are treated with the bispecific anti-tumor antagonist of the present invention.

[0253] In certain embodiments, the anti-tumor antagonist comprises a dominant-negative protein region of an immune checkpoint modulator. In certain embodiments, the dominant-negative protein comprises an extracellular domain derived from the group consisting of PD-L1, PD-L2, PD-1, B7-1, B7-2, B7H3, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, VISTA, CD70, and combinations thereof. In some specific embodiments, these extracellular domains are fused to an immunoglobulin constant region or Fc receptor of an antibody described herein. Such mutations are capable of binding to endogenous receptors to form complexes that are deficient in signal transduction. In certain embodiments, the extracellular domain is fused to an immunoglobulin constant region or Fc receptor, or to a monomer of an oligoprotein complex.

[0254] In certain embodiments, the dominant-negative PD-L1 antagonist comprises the extracellular domain of PD-L1, PD-L2, or PD-1. In another embodiment, a dominant-negative PD-1 antagonist is used that has a mutation that prevents it from binding to PD-L1. An exemplary dominant-negative protein is AMP-224 (co-developed by Glaxo Smith Kline and Amplimmune), a recombinant fusion protein comprising the extracellular domain of PD-L2 and the Fc region of human IgG.

[0255] Exemplary immune checkpoint modulator agonists include, but are not limited to, tumor necrosis factor (TNF) receptor superfamily members (e.g., CD27, CD40, OX40, GITR, and 4-1BB (CD137)) and their ligands, or B7-CD28 superfamily members (including CD28 and ICOS (CD278)). Other checkpoint modulator agonists include CD2, CDS, ICAM-1, LFA-1 (CD11a / CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligands. Immune checkpoint agonists can include antibodies or soluble fusion protein agonists comprising one or more costimulatory domains. Agonist antibodies include, but are not limited to, anti-CD40 mAbs such as, for example, CP-870 and 893, lucatumumab, and dacetuzumab; anti-CD137 mAbs such as, for example, BMS-663513, urelumab, and PF-05082566; anti-OX40 mAbs; anti-GITR mAbs such as, for example, TRX518; anti-CD27 mAbs such as, for example, CDX-1127; and anti-ICOS mAbs.

[0256] Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as, for example, GITR fusion proteins described in U.S. Pat. Nos. 6,111,090 and 8,586,023, European Patent No. 090505B1, International Patent Application Publication Nos. WO 2010 / 003118 and WO 2011 / 090754, etc. Anti-GITE antibodies are described, for example, in U.S. Patent Nos. 7,025,962, 7,618,632, 7,812,135, 8,388,967, and 8,591,886, European Patent Nos. 1,947,183B1 and 1,866,339, and International Patent Application Publication Nos. WO 2011 / 028683, WO 2013 / 039954, WO 2005 / 007190, WO 2007 / 133822, WO 2005 / 055808, WO 99 / 40196, WO 2001 / 03720, WO 99 / 20758, WO 2006 / 083289, WO 2005 / 115451, and WO 2011 / 051726. An exemplary anti-GITR antibody is TRX518.

[0257] Other families of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors include CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 / 4-1BB, TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, and lymphotoxin α / TNF. TNF family molecules that are bound by cognate TNF receptor family members, including TNF-γ, TNFR2, TNF-α, LTβR, lymphotoxin α1 (32), FAS, FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey, MG et al., (2009) Drug Discovery Today, 14(23-24):1082-1088).

[0258] Immune checkpoint agonists or costimulatory molecules include MHC class I molecules, MHC class II molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD5, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, and IL7R. α, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACA These include cell surface molecules other than antigen receptors or their ligands necessary for an effective immune response, including, but not limited to, ligands that specifically bind to M1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0259] In one embodiment, the T cell response is an anti-PD-1 or anti-PD-L1 T cell response of the present invention. Stimulation via mAbs can be achieved by a combination of one or more of: (i) antagonists (e.g., immune checkpoint inhibitors) of proteins that inhibit T cell activation, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-1, CD96, and TIM-4; and (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0260] Exemplary agents that modulate one of the above proteins can be used in combination with the anti-PD-1 or anti-PD-L1 antibodies of the invention to treat cancer. These exemplary agents include, for example, YERVOY 商標 / ipilimumab or tremelimumab (for CTLA-4), galiximab (for B7.1), OPDIVO 商標 / nivolumab / BMS-936558 (for PD-1), pidilizumab / CT-011 (for PD-1), KEYTRUDA 商標 / pembrolizumab / MK-3475 (for PD-1), AMP224 (for B7-DC / PD-L2), BMS-936559 (for B7-H1), MPDL3280A (for B7-H1), MEDI-570 (for ICOS), AMG557 (for B7H2), MGA271 (for B7H3), IMP321 (for LAG-3), urelumab / BMS-663513 and PF-05082566 (CD1 These include 37 / 4-1BB), CDX-1127 (for CD27), MEDI-6383 and MEDI-6469 (for OX40), RG-7888 (for OX40L), Atacicept (for TACI), CP-870893 (for CD40), lucatumumab (for CD40), dacetuzumab (for CD40), and muromonab-CD3 (for CD3).

[0261] Molecules that can be used in combination with the anti-tumor antagonists described herein in cancer treatment include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, antagonist anti-PD-1 and / or anti-PD-L1 antibodies can be used in combination with KIR (e.g., lirilumab), CSF-1R antagonists (e.g., RG7155) antagonists.

[0262] Tumors evade host immune monitoring through a variety of mechanisms, many of which can be overcome by inactivating tumor-expressed immunosuppressive proteins, including TGF-β, IL-10, and Fas ligand. Antibodies against each of these entities can be used in combination with the anti-tumor antagonists described herein to counteract the effects of immunosuppressants and promote host tumor immune responses.

[0263] Other antibodies that activate host immune responses can be used in combination with the anti-tumor inhibitors described herein. This includes dendritic cell surface molecules that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively replace T cell accessory activity and can be used in combination with the anti-tumor antagonists described herein. For example, antibodies that activate T cell costimulatory molecules such as OX-40, CD137 / 4-1BB, and ICOS can enhance T cell activation levels.

[0264] In certain embodiments, the anti-tumor antagonists described herein can be used in combination with one or more other therapeutic agents, such as, for example, anti-cancer agents, radiotoxic agents, or immunosuppressants. Such combinations can overcome problems caused by drug resistance, changes in tumor antigenicity that render the antagonist ineffective, and toxicity (by administering low doses of one or more agents).

[0265] The anti-tumor antagonists described herein can be linked to the agent (as an immunoconjugate) or administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after, or simultaneously with the agent, or can be administered along with other known therapies, such as anti-tumor therapies (e.g., radiation therapy). The anti-tumor antagonists described herein can be administered simultaneously with one or more anti-cancer agents to provide two anti-cancer agents that act synergistically through different mechanisms to produce a cytotoxic effect on human cancer cells.

[0266] The anti-tumor antagonists described herein can be combined with anti-cancer agents such as, for example, alkylating agents, anthracyclines, antimetabolites, antidotes, interferons, polyclonal or monoclonal antibodies, EGFR inhibitors, HER2 inhibitors, histone deacetylase inhibitors, hormones, mitotic inhibitors, phosphatidyl alcohol-3-kinase (PI3K) inhibitors, Akt inhibitors, mammalian target of rapamycin (mTOR) inhibitors, proteasome inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, Ras / MAPK pathway inhibitors, centrosome declustering agents, multikinase inhibitors, serine / threonine kinase inhibitors, tyrosine kinase inhibitors, VEGF / VEGFR inhibitors, taxanes or taxane derivatives, aromatase inhibitors, anthracyclines, microtubule targeting agents, topoisomerase poisons, molecular targeting or enzyme inhibitors (e.g., kinases or protein jacketsases), cytidine analogs, or combinations thereof.

[0267] Exemplary alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan, Neosar), chlorambucil (Leukeran), melphalan (Alkeran), carmustine (BiCNU), busulfan (Busulfex), lomustine (CeeNU), dacarbazine (DTIC-Dome), oxaliplatin (Eloxatin), carmustine (Gliadel), ifosfamide (Ifex), dichloroethylmethylamine (Mustargen), busulfan (Myleran), carboplatin (Paraplatin), cisplatin (CDDP, Platinol), temozolomide (Temodar), thioplex (Thioplex), bendamustine (Treanda), or streptozotocin (Zanosar).

[0268] Exemplary anthracyclines include, but are not limited to, doxorubicin (Adriamycin), doxorubicin liposomal (Doxil), mitoxantrone (Novantrone), bleomycin (Blenoxane), daunorubicin (Cerubidine), daunorubicin liposomal (DaunoXome), actinomycin (Cosmegen), epirubicin (Ellence), idarubicin (Idamycin), purkamycin (Mithracin), ceromycin (Mutamycin), pentostatin (Nipent), or valbicin (Valstar).

[0269] Exemplary antimetabolites include fluorouracil (Adrucil), fluorouracil capecitabine (Xeloda), hydroxyurea (Hydrea), mercaptopurine (Purinol), pemetrexed (Alimta), fludarabine (Fludara), nelarabine (Arranon), cladribine (Cladribine), and the like. Novaplus), clofarabine (Clolar), cytarabine (Cytosar-U), decitabine (Dacogen), cytarabine liposomal (DepoCyt), hydroxyurea (Droxia), pralatrexate (Folotyn), fludarabine (FUDR), gemcitabine (Gemzar), cladribine (Leustatin), fludarabine (Oforta), methotrexate (MTX, Rheumatrex), methotrexate (Trexall), thioguanine (Tabloid), TS-1 or cytarabine (Tarabine PFS).

[0270] Exemplary antidotes include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

[0271] Exemplary interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha-2a (Roferon-A).

[0272] Exemplary polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin), ofatumumab (Arzerra), bevacizumab (Avastin), rituximab (Rituxan), cetuximab (Erbitux), panitumumab (Vectibix), tositumumab / iodine-131 tositumumab (Bexxar), alemtuzumab (Campath), ibritumomab (Zevalin, In-111, Y-90 Zevalin), gemtuzumab (Mylotarg), eculizumab (Soliris), or denosumab.

[0273] Exemplary EGFR inhibitors include, but are not limited to, gefitinib (Iressa), lapatinib (Tykerb), cetuximab (Erbitux), erlotinib (Tarceva), panitumumab (Vectibix), PKI-166, canutinib (CI-1033), matuzumab (Emd7200) or EKB-569.

[0274] Exemplary HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin), lapatinib (Tykerb) or AC-480.

[0275] Exemplary histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, abexinostat, gubinostat, and moxetinostat.

[0276] Exemplary hormones include tamoxifen (Soltamox, Nolvadex), raloxifene (Evista), megestrol (Megace), leuprorelin (Lupron, Lupron Depot, Eligard, Viadur), fulvestrant (Faslodex), letrozole (Femara), triptorelin (Trelstar LA, Trelstar These include, but are not limited to, flumethasone (Depot), exemestane (Aromasin), goserelin (Zoladex), bicalutamide (Casodex), anastrozole (Arimidex), flumethyltestosterone (Androxy, Halotestin), medroxyprogesterone (Provera, Depo-Provera), estramustine (Emcyt), flutamide (Eulexin), toremifene (Fareston), degarelix (Firmagon), nilutamide (Nilandron), abrelix (Plenaxis), or testosterone (Teslac).

[0277] Exemplary antimitotic agents include, but are not limited to, taxol (Taxol, Onxol, Abraxane), docetaxel (Taxotere), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), etoposide (Toposar, Etopophos, VePesid), teniposide (Vumon), ixabepilone (Ixempra), nocodazole, epothilone, vinorelbine (Navelbine), camptothecin (CPT), irinotecan (Camptosar), topotecan (Hycamtin), amacridine or lamellarin D (LAM-D).

[0278] Exemplary phosphatidylinositol-3-kinase (PI3K) inhibitors include, but are not limited to, the irreversible PI3K inhibitor wortmannin, the wortmannin derivative demethoxypyridine LY294002, the reversible PI3K inhibitor BKM120 (Buparlisib), Idelalisib (a PI3K delta inhibitor), duvelisib (IPI-145, a PI3K delta and gamma inhibitor), alpelisib (BYL719), an alpha-specific PI3K inhibitor, TGR 1202 (formerly known as RP5264), an oral PI3K delta inhibitor, and copanlisib (BAY 80-6946), a primarily PI3K alpha, delta isotype inhibitor.

[0279] Exemplary Akt inhibitors include, but are not limited to, mitifox, AZD5363, GDC-0068, MK2206, Perifor, RX-0201, PBI-05204, GSK2141795, and SR13668.

[0280] Exemplary MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel), rapamune, ridaforolimus, ridaforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128, BEZ235, PI-103, Torin 1, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE-354, and CC-223.

[0281] Exemplary proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, opozomib (ONX-0912), marizomib (NPI-0052), and disufiram.

[0282] Exemplary PARP inhibitors include, but are not limited to, olaparib, iparib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338, and combinations of the foregoing.

[0283] Exemplary Ras / MAPK inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026, RO4987655, RO5068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162, and GDC-0973.

[0284] Exemplary centrosome declustering agents include, but are not limited to, griseofulvin, noscapine, e.g., noscapine bromide (e.g., 9-bromonoscapine), reduced noscapine bromide (RBN), noscapine derivatives such as N-(3-bromobenzyl)noscapine, amine noscapine and its water-soluble derivatives, CW069, a phenanthrene-derived poly(ADP-ribose) polymerase inhibitor, PJ-34, N2-(3-pyridylmethyl)-5-nitro-2-furamide, N2-(2-thienylmethyl)-5-nitro-2-furamide, and N2-benzyl-5-nitro-2-furamide.

[0285] Exemplary multikinase inhibitors include, but are not limited to, regorafenib, sorafenib (Nexavar), sunitinib (Sutent), BIBW2992, E7080, Zd6474, PKC-412, motesanib, or AP24534.

[0286] Exemplary serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin, eril / esudil hydrochloride, flavonoid antitumor drugs, seliciclib (CYC202, Roscovitrine), SNS-032 (BMS-387032), Pkc412, bryostatin, KAI-9803, SF1126, VX-680, Azd1152, Arry-142886 (AZD-6244), SCIO-469, GW681323, CC-401, CEP-1707, or PD332991.

[0287] Exemplary tyrosine kinase inhibitors include erlotinib (Tarceva), gefitinib (Iressa), imatinib (Gleevec), sorafenib (Nexavar), sunitinib (Sutent), trastuzumab (Heaping), bevacizumab (Avastin), rituximab (Rituxan), lapatinib (Tykerb), cetuximab (Erbitux), panitumumab (Vectibix), everolimus (Afini), and rifacillin. In some embodiments, the therapeutic agent may be a vasodilator, such as, but not limited to, rivaroxaban (Renault), rivaroxaban (Renault Azur Lane), rivaroxaban (Renault Razorback ...

[0288] Exemplary VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin), sorafenib (Nexavar), sunitinib (Sutent), ranibizumab, pegaptanib, or vandetinib.

[0289] Exemplary microtubule-targeting agents include, but are not limited to, taxol, docetaxel, vincristin, vinblastin, nocodazole, epothilones, and novivon.

[0290] Exemplary topoisomerase poisons include, but are not limited to, teniposide, etoposide, doxorubicin, camptothecin, daunorubicin, actinomycin D, mitoxantrone, amacridine, epirubicin, and idarubicin.

[0291] Exemplary taxanes or taxane derivatives include, but are not limited to, taxol and docetaxel.

[0292] Exemplary common chemotherapeutic, anti-cancer, and anti-proliferative agents include hexamethylmelamine (Hexalen), isotretinoin (Accutane, Amnesteem, Claravis, Sotret), retinoic acid (Vesanoid), azacitidine (Vidaza), bortezomib (Velcade), asparaginase (Elspar), levamisole (Ergamisol), mitotane (Lysodren), procarbazole (Matulane), and penicillin-resistant Staphylococcus aureus (Pag). These include, but are not limited to, guaspargase (Oncaspar), denileukin-toxin conjugate (Ontak), photofrin, aldesleukin (Proleukin), rethalidomide (Revlimid), bexarotene (Targretin), thalidomide (Thalomid), sirolimus (Torisel), arsenic trioxide (Trisenox), verteporfin (Visudyne), mimosine (Leucenol), (1 M tegafur-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 M potassium oxazine) or lovastatin.

[0293] In certain embodiments, the anti-tumor antagonist described herein is administered at a sub-therapeutic dose, the other anti-immune checkpoint modulator antibody or antagonist is administered at a sub-therapeutic dose, the angiogenesis antagonist is administered at a sub-therapeutic dose, or any antagonists in combination therewith are each administered at a sub-therapeutic dose.

[0294] In certain embodiments, TGFβ / TGFβ RII, checkpoint regulators, and / or angiogenesis pathway inhibition can be combined with standard cancer treatments (e.g., surgery, radiation therapy, and chemotherapy) according to common chemotherapy regimens. In these cases, it may be possible to reduce the dose of the chemotherapeutic agent administered. An example of such a combination is combining a checkpoint regulator antagonist of the present invention with dacarbazine in the treatment of melanoma. Another example of such a combination is combining a checkpoint regulator antagonist of the present invention with interleukin-2 (IL-2) in the treatment of melanoma. It is believed that combining checkpoint regulator inhibition with chemotherapy may enhance apoptosis of cytotoxic immune responses and increase tumor antigen presentation. Other synergistic combination therapies include checkpoint regulator inhibition via cell death when combined with radiation therapy, surgery, or androgen deprivation. Each of these approaches creates a source of tumor antigens for the host.

[0295] In certain embodiments, the checkpoint regulator antagonists described herein can be used in multispecific antagonists or in conjunction with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to cancer cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can target two independent antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies can be used to target macrophages to cancer cells or tumors. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses can be enhanced by inhibition of checkpoint regulators. Alternatively, bispecific antibodies can be used to deliver antigens directly to DCs, where the bispecific antibody binds to a tumor antigen and a specific cell surface marker of dendritic cells.

[0296] III. Nucleic Acids and Host Cells for Expressing Anti-Tumor Antagonists In another aspect, the invention provides nucleic acids encoding the anti-tumor antagonists of the invention, including heavy and light chains, and expression vectors comprising these nucleic acids, particularly nucleic acids encoding one or more HCDRs, LCDRs, HCVRs and / or LCVRs for any of the antibodies, antagonists, or fragments described herein.

[0297] Now, in one aspect, the invention provides one or more nucleic acids encoding any of the anti-tumor antagonists, antibodies or antigen-binding portions thereof described herein.

[0298] In another aspect, the invention provides one or more expression vectors comprising one or more nucleic acids encoding any of the anti-tumor antagonists, antibodies or antigen-binding portions thereof described herein.

[0299] In another aspect, the invention provides the use of one or more such host cells transfected with an expression vector, wherein the expression vector comprises one or more nucleic acids encoding any of the anti-tumor antagonists, antibodies or antigen-binding portions thereof described herein.

[0300] The DNA(s) of the antigen-binding site can be isolated and sequenced from monoclonal antibodies produced by hybridoma tumor cells using conventional procedures (e.g., using oligonucleotide probes that specifically bind to genes encoding the monoclonal antibody heavy and light chains). Alternatively, the amino acid sequence from the immunoglobulin of interest can be determined by direct protein sequencing, and an appropriate coding nucleotide sequence can be designed according to a universal codon table. Alternatively, the nucleotide and amino acid sequences of antigen-binding sites or other immunoglobulin sequences (including constant regions, hinge regions, etc.) can be obtained from published sources known in the art.

[0301] In one embodiment, any of the binding antagonist fragments of the invention may involve the use of codon-optimized synthetic DNA fragments corresponding to, for example, antibody variable regions. By way of example, the codons of cDNA sequences encoding immunoglobulin VH, VL, HC, LC, CH1, CH2, CH3 and / or framework regions may be optimized for expression in various human, primate or mammalian cells (e.g., HEK or CHO cells). The invention features nucleic acid molecules comprising one or two nucleotide sequences encoding the heavy and light chain variable regions, CDRs, hypervariable loops, and framework regions of an anti-PD-L1 antibody molecule described herein.

[0302] Expression vectors encoding particular bispecific anti-tumor antagonists can be used to synthesize the anti-tumor antagonists herein in cultured cells in vitro, or can be administered directly to a patient to express the anti-tumor antagonist in vivo or ex vivo. As used herein, "expression vector" refers to a viral or non-viral vector that contains a polynucleotide encoding one or more polypeptide chains corresponding to the bispecific anti-tumor antagonists herein and presents the polynucleotide in a form suitable for expression in a host for antibody preparation or direct administration as a therapeutic agent.

[0303] A nucleic acid sequence is "operably linked" to another nucleic acid sequence when it is placed into a functional relationship with the other. For example, a presequence or signal peptide DNA is operably linked to a polypeptide DNA if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned for convenient translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in reading phase, in the case of a signal peptide. However, enhancers need not be contiguous. Linking can be achieved by linkage at a convenient restriction site. If such sites are not present, oligonucleotide adapters or linking peptides synthesized in accordance with conventional procedures can be used.

[0304] The nucleic acid sequence used to express checkpoint regulator antagonists usually contains an amino-terminal signal peptide sequence that is removed from the mature protein.Because the signal peptide sequence can affect the expression level, the polynucleotide can encode any of a variety of different N-terminal signal peptide sequences.Those skilled in the art will understand that the design of the expression vector can depend on factors such as the selection of the host cell to be transfected, the desired protein expression level, etc.

[0305] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in one or more host organisms. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that support constitutive expression of a nucleotide sequence in many classes of these host cells, or those that support expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Expression vectors usually contain a sequence for transcription termination and may further contain one or more elements that positively affect mRNA stability.

[0306] The expression vector contains one or more transcriptional regulatory elements, including a promoter and / or enhancer, to support the expression of the anti-tumor antagonist. A promoter contains a DNA sequence that initiates transcription from a transcription initiation site relative to a fixed position. A promoter contains core elements required for essential interaction between RNA polymerase and transcription factors, and can be scanned along with other upstream and response elements.

[0307] As used herein, the term "promoter" should be used in its broadest sense and includes a transcriptional regulatory element (TRE) from a genomic gene or its chimeric TRE, including a TATA box or initiation subunit for accurate transcription initiation, with or without additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers, and silencers), which regulate the activation or inhibition of genes to which it can be operably linked, as well as trans-acting regulatory proteins or nucleic acids, in response to developmental and / or external stimuli. Promoters can include genomic fragments or chimeric combinations of one or more TREs.

[0308] Preferred promoters are those capable of supporting high-level expression in target cells. Promoters can include constitutive promoters (e.g., HCMV, SV4, elongation factor-1α (EF-1α)) or promoters that show optimal expression in these specific target cell classes. Enhancers generally refer to DNA sequences that function away from the transcription start site and can be 5' or 3' to the transcription unit. Enhancers can also be found within introns and coding sequences. They are typically between 10 and 300 bp in length and act in cis. The function of an enhancer is to increase and / or regulate transcription of nearby promoters. Preferred enhancers are those that support high-level expression in antibody-producing cells. Cell- or tissue-specific transcriptional regulatory elements (TREs) can be introduced into expression vectors to restrict expression to the desired cell class. Pol III promoters (H1 or U6) are particularly suitable for shRNAs expressing siRNAs. Expression vectors can be designed to drive expression of anti-tumor antagonists in one or more cell classes.

[0309] In certain embodiments, one or more expression vectors can be designed to express an anti-tumor antagonist and one or more siRNAs targeting the Tie2 pathway, the VEGF pathway, or an immune checkpoint modulator.

[0310] siRNAs are double-stranded RNAs that can be designed to induce mRNA sequence-specific post-transcriptional gene silencing. Synthetically produced siRNAs structurally mimic the siRNA class normally processed in cells by the Dicer enzyme. When expressed from an expression vector, the expression vector is designed to transcribe short, double-stranded hairpin-like RNAs (shRNAs) that are processed into cellular targeting siRNAs. Synthetic siRNAs and shRNAs can be designed using well-known algorithms and synthesized using standard DNA / RNA synthesizers.

[0311] To co-express each chain of the anti-tumor antagonist, appropriate splice donor and splice acceptor sequences can be introduced to express two products. Alternatively, an internal ribosome binding sequence (IRES) or a 2A peptide sequence can be used to express multiple products from a single promoter. The IRES provides a structure to which ribosomes can bind without binding to the 5' end of the mRNA, thus directing the ribosome to initiate translation at the second start codon of the mRNA, producing multiple polypeptides from a single mRNA. The 2A peptide contains a short sequence that mediates simultaneous translational self-cleavage from peptides upstream and downstream of the 2A site, allowing the production of two equimolar amounts of different proteins from a single transcription product. CHYSEL is a non-limiting example of a 2A peptide that directs eukaryotic translating ribosomes to release a growing polypeptide chain without dissociating from the mRNA. The ribosomes then undergo continuous translation to produce a second polypeptide.

[0312] Expression vectors can include viral or non-viral vectors. Viral vectors can be derived from adeno-associated viruses (AAV), adenoviruses, herpesviruses, vaccinia viruses, polioviruses, poxviruses, retroviruses (including, e.g., lentiviruses such as HIV-1 and HIV-2), Sindbis and other RNA viruses, alphaviruses, astroviruses, coronaviruses, orthomyxoviruses, milk polyvesicle viruses, paramyxoviruses, parvoviruses, picornaviruses, togaviruses, and the like. Non-viral vectors are simply "naked" expression vectors that are not packaged with viral-derived components (e.g., capsid and / or envelope).

[0313] In some cases, the targeting function inherent in the viral vector or incorporated into the viral vector can be used to design these vectors to target specific diseases or cell populations. For the delivery and expression of polynucleotides, specific cells can be "targeted". Therefore, in this case, the term "targeting" can be based on the capsid, envelope protein, endogenous and heterologous binding agents in the form of antibodies that are delivered to specific cells, tissue-specific regulatory components that limit expression to a specific subset of cells, or the aforementioned therapies.

[0314] In some embodiments, expression of the antibody chain is under the control of a regulatory element, such as a tissue-specific or ubiquitous promoter. In some embodiments, a ubiquitous promoter (e.g., a CMV promoter, a CMV-chicken β-actin hybrid (CAG) activator, a tissue-specific or tumor-specific promoter) controls expression of the heavy chain, or light chain, or single-chain derivative of the particular antibody from which it is derived.

[0315] Non-viral expression vectors can be used for non-viral gene transfer by directly injecting naked DNA or by packaging polynucleotides encoding anti-tumor antagonists into liposomes, microparticles, microcapsules, virus-like particles, or erythrocyte ghosts. These compositions can be further linked to targeting domains by chemical conjugation to facilitate targeted delivery and / or entry of the nucleic acid into desired cells of interest. Additionally, plasmid vectors can be co-cultured with synthetic gene transfer molecules such as polymeric DNA-binding cations (e.g., polylysine, protamine, and albumin) and linked to cell-targeting ligands (e.g., non-salivary mucoids, insulin, galactose, lactose, or transferrin).

[0316] Alternatively, naked DNA can be used. The uptake efficiency of naked DNA can be improved by compaction or by using biodegradable latex beads. These delivery methods can be further improved by treating the beads to increase their hydrophobicity, promoting endosome disruption and release of the DNA into the cytoplasm.

[0317] IV. Methods of Making Bispecific Antagonists In another aspect, the present invention provides a host cell transfected with a nucleic acid or expression vector encoding the bispecific anti-tumor antagonist of the present invention. The host cell may be any eukaryotic or prokaryotic cell capable of expressing the bispecific anti-tumor antagonist of the present invention, including its immunoglobulin heavy or light chain.

[0318] In another embodiment, a method of making an anti-tumor antagonist comprises culturing a host cell transfected with one or more nucleic acids or expression vectors encoding the bispecific anti-tumor antagonist of the present invention under conditions that allow for the production and purification of the antagonist, antibody or fragment thereof suitable for expression in the cell.

[0319] In another aspect, the present invention provides a method for producing an antibody, comprising culturing cells transiently or stably expressing one or more structures encoding one or more polypeptide chains in the antibody, and purifying the antibody from the cultured cells. Any cells capable of producing a functional antibody can be used. In a preferred embodiment, the antibody-expressing cells are eukaryotic or mammalian (preferably human) cells. Cells of various tissue morphologies can be used to express antibodies. In another embodiment, the cells are yeast cells, insect cells, or bacterial cells. Preferably, the antibody-producing cells are stably transfected with an antibody expression vector.

[0320] For example, one or more expression vectors encoding antibody heavy and light chains can be introduced into cells by any common method, such as naked DNA techniques, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, viral-mediated infection, physical or chemical agents or treatments, electroporation, etc. Additionally, one or more expression vectors and a selectable marker can be transfected into cells to facilitate selection of stably transfected clones expressing the antibody. Antibodies produced by cells can be purified and / or collected according to techniques known in the art, for example, via centrifugation, chromatography, etc.

[0321] Examples of selectable markers suitable for use in mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hycomycin, and puromycin. When a selectable marker is successfully transferred to a mammalian host cell, the transformed mammalian host cell can survive when placed under selection pressure. There are two distinct classes of selectable systems in widespread use. The first class is based on cellular metabolism and uses mutant cell lines that lack the ability to grow independently of a supplemented medium. Two examples are CHO DHFR- cells and mouse LTK- cells. These cells lack the ability to grow without added nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented medium. A supplemented medium substitution strategy can be used to introduce an intact DHFR or TK gene into cells lacking the corresponding gene to alter their growth requirements. Cells of individuals that have not been transformed with the DHFR or TK genes cannot survive in unsupplemented media.

[0322] The second class is dominant selection, which refers to selection strategies used with any cell class and does not require the use of mutant cell lines. These strategies typically use drugs to block host cell growth. These cells, carrying the new gene, express a resistance protein that allows them to survive selection. Examples of this dominant selection are the use of the drugs neomycin, mycophenolic acid, or hygromycin. These three examples use bacterial genes under the control of eukaryotic cells to express resistance to the appropriate drug, G418, or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Other examples include the neomycin analog G418 and puromycin.

[0323] Examples of antibody-expressing cells include human Jurkat, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO) cells, mouse WEHI fibrosarcoma cells, and unicellular protozoan species such as Leishmania. Primary cells can also be immortalized using c-myc or other immortalizing agents to generate stably transformed, antibody-producing cell lines.

[0324] In one embodiment, the cell line comprises a stably transformed Leishmania cell line, such as Leishmania tarentolae, which is known to be a potent, rapidly growing unicellular host that highly expresses eukaryotic proteins with mammalian glycosylation patterns. A commercially available Leishmania expression set (Jena Bioscience GmbH, Jena, Germany) can be used.

[0325] In some embodiments, the cell line expresses at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg of antibody per liter of medium.

[0326] The antibodies of the present invention can be cultured in any suitable medium (e.g., RPMI, DMEM, and AIM V). R ), and then isolated from antibody-expressing cells. Antibodies can be purified using common protein purification methods (e.g., parent purification, chromatography, etc.), including immunoparent purification using Protein A or Protein G. In some embodiments, the antibodies are designed to be secreted into the isolated culture supernatant.

[0327] V. Pharmaceutical Compositions and Methods of Treatment Another aspect of the present invention relates to pharmaceutical compositions and methods for treating cell proliferative disorders (e.g., cancer, chronic infection, or immunodeficiency disease states). In one embodiment, the pharmaceutical composition comprises one or more anti-tumor antagonists of the present invention. In some embodiments, the anti-tumor antagonist comprises one or more TGF-β1 inhibitors or TGF-β1 RII inhibitors linked to one or more angiogenesis inhibitors, such as (1) a PD-1 inhibitor or a PD-L1 inhibitor, or (2) a VEGF inhibitor, a VEGFR2 inhibitor, an angiopoietin-1 / 2 inhibitor, and a Tie2R inhibitor. The antagonist is formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may comprise one or more different antibodies, one or more multispecific antibodies, one or more immunoconjugates, or combinations of the foregoing, as described herein.

[0328] As noted above, the method of using the pharmaceutical compositions as described herein comprises administering to an individual in need thereof an effective amount of a pharmaceutical composition according to the present invention.

[0329] Any suitable route or mode of administration can be used to provide a patient with a therapeutically or prophylactically effective amount of the antibody or antagonist. Exemplary routes or modes of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumor), oral, topical (intranasal, transdermal, intradermal, or intraocular), mucosal (e.g., nasal, sublingual, oral, rectal, vaginal), inhalation, intralymphatic, intraspinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, intraintestinal, intrapulmonary, intralymphatic, intravascular, intraorbital, intracapsular, and transurethral, ​​as well as local delivery via a catheter or stent.

[0330] Pharmaceutical compositions containing antibodies or antagonists according to the present invention can be prepared with any pharmaceutically acceptable carrier or excipient. As used herein, the term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Pharmaceutical compositions may contain suitable solid- or gel-phase carriers or excipients. Exemplary carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Exemplary pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of the foregoing. Often, compositions preferably include an isotonic agent, such as a sugar, e.g., a polyol such as mannitol or sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the therapeutic agent.

[0331] Checkpoint modulator antagonists can be incorporated into pharmaceutical compositions suitable for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate. Sodium chloride can be used to modify the toxicity of solutions to concentrations of 0-300 millimolar (mM) (150 mM is optimal for liquid dosage forms). A cryoprotectant can be included in lyophilized dosage forms, primarily 0-10% sucrose (most preferably 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Lyophilized dosage forms can contain a bulking agent primarily of 1-10% mannitol (most preferably 2-4%). ​​Liquid and lyophilized dosage forms can use a stabilizer, primarily L-methionine at concentrations of 1-50 mM (most preferably 5-10 mM). Other suitable bulking agents include glycine, arginine, which may be included in the form of 0-0.05% polysorbate-80 (most preferably 0.005-0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.

[0332] Therapeutic anti-tumor antagonist formulations can be lyophilized and stored as a sterile powder, preferably stored under vacuum, and then reconstituted with bacteriostatic water (e.g., benzyl alcohol preservative) or sterile water prior to injection. Pharmaceutical compositions can be formulated for parenteral administration by injection, e.g., a single bolus injection or continuous infusion.

[0333] The therapeutic agent in the pharmaceutical composition can be prepared in a "therapeutically effective amount" or a "prophylactically effective amount." A "therapeutically effective amount" refers to an amount effective at the dosage and for the time period necessary to achieve the desired therapeutic result. The therapeutically effective amount of the recombinant vector can be based on the disease being treated, the severity and course of the disease, the mode of administration, whether the antibody or drug is administered topically or therapeutically, the bioavailability of the particular reagent, the ability of the anti-tumor antagonist to elicit the desired response in the individual, previous therapy, the patient's age, weight, and sex, the patient's medical history and response to the antibody, the class of anti-tumor antagonist used, and considerations of the attending physician. A therapeutically effective amount is also an amount in which the toxic or harmful effects of the recombinant vector outweigh the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at the dosage and for the time period necessary to achieve the desired prophylactic result.

[0334] Preferably, the polypeptide region of the anti-tumor antagonist is derived from the same host to which it is administered in order to reduce the inflammatory response to the administered therapeutic agent.

[0335] The anti-tumor antagonist is suitable for administration to the patient at one time or over a series of treatments and can be administered to the patient at any time from diagnosis. The anti-tumor antagonist can be administered as a monotherapy or in combination with other drugs or therapies used to treat the disease.

[0336] As a general proposition, a therapeutically or prophylactically effective amount of an anti-tumor antagonist, whether administered in a single dose or multiple doses, ranges from about 1 ng / kg body weight / day to about 100 mg / kg body weight / day.In certain embodiments, each anti-tumor antagonist is administered at a dose of about 1 ng / kg body weight / day to about 10 mg / kg body weight / day, about 1 ng / kg body weight / day to about 1 mg / kg body weight / day, about 1 ng / kg body weight / day to about 100 μg / kg body weight / day, about 1 ng / kg body weight / day to about 10 μg / kg body weight / day, about 1 ng / kg body weight / day to about 1 μg / kg body weight / day, about 1 ng / kg body weight / day to about 100 ng / kg body weight / day, about 1 ng / kg body weight / day to about 10 ng / kg body weight / day, about 10 ng / kg body weight / day to about 100 mg / kg body weight / day, or about 10 ng / kg body weight / day Body weight / day ~ approx. 10 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 mg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 100 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approx. 10 ng / kg body weight / day ~ approx. 1 μg / kg body weight weight / day, 10ng / kg body weight / day ~ approx. 100 ng / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. mg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. Body weight / day ~ approx. 100 μg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 10 μg / kg body weight / day, approx. 100 ng / kg body weight / day ~ approx. 1 μg / kg body weight / day, approx. 1 μg / kg body weight / day ~ approx. 100 mg / kg body weight / day, approx. 1 μg / kg body weight / day ~ approx. 10 mg / k g body weight / day, approximately 1 μg / kg body weight / day to approximately 1 mg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 100 μg / kg body weight / day, approximately 1 μg / kg body weight / day to approximately 10 μg / kg body weight / day, approximately 10 μg / kg body weight / day to approximately 100 mg / kg body weight / day, approximately 10 μg / kg body weight / day to about 10 mg / kg body weight / day, about 10 μg / kg body weight / day to about 1 mg / kg body weight / day, about 10 μg / kg body weight / day to about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 100 mg / kg body weight / day, about 100 μg / kg body weight / day to about 10 mg / kg body weight / day, about 100 μg / kg body weight / day to about 1 mg / kg body weight / day, about 1 mg / kg body weight / day to about 100 mg / kg body weight / day, about 1 mg / kg body weight / day to about 10 mg / kg body weight / day, about 10 mg / kg body weight / day to about 100 mg / kg body weight / day.

[0337] In other embodiments, the anti-tumor antagonist is administered at a dose of 500 μg to 20 g every three days, or 25 mg / kg body weight every three days.

[0338] In other embodiments, each anti-tumor antagonist is administered in a single dose of about 10 ng to about 100 ng, about 10 ng to about 1 μg, about 10 ng to about 10 μg, about 10 ng to about 100 μg, about 10 ng to about 1 mg, about 10 ng to about 10 mg, about 10 ng to about 100 mg, about 10 ng to about 1000 mg, about 10 ng to about 10,000 mg, about 100 ng to about 1 μg, or about 1 mg per single dose. About 100ng to about 10μg, about 100ng to about 100μg per single dose, about 100ng to about 1mg per single dose, about 100ng to about 10mg per single dose, about 100ng to about 100mg per single dose, about 100ng to about 1000mg per single dose, about 100ng to about 10000mg per single dose, about 1μg to about 10μg per single dose, about 1μg to about 100μg per single dose, about 1μg to about 1mg per single dose, about 1μg to about 10mg per single dose, about 1μg to about 100mg per single dose g, about 1 μg to about 1000 mg per dose, about 1 μg to about 10,000 mg per dose, about 10 μg to about 100 μg per dose, about 10 μg to about 1 mg per dose, about 10 μg to about 10 mg per dose, about 10 μg to about 100 mg per dose, about 10 μg to about 1000 mg per dose, about 10 μg to about 10,000 mg per dose, about 100 μg to about 1 mg per dose, about 100 μg to about 10 mg per dose, about 100 μg to about 100 mg per dose, about 100 μg to about 1000 mg per single dose, about 100 μg to about 10,000 mg per single dose, about 1 mg to about 10 mg per single dose, about 1 mg to about 100 mg per single dose, about 1 mg to about 1,000 mg per single dose, about 1 mg to about 10,000 mg per single dose, about 10 mg to about 100 mg per single dose, about 10 mg to about 1,000 mg per single dose, about 100 mg to about 1,000 mg per single dose, about 100 mg to about 10,000 mg per single dose, and 単回投与当たりIt is administered in the range of about 1000 mg to about 10000 mg, etc. The anti-tumor antagonist can be administered daily, every 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3 or 4 weeks.

[0339] In other specific embodiments, the anti-tumor antagonist can be administered at a dose of about 0.0006 mg / day, 0.001 mg / day, 0.003 mg / day, 0.006 mg / day, 0.01 mg / day, 0.03 mg / day, 0.06 mg / day, 0.1 mg / day, 0.3 mg / day, 0.6 mg / day, 1 mg / day, 3 mg / day, 6 mg / day, 10 mg / day, 30 mg / day, 60 mg / day, 100 mg / day, 300 mg / day, 600 mg / day, 1000 mg / day, 2000 mg / day, 5000 mg / day, or 10000 mg / day, etc. As expected, the dose will depend on the disease, size, age, and condition of the patient.

[0340] In certain embodiments, the sequence encoding the anti-tumor antagonist is introduced into a suitable expression vector (e.g., a viral or non-viral vector) to express an effective amount of the anti-tumor antagonist in a patient suffering from a cell proliferative disorder. For example, in certain embodiments involving administration of one or more recombinant AAV (rAAV) viruses, the pharmaceutical composition comprises at least 1000 rAAV / kg of anti-tumor antagonist. 10 , at least 10 11 , at least 10 12 , at least 10 13 , or at least 10 14 In certain embodiments, the pharmaceutical composition may contain at least 10 genome copies (GC) or recombinant viral particles of rAAV per individual, or any range thereof. 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 The recombinant virus (e.g., rAAV) comprises an effective amount of a recombinant virus (e.g., rAAV) in an amount including 100 genome copies or 100 genome copies of the recombinant virus particle, or any range thereof.

[0341] Dosages can be tested in multiple, art-accepted animal models appropriate for the particular cell proliferative disorder.

[0342] Delivery methods may also include polycationically condensed DNA linked or not to killed viruses, ligand-conjugated DNA, liposomes, eukaryotic cell delivery vector cells, precipitation of photopolymerizable hydrogel materials, use of portable gene transfer particle guns, ionizing radiation, nuclear charge neutralization or fusion with cell membranes, particle-mediated gene transfer, etc.

[0343] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications, and figures and tables cited throughout this specification are hereby incorporated by reference.

[0344] Example Example 1: Generation of monoclonal antibodies Monoclonal antibodies (mAbs) of the present invention can be produced and screened using techniques known in the art, see, for example, Harlow and Lane (1988) "Antibodies, A Laboratory Manual", Cold Spring Harbor Publications, New York. Antigen-specific fusion tumor monoclonal antibodies can be cloned, sequenced, and designed using techniques known in the art, see, for example, Lo. BKC, "Methods of Molecular Biology", vol. 商標 (Methods in Molecular Biology 商標 ),” Vol. 248 2004, Antibody Engineering.

[0345] Example 2: Design of bispecific antitumor antagonists Figures 5A and 5B show two bispecific antitumor antagonists, Bi-PB-1 (or Bi-PLB-1) and Bi-PB-1 (or Bi-PLB-2), respectively. These antagonists comprise a checkpoint regulator antibody scaffold (anti-PD-1 or anti-PD-L1) bearing the TGF-β RII extracellular domain (TGF-β-RII ECD) either (i) fused to the carboxy terminus of the CH3 region of each of the two heavy chains (Figure 5A) or (ii) inserted within the CH3 region of the Fc loop (Figure 5B).

[0346] 5A and 5B, the bispecific antibody can have an IgG1 or IgG4 scaffold, and any one or more antibody specificities can be replaced with any other checkpoint regulator antagonist specificity and / or any tumor-targeting antibody specificity (e.g., CD20, EGFR, etc.).

[0347] FIG. 6 shows exemplary functional region sequences corresponding to the bispecific antibodies of FIGS. 5A and 5B.

[0348] 7A-7B show exemplary heavy chains (HC) and light chains (LC) corresponding to the bispecific antibodies of FIGS. 5A and 5B.

[0349] Example 3: Design of other bispecific antagonists Figures 8A-8C show the design of three different bispecific antagonists, Bi-AB-1, Bi-A1B-1, and Bi-ZB-1, each containing the carboxy-terminal TGF-β1 RII extracellular domain (ECD) in a mutant IgG1 (K447A) scaffold. Bi-AB-1 and Bi-A1B-1 all contain amino-terminal anti-VEGF variable regions (VH1, VL1) derived from Avastin / bevacizumab, with Bi-A1B-1 containing two amino acid substitutions (E6Q, L11V) in the VH region. Bi-ZB-1 contains the amino-terminal aflibercept region upstream of the IgG1 Fc (K447A) region. In other embodiments, as an alternative to the mutated IgG1(K447A) scaffold, the bispecific antagonist comprises a wild-type IgG1 scaffold, a variety of mutated IgG1 scaffolds, an IgG2 scaffold, a mutated IgG2 scaffold, an IgG4 scaffold, or a mutated IgG4 scaffold.

[0350] Figures 9A and 9B show the various functional region sequences present in the bispecific antagonists shown in Figures 8A-8C.

[0351] FIG. 10 shows the amino acid sequences of the heavy chain (HC) and light chain (LC) corresponding to the bispecific antagonists shown in FIGS. 8A-8C.

[0352] FIG. 11 summarizes the arrangement of functional regions of the bispecific antagonists shown in FIGS. 8A-8C.

[0353] Example 4: Expression and purification of antagonists of Figures 10-11 Figure 12 is a Coomassie Brilliant Blue-stained polyacrylamide gel showing the increased expression levels of wild-type VEGF-A or antagonists containing the E6Q and L11V mutations (i.e., Bi-AB-1 and Bi-A1B-1, respectively) when transiently transfected into HEK293 cells, as determined by non-reducing polyacrylamide gel electrophoresis (PAGE). The titers shown in Figure 12 were determined by quantification of cell supernatants using a POROS A column (Applied Biosystems). The results of this analysis indicate a 167% increase in Bi-A1B-1 expression levels compared to Bi-AB-1.

[0354] Figure 13A shows size-exclusion chromatography (SEC) graphs showing the relative levels of high molecular weight (HMW), low molecular weight (LMW) species, and dimers obtained from Bi-AB-1 and Bi-A1B-1 purified by Protein A. Figure 13B shows that the percentage of purified dimers (98.6% for Bi-AB-1 and 98.7% for Bi-A1B-1) was determined by SE-UPLC using a Tosoh TSKgel UP-G3000SWXL column to be much greater than the HMW (1%) and LMW (0.4%) species. These results indicate that fusion of the TGF-β1 RII extracellular domain (ECD) to Bi-AB-1 and Bi-A1B-1 did not result in significant levels of high molecular weight (HMW) or low molecular weight (LMW) species.

[0355] Figures 14A-14B show Coomassie Brilliant Blue-stained PAGE analysis of supernatants obtained after transient transfection of Bi-ZB-1 under non-reducing and reducing conditions, respectively, demonstrating good transient expression levels of Bi-ZB-1 (220 μg / ml) after 8 days of cell growth in vitro.

[0356] Figure 15 shows a size exclusion chromatography (SEC) graph, which shows that compared to the dimer (96.1%), Bi-ZB-1 purified by Protein A has low levels of high molecular weight (HMW, 3.4%) and low molecular weight (LMW, 0.5%) species.

[0357] Example 5: Functional properties of the anti-tumor antagonists of Figures 8A-8C To assess the ability of the anti-VEGF antagonist (Bi-A1B-1) to antagonize VEGF-mediated activation of VEGFR2 (i.e., its cognate receptor), a cell-based luciferase assay was performed. This experiment demonstrates that huVEGF165 is used to stimulate recombinant HEK-293 cells expressing human VEGFR2 and firefly luciferase under the control of an NFAT-responsive element in the presence of serially diluted anti-VEGF antagonist (Bi-A1B-1). The results of this assay are shown in Figure 16. As expected, increasing concentrations of the anti-VEGF antagonist gradually neutralized the ability of VEGF165 to activate VEGFR2 and induce NFAT-mediated luciferase activity.

[0358] To evaluate the ability of the bispecific antagonist (Bi-A1B-1) to simultaneously bind to TGF-β1 and VEGF165, a sandwich ELISA test was performed. In this experiment, huTGF-β1 was coated onto a 96-well plate and blocked with 5% BSA. Subsequently, Bi-A1B-1 was added to serially diluted samples and incubated, followed by the addition of biotinylated huVEGF165. Bound molecules were detected by streptavidin-HRP using TMB substrate. The analytical results in Figure 17 demonstrate that the bispecific antagonist (Bi-A1B-1) can simultaneously bind to TGF-β1 and VEGF165.

[0359] To assess the ability of TGF-β1 RII ECD to antagonize TGF-β1-mediated activation of human TGF-β1 RII (i.e., its cognate receptor), a cellular luciferase assay was performed. This experiment demonstrates that, in the presence of serially diluted antibodies fused to TGF-β1 RII ECD (Bi-A1B-1, Bi-ZB-1), huTGF-β1 stimulates recombinant HEK-293 cells expressing the human TGF-β1 RII receptor and firefly luciferase under the control of an SMAD-responsive element. The results are shown in Figure 18. As expected, increasing concentrations of antibodies containing the TGF-β1 RII ECD gradually neutralize TGF-β1's ability to activate human TGF-β1 RII and induce SMAD-mediated luciferase activity. In this case, Bi-A1B-1 and Bi-ZB-1 exhibit similar antagonistic potencies as reflected by an IC50 of 0.32 nM for Bi-A1B-1 and 0.31 nM for Bi-ZB-1, etc. These IC50s are comparable to the control TGFBR2 fusion product (IC50 = 0.20 nM).

[0360] To evaluate the pharmacokinetic properties of Bi-A1B-1 in vivo, a pharmacokinetic curve was performed. Briefly, 10 mg / kg of each antagonist was intravenously injected into the tail vein of 6-10-week-old female CD1 mice (n = 2 mice for each molecule). Serum was collected 3 min, 3 h, 1 day, 3 days, 7 days, and 10 days after injection. To detect antibodies in the serum, 5 μg / kg of goat anti-human IgG F(ab')2 fragment was used to coat a 96-well plate and then blocked with 5% milk (prepared in PBS). Serial dilutions of mouse serum in 5% milk and serial dilutions of purified protein molecules as standards were added to the 96-well plate. After incubation with peroxidase-covalently conjugated mouse anti-human IgG and further washing, the bispecific antagonist Bi-A1B-1 (Figure 19) antibodies were detected using a TMB-ELISA matrix. The results of this analysis show that the half-life (T 1 / 2 ) indicates that the average lifespan is 7.4 to 16 days.

[0361] Example 6: Design of bispecific PD-1 / PD-L1 antagonists with TGF-β1 ECDs Figures 20A-20B show two bispecific anti-tumor antagonists, Bi-PB-1.2 (Figure 20A) and Bi-PLB-1.2 (Figure 20B), each comprising an antibody scaffold (IgG4 K447A or IgG1 K447A) and variable regions derived from anti-PD-1 and anti-PD-L1, respectively, and further comprising the TGF-β-RII ECD fused to the carboxy terminus of each heavy chain CH3 domain.

[0362] Figure 21 shows the functional region sequences present in the bispecific antibody of Figures 20A and 20B. Figure 22 shows the amino acid sequences of the heavy chain (HC) and light chain (LC) corresponding to the bispecific antagonist shown in Figures 20A and 20B. Figure 23 summarizes the arrangement of the functional regions of the bispecific antagonist shown in Figures 20A and 20B.

[0363] Example 7: Expression and purification of the bispecific antagonist of Figures 20A-20B Figure 24A shows native polyacrylamide gel (PAGE) analysis, which shows the expression levels of Bi-PB-1.2 and Bi-PLB-1.2 in a transient expression system compared to 1 μg of purified parental control antibody (2P17). Figure 24B shows size-exclusion chromatography (SEC) graphs, which show that Bi-PB-1.2, Bi-PLB-1.2, and anti-PDL1 TGF-β1 RII ECD fusion antibody (benchmark) purified by Protein A from transiently transfected HEK293 cells have low levels of high-molecular-weight (HMW) and low-molecular-weight (LMW) species compared to the dimerized molecule ("dimer"), as shown in Figure 24C. Figures 25A-25B show that the dimeric, HMW, and LMW forms of Bi-PB-1.2 and Bi-PLB-1.2 exhibit good stability for at least 4 weeks at 4°C.

[0364] Example 8: Functional properties of Bi-PB-1.2 and Bi-PLB-1.2 antagonists in Figures 20A-20B 1. Detection of Bi-PB-1.2 binding to PD-1 and TGF-β1 using bio-layer interferometry Bio-light interferometry was performed using an Octet RED96 system (ForteBio Octet RED96 system) to measure the binding kinetics of antibodies to His-tagged human PD-1 protein or His-tagged TGF-β1. 20 nM antibody was incorporated into an anti-human IgG capture biosensor. The sensor was placed in wells containing 2- or 3-fold serial dilutions of the analyte (maximum concentration of 72 nM) for 5 minutes to observe binding of the analyte (His-tagged human PD-1 or His-tagged TGF-β1 protein). After transferring the biosensor alone to kinetic buffer and monitoring the interferometer signal for 10 minutes, the degree of dissociation was measured. A 1:1 binding global fit model, including at least five test concentrations, was used to fit the observed onset and termination rates (Ka and Kd), and then the equilibrium binding constant, KD, was calculated.

[0365] Figures 26A-26E show the binding results of PD-1 and TGF-β1 to Bi-PB-1.2 (Figures 26A and 26B, respectively) and the corresponding anti-PD-1 antibody and benchmark (M7824 purchased from Merck KGA) (Figures 26C and 26D, respectively), along with the resulting binding constants (Figure 26E). The results demonstrate that Bi-PB-1.2 has superior binding affinity to PD-1 and TGF-β1 compared to the anti-PD-1 benchmark and M7824 benchmark.

[0366] 2. Detection of Bi-PLB-1.2 binding to PD-L1 and TGF-β1 using biolayer interferometry Biolayer interferometry was used to detect the binding of Bi-PLB-1.2 to PD-L1 and TGF-β1, as previously described for Bi-PLB-1.2. Figures 27A-27E show the binding of PD-L1 and TGF-β1 to Bi-PLB-1.2 (Figures 27A and 27B, respectively) and the corresponding anti-PD-L1 and anti-TGF-β-RII ECD fusion antibodies (TGF-β1 benchmarks) (Figures 27C and 27D, respectively), along with the resulting binding constants (Figure 27E). The results demonstrate that Bi-PLB-1.2 binding to TGF-β1 is superior to that of the TGF-β1 ECD benchmark, and that Bi-PLB-1.2 binding to PD-L1 is comparable to that of the anti-PD-L1 benchmark.

[0367] 3. Simultaneous binding to PD-1 / PD-L1 using Bi-PB-1.2 and Bi-PLB-1.2 Figure 28A shows an ELISA test using Bi-PB-1.2 for simultaneous binding of TGF-β1 and PD-1, in which a 96-well plate coated with huTGF-β1 was incubated with serially diluted samples of Bi-PB-1.2 followed by His-tagged huPD-1, and bound molecules were detected by anti-His-tagged HRP with TMB substrate. Figure 28B shows an ELISA test using Bi-PLB-1.2 for simultaneous binding of TGF-β1 and PD-L1, in which a 96-well plate coated with huTGF-β1 was incubated with serially diluted samples of Bi-PLB-1.2 followed by His-tagged huPD-L1, and bound molecules were detected by anti-His-tagged HRP with TMB substrate. EC 50 Values ​​reflect half-maximal effector concentrations achieved midway through the binding reaction for baseline and maximal responses of human PD-1 or PD-L1, respectively.

[0368] 4. Bi-PB-1.2 effectively blocks the binding of PD-L1 and TGF-β1 to their receptors In Figure 29A, a blocking assay was performed to calculate the IC50 value, which corresponds to the ability of Bi-PB-1.2 to block PD-1 binding to PD-L1, compared to the anti-PD-1 benchmark antibody. Briefly, two-fold serial dilutions of Bi-PB-1.2 or benchmark anti-PD-1 antibodies were prepared (maximum Ab concentration was 128 nM, with each mAb in triplicate). CHO-K1 cells transfected with human PD-1 were washed using FACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) and then incubated for 10 min at RT. 6 The cells were resuspended at a concentration of 1000 cells / ml. FITC-labeled human PD-L1-Fc protein was added to the human PD-1-transfected CHO-K1 cells at a final concentration of 7μg / ml and mixed evenly. In the absence of culture, 2000 CHO-K1 cells (containing PD-L1 protein) in 20μl of FACS buffer were immediately added to a 96-well round dish, and 20μl or two-fold serial dilutions of antibody were immediately added to the cells and incubated at 4℃ for 30 minutes. The cells were then washed and resuspended in 30μl of 7AAD solution, followed by the addition of 35μl of 10% neutral buffered formalin solution and incubation for 15 minutes before analysis using the iQue intellicyt system. As expected, the analytical results in Figure 29A show that increasing concentrations of Bi-PB-1.2 gradually blocked the ability of PD-L1 to bind to its receptor. Furthermore, it was found that the IC50 of Bi-PB-1.2 against PD-L1 was calculated to be comparable to the IC50 of the anti-PD-1 benchmark against PD-L1.

[0369] A cellular luciferase assay was performed to assess the ability of TGF-β1 RII ECD to antagonize TGF-β1-mediated activation of human TGF-β1 RII (i.e., its cognate receptor). In this experiment, huTGF-β1 was used to stimulate recombinant HEK-293 cells expressing the human TGF-β1 RII receptor and firefly luciferase under the control of an SMAD-responsive element in the presence of serial dilutions of Bi-PB-1.2 with TGF-β1 RII ECD. Biological activity was determined by measuring the decrease in luciferase-mediated fluorescence. The results of this assay are shown in Figure 29B. As expected, increasing concentrations of Bi-PB-1.2 and the benchmark anti-PD-L1 TGF-β1 RII ECD gradually neutralized the ability of TGF-β1 to activate human TGF-β1 RII and induce SMAD-mediated luciferase activity. Furthermore, it was found that the IC50 of Bi-PB-1.2 against TGF-β1 was calculated to be comparable to the IC50 of the anti-PD-L1 TGF-β1 RII ECD benchmark against TGF-β1.

[0370] 5. Bi-PB-1.2 binds to human and crab-eating monkey PD-1 with similar activity To assess the ability of Bi-PB-1.2 to bind to human and cyno PD-1, serial dilutions of Bi-PB-1.2 were added to CHO-K1 cells (20,000 cells / well) overexpressing human or cyno PD-1. The mixtures were incubated for 20 minutes at 4°C, washed three times, and stained with a secondary antibody (PE-labeled F(ab')2-goat anti-human IgG Fc, Thermo H10104) for 20 minutes at 4°C. The cells were washed with 7AAD solution, resuspended in 7AAD solution, and fixed in 10% neutral buffered formalin for 15 minutes before analysis using the iQue Intellicyt system. Figures 30A-30B show that Bi-PB-1.2 binds to both human PD-1 (Figure 30A) and cyno PD-1 (Figure 30B). As expected, the ability of the anti-human PD-1 benchmark antibodies to bind to human PD-1 (Figure 30A) was found to be similar to their ability to bind to crab-eating monkey PD-1 (Figure 30B). The half-maximal effector concentration (EC 50 ) against EC 50 EC values ​​are also determined, which are generated midway through the response binding to human PD-1 and cyno PD-L1 at baseline and maximum responses, respectively. 50 exhibits similar binding properties to the Bi-PB-1.2 benchmark antibody.

[0371] 6. Bi-PLB-1.2 effectively blocks the binding of PD-1 and TGF-β1 to their receptors Blocking studies were performed as described in Section 4 of Example 8 above to calculate IC50 values ​​corresponding to the binding between Bi-PLB-1.2 and PD-L1 compared to the anti-PD-L1 benchmark antibodies. Figure 31A shows that increasing the concentration of Bi-PLB-1.2 or the anti-PD-L1 benchmark antibodies gradually blocks the binding of PD-1 to PD-L1. As reflected by the resulting IC50 values, the bispecific antibodies compare favorably with the anti-PD-L1 benchmarks.

[0372] To calculate IC50 values ​​for binding between Bi-PLB-1.2 and TGF-β1 (which are compared to anti-TGF-β1 benchmark antibodies), cell-based studies are performed as described in section 4 of Example 8. Figure 31B shows that serial dilutions of Bi-PLB-1.2 and the anti-PDL1-TGF-β1 RII ECD benchmark block the ability of TGF-β1 to activate luciferase expression under SMAD response element control. As reflected by the resulting IC50 values, the bispecific antibody exhibits similar binding characteristics to the anti-PDL1-TGF-β1 RII ECD benchmark.

[0373] 7. Bi-PLB-1.2 binds to human and crab-eating monkey PD-1 with similar activity To assess the ability of Bi-PLB-1.2 to bind to human and cyno PD-1, serial dilutions of Bi-PLB-1.2 were added to CHO-K1 cells (20,000 cells / well) overexpressing human or cyno PD-1, as described in Section 5 of Example 9. Figures 32A-32B show that Bi-PLB-1.2 binds to human PD-L1 (Figure 32A) and cyno PD-L1 (Figure 32B). The resulting EC 50 Values ​​are expressed as half the maximal effector concentration (EC 50 ), which reflects the baseline and maximum responses generated during the binding response to human PD-1 and cyno PD-L1, respectively.

[0374] 8. Bi-PB-1.2 enhances T cell activation A human T cell-based study was conducted to demonstrate the efficacy of the Bi-PB-1.2 antibody. Briefly, normal healthy human PBMCs collected from donors 1 and 2 were activated using SEB (Toxin Technology, Cat#: BT202). To detect IFN-γ production, 100,000 cells were stimulated with 0.5 μg / ml SEB in a 96-well plate. 25,000 tumor-inhibiting and SHP77 cells were added to provide an inhibitory signal. 66 nM of Bi-PB-1.2 mAbs or isotype control Abs were added. Five days later, IFN-γ and IL-2 were checked in the supernatants by ELISA. Figures 33A-33D show the increase in IFN-γ (donor 1, Figure 33A; donor 2, Figure 33B) and IL-2 (donor 1, Figure 33C; donor 2, Figure 33D) by Bi-PB-1.2 compared to the negative control treatment.

[0375] 9. Bi-PLB-1.2 outperforms parental anti-PD-L1 antibodies in enhancing T cell activation and overcoming tumor cell T cell inhibition Figures 34A-34B show increased IFN-γ secretion by human PBMCs treated with Bi-PLB-1.2 compared to negative control treatment and the parental anti-PD-L1 antibody (donor 8, Figure 34A; donor 9, Figure 34B). Figures 34C-34D show increased IL-2 secretion by human PBMCs treated with Bi-PLB-1.2 compared to negative control treatment and the parental anti-PD-L1 antibody (donor 8, Figure 34C; donor 9, Figure 34D). These results are consistent with the idea that Bi-PLB-1.2 overcomes T cell inhibition of tumor cells better than the parental anti-PD-L1 antibody.

[0376] 10. Bi-PB-1.2 and Bi-PLB-1.2 Improve Pharmacokinetic Curves in Mice Compared to Benchmark Antibodies Pharmacokinetic curves were generated to evaluate the in vivo pharmacokinetic properties of Bi-PB-1.2, Bi-PLB-1.2, and the benchmark antibody. Briefly, 10 mg / kg of each antagonist was intravenously injected into the tail vein of 6-10 week-old female CD1 mice (n = 2 mice per molecule). Serum was collected 3 min, 3 h, 1 day, 3 days, 7 days, and 10 days after injection. To detect antibodies in the serum, 96-well ELISA plates were coated with 5 μg / kg of goat anti-human IgG F(ab')2 fragment and then blocked with 5% milk (prepared in PBS). Serial dilutions of mouse serum in 5% milk, as well as serial dilutions of purified protein molecules as standards, were added to the plates.

[0377] After incubation with peroxidase-covalently conjugated mouse anti-human IgG and further washing, TMB-ELISA substrate was added and Bi-PB-1.2 (Figure 35A), Bi-PLB-1.2 (Figure 35C), and anti-PDL1-TGF-β1 RII ECD benchmark (M8724) (Figure 35E) were detected. The integrity and clipping levels of injected antibodies Bi-PB-1.2 (Figure 35B), Bi-PLB-1.2 (Figure 35D), and benchmark (Figure 35F) in mouse serum were also analyzed by anti-human Fc Western blotting. The results of this analysis show improved mouse pharmacokinetic curves of Bi-PB-1.2 and Bi-PLB-1.2 compared to the benchmark antibodies.

[0378] Example 9: Probing protein clipping of the TGFβ1 RII ECD region The present inventors have discovered that antagonists containing the TGFβ1 RII ECD region, when produced by stably transfected CHO cells, exhibit unacceptable levels of proteolysis or clipping over time after storage. This is reflected in Figure 36, which shows that the percentage of low molecular weight (LMW) species determined by SE-UPLC using a Tosoh TSKgel UP-G3000SWXL column for Bi-PB-1.2 (PD1 / TGFβ1 RII ECD, see Figure 20A) improves with storage time at 4°C, while the percentage of dimeric species decreases over time.

[0379] Because no LMW was observed when the PD1 / PDL1-TGFB RII ECD molecule was produced by transient transfection of HEK293 cells, SEC analysis was performed to compare the benchmark and Bi-PB-1.2 produced by stable CHO derived from two different CHO transfection pools with Bi-PB-1.2 produced by HEK293 cells (Figure 37). As shown in Figure 37, the SEC graph shows a shoulder near the main peak and a smaller secondary peak for the molecule produced using CHO cells rather than HEK293 cells, consistent with improved proteolytic cleavage (or clipping) for the CHO-produced molecule.

[0380] To further investigate the specific nature of the cleavage, we expressed the Bi-PB-1.2 antagonist in CHO-K1 cells and performed cation exchange column chromatography (CEX) to generate fractions for liquid chromatography-mass spectrometry (LC-MS) to determine the moiety associated with clipping (Figure 38). The same analysis was performed on Bi-A1B-1 (data not shown). For LC-MS analysis, the sample was enzymatically deglycosylated using PNGase F. The reduced intact protein was then separated by reverse ultra-performance liquid chromatography (RP-UPLC) using TSKgel Phenyl-5PW. Mobile phase A (MPA) and mobile phase B (MPB) were 0.05% formic acid in water and 0.05% formic acid in acetonitrile, respectively. The column effluent passed directly through an online UV detector and then into the ESI source of an ESI-TOF (time-of-flight) mass spectrometer. The data are deconvoluted using Waters Masslynx software. To confirm sequence accuracy, the experimental mass of the reduced, deglycosylated molecule is compared with the theoretical mass calculated by the primary amino acid sequence.

[0381] Results from LC-MS (data not shown) identify several protein cleavage fragments (Figure 39). More specifically, these analytical results indicate that Bi-PB-1.2 and Bi-AB-1 have similar cleavage sites in their respective heavy chains, as further shown in Figures 40A and 40B, respectively.

[0382] Example 10: Reduced Clipping Properties of TGFβ1 RII ECD Mutants To develop mutant molecules containing the TGFβ1 RII ECD that exhibit reduced clipping, various ECD mutations were designed, as shown in Figures 41A-41C. The mutations were designed to target the proposed clipping sites. Figures 42A-42H, 43, and 44A-44D show the production and expression of the heavy chain sequences of Bi-PB-1.2, Bi-PLB-1.2, and Bi-A1B-1, respectively, which contain various ECD mutations as shown in Figures 41A-41C. Figure 45 shows the targeting of proposed clipping sites, including specific amino acid deletion mutations without additional substitutions (mutants Δ7, Δ12, and Δ13), specific amino acid deletion mutations without additional substitutions (mutants B, C, and I-B1), and complete substitution mutations in longer molecules (mutant DH).

[0383] The resulting molecule and parental purified protein A were transiently expressed in CHO cells. In Figures 46A-46C, reducing PAGE analysis of the expression products in Figures 42A-42H shows reduced clipping of the mutant molecule. Consistent with these results, SEC graphs of the expression products shown in Figures 47A and 47B lack the "shoulder" and second peak seen in Bi-PB-1.2 or observed in Figure 37. In Figure 48, the peaks in the SEC graphs are quantified, and the mutants show a lower percentage of high molecular weight forms compared to the parent molecule.

[0384] Example 11: Functional activity of TGFβ1 RII ECD mutations To confirm that the reduced clipping in the mutants did not compromise functional activity, we performed cell-based assays to assess the functional activity of the ECD mutants (Figure 49). Briefly, Bi-PB-1.2 and various TGFβ1 RII ECD mutants produced in HEK293 cells were serially diluted and tested (along with a negative control antibody) to determine whether the antagonists could block TGF-β1's ability to activate luciferase expression under conditions controlled by the SMAD response element described above. Biological activity was determined by measuring the decrease in luciferase-mediated fluorescence. The IC50 results from this assay are summarized in Figure 50 and show that all mutants, except for Bi-A1B-1W, Bi-PB-1.2Δ15, and Bi-PB-1.2Δ20, retain similar biological activity to the parental Bi-PB-1.2 and Bi-A1B-1.

[0385] Example 12: Pharmacokinetics of Bi-PB-1C and Bi-PB-1D To evaluate the in vivo pharmacokinetic properties of Bi-PB-1.2C and Bi-PB-1.2D compared to Bi-PB-1.2 (wt), pharmacokinetic curves were generated. Briefly, 10 mg / kg of each antagonist was intravenously injected into the tail vein of 6-10 week-old female CD1 mice (n = 2 mice per molecule). Serum was collected 3 min, 3 h, 1 day, 3 days, 7 days, and 10 days after injection. To detect antibodies in the serum, a 96-well ELISA plate was coated with 5 μg / ml goat anti-human IgG F(ab')2 fragment (Sigma, #SAB3701274) and then blocked using 5% milk (prepared in PBS). Serial dilutions of mouse serum in 5% milk and serial dilutions of purified protein molecules as standards were added to the dishes. After incubation with Peroxidase AffiniPure Mouse Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, #209-035-098) and further washing, Bi-PB-1.2 (Figure 51A), Bi-PB-1.2C (Figure 51B), and Bi-PB-1.2D (Figure 51C) antagonists were detected after incubation with TMB-ELISA substrate solution (Thermo-Fisher, #34029) and quantified by OD650 signal using a Perkin Elmer multifunction plate detector. The results of this analysis showed that the pharmacokinetic curves of Bi-PB-1.2C and Bi-PB-1.2D in mice were comparable to those of Bi-PB-1.2, with in vivo half-lives (T 1 / 2 ) indicates that

[0386] Example 13: Production from a stable CHO cell pool C mutant molecules corresponding to the parental Bi-PB-1.2, Bi-PLB-1, and Bi-A1B-1 molecules, and U mutants of the Bi-PB-1.2 and Bi-A1B-1 molecules were generated from CHO-K1 stable cell pools, and their clipping levels were compared with their wild-type counterparts and / or counterparts carrying the C mutants. The CHO-K1 stable cell pools were grown under fed-batch conditions for 14 days and purified by protein A chromatography. Clipping levels were determined by reducing PAGE analysis of the purified mutant and variant proteins. The results of this analysis show that clipping is significantly reduced in molecules carrying the C and U mutants compared to the parental wild-type molecules (Figures 52A-52B).

[0387] The foregoing description is intended to teach those skilled in the art how to carry out the present invention, and does not intend to describe in detail all of those obvious modifications and variations that will become apparent to those skilled in the art upon reading the specification. However, all such obvious modifications and variations are intended to be included within the scope of the present invention as defined by the appended claims. Unless the context clearly dictates otherwise, the intention is to cover the components and steps necessary in any order necessary to achieve the desired objectives.

Claims

1. an anti-tumor antagonist, a first targeting domain comprising a mutated TGF-β1 RII extracellular domain (mutated ECD); and an immunoglobulin scaffold having an amino terminus and a carboxy terminus; wherein the mutated ECD comprises one or more substitution mutations at positions 6, 7, 13, 16, 17, 20, 22, and / or 23 of SEQ ID NO: 89, or one or more deletion mutations in the region of positions 1 to 20 of SEQ ID NO: 89; and wherein the one or more mutations reduce proteolytic cleavage of the anti-tumor antagonist.

2. The one or more mutations are characterized in that they comprise a substitution of a non-polar amino acid residue at positions 6, 7, 16, 17, 20, 22 and / or 23 of SEQ ID NO: 89 with a polar or hydrophobic amino acid. The anti-tumor antagonist of claim 1.

3. the one or more mutations comprise a deletion of amino acid residues 1-7, 1-12, 1-13, 1-15, 1-20, 7-12, 7-13, 7-15, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, or 17-20 The anti-tumor antagonist of claim 1.

4. (1) one or more substitution mutations at positions 6, 7, 13, 16, 17, 20, 22, and / or 23 of SEQ ID NO: 89; and (2) A vector containing one or more deletion mutations in the region of sites 1 to 20 of SEQ ID NO:

89. The anti-tumor antagonist of claim 1.

5. The mutated ECD is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 114-123 and 251-270. The anti-tumor antagonist of claim 1.

6. Further comprising a second targeting domain. The antitumor antagonist according to any one of claims 1 to 5.

7. The antibody further comprises a second targeting domain, wherein the second targeting domain comprises an immune checkpoint modulator antagonist, a VEGF binding antagonist, a PD1 binding antagonist, or a PD-L1 binding antagonist. The antitumor antagonist of claim 6.

8. The first targeting domain is linked to the immunoglobulin scaffold by a peptide linker. The antitumor antagonist of claim 6.

9. The peptide linker is characterized in that it contains the amino acid sequence of SEQ ID NO: 101 or SEQ ID NO:

156. The anti-tumor antagonist of claim 8.

10. The second targeting domain specifically binds to PD-1. The antitumor antagonist according to any one of claims 6 to 9.

11. The second targeting domain comprises: (1) an immunoglobulin heavy chain variable region (HCVR) comprising three complementarity determining regions (HCDRs): HCDR1, HCDR2, and HCDR3; and (2) an immunoglobulin light chain variable region (LCVR) comprising three complementarity-determining regions (LCDRs): LCDR1, LCDR2, and HCDR3; In the above (1), wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 7, 9 and 12; wherein the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 10 and 13; and wherein the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 8, 11 and 14; In the above (2), wherein the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21-23 and 26; wherein the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 19, 24 and 27; and wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 20, 25 and 28. The anti-tumor antagonist of claim 10.

12. an HCVR having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, 35, 37 and 39, and / or SEQ ID NOs: 30, 32, 34, 36, 38 and 40.

12. The antitumor antagonist of claim 10 or 11.

13. an immunoglobulin HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33, 35, 37 and 39, and / or SEQ ID NOs: 30, 32, 34, 36, 38 and 40. The anti-tumor antagonist of claim 12.

14. an immunoglobulin HCVR having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 39, and / or and comprising an immunoglobulin LCVR having at least 90% identity with the amino acid sequence set forth in SEQ ID NO:

40.

12. The antitumor antagonist of claim 10 or 11.

15. an immunoglobulin HCVR having the amino acid sequence shown in SEQ ID NO: 39, and / or The immunoglobulin LCVR has the amino acid sequence shown in SEQ ID NO:

40. The anti-tumor antagonist of claim 14.

16. The second targeting domain is characterized by specifically binding to PD-L1. The antitumor antagonist according to any one of claims 6 to 9.

17. The second targeting domain comprises: (1) an immunoglobulin HCVR comprising three complementarity-determining regions (HCDRs), such as HCDR1, HCDR2, and HCDR3; and (2) an immunoglobulin LCVR comprising three complementarity-determining regions (LCDRs): LCDR1, LCDR2, and LCDR3; In the above (1), wherein the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 44, 50 and 53; wherein the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 45, 47, 49, 51 and 54; and wherein the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 46, 48, 52 and 55; In the above (2), wherein the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 59, 63, 66 and 69; wherein the LCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 60, 64, 67 and 70; and wherein the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 61, 62, 65, 68 and 71.

17. The anti-tumor antagonist of claim 16.

18. an immunoglobulin HCVR having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84 and 86, and / or and comprising an immunoglobulin LCVR having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 73, 75, 77, 79, 81, 83, 85, and 87.

18. The anti-tumor antagonist of claim 17.

19. an immunoglobulin HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 72, 74, 76, 78, 80, 82, 84 and 86, and / or SEQ ID Nos: 73, 75, 77, 79, 81, 83, 85 and 87.

18. The anti-tumor antagonist of claim 17.

20. an immunoglobulin HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 86, and / or SEQ ID NO:

87.

18. The anti-tumor antagonist of claim 17.

21. The second targeting domain is characterized by specifically binding to VEGF. The antitumor antagonist according to any one of claims 6 to 9.

22. The second targeting domain is characterized by comprising an amino acid sequence such as (1) SEQ ID NO: 88, (2) SEQ ID NOs: 90 and 91, or (3) SEQ ID NOs: 96 and 91.

22. The anti-tumor antagonist of claim 21.

23. The immunoglobulin scaffold is an IgG1, IgG2, or IgG4 scaffold. The antitumor antagonist according to any one of claims 1 to 22.

24. The immunoglobulin scaffold is characterized in that it comprises an N297A mutation, a K447A mutation, or both.

24. The anti-tumor antagonist of claim 23.

25. The immunoglobulin scaffold is characterized in that it comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 100, 159-166 and 173-175.

25. An antitumor antagonist according to claim 23 or 24.

26. SEQ ID NOs: 124-133, 145, 147, 148, 150, and 271-303. The anti-tumor antagonist of claim 1.

27. (1) a heavy chain amino acid sequence shown in SEQ ID NO: 126 or SEQ ID NO: 283 that pairs with a light chain amino acid sequence shown in SEQ ID NO: 93; (2) the heavy chain amino acid sequence shown in SEQ ID NO: 145 or SEQ ID NO: 303 paired with the light chain amino acid sequence shown in SEQ ID NO: 95; or (3) A light chain amino acid sequence shown in SEQ ID NO: 103 and a heavy chain amino acid sequence shown in SEQ ID NO: 148 or SEQ ID NO:

295. The anti-tumor antagonist of claim 1.

28. 1. A method of treating a cell proliferative disorder in a subject, comprising: A method of treating a cell proliferative disorder in a subject in need thereof, comprising administering to said subject an effective amount of the anti-tumor antagonist of any one of claims 1-27.

29. one or more nucleotides, Said one or more nucleotides, characterized in that they code for an antitumor antagonist according to any one of claims 1 to 27.

30. one or more vectors, 30. The one or more vectors, characterized in that they comprise one or more nucleotides according to claim 29.

31. A host cell comprising: A host cell transformed with one or more vectors according to claim 30.

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