IL-2 and anti-PD-1 based therapeutics and methods of use thereof

Abstract

The present disclosure relates to fusion proteins comprising an antigen-binding portion that specifically binds human PD-1 and an IL2 portion, and methods of use thereof.

Description

[Technical field]

[0001] The present disclosure relates generally to IL2-based therapeutics and methods of use thereof, and more specifically to fusion proteins comprising an IL2 moiety and an antigen-binding moiety that specifically binds to human PD-1, and methods of use thereof. [Background technology]

[0002] Interleukin 2 (IL-2 or IL2) is a multipotent cytokine produced primarily by activated T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphocyte-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, promotes the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells (see Non-Patent Document 1 and Non-Patent Document 2). IL2 is involved in the maintenance of peripheral CD4+CD25+ regulatory T (Treg) cells, also known as suppressor T cells (see, for example, Non-Patent Document 3). Treg cells inhibit effector T cells from destroying (self) targets by cell-to-cell contact by inhibiting the help and activation of T cells, or by the release of immunosuppressive cytokines such as IL-10 and TGFβ. Depletion of Treg cells has been shown to enhance antitumor immunity induced by IL2 (Non-Patent Document 4).

[0003] However, due to its pleiotropic effects, IL2 is not optimal for suppressing tumor growth. The use of IL2 as an antitumor agent has been limited by the severe toxicity associated with the doses required for tumor response. Proleukin® (sold by Prometheus Laboratories, San Diego, Calif.) is a recombinant form of IL2 that is approved for the treatment of metastatic melanoma and metastatic renal carcinoma, but its side effects are so severe that it is only recommended for use in hospitals with intensive care. The main side effect of IL2 therapy is vascular leak syndrome (VLS), which is the accumulation of interstitial fluid in the lungs and liver, causing pulmonary edema and liver damage. The only treatment for VLS is to discontinue IL2. IL2-induced pulmonary edema has been shown to result from direct binding of IL2 to pulmonary endothelial cells, which express low to moderate levels of functional high affinity IL2 receptors (Non-Patent Document 5). Despite the general acceptance of CD122-directed IL2 therapeutics being developed for cancer treatment, it has surprisingly been found that such molecules have a low therapeutic index for cancer treatment, requiring high and toxic doses to achieve even modest anticancer effects. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Waldmann, 2006, Nat Rev Immunol 6:595~601 [Non-Patent Document 2] Malek, 2008, Annu Rev Immunol 26:453~79 [Non-Patent Document 3] Fontenot et al., 2005, Nature Immunol 6:1142-51 [Non-Patent Document 4] Imai et al., 2007, Cancer Sci 98:416~23 [Non-Patent Document 5] Krieg et al., 2010, Proc Nat Acad Sci USA 107:11906-11 Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is an urgent need in the art for new IL2 therapies with improved therapeutic efficacy and safety profiles. [Means for solving the problem]

[0006] The present disclosure addresses one or more of the above needs. The fusion protein of the present disclosure comprises an antigen-binding portion that specifically binds to human PD-1 to target tumor-reactive T cells. The antigen-binding portion is useful for selectively reconstituting activity on tumor-reactive T cells. The fusion protein further comprises an IL2 portion, the IL2 portion comprising IL2 bound to IL2Rα. In certain embodiments, the fusion protein comprises an unmodified IL2 sequence. In certain embodiments, the fusion protein retains the ability to associate with endogenous IL2Rα. The fusion protein comprises an IL2 portion in a trans-sequestered conformation. This maintains engagement with activated CD8+ T cells; however, the bound conformation of the IL2 portion serves to mask IL2 and attenuate its activity, thus leading to reduced systemic toxicity. For example, the fusion protein of the present disclosure does not induce acute pulmonary edema (vascular leakage) in mice compared to wild-type IL2. The fusion proteins of the present disclosure provide enhanced anti-tumor efficacy and improved therapeutic index compared to IL2 alone or in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof).

[0007] In one aspect, the disclosed technology relates to a fusion protein comprising: (i) an antigen-binding portion that specifically binds to human programmed cell death protein 1 (PD-1), and (ii) an interleukin 2 (IL2) portion. In some embodiments, the antigen-binding portion comprises an antibody or antigen-binding fragment thereof that specifically binds to human PD-1. In some embodiments, the antibody or antigen-binding fragment thereof that binds to human PD-1 is a human monoclonal antibody. In some embodiments, the antigen-binding portion comprises three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2 and HCDR3), and three light chain CDRs (LCDR1, LCDR2 and LCDR3): HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, 4, or 24; HCDR2 comprises the amino acid sequence of SEQ ID NO: 45, 6, or 26; HCDR3 comprises the amino acid sequence of SEQ ID NO: 47, 8, or 28; LCDR1 comprises the amino acid sequence of SEQ ID NO: 12 or 32; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or 35.

[0008] In some embodiments, the antigen-binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively; (ii) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; or (iii) SEQ ID NOs: 24, 26, 28, 32, 14, and 35, respectively. In some embodiments, the antigen-binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively. In some embodiments, the antigen-binding portion comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 41, 2, or 22; and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10 or 30. In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO: 2, and the LCVR comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO:22 and the LCVR comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO:41 and the LCVR comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the antigen-binding portion comprises a heavy chain constant region of SEQ ID NO:55 and a light chain constant region of SEQ ID NO:56. In some embodiments, the antigen-binding portion comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:61, 57, or 59 and the light chain comprises the amino acid sequence of SEQ ID NO:62, 58, or 60. In some embodiments, the antigen-binding portion comprises a heavy chain / light chain sequence pair of SEQ ID NO:61 / 62, 57 / 58, or 59 / 60.

[0009] In some embodiments, the antigen-binding portion comprises the heavy / light chain sequence pair of SEQ ID NO:61 / 62. In some embodiments, the IL2 portion comprises (i) IL2 or a fragment thereof; and (ii) IL2 receptor alpha (IL2Rα) or a fragment thereof. In some embodiments, the IL2 or a fragment thereof is human IL2 (hIL2) or a fragment thereof. In some embodiments, the IL2Rα or a fragment thereof is human IL2Rα (hIL2Rα) or a fragment thereof. In some embodiments, the IL2 or a fragment thereof comprises the amino acid sequence of SEQ ID NO:53. In some embodiments, the IL2Rα or a fragment thereof comprises the amino acid sequence of SEQ ID NO:51. In some embodiments, the IL2 or a fragment thereof is connected to the C-terminus of the IL2Rα or a fragment thereof via a linker. In some embodiments, the IL2 portion is connected to the C-terminus of the heavy chain constant region of the antigen-binding portion via a linker. In some embodiments, the linker comprises an amino acid sequence of one or more repeats of GGGGS (SEQ ID NO:67). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:50 or 52. In some embodiments, the IL2 portion comprises the amino acid sequence of SEQ ID NO:54.

[0010] In another aspect, the disclosed technology relates to a fusion protein comprising: (i) a first polypeptide comprising a light chain variable region (LCVR) of a human antibody that specifically binds to human PD-1; and (ii) a second polypeptide comprising (a) a heavy chain variable region (HCVR) of an antibody that specifically binds to human PD-1 and (b) an IL2 portion. In some embodiments, the HCVR comprises three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2, and HCDR3), and the LCVR comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3): HCDR1 comprises the amino acid sequence of SEQ ID NO: 43, 4, or 24; HCDR2 comprises the amino acid sequence of SEQ ID NO: 45, 6, or 26; HCDR3 comprises the amino acid sequence of SEQ ID NO: 47, 8, or 28; LCDR1 comprises the amino acid sequence of SEQ ID NO: 12 or 32; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or 35. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of: (i) SEQ ID NOs: 43, 45, 47, 12, 14, and 16; (ii) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; or (iii) SEQ ID NOs: 24, 26, 28, 32, 14, and 35. In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of: 43, 45, 47, 12, 14, and 16, respectively.

[0011] In some embodiments, the HCVR and LCVR comprise the amino acid sequences of (i) SEQ ID NOs: 41 and 10; (ii) SEQ ID NOs: 2 and 10; or (iii) SEQ ID NOs: 22 and 30. In some embodiments, the first polypeptide comprises a light chain constant region linked to the LCVR. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the second polypeptide comprises a heavy chain constant region linked to the HCVR, and the IL2 moiety is linked to the C-terminus of the heavy chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the first polypeptide comprises a light chain sequence of SEQ ID NOs: 62, 58, or 60. In some embodiments, the second polypeptide comprises a heavy chain sequence of SEQ ID NOs: 61, 57, or 59.

[0012] In some embodiments, the fusion protein comprises a heavy chain / light chain sequence pair of SEQ ID NO: 61 / 62, 57 / 58, or 59 / 60. In some embodiments, the fusion protein comprises a heavy chain / light chain sequence pair of SEQ ID NO: 61 / 62. In some embodiments, the IL2 portion comprises (i) IL2 or a fragment thereof; and (ii) IL2Rα or a fragment thereof. In some embodiments, the IL2 portion is connected to the C-terminus of the heavy chain constant region via a linker. In some embodiments, the IL2 or a fragment thereof is connected to the C-terminus of the IL2Rα or a fragment thereof via a linker. In some embodiments, the IL2 or a fragment thereof is human IL2 (hIL2) or a fragment thereof. In some embodiments, the IL2Rα or a fragment thereof is human IL2Rα (hIL2Rα) or a fragment thereof. In some embodiments, the IL2 or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the IL2Rα or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the linker comprises one or more repeats of the amino acid sequence of GGGGS (SEQ ID NO: 67). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 50 or 52. In some embodiments, the IL2 portion comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 62, 58 or 60.

[0013] In some embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 63, 64 or 65. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 58 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 62 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the fusion protein forms a dimeric fusion protein. In some embodiments, the fusion protein dimerizes via the respective heavy chain constant regions.

[0014] In some embodiments, the fusion protein does not cross-compete with REGN2810, pembrolizumab, or nivolumab for binding to PD-1. In some embodiments, the fusion protein exhibits reduced activity to activate human IL2Rα / β / γ and IL2Rβ / γ complexes compared to IL2. In some embodiments, the fusion protein exhibits increased activity to activate human IL2Rα compared to non-targeted IL2Rα-IL2. In some embodiments, the fusion protein exhibits increased activity to stimulate T cells as measured by the level of IFN-γ release compared to wild-type human IL2. In some embodiments, the fusion protein exhibits attenuated binding to IL2Rα, IL2Rβ, and IL2Rγ.

[0015] In another aspect, the disclosed technology relates to a nucleic acid or a plurality of nucleic acids comprising a polynucleotide sequence encoding a fusion protein disclosed herein. In another aspect, the disclosed technology relates to a vector comprising a nucleic acid or a plurality of nucleic acids disclosed herein. In another aspect, the disclosed technology relates to a host cell comprising a nucleic acid or a plurality of nucleic acids or vectors disclosed herein. In some embodiments, the host cell expresses a first vector comprising a polynucleotide sequence encoding a first polypeptide of a fusion protein disclosed herein and a second vector comprising a polynucleotide sequence encoding a second polypeptide of a fusion protein disclosed herein.

[0016] In another aspect, the disclosed technology relates to a method of producing a fusion protein disclosed herein, comprising culturing a host cell disclosed herein under conditions allowing the production of the fusion protein or fragment, and recovering the fusion protein or fragment thereof so produced.

[0017] In another aspect, the disclosed technology relates to a pharmaceutical composition comprising the fusion protein disclosed herein. In some embodiments, the pharmaceutical composition further comprises an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof does not cross-compete with the fusion protein for binding to PD-1.

[0018] In another aspect, the disclosed technology relates to a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a fusion protein or pharmaceutical composition disclosed herein. In some embodiments, the method comprises administering to the subject the fusion protein in an amount between 0.005 mg / kg and 10 mg / kg based on the subject's body weight. In some embodiments, the method further comprises administering to the subject a second therapeutic agent or therapy. [Brief description of the drawings]

[0019] [Figure 1A] Graphs showing the results of the in vivo study described in Example 6. Figure 1A and Figure 1B show the tumor volume (mm3) (Figure 1A) and survival rate (Figure 1B) of mice treated with isotype control Ab, isotype control hIL2Rα-IL2 + anti-PD-1, and REGN10486 + isotype control Ab. Figure 1C and Figure 1D show the tumor volume (mm3) (Figure 1C) and survival rate (Figure 1D) of mice treated with isotype control, REGN10486, REGN10595, and REGN10597. [Figure 1B] Graphs showing the results of the in vivo study described in Example 6. Figure 1A and Figure 1B show the tumor volume (mm3) (Figure 1A) and survival rate (Figure 1B) of mice treated with isotype control Ab, isotype control hIL2Rα-IL2 + anti-PD-1, and REGN10486 + isotype control Ab. Figure 1C and Figure 1D show the tumor volume (mm3) (Figure 1C) and survival rate (Figure 1D) of mice treated with isotype control, REGN10486, REGN10595, and REGN10597. [Figure 1C] Graphs showing the results of the in vivo study described in Example 6. Figure 1A and Figure 1B show the tumor volume (mm3) (Figure 1A) and survival rate (Figure 1B) of mice treated with isotype control Ab, isotype control hIL2Rα-IL2 + anti-PD-1, and REGN10486 + isotype control Ab. Figure 1C and Figure 1D show the tumor volume (mm3) (Figure 1C) and survival rate (Figure 1D) of mice treated with isotype control, REGN10486, REGN10595, and REGN10597. [Figure 1D] Graphs showing the results of the in vivo study described in Example 6. Figure 1A and Figure 1B show the tumor volume (mm3) (Figure 1A) and survival rate (Figure 1B) of mice treated with isotype control Ab, isotype control hIL2Rα-IL2 + anti-PD-1, and REGN10486 + isotype control Ab. Figure 1C and Figure 1D show the tumor volume (mm3) (Figure 1C) and survival rate (Figure 1D) of mice treated with isotype control, REGN10486, REGN10595, and REGN10597. [Figure 2A] FIG. 2B is a set of graphs showing the results of a FACS binding assay comparing the binding of the anti-α-hPD1 antibody ("αhPD1" or "ahPD1") parent antibody to PD1+ cells (FIG. 2A) and the binding of the anti-αhPD1 antibody-IL2Rα-IL2 fusion construct to PD1+ cells (FIG. 2B), as described in Example 8. [Figure 2B] FIG. 2B is a set of graphs showing the results of a FACS binding assay comparing the binding of the anti-α-hPD1 antibody ("αhPD1" or "ahPD1") parent antibody to PD1+ cells (FIG. 2A) and the binding of the anti-αhPD1 antibody-IL2Rα-IL2 fusion construct to PD1+ cells (FIG. 2B), as described in Example 8. [Figure 3A] FIG. 3A shows the experimental design (FIG. 3B) and resulting native mass spectra (FIG. 3B) of a mixture of anti-PD-1-IL2Ra-IL2+hIgG4 antibodies compared to specific antibody mixtures as described in Example 9. [Figure 3B-1] FIG. 3A shows the experimental design (FIG. 3B) and resulting native mass spectra (FIG. 3B) of a mixture of anti-PD-1-IL2Ra-IL2+hIgG4 antibodies compared to specific antibody mixtures as described in Example 9. [Figure 3B-2] FIG. 3A shows the experimental design (FIG. 3B) and resulting native mass spectra (FIG. 3B) of a mixture of anti-PD-1-IL2Ra-IL2+hIgG4 antibodies compared to specific antibody mixtures as described in Example 9. [Figure 4A] FIG. 4A is a set of graphs showing IFNγ release and proliferation of T cells in response to antibody or cytokine titration, as described in Example 10. Allogeneic PBMCs mixed with T cells were treated with dose titrations of recombinant IL2, anti-PD-1-IL2Rα-IL2 (REGN10597, REGN10595, and REGN10486) or isotype-IL2Rα-IL2 control (REGN9903), and IFNγ release (FIG. 4A) and proliferation (FIG. 4B) were assessed. [Figure 4B]FIG. 4A is a set of graphs showing IFNγ release and proliferation of T cells in response to antibody or cytokine titration, as described in Example 10. Allogeneic PBMCs mixed with T cells were treated with dose titrations of recombinant IL2, anti-PD-1-IL2Rα-IL2 (REGN10597, REGN10595, and REGN10486) or isotype-IL2Rα-IL2 control (REGN9903), and IFNγ release (FIG. 4A) and proliferation (FIG. 4B) were assessed. [Figure 5A] FIG. 5B is a set of graphs showing IFNγ release of T cells in response to antibody or cytokine titration in the absence (FIG. 5A) or presence (FIG. 5B) of REGN2810, as described in Example 11. RAJI / CD80 KO / CD86 KO / PDL1 cells were incubated with T cells and treated with a fixed dose of anti-CD3×CD20 (REGN1979) and dose titrations of isotype control (REGN7540), isotype-IL2Rα-IL2 antibody (REGN9903), anti-PD1 antibody with the variable region of mAb9048 (REGN15187), anti-PD1-IL2Rα-IL2 (REGN10597), or a combination of REGN9903+REGN15187, in the absence (FIG. 5A) or presence (FIG. 5B) of REGN2810. IFNγ release was measured. [Figure 5B] FIG. 5B is a set of graphs showing IFNγ release of T cells in response to antibody or cytokine titration in the absence (FIG. 5A) or presence (FIG. 5B) of REGN2810, as described in Example 11. RAJI / CD80 KO / CD86 KO / PDL1 cells were incubated with T cells and treated with a fixed dose of anti-CD3×CD20 (REGN1979) and dose titrations of isotype control (REGN7540), isotype-IL2Rα-IL2 antibody (REGN9903), anti-PD1 antibody with the variable region of mAb9048 (REGN15187), anti-PD1-IL2Rα-IL2 (REGN10597), or a combination of REGN9903+REGN15187, in the absence (FIG. 5A) or presence (FIG. 5B) of REGN2810. IFNγ release was measured. [Figure 6A]FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6B-1] FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6B-2] FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6C]FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6D] FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6E] FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6F]FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6G] FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 6H] FIG. 6 is a graph showing in vivo activity evaluation of the anti-PD1-IL2Rα-IL2 fusion construct REGN10597 in PD1×LAG3 humanized mice, as described in Example 12, primarily in comparison to: the non-targeting control REGN9904 in combination with the PD-1 blocking antibody REGN2810 (FIGS. 6A and 6B); the comparative anti-PD1 targeted "non-α" (3m) molecule REGN13233 (FIGS. 6C, 6D, 6E, and 6F); and a construct containing all the same components as REGN10597, but in which IL2 and IL2Rα are fused to each other in the reverse order (FIGS. 6G and 6H). [Figure 7A]Figure 7 shows a graph of the IL2 binding assay described in Example 13. YT / STAT5-Luc / IL2Ra KO (Figure 7A) or YT / STAT5-Luc / IL2Ra OE (Figure 7B) were incubated with titrations of an antibody fused in tandem to IL2 (REGN8512; dashed grey open circle) or an antibody fused in tandem to IL2Ra and IL2 (REGN9904; solid black closed circle). Cells were washed, stained with secondary antibody AF647 anti-human IgG, washed, fixed and acquired on an iQue Plus flow cytometer. [Figure 7B] Figure 7 shows a graph of the IL2 binding assay described in Example 13. YT / STAT5-Luc / IL2Ra KO (Figure 7A) or YT / STAT5-Luc / IL2Ra OE (Figure 7B) were incubated with titrations of an antibody fused in tandem to IL2 (REGN8512; dashed grey open circle) or an antibody fused in tandem to IL2Ra and IL2 (REGN9904; solid black closed circle). Cells were washed, stained with secondary antibody AF647 anti-human IgG, washed, fixed and acquired on an iQue Plus flow cytometer. [Figure 8A] FIG. 8B is a graph showing IL2 reporter assay comparison of Ab-IL2Ra-IL2 and Ab-IL2-IL2Ra as described in Example 14. YT / STAT5-Luc / IL2Ra KO / hPD1 (FIG. 8A) or YT / STAT5-Luc / hIL2Ra / hPD1 (FIG. 8B) were incubated with titrations of non-targeting IL2Ra-IL2 (black dashed line with black open circle symbols, REGN9903), non-targeting IL2-IL2Ra (grey dashed line with grey open square symbols), anti-PD1-IL2Ra-IL2 (black solid line with black closed circle symbols, REGN10597), or anti-PD1-IL2-IL2Ra (grey solid line with grey closed square symbols). After 4 hours and 30 minutes, STAT5 activity was assessed by luminescence measurements. [Figure 8B]FIG. 8B is a graph showing IL2 reporter assay comparison of Ab-IL2Ra-IL2 and Ab-IL2-IL2Ra as described in Example 14. YT / STAT5-Luc / IL2Ra KO / hPD1 (FIG. 8A) or YT / STAT5-Luc / hIL2Ra / hPD1 (FIG. 8B) were incubated with titrations of non-targeting IL2Ra-IL2 (black dashed line with black open circle symbols, REGN9903), non-targeting IL2-IL2Ra (grey dashed line with grey open square symbols), anti-PD1-IL2Ra-IL2 (black solid line with black closed circle symbols, REGN10597), or anti-PD1-IL2-IL2Ra (grey solid line with grey closed square symbols). After 4 hours and 30 minutes, STAT5 activity was assessed by luminescence measurements. [Figure 9A] FIG. 9B is a graph showing IL2 reporter assay comparison of Ab-IL2Ra-IL2 and Ab-IL2(3m) as described in Example 14. YT / STAT5-Luc / IL2Ra KO / hPD1 (FIG. 9A) or YT / STAT5-Luc / hIL2Ra / hPD1 (FIG. 9B) were incubated with titrations of non-targeting IL2Ra-IL2 (black open circle symbols with black dashed line, REGN9904), non-targeting IL2(3m) (grey dashed line with grey diamond open symbols), anti-PD1-IL2Ra-IL2 (black solid line with black closed circle symbols, REGN10597), or anti-PD1-IL2(3m) (grey solid line with grey diamond closed symbols). After 4 hours, STAT5 activity was assessed by luminescence measurements. [Figure 9B] FIG. 9B is a graph showing IL2 reporter assay comparison of Ab-IL2Ra-IL2 and Ab-IL2(3m) as described in Example 14. YT / STAT5-Luc / IL2Ra KO / hPD1 (FIG. 9A) or YT / STAT5-Luc / hIL2Ra / hPD1 (FIG. 9B) were incubated with titrations of non-targeting IL2Ra-IL2 (black open circle symbols with black dashed line, REGN9904), non-targeting IL2(3m) (grey dashed line with grey diamond open symbols), anti-PD1-IL2Ra-IL2 (black solid line with black closed circle symbols, REGN10597), or anti-PD1-IL2(3m) (grey solid line with grey diamond closed symbols). After 4 hours, STAT5 activity was assessed by luminescence measurements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] It is to be understood that the disclosure is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It is also to be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting, and the scope of the disclosure is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety unless otherwise indicated.

[0021] Fusion proteins In one aspect, the present disclosure provides a fusion protein comprising: (i) an antigen-binding portion that specifically binds to human PD-1, and (ii) an IL2 portion. The disclosed fusion protein is surprisingly effective in stimulating T cells and inhibiting tumor growth compared to anti-hPD1 antibodies (such as REGN2810). In certain embodiments, the fusion protein exhibits a better safety profile compared to anti-PD-1 targeting IL2 molecules (such as REGN13233).

[0022] The disclosed fusion proteins are monomeric or multimeric, for example, dimeric (homodimer or heterodimer) or higher order complex.For simplicity, fusion proteins that are homodimeric (or higher order multimers of the same polypeptide) are described by their constituent monomers; however, upon recombinant expression of the constituent monomers in an appropriate cell line, homodimeric (or higher order multimeric) molecules are produced.

[0023] In some embodiments, the antigen-binding moiety comprises an antibody or antigen-binding fragment thereof that specifically binds to human PD-1. In some embodiments, the antigen-binding moiety that binds to human PD-1 is a human monoclonal antibody.

[0024] As used herein, the term "antibody" refers to an immunoglobulin molecule (i.e., a "complete antibody molecule") that is made up of four polypeptide chains, two heavy (H) and two light (L) chains, linked by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain comprises a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (comprising domains CH1, CH2, and CH3). Each light chain comprises a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions can be further subdivided into more conserved regions, called framework regions (FR), interspersed with regions of hypervariability, called complementarity determining regions (CDRs). Each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of an antibody (or an antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a comparative analysis of two or more CDRs. The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules.

[0025] As used herein, the terms "antigen-binding fragment" of an antibody, "antigen-binding portion" of an antibody, and the like include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion and recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated using chemical or molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids, and the like.

[0026] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragments" as used herein.

[0027] Antigen-binding fragments of antibodies generally contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or co-located with one or more framework sequences. L V associated with domain H For antigen-binding fragments containing domains, V H Domains and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a dimer of monomeric V H or V L It may also include a domain.

[0028] In some embodiments, an antigen-binding fragment of an antibody can comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains found in antigen-binding fragments of an antibody of the present disclosure include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L-C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations above, the variable and constant domains are directly linked to each other or linked by a full or partial hinge or linker region. The hinge region is composed of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, thereby providing a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure can be homodimers or heterodimers (or other multimers) of any of the above variable and constant domain configurations in non-covalent association with each other and / or with one or more monomeric Vs. H Or V L It may be included in association with the domain (eg, by a disulfide bond).

[0029] The antibody or antigen-binding fragment thereof contained in the fusion protein disclosed herein may be a human antibody or antigen-binding fragment thereof. As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. The human antibody or antigen-binding fragment thereof contained in the fusion protein of the present disclosure may nevertheless contain amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs and particularly in CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mouse, are grafted onto human framework sequences.

[0030] The antibody contained in the fusion protein disclosed herein may be a recombinant human antibody. As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) introduced with human immunoglobulin genes (see, e.g., Taylor et al., (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) to thereby modify the V and V regions of the recombinant antibody. H and V LThe amino acid sequence of the region is H and V L It is a sequence derived from and related to the sequence, but which may not naturally occur within the human antibody germline repertoire in vivo.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to human PD-1. As used herein, terms such as "specifically bind" mean that the antibody or antigen-binding fragment thereof forms a complex with the antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" to human PD-1 as used in the context of this disclosure has a K of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM as measured by a surface plasmon resonance assay. D The term "antibody" includes antibodies that specifically bind to human PD-1 or a portion thereof. However, an isolated antibody that specifically binds human PD-1 may exhibit cross-reactivity to other antigens, such as PD-1 molecules from other (non-human) species.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2 and HCDR3) and three light chain CDRs (LCDR1, LCDR2 and LCDR3): HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, 24, or 43; HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, 26, or 45; HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, 28, or 47; LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, or 32; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or 35.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of: (i) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; (ii) SEQ ID NOs: 24, 26, 28, 32, 14, and 35; or (iii) SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, 22, or 41, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, 22, or 41; and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 10, or 30, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, or 30.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:2, and a LCVR comprising the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:10.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:22, and a LCVR comprising the amino acid sequence of SEQ ID NO:30, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:30.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence of SEQ ID NO:41, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:41, and a LCVR comprising the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:10.

[0039] In some embodiments, the fusion protein further comprises a heavy chain constant region of SEQ ID NO:55, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:55, and a light chain constant region of SEQ ID NO:56, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:56.

[0040] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:57, 59, or 61, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:57, 59, or 61, and the light chain has the amino acid sequence of SEQ ID NO:58, 60, or 62, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:58, 60, or 62.

[0041] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:57, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:57, and the light chain comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:58.

[0042] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:59, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:59, and the light chain comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:61.

[0043] In some embodiments, the fusion protein comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:61, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:61, and the light chain comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:62.

[0044] In some embodiments, the fusion protein comprises the heavy / light chain sequence pair of SEQ ID NOs: 57 / 58, 59 / 60, or 61 / 62. In some embodiments, the fusion protein comprises the heavy / light chain sequence pair of SEQ ID NOs: 61 / 62.

[0045] In some embodiments, the IL2 portion comprises: (i) IL2, or a fragment thereof; and (ii) IL2 receptor alpha (IL2Rα), or a fragment thereof.

[0046] In some embodiments, the IL2 moiety may comprise a wild-type or variant IL2 domain, which is fused, optionally via a linker, to the IL2 binding domain of IL-2Rα. The IL2 binding domain of IL-2Rα is N-terminal or C-terminal to the wild-type or variant IL2 domain. In some embodiments, the IL2 binding domain of IL-2Rα is N-terminal to the wild-type or variant IL2 domain.

[0047] In some embodiments, the IL2 portion comprises the amino acid sequence of SEQ ID NO:54, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:54.

[0048] In eukaryotic cells, human IL2 is synthesized as a 153 amino acid precursor polypeptide from which 20 amino acids are removed to yield the mature secreted IL2 (Taniguchi et al., 1983, Nature 302(5906):305-10). Mature human IL2 has the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 75) has.

[0049] In some embodiments, the IL2 moieties are directed against IL2Ra, e.g., they have one or more amino acid substitutions in the IL2 domain that cause them to preferentially bind to IL-2Rα compared to IL-2Rβ. In some embodiments, the IL2 moieties are not directed against CD122, e.g., they do not have amino acid substitutions in the IL2 domain that cause them to preferentially bind to IL-2Rβ compared to IL-2Rα.

[0050] In some embodiments, the fusion proteins of the present disclosure have one or more amino acid substitutions in the IL2 domain that reduce binding to IL-2Rβ. For example, in some embodiments, the IL2 moiety may have up to 50-fold (in some embodiments, up to 100-fold) to 1,000-fold reduced binding to human IL-2Rβ compared to wild-type human IL2. In some embodiments, the IL2 moiety with reduced binding to IL-2Rβ may retain affinity to IL-2Rα or may have reduced binding to IL-2Rα. For example, in some embodiments, the IL2 moiety may have up to 50-fold reduced binding to human IL2-Rα compared to wild-type human IL2. In one embodiment, the IL2 domain includes one or more amino acid substitutions that reduce affinity to IL-2Rβ and maintain affinity to IL2-Rα. An exemplary amino acid substitution is N88D. Other amino acid substitutions that reduce or eliminate the affinity of IL2 for IL-2Rβ are D20T, N88R, N88D or Q126D (see, eg, US Patent Publication No. 2007 / 0036752).

[0051] In some embodiments, the IL2 domain contains one or more amino acid substitutions that reduce affinity for IL2-Rα and maintain or to a lesser extent reduce affinity for IL-2Rβ, resulting in a CD122-directed IL2 moiety. An exemplary CD122-directed IL2 domain is one that contains both the H16A and F42A substitutions. Thus, in some embodiments, the IL2 moiety comprises the amino acid sequence of human IL2 with the H16A and F42A substitutions.

[0052] In some embodiments, the IL2 portion comprises an amino acid substitution that removes the O-glycosylation site of IL2 at a position corresponding to residue 3 of human IL2. Exemplary amino acid substitutions in T3 are T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In certain embodiments, the substitution is T3A.

[0053] In some embodiments, the IL2 domain may include a substitution at C125, which may be substituted with S, V, or A to reduce protein aggregation, as described in U.S. Patent No. 4,518,584. In some embodiments, the IL2 domain may include a substitution of methionine 104 with a neutral amino acid, such as alanine, as described in U.S. Patent No. 5,206,344. In some embodiments, the IL2 domain may delete the N-terminal alanine residue of IL2, resulting in des-A1 IL2.

[0054] In some embodiments, the IL2 domain can have amino acid deletions and / or substitutions selected from des-A1 M104A IL2, des-A1 M104A C125S IL2, M104A IL2, M104A C125A IL2, des-A1 M104A C125A IL2, and M104A C125S IL2, in addition to other mutations, which can alter the binding of IL2 to its receptor. These and other mutants are described in U.S. Patent No. 5,116,943 and Weiger et al., 1989, Eur J Biochem 180:295-300.

[0055] In some embodiments, the IL2 domain may comprise an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to mature human IL2.

[0056] In some embodiments, the IL2 or fragment thereof comprises the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:53.

[0057] In some embodiments, the IL2 moiety further comprises an IL2 binding domain of IL-2Rα (referred to as an "IL2-Rα domain"), e.g., the extracellular domain of IL-2Rα fused at the C-terminus or N-terminus of IL2, optionally via a linker. In some embodiments, the IL2Rα or fragment thereof is human IL2Rα (hIL2Rα) or a fragment thereof. In some embodiments, the IL2-Rα domain may comprise a mature human IL-2Rα extracellular domain (corresponding to amino acids 22-272 of human IL-Rα). In some embodiments, the IL2-Rα domain may comprise an IL2 binding portion of the human IL-2Rα extracellular domain (including two "sushi" domains corresponding to amino acids 22-186 of human IL-2Rα). In some embodiments, the IL2-Rα domain may comprise an alternative IL2 binding portion of the human IL-2Rα extracellular domain corresponding to amino acids 22-240 of human IL-2Rα.

[0058] In some embodiments, the IL2-Rα domain or the IL2-binding portion of the IL-2Rα extracellular domain has an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to any one of the above sequences, i.e., amino acids 22-186 of IL-2Rα, amino acids 22-240 of IL-2Rα, or amino acids 22-272 of IL-2Rα, or an IL2-binding portion thereof.

[0059] In some embodiments, the IL2-Rα domain or IL2 binding portion can comprise or consist of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the IL2 binding portion of human IL-2Rα, optionally where the binding portion has an amino acid sequence of (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids or at least 165 amino acids, and / or (b) up to 251, up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180 or up to 170 amino acids of the extracellular domain of human IL2-Rα. In certain embodiments, the portion of human IL-2Rα is linked by any one of (a) and (b) above, e.g., at least 160 and up to 180 amino acids from human IL-2Rα, at least 162 and up to 200 amino acids from human IL-2Rα, at least 160 and up to 220 amino acids from human IL-2Rα, at least 164 and up to 190 amino acids from human IL-2Rα, etc.

[0060] In some embodiments, the IL2-Rα domain or IL2 binding portion comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to amino acids 22-186 of IL2-Rα, with or without up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 40 additional amino acids C-terminal to amino acid residue 186.

[0061] In some embodiments, the IL2-Rα domain or IL-2Rα extracellular domain has at least one less O-glycosylation and / or N-glycosylation compared to the extracellular domain of native IL-2Rα, e.g., by substitution at one or more of amino acids N49, N68, T74, T85, T197, T203, T208, and T216. In some embodiments, the one or more substitutions are for an asparagine with an amino acid selected from alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are for threonine with an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine. In some embodiments, the one or more substitutions are at amino acid S50 (e.g., S50P), amino acid S51 (e.g., S51R, S51N, S51D, S51C, S51Q, S51E, S51G, S51H, S51I, S51L, S51K, S51M, S51F, S51P, S51W, S51Y, or S51V), amino acid T69 (e.g., T69P), amino acid T70 (e.g., T70R, T70N, T70D, T70C, T70Q, T70E, T70G, T70H, T70I, T70L, T70K, T70M, T70F, T70P, T70W, T70Y, or T70V, amino acid C192 (e.g., C192R, C192N, C192D, C192Q, C192E, C192G, C192H, C192I, C192L, C192K, C192M, C192F, C192P, C192W, C192Y, or C192V), or any combination thereof.

[0062] In some embodiments, the IL2Rα or a fragment thereof comprises the amino acid sequence of SEQ ID NO:51, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:51.

[0063] A fusion protein can include one or more linkers (e.g., peptide or non-peptide linkers) that connect various components of the molecule. In some embodiments, two or more components of a fusion protein are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect (a) an IL2 moiety and an antigen-binding moiety; (b) different domains within an IL2 moiety (e.g., an IL2 domain and an IL-Rα domain); or (c) different domains within an antigen-binding moiety (e.g., different components of an anti-PD-1 antibody).

[0064] Peptide linkers can range from 2 amino acids to 60 amino acids or more, in some embodiments, peptide linkers range in length from 3 amino acids to 50 amino acids, 4 amino acids to 30 amino acids, 5 amino acids to 25 amino acids, 10 amino acids to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids.

[0065] In some embodiments, the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length. In some embodiments, the linker is in the range of 5 amino acids to 50 amino acids in length, e.g., 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker is in the range of 6 amino acids to 50 amino acids in length, e.g., 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker is in the range of 7 amino acids to 50 amino acids in length, e.g., 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids in length.

[0066] In some embodiments, the linker comprises a polar (e.g., serine (S)) or charged (e.g., lysine (K)) residue. In some embodiments, the linker is a flexible linker, e.g., comprising one or more glycine (G) or serine (S) residues.

[0067] Examples of flexible linkers that can be used in the fusion proteins of the present disclosure include those disclosed by Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369, and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers are or include glycine and serine repeats, such as GnS or SGn monomers or multimers, where n is an integer between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is or includes G4S(GGGGS; SEQ ID NO:67) repeat monomers or multimers, such as (GGGGS)n.

[0068] Polyglycine linkers can be suitably used in the fusion proteins of the present disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO:68), five consecutive glycines (5Gly) (SEQ ID NO:69), six consecutive glycines (6Gly) (SEQ ID NO:70), seven consecutive glycines (7Gly) (SEQ ID NO:71), eight consecutive glycines (8Gly) (SEQ ID NO:72) or nine consecutive glycines (9Gly) (SEQ ID NO:73).

[0069] In some embodiments, the IL2 moiety and the antigen-binding moiety are connected via a linker. In some embodiments, the linker comprises one or more repeats of the amino acid sequence of GGGGS (SEQ ID NO: 67). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 50 or 52. In some embodiments, the IL2 moiety is linked to the C-terminus of the antigen-binding moiety via a peptidyl linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 50.

[0070] In another aspect, the disclosure provides a fusion protein comprising: (i) a first polypeptide comprising a light chain variable region (LCVR) of a human antibody that specifically binds human PD-1; and (ii) a second polypeptide comprising (a) a heavy chain variable region (HCVR) of an antibody that specifically binds human PD-1; and (b) an IL2 portion.

[0071] In some embodiments, the HCVR comprises three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2 and HCDR3) and the LCVR comprises three light chain CDRs (LCDR1, LCDR2 and LCDR3): HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, 24, or 43; HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, 26, or 45; HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, 28, or 47; LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, or 32; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or 35.

[0072] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of: (i) SEQ ID NOs: 4, 6, 8, 12, 14, and 16; (ii) SEQ ID NOs: 24, 26, 28, 32, 14, and 35; or (iii) SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively.

[0073] In some embodiments, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively.

[0074] In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO: 2, 22, or 41, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2, 22, or 41. In some embodiments, the LCVR comprises the amino acid sequence of SEQ ID NO: 10, or 30, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10, or 30.

[0075] In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, and the LCVR comprises the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:10.

[0076] In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:22, and the LCVR comprises the amino acid sequence of SEQ ID NO:30, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:30.

[0077] In some embodiments, the HCVR comprises the amino acid sequence of SEQ ID NO:41, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:41, and the LCVR comprises the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:10.

[0078] In some embodiments, the HCVR and LCVR comprise the amino acid sequences of: (i) SEQ ID NOs: 2 and 10; (ii) SEQ ID NOs: 22 and 30; or (iii) SEQ ID NOs: 41 and 10, respectively. In some embodiments, the HCVR and LCVR comprise the amino acid sequences of SEQ ID NOs: 41 and 10, respectively.

[0079] In some embodiments, the first polypeptide further comprises a light chain constant region linked to the LCVR. In some embodiments, the light chain constant region comprises an amino acid sequence of SEQ ID NO: 56, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56.

[0080] In some embodiments, the second polypeptide further comprises a heavy chain constant region disposed between the HCVR and the IL2 portion. In some embodiments, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO:55, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:55.

[0081] In some embodiments, the first polypeptide comprises a light chain amino acid sequence of SEQ ID NO:58, 60, or 62, or a light chain amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:58, 60, or 62.

[0082] In some embodiments, the second polypeptide comprises a heavy chain amino acid sequence of SEQ ID NO:57, 59, or 61, or a heavy chain amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57, 59, or 61.

[0083] In some embodiments, the fusion protein comprises the heavy / light chain sequence pair of SEQ ID NOs: 57 / 58, 59 / 60, or 61 / 62. In some embodiments, the fusion protein comprises the heavy / light chain amino acid sequence pair of SEQ ID NOs: 61 / 62.

[0084] In some embodiments, the IL2 portion is connected to the C-terminus of the heavy chain constant region via a linker. In some embodiments, the linker comprises one or more repeats of the amino acid sequence of GGGGS (SEQ ID NO: 67). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 50 or 52. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO: 50.

[0085] In some embodiments, the IL2 portion comprises: (i) IL2, or a fragment thereof; and (ii) IL2 receptor alpha (IL2Rα), or a fragment thereof.

[0086] In some embodiments, the IL2 or fragment thereof is human IL2 (hIL2) or a fragment thereof. In some embodiments, the IL2 or fragment thereof comprises the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:53.

[0087] In some embodiments, the IL2Rα or fragment thereof is human IL2Rα (hIL2Rα) or a fragment thereof. In some embodiments, the IL2Rα or fragment thereof comprises the amino acid sequence of SEQ ID NO:51, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:51.

[0088] In some embodiments, the IL2 portion comprises the amino acid sequence of SEQ ID NO:54, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:54.

[0089] In some embodiments, the IL2 or fragment thereof is linked to the C-terminus of the IL2Rα or fragment thereof via a linker. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:52.

[0090] In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:58, 60, or 62, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:58, 60, or 62. In some embodiments, the second polypeptide comprises the amino acid sequence of SEQ ID NO:63, 64, or 65, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:63, 64, or 65.

[0091] In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:58, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:58, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:64, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:64.

[0092] In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:60, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:63, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:63.

[0093] In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:62, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:65, or an amino acid sequence having 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO:65.

[0094] Also provided in the present disclosure is a dimeric fusion protein formed by the fusion protein described herein. In some embodiments, the dimeric fusion protein is a homodimeric fusion protein, and each of the constituent monomers comprises a fusion protein described herein. In some embodiments, each of the constituent monomers comprises a fusion protein described herein. In some embodiments, the monomers of the dimeric fusion protein dimerize via the heavy chain constant region of each monomer.

[0095] biological activity The fusion proteins of the present disclosure exhibit attenuated binding to IL2Rα, IL2Rβ, and IL2Rγ. In some embodiments, the fusion proteins do not compete with REGN2810, pembrolizumab, or nivolumab. In some embodiments, the fusion proteins exhibit reduced activity to activate human IL2Rα / β / γ trimer and IL2Rβ / γ dimer receptor complexes compared to IL2, and exhibit increased activity to activate human IL2Rα / β / γ trimer and IL2Rβ / γ dimer receptor complexes compared to non-targeted IL2Rα-IL2 constructs. In some embodiments, the fusion proteins exhibit increased activity to stimulate antigen-activated T cells compared to wild-type human IL2, as measured by the level of IFN-γ release. In some embodiments, the fusion proteins exhibit enhanced anti-tumor efficacy compared to IL2 alone or in combination with an anti-PD-1 antibody.

[0096] Nucleic Acids and Host Cells In another aspect, the disclosure provides an isolated nucleic acid or nucleic acids comprising one or more polynucleotide sequences encoding a fusion protein of the disclosure. In some embodiments, the fusion protein is encoded by a single nucleic acid. In other embodiments, the fusion protein is encoded by multiple (e.g., two, three, four or more) nucleic acids, for example, in the case of heterodimeric molecules or molecules consisting of an anti-PD-1 antibody comprising two or more polypeptide chains.

[0097] A single nucleic acid can encode a portion of a fusion protein containing a single polypeptide chain, a fusion protein containing two or more polypeptide chains, or a fusion protein containing three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a fusion protein containing three, four or more polypeptide chains, or three polypeptide chains of a fusion protein containing four or more polypeptide chains). In order to separately control expression, the open reading frames encoding the two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Alternatively, the open reading frames encoding the two or more polypeptides are controlled by the same transcriptional regulatory elements and separated by an internal ribosome entry site (IRES) sequence that allows translation into separate polypeptides.

[0098] In some embodiments, a fusion protein comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the fusion protein is equal to or less than the number of polypeptide chains in the fusion protein (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0099] The nucleic acids of the disclosure can be DNA or RNA (eg, mRNA).

[0100] In another aspect, the disclosure provides host cells and vectors comprising the nucleic acids of the disclosure. The nucleic acids can be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in more detail herein below.

[0101] In some embodiments, the host cell expresses a first vector comprising a polynucleotide sequence encoding the HCVR of an antibody described herein, and a second vector comprising a polynucleotide sequence encoding the LCVR of an antibody described herein.

[0102] In some embodiments, the host cell expresses a first vector comprising a polynucleotide sequence encoding a first polypeptide of a fusion protein described herein, and a second vector comprising a polynucleotide sequence encoding a second polypeptide of a fusion protein described herein.

[0103] The present disclosure provides a vector comprising a nucleotide sequence encoding one or two of the polypeptide chains of a fusion protein or fusion protein components described herein, such as a half antibody. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0104] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern equine encephalitis virus, flaviviruses, etc.

[0105] Furthermore, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide, for example, prototrophy to heterotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may be required for optimal synthesis of mRNA. These elements include splice signals, transcription promoters, enhancers, termination signals, etc.

[0106] Once the expression vector or DNA sequence containing the construct is prepared for expression, the expression vector is transfected or introduced into a suitable host cell.To achieve this, various techniques can be adopted, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, and other conventional techniques.The method and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and are changed or optimized according to the specific expression vector and mammalian host cell adopted based on this specification.

[0107] The present disclosure also provides a host cell comprising the nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to comprise one or more nucleic acids described herein. In one embodiment, the host cell is genetically engineered by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host that is compatible with such sequence. Such a cassette can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for expression, such as, for example, an inducible promoter, may also be used.

[0108] The present disclosure also provides a host cell comprising the vectors described herein.

[0109] The cell can be, but is not limited to, eukaryotic cell, bacterial cell, insect cell, or human cell.Suitable eukaryotic cells include, but are not limited to, Vero cell, HeLa cell, COS cell, CHO cell, HEK293 cell, BHK cell, and MDCKII cell.Suitable insect cells include, but are not limited to, Sf9 cell.

[0110] Pharmaceutical Compositions The fusion proteins or dimeric fusion proteins disclosed herein may be in the form of a composition comprising the fusion protein (e.g., a dimeric fusion protein) and one or more carriers, excipients and / or diluents.

[0111] The composition is formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers used will depend on the intended use of the fusion protein or dimeric fusion protein and, in the case of therapeutic use, the mode of administration.

[0112] For therapeutic use, the composition is supplied as part of a sterile pharmaceutical composition that includes a pharma- ceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition is administered to the patient by a variety of routes, including oral, transdermal, subcutaneous, nasal, intravenous, intramuscular, intratumoral, intrathecal, topical or localized. The most suitable route of administration in any given case depends on the particular antibody, the subject, the nature and severity of the disease, and the physical condition of the subject. In some embodiments, the pharmaceutical composition is administered intravenously or subcutaneously.

[0113] The pharmaceutical composition is conveniently presented in a unit dosage form, which contains a predetermined amount of the fusion protein of the present disclosure per dose. The amount of fusion protein contained in the unit dosage depends on the disease to be treated, as well as other factors well known in the art. Such unit dosage may be in the form of a lyophilized powder, which contains an amount of fusion protein suitable for one administration, or in the form of a liquid. The dry powder unit dosage form is packaged in a kit with a syringe, an appropriate amount of diluent, and / or other components useful for administration. The unit dosage form in the form of a liquid is conveniently supplied in the form of a syringe pre-filled with an amount of fusion protein suitable for one administration. The pharmaceutical composition is also supplied in bulk, which contains an amount of fusion protein suitable for multiple administrations.

[0114] Pharmaceutical compositions are prepared by combining the fusion protein of the desired purity for storage as a lyophilized formulation or in an aqueous solution with any pharma- ceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as "carriers") commonly used in the art, including buffers, stabilizers, preservatives, isotonicity agents (isotonifiers), non-ionic surfactants, antioxidants, and various other additives (see Remington's Pharmaceutical Sciences, 16th ed. (Osol, ed., 1980)). Such additives should be nontoxic to recipients at the dosages and concentrations employed.

[0115] How to use The fusion proteins of the present disclosure are useful for treating disease conditions in which stimulation of the host's immune system is beneficial, particularly conditions in which an enhanced cellular immune response is desirable. These may include disease conditions in which the host's immune response is insufficient or deficient. Disease conditions in which the fusion proteins of the present disclosure can be administered include, for example, tumors or infectious diseases in which the cellular immune response is an important mechanism of specific immunity. Particular disease conditions in which the fusion proteins of the present disclosure can be employed include cancer, such as renal cell carcinoma or melanoma; immunodeficiencies, particularly HIV-positive patients, immunosuppressed patients, chronic infections, and the like. The fusion proteins of the present disclosure can be administered by themselves or in any suitable pharmaceutical composition.

[0116] In one aspect, the fusion protein of the present disclosure is provided for use as a medicament. In a further aspect, the fusion protein of the present disclosure is provided for use in the treatment of a disease. In some embodiments, the fusion protein of the present disclosure is provided for use in a method of treatment. In some embodiments, the present disclosure provides a fusion protein as described herein for use in the treatment of a disease in a subject in need thereof. In some embodiments, the present disclosure provides a fusion protein for use in a method of treating a subject having a disease, comprising administering a therapeutically effective amount of the fusion protein to the subject. In some embodiments, the disease to be treated is a proliferative disorder. In some embodiments, the disease is cancer.

[0117] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent when the disease to be treated is cancer.In further embodiments, the present disclosure provides a fusion protein for use in stimulating the immune system of a subject.In some embodiments, the present disclosure provides a fusion protein for use in a method for stimulating the immune system of a subject, comprising administering to the subject an effective amount of the fusion protein to stimulate the immune system.

[0118] "Stimulation of the immune system" according to any of the embodiments herein may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, restoration of lymphocyte function, an increase in expression of the IL-2 receptor, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0119] In a further aspect, the present disclosure provides the use of the fusion protein of the present disclosure in the manufacture or preparation of a medicament for the treatment of a disease in a subject in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to a subject having the disease. In some embodiments, the disease being treated is a proliferative disorder. In some embodiments, the disease is cancer. In one such embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent, if the disease being treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system of a subject, comprising administering to the subject an effective amount of the medicament to stimulate the immune system.

[0120] In some embodiments, the disease to be treated is a proliferative disorder, preferably cancer.Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferative disorders that can be treated with the fusion protein of the present disclosure include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest region, and genitourinary system.Also included are precancerous conditions or lesions and cancer metastasis. In some embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain tumor, head and neck cancer. Similarly, other cell proliferation disorders can also be treated by the fusion proteins of the present disclosure. Examples of such cell proliferation disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstrom's macroglobulinemia, Gaucher's disease, histiocytosis, and any other cell proliferation disorder other than neoplasms located in the organ systems listed above. In another embodiment, the disease is associated with autoimmunity, transplant rejection, post-traumatic immune response, and infectious diseases (e.g., HIV). More specifically, the fusion proteins can be used to eliminate cells involved in immune cell-mediated disorders including lymphoma; autoimmunity, transplant rejection, graft-versus-host disease, ischemia, and stroke. Those skilled in the art will readily recognize that in many cases, fusion proteins may not provide a cure, but only a partial benefit. In some embodiments, a physiological change that has some benefit is also considered to be therapeutically beneficial. Thus, in some embodiments, the amount of fusion protein that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject or patient in need of treatment is typically a mammal, more particularly a human.

[0121] A variety of administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusions.

[0122] The fusion proteins of the present disclosure are generally used in an amount effective to achieve the intended purpose. When used to treat or prevent a disease state, the fusion proteins of the present disclosure or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.

[0123] A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more. In the case of repeated administration over several days or more, depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the fusion protein would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, the dosage may also include about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg body weight, about 200 μg / kg / body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, or about 100 mg / kg body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numerical values ​​recited herein, ranges such as about 5 mg / kg body weight to about 50 mg / kg body weight, about 5 μg / kg body weight to about 100 mg / kg body weight, etc., based on the numerical values ​​above, can be administered. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient receives about 2 to about 20, or, for example, about 6 doses of the fusion protein). An initial high loading dose is administered, followed by one or more lower doses. However, other dosage regimes are also useful. The progress of this treatment can be easily monitored by conventional techniques and assays.

[0124] A therapeutically effective dose of the fusion proteins described herein generally provides a therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion proteins are determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies are used to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic efficacy is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Fusion proteins that exhibit large therapeutic indices are preferred. In one embodiment, the fusion proteins described in the present disclosure exhibit high therapeutic indices. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for human use. Dosages are preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on various factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety).

[0125] Due to the lower toxicity, the fusion proteins of the present disclosure can have a higher maximum therapeutic dose than wild-type IL2.

[0126] The fusion proteins described in the present disclosure are administered in combination with one or more other agents or therapies. For example, the fusion proteins of the present disclosure are co-administered with at least one additional therapeutic agent or therapy. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in a subject in need of such treatment. Such additional therapeutic agents are comprised of any active ingredient suitable for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent is an immunomodulatory agent, a cell proliferation inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that enhances the sensitivity of cells to apoptosis inducers. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy agent, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0127] In certain embodiments, the second therapeutic agent or treatment is: radiation, surgery, a chemotherapeutic agent, an oncolytic virus, a cancer vaccine, a PD-1 inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a lymphocyte activation gene 3 (LAG3) inhibitor, a T-cell membrane protein 3 (TIM3) inhibitor, a galectin 9 (GAL9) inhibitor, a V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA) inhibitor, a killer cell immunoglobulin-like receptor (KIR) inhibitor, a B- and T-lymphocyte attenuator (BTLA) inhibitor, an Ig and ITIM domain-containing inhibitor ... PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD-1 inhibitor, a PD- T-cell immunoreceptor (TIGIT) inhibitors, CTLA4 inhibitors, CD38 inhibitors, CD47 inhibitors, CD28 activators, 4-1BB activators, GITR agonists, CD40 agonists, OX40 modulators, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, angiopoietin-2 (Ang2) inhibitors, transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens, Bacillus Calmette-Guerin (Bacillus Calmette-Guerin vaccine, granulocyte-macrophage colony stimulating factor, cytotoxins, interleukin 6 receptor (IL-6R) inhibitors, interleukin 4 receptor (IL-4R) inhibitors, IL-10 inhibitors, IL-7, IL-12, IL-21, IL-15, IL-18, type I interferons, antibody drug conjugates, anti-inflammatory drugs, and combinations thereof.

[0128] In certain embodiments, the fusion proteins of the disclosure are used in combination with an anti-PD-1 antibody, where the antibody does not cross-compete with the fusion protein for binding to human PD-1.

[0129] Such other drugs are preferably present in combination in an amount effective for the intended purpose. The effective amount of such other drugs depends on the amount of fusion protein used, the type of disorder or treatment, and other factors mentioned above. Fusion proteins are generally used in the same doses and routes of administration as described herein, or at about 1-99% of the doses described herein, or at any dose and by any route determined empirically / clinically appropriate.

[0130] Such combination therapy as described above includes combined administration (wherein the two or more therapeutic agents are included in the same composition or in separate compositions) and separate administration, where administration of the fusion protein of the present disclosure may occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The fusion protein of the present disclosure may also be used in combination with radiation therapy and / or surgery.

[0131] Further definitions To aid in the understanding of the detailed description of the compositions and methods described in this disclosure, some explicit definitions (in addition to those disclosed elsewhere herein) are provided to facilitate clear disclosure of the various aspects of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0132] As used herein, the term "fusion protein" or "fusion polypeptide" refers to a protein comprising two or more polypeptide sequences that are covalently or non-covalently linked. For example, a fusion polypeptide encompassed by the present disclosure can comprise the translation product of a chimeric gene construct that combines a nucleic acid sequence encoding a first polypeptide with a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. Alternatively, a fusion protein may be encoded by two or more gene constructs on separate vectors that can be co-expressed in a host cell. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins that are linked by a peptide bond or through several peptides. In some embodiments, the fusion protein may also include a peptide linker between the two domains.

[0133] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs, and peptidomimetics.

[0134] As used herein, a "wild type" form of IL-2 is a form of IL-2 that is the same as the mutant IL-2 polypeptide, except that the wild type form has a wild type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 mutant is a full-length IL-2 (i.e., IL-2 that is not fused or linked to any other molecule), the wild type form of the mutant is the full-length native IL-2. If the IL-2 mutant is a fusion of IL-2 with another polypeptide (e.g., an antibody chain) encoded downstream of IL-2, the wild type form of the IL-2 mutant is an IL-2 with the wild type amino acid sequence fused to the same downstream polypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (a mutated or modified sequence within the non-truncated portion of IL-2), the wild type form of the IL-2 mutant is a similarly truncated IL-2 with the wild type sequence.

[0135] The fusion proteins disclosed herein may contain one or more conservative modifications. Fusion proteins with one or more conservative modifications may retain desired functional properties, which can be tested using functional assays known in the art. As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Cas proteins contain one or more conservative modifications. Cas proteins with one or more conservative modifications may retain desired functional properties, which can be tested using functional assays known in the art. As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or change the binding properties of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0136] As used herein, the percentage homology between two amino acid sequences corresponds to the percentage identity between the two sequences. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). Comparison of sequences and determination of the percentage identity between two sequences can be achieved using a mathematical algorithm, as described in the following non-limiting examples.

[0137] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as implemented into the ALIGN program (version 2.0) using a PAM120 weighted residue table with a gap length penalty of 12 and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as implemented into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0138] Additionally or alternatively, the protein sequences of the present disclosure can be further used as a "query sequence" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the antibody molecules of the present disclosure. To obtain gapped alignments for comparison, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see www.ncbi.nlm.nih.gov).

[0139] As used herein, when referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantially identical" refers to nucleotide sequence identity at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as described below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in some instances, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule. When applied to a polypeptide, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 98% or 99% sequence identity, when optimally aligned, such as by the GAP or BESTFIT programs with default gap weighting. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.

[0140] As used herein, the term "recombinant" refers to a protein of the present disclosure or a fragment thereof that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term refers to a fusion protein expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse), or cell (e.g., a CHO cell) expression system, or a fusion protein isolated from a recombinant combinatorial human antibody library.

[0141] As used herein, the term "associated" in the context of a fusion protein or a component thereof (e.g., a targeting moiety such as an antibody) refers to a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example, non-covalently through molecular interactions, or covalently through one or more disulfide bridges or chemical crosslinks, to produce a functional fusion protein. Examples of associations that may be present in the fusion proteins of the present disclosure include (but are not limited to) the association between homodimeric or heterodimeric Fc domains in the Fc region, the association between the VH and VL regions in a Fab or scFv, the association between CH1 and CL in a Fab, and the association between CH3 and CH3 in a domain-substituted Fab.

[0142] As used herein, the term "monovalent" as used herein with respect to an IL2 moiety and / or a targeting moiety in a fusion protein means a fusion protein having only a single IL2 moiety and / or targeting moiety, respectively (e.g., an anti-PD-1 antibody or antigen-binding portion thereof).

[0143] As used herein, the term "bivalent" as used herein with respect to an IL2 moiety and / or a targeting moiety in a fusion protein refers to a fusion protein having two IL2 moieties and / or targeting moieties (e.g., anti-PD-1 antibodies or antigen-binding portions thereof), respectively. Typically, a fusion protein that is bivalent with respect to an IL2 moiety and / or a targeting moiety is a dimer (either a homodimer or a heterodimer).

[0144] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid sequence in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, HCDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. See, e.g., Kabat, 1991, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (Kabat numbering system); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 (Chothia numbering system); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABM numbering system); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering system). Public databases for identifying CDR sequences in antibodies are also available.

[0145] As used herein, the term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. The term "Fc region" refers to the region of an antibody-based binding molecule that is formed by the association of two heavy chain Fc domains. The two Fc domains in an Fc region may be the same or different from each other. In natural antibodies, the Fc domains are usually identical, but one or both Fc domains may be advantageously modified to allow heterodimerization, for example, via knob-in-hole interactions.

[0146] As used herein, the term "EC50" refers to the half-maximal effective concentration of a molecule (such as a fusion protein) that induces a response halfway between baseline and maximum after a particular exposure time. EC50 essentially represents the concentration of an antibody or fusion protein at which 50% of the maximum effect is observed. In some embodiments, the EC50 value is equal to the concentration of the fusion protein that gives half-maximal STAT5 activation in the assay.

[0147] An epitope, or antigenic determinant, is a portion of an antigen (e.g., a target molecule) that is recognized by an antibody or other antigen-binding moiety, as described herein. Epitopes can be linear or conformational.

[0148] As used herein, the term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0149] As used herein, the term "target molecule" as used herein refers to any biological molecule (e.g., a protein, carbohydrate, lipid, or combination thereof) expressed on a cell surface or in the extracellular matrix that is specifically bound by the targeting moiety of a fusion protein of the present disclosure.

[0150] As used herein, the terms "treat", "treatment", and "treating" refer to a reduction or amelioration of the progression, severity and / or duration of a proliferative disorder, or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from administration of one or more fusion proteins of the present disclosure. In certain embodiments, the terms "treat", "treatment", and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, but not necessarily discernible by the patient. In other embodiments, the terms "treat", "treatment", and "treating" refer to an inhibition of the progression of a proliferative disorder, either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat", "treatment", and "treating" refer to a reduction or stabilization of tumor size or cancer cell number.

[0151] As used herein, the term "cancer" refers to a disease characterized by uncontrolled (often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream or lymphatic system. Examples of various cancers are described herein, including, but not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive cancer, colon cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, and the like.

[0152] As used herein, the term "tumor" is used interchangeably with the term "cancer", e.g., both terms encompass solid tumors and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant cancers and tumors, as well as malignant cancers and tumors.

[0153] As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present disclosure is introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer to the particular subject cell and the progeny or potential progeny of such a cell. Since certain modifications occur in successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term herein. Exemplary host cells are eukaryotic host cells, such as mammalian host cells.

[0154] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression includes splicing of the mRNA in eukaryotic cells.

[0155] As used herein, an "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a therapeutic polypeptide contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides that have been purified (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule that is normally contained in a cell that contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present disclosure, as well as positive and negative stranded forms, and double-stranded forms. An isolated polynucleotide or nucleic acid according to the present disclosure further includes such molecules that have been produced synthetically. Additionally, a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

[0156] As used herein, the term "disease" is generally intended to be synonymous with, and used interchangeably with, the terms "disorder" and "condition" (in medical conditions), in that both reflect an abnormal condition of the human or animal body or parts thereof that impairs normal functioning, is typically manifested by characteristic signs and symptoms, and reduces the duration or quality of a human or animal's life.

[0157] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one component useful with a fusion protein of the present disclosure in combination with other components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. A pharmaceutical composition facilitates administration of one or more components of the present disclosure to an organism.

[0158] As used herein, the term "pharmaceutical acceptable" refers to a material, such as a carrier or diluent, that does not destroy the biological activity or properties of the composition and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0159] As used herein, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, involved in carrying or transporting a compound of the present disclosure into or to a subject so that it performs its intended function. Typically, such compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Examples of substances which can function as pharma- ceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; glycerin, sorbitol, mannitol, polyethylene glycols, and the like. Examples of suitable non-toxic and compatible substances include polyols such as ethyl oleate, ethyl laurate, and the like; esters such as ethyl oleate, ethyl laurate, and the like; agar; buffers such as magnesium hydroxide, aluminum hydroxide, and the like; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; coloring agents; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickening agents; hardening agents; solidifying agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic and compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutical acceptable carrier" also includes any and all coating agents, antibacterial and antifungal agents, and absorption retardants, etc., that are compatible with the activity of one or more components of the present disclosure and are physiologically acceptable to the subject. Supplementary active compounds are also incorporated into the composition.

[0160] As used herein, the term "modulate" is meant to refer to any change in a biological state, ie, an increase, a decrease, and the like.

[0161] As used herein, the terms "increased," "increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of doubt, the terms "increased," "increase" or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20% compared to a reference level, or an increase of at least about 30%, or an increase of at least about 40%, or an increase of at least about 50%, or an increase of at least about 60%, or an increase of at least about 70%, or an increase of at least about 80%, or an increase of at least about 90%, or an increase up to and including 100%, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to a reference level, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0162] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0163] As used herein, the terms "including," "comprising," "containing," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof, as well as additional subject matter, unless expressly stated otherwise.

[0164] As used herein, the phrases "in one embodiment," "in various embodiments," "in some embodiments," and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may, unless context dictates otherwise.

[0165] As used herein, the term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0166] As used herein, the term "substantially" does not exclude "completely", e.g. a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the term "substantially" may be removed from the definition.

[0167] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction (greater or less) of the stated reference value, unless otherwise stated or clear from the context (unless such number exceeds 100% of the possible values). Unless otherwise indicated in the specification, the term "about" is intended to include values, e.g., weight percent, close to the stated range that are equivalent in terms of the functionality of the individual components, compositions, or embodiments.

[0168] As disclosed herein, a number of ranges of values ​​are provided. Between the upper and lower limits of the range, each intervening value, to the tenth of the unit of the lower limit, is understood to be specifically disclosed, unless the context clearly dictates otherwise. Each subrange between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed in the disclosure. The upper and lower limits of these smaller ranges may each be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed in the disclosure, subject to the specifically excluded limit in the stated range. If a stated range includes one or both limits, ranges excluding one or both of those included limits are also encompassed in the disclosure.

[0169] As used herein, the term "each," when used in connection with a collection of items, is intended to identify an individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions may occur where explicit disclosure or context clearly dictates.

[0170] Any examples provided herein, or the use of illustrative language (e.g., "such as"), are intended merely to better describe the invention and are not intended to limit the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0171] Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. For any method provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order unless otherwise specified. When the method includes a combination of steps, each combination or subcombination of steps is included within the scope of the present disclosure, unless otherwise specified herein.

[0172] Each publication, patent application, patent document, and other reference cited herein is incorporated herein by reference in its entirety, unless inconsistent with this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein should be construed as an admission that the present disclosure is not entitled to supersede such publication by prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0173] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. EXAMPLES

[0174] The following examples are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors consider to be the invention. Likewise, the disclosure is not limited to any particular preferred embodiment described herein. Indeed, modifications and variations of the embodiments will be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, room temperature is about 25° C., and pressure is at or near atmospheric pressure. EXAMPLES

[0175] Generation of human antibodies against human PD-1 Human antibodies against the PD-1 protein were generated using VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions. The mice were immunized with a PD-1 fragment of approximately 25-170 amino acids from GenBank accession NP_005009.2 in the presence of adjuvant. Antibody immune responses were monitored by PD-1-specific immunoassays. When the desired immune response was obtained, spleen cells were harvested and fused with mouse myeloma cells to maintain viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing PD-1-specific antibodies.

[0176] As described in U.S. Pat. No. 7,582,298, which is specifically incorporated by reference herein in its entirety, anti-PD-1 antibodies were isolated directly from antigen-positive mouse B cells without fusion with myeloma cells.

[0177] Using this method, several fully human anti-PD-1 antibodies (i.e., antibodies with human variable domains and human constant domains) have been obtained.

[0178] Exemplary antibodies generated as disclosed above are designated mAb29512, mAb7798, mAb9048 and have the amino acid and nucleic acid sequences of HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCDR1, LCDR2, LCDR3 identified in Tables 1 and 2.

[0179] Exemplary anti-PD-1 antibodies were used in constructing IL2-based reagents using standard molecular biology techniques known in the art. The amino acid sequences of the reagents are disclosed in Table 4.

[0180] The biological properties of exemplary proteins made according to the methods of this example are described in detail in the Examples set forth below. EXAMPLES

[0181] Amino acid and nucleotide sequences of the heavy and light chain variable regions Table 1 shows the heavy and light chain variable region sequences of exemplary anti-PD-1 antibodies, and the amino acid sequence identifiers of the CDR sequences.

[0182] [Table 1]

[0183] The corresponding nucleic acid sequence identifiers for exemplary anti-PD-1 antibodies are shown in Table 2.

[0184] [Table 2]

[0185] Antibodies may have a human or mouse Fc isotype. As will be appreciated by those of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG1 or human IgG4 Fc, etc.), but in any event, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 will remain the same, and the binding characteristics to the antigen will be expected to be the same or substantially similar, regardless of the nature of the Fc domain.

[0186] Exemplary antibodies mAb29512, mAb7798, and mAb9048, which contain a human IgG4 Fc with a serine to proline mutation (S108P) in the hinge region, were named H4H29512, REGN2810, and H4H9048, respectively. Table 3 shows the amino acid sequence identifiers for the full-length heavy and light chain sequences of these antibodies.

[0187] [Table 3]

[0188] REGN2810 (also known as semipilimab; LIBTAYO®) was first disclosed in U.S. Patent No. 9,987,500 and has since been approved for the treatment of cutaneous squamous cell carcinoma, basal cell carcinoma, and non-small cell lung cancer.

[0189] Table 4 shows the amino acid sequence identifiers of the anti-PD-1-IL2Rα-IL2 reagents.

[0190] [Table 4]

[0191] The IL2 portion is connected to the C-terminus of the heavy chain constant region (SEQ ID NO:55) via a linker comprising the amino acid sequence of SEQ ID NO:50. For REGN10595, the heavy chain (HC) SEQ ID NO:57 comprises the amino acid sequences of the HCVR (SEQ ID NO:2), the heavy chain constant region (SEQ ID NO:55), the linker (SEQ ID NO:50), and the IL2 portion (SEQ ID NO:54). For REGN10486, the heavy chain (HC) SEQ ID NO:59 comprises the amino acid sequences of the HCVR (SEQ ID NO:22), the heavy chain constant region (SEQ ID NO:55), the linker (SEQ ID NO:50), and the IL2 portion (SEQ ID NO:54). For REGN10597, the heavy chain (HC) SEQ ID NO:61 comprises the amino acid sequences of the HCVR (SEQ ID NO:41), the heavy chain constant region (SEQ ID NO:55), the linker (SEQ ID NO:50), and the IL2 portion (SEQ ID NO:54).

[0192] Control constructs used in the following examples: For comparison, the following control constructs are included in the following examples: "Comp 1": the V of the antibody "MK-3475" described in WO2008 / 156712 (Merck Sharp & Dohme) H / V L "Comp 2": a monoclonal anti-PD-1 antibody having the sequence of antibody "5C4" described in WO2006 / 121168 (Medarex, Inc / ER Squibb); H / V L and "REGN13233": an anti-PD-1 antibody comprising the VH / VL sequences of SEQ ID NO: 41 / 10 and linked to an IL2 variant with abolished CD25 binding (IL2(3m)) (Klein et al., 2017, Oncoimmunology). EXAMPLES

[0193] Binding kinetics of anti-PD1-IL2Rα-IL2 fusion constructs with parental bivalent anti-PD1 antibodies To assess the binding kinetics of the anti-PD1-IL2Rα-IL2 fusion construct with the parental bivalent anti-PD1 antibody, the equilibrium dissociation constants (K ) of binding of human PD-1 expressed with a C-terminal myc-myc-hexahistidine tag (hPD-1.mmH, SEQ ID NO: 66) to purified anti-PD-1 parental mAb and the anti-PD1-IL2Rα-IL2 fusion construct were calculated. D The binding activity (p-value) was measured using a real-time surface plasmon resonance biosensor with a Biacore 3000 or 4000 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, #BR-1008-39). All Biacore binding studies were performed in a buffer containing 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). Different concentrations of hPD-1.mmH (ranging from 100 to 3.7 nM in 3-fold dilutions, or 90 nM to 3.33 nM in 3-fold dilutions) prepared in HBS-EP running buffer were injected over the capture surface of anti-PD-1 antibody or anti-PD1-IL2Rα-IL2 fusion construct at a flow rate of 30 or 50 μL / min. Antibody-reagent association was monitored for 4 or 5 min, and dissociation was monitored for 10 min in HBS-EP running buffer. At the end of each cycle, the anti-PD-1 mAb or anti-PD1-IL2Rα-IL2 fusion construct capture surfaces were regenerated with a 12 s injection of 20 mM phosphoric acid. All binding kinetics experiments were performed at 25°C.

[0194] Kinetic coupling (k a ) and dissociation (k d The binding / dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

number

[0195] The binding kinetics of the anti-PD1-IL2Rα-IL2 fusion constructs and the parental bivalent anti-PD1 antibodies are summarized in Table 5.

[0196] [Table 5] EXAMPLES

[0197] Cross-competition of anti-PD-1 mAb with anti-PD1-IL2Rα-IL2 fusion construct To assess cross-competition between anti-PD-1 mAbs and anti-PD1-IL2Rα-IL2 fusion constructs, binding competition between anti-PD1-IL2Rα-IL2 and commercial anti-PD-1 mAbs was determined using a real-time, label-free biolayer interferometry (BLI) assay with an Octet HTX biosensor (ForteBio Corp., A Division of Sartorius). The entire experiment was performed at 25°C in a buffer containing 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant P20, 0.1mg / mL BSA (Octet HBS-EP buffer), and the plate was shaken at a speed of 1000 rpm. To assess whether the two antibodies could compete with each other for binding to their respective epitopes on hPD-1.mmH (SEQ ID NO: 66), approximately 0.34 nm of hPD-1.mmH was first captured on a HIS1K antibody-coated Octet biosensor (Fortebio Inc, #18-5120) by immersing the biosensor in a well containing a 20 μg / mL solution of hPD-1.mmH for 2 min. The antigen-captured biosensor was then saturated with the primary anti-PD-1 antibody or anti-PD1-IL2Rα-IL2 fusion construct (hereafter referred to as mAb-1) by immersing the biosensor in a well containing a 50 μg / mL solution of mAb-1 for 5 min. The biosensor was then immersed in a well containing a 50 μg / mL solution of the secondary anti-PD-1 antibody or anti-PD1-IL2Rα-IL2 fusion construct (hereafter referred to as mAb-2) for 3 min. Between each step of the experiment, all biosensors were washed with Octet HBS-EP buffer. Real-time binding responses were monitored throughout the experiment and recorded at the end of each step. The binding responses of mAb-2 to hPD-1.mmH precomplexed with mAb-1 were compared, and the competitive / noncompetitive behavior of the different anti-PD-1 antibodies was determined using a 50% inhibition threshold.

[0198] Table 6 defines the relationships of competing antibodies in both orientations, regardless of the order of binding.

[0199] [Table 6]

[0200] Table 6 shows that REGN10486 and its parent antibody REGN2810 did not cross-compete with REGN10595 or REGN10597 for binding to hPD-1. EXAMPLES

[0201] Cell line engineering and in vitro functional characterization of anti-PD1-IL2Rα-IL2 constructs Engineering of YT / Reporter Cells: Human T / NK cell leukemia YT cell line was electroporated with a signal transducer and activator of transcription 5 (STAT5)-luciferase reporter construct and maintained in Iscoves+20%FBS+P / S / G+200μg / mL hygromycin. A single cell clone showing high responsiveness to IL-2 was identified and newly named YT / Stat5-Luc cl.4. IL2Rα (CD25) was knocked out in this clone using CRISPR-Cas9 technology and the resulting cell line YT / STAT5-Luc / IL2Rα KO was validated by flow cytometry. Human IL2Rα was then stably reintroduced into the YT / STAT5-Luc / IL2Rα KO cell line (amino acids M1-I272 in accession number NP_000408.1) and the resulting cell line, YT / STAT5-Luc / hIL2Rα, was verified by flow cytometry and maintained in Iscoves+20%FBS+P / S / G+200μg / mL hygromycin+15μg / mL blasticidin. YT / Stat5-Luc cl.4, YT / Stat5-Luc / IL2Rα KO, and YT / Stat5-Luc / hIL2Rα cells were engineered to stably express human PD1 (amino acids M1-L288 in accession number NP_005009.2, 2Q=>E mutation) and cells were selected in 1mg / mL G418-supplemented medium. The cells were verified by flow cytometry and newly named YT / STAT5-Luc / hPD1, YT / STAT5-Luc / IL2Rα KO / hPD1, and YT / STAT5-Luc / hIL2Rα / hPD1.

[0202] Engineering of PD1 reporter cells: Jurkat E6-1 cells were engineered to stably express an activator protein 1 (AP1) luciferase reporter construct, antibiotic-resistant cells were selected and maintained in RPMI + 10% FBS + P / S / G + 1 μg / mL puromycin. The resulting pool of reporter cells was engineered to express human PD1 (amino acid M1-L288, 2Q=>E mutation in accession number NP_005009.2), and a high PD-1 expressing clonal cell line was isolated by fluorescence-activated cell sorting and maintained in RPMI + 10% FBS + P / S / G + 1 μg / mL puromycin. The resulting clone was newly named Jurkat / AP1-Luc / hPD1 cl.4E5.

[0203] Engineering HEK293 / anti-CD3 / PD-L1 cells: HEK293 were transduced with lentiviral vectors encoding human CD79a (hIga:M1-P226 from accession no. NP_001774.1), human CD79b (hIgb:M1-E229 from accession no. NP_000617.1), anti-CD3 variable domains (anti-CD3 mIgE heavy chain, anti-CD3 kappa light chain; clone 2706N, IgE genbank#AAB59424.1, CemX-migis-Cyto / TM PIR#PIH1215) and human PD-L1 (amino acids M1-T290 from accession no. NP_054862.1), then single-cell sorted and cultured in DMEM+10%FBS+P / S / G+500 μg / mL The cells were maintained in G418 + 100 μg / mL hygromycin + 1 μg / mL puromycin. Anti-CD3 and PD-L1 expression was confirmed by flow cytometry, and the resulting clonal line was newly named 293 / aCD3 / hPD-L1 cl.A3.

[0204] Engineering Raji / CD80 KO / CD86 KO and Raji / CD80 KO / CD86 KO / PD-L1 cells: CRISPR-Cas9 technology was used to ablate the expression of CD80 and CD86 in Raji cells. Raji / CD80 KO / CD86 KO cells were single cell cloned and the resulting clone (Raji / CD80 KO / CD86 KO cl.1D6) was engineered to express human PD-L1 (amino acids M1-T290 of accession number NP_054862.1). The resulting cell line, Raji / CD80 KO / CD86 KO / hPD-L1, was validated by flow cytometry and maintained in RPMI-1640 + 10% FBS + HEPES + NaPyr + P / S / G + 0.5 μg / mL puromycin.

[0205] PD1 antagonist assay HEK293 cells expressing membrane-bound anti-hCD3ε and human PD-L1 were incubated with Jurkat / AP1-Luc / hPD-1 reporter T cells. Clustering of CD3 complexed with the T cell receptor on the reporter cells via anti-CD3 on HEK293 cells activates the transcription factor AP1, driving expression of the luciferase reporter gene. Luciferase expression in the reporter T cells can be suppressed by interaction of the inhibitory receptor PD-1 with PD-L1 on HEK293 cells, but this can be overcome by the addition of antibodies that block the PD1 / PD-L1 axis.

[0206] RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium to prepare cell suspensions and antibody dilutions. The day before screening, engineered Jurkat / AP1-Luc / hPD1 reporter cells were incubated at 3 × 10 5 On the day of the assay, cells were spun down, resuspended in assay medium, and plated in a 96-well white flat-bottom plate at 2.5 × 10 293 / aCD3 / hPD-L1 cells. 42.5 × 10 Jurkat / AP1-Luc / hPD1 reporter cells were then plated at 2.5 × 10 cells / well. 11 titration ranges (500 nM to 477 fM) of serially diluted (1:4) non-targeting IL2Rα-IL2 [anti-PSMA-IL2Rα-IL2 (IL2Rα-IL2 fused to an irrelevant (PSMA) antibody), anti-PD1 (REGN2810, H4H29512, or H4H9048), anti-PD1-IL2Rα-IL2 (REGN10486, REGN10595, REGN10597) or a 12th titration range of isotype controls without recombinant protein (isotype control 1 or isotype control 2) were then added to the plated cells, followed by 2.5 × 10 Jurkat / AP1-Luc / hPD1 reporter cells. 4 After incubating the plate at 37°C / 5% CO2 for 5 hours, 100 μL of ONE-Glo™ (Promega) reagent was added to the wells to lyse the cells and detect luciferase activity. The light emitted was measured in relative light units (RLU) using a multilabel plate reader Envision (PerkinElmer). The EC 50 Values ​​were determined from a four-parameter logistic equation on a 12-point dose-response curve using GraphPad Prism™ software. Fold induction was calculated using the following formula:

number

[0207] IL2 reporter assay In this experiment, engineered YT reporter cells were stimulated via either recombinant IL2-Fc, non-targeted IL2Rα-IL2, or an anti-PD1-IL2Rα-IL2 fusion construct. Functional IL2 receptors are formed by differential assembly of IL2R subunits (IL2Rα, IL2Rβ, and IL2Rγ), with the assembly of IL2Rβ / IL2Rγ subunits comprising low affinity receptors and receptors containing all three subunits (IL2Rα / IL2Rβ / IL2Rγ) forming high affinity receptors. Binding of cytokines by IL2R leads to activation of STAT5, which drives luciferase production in engineered cell lines. To assess the relative potency of anti-PD1-IL2Rα-IL2 in the presence of low affinity IL2 receptors (IL2Rβ / γ) or high affinity IL2 receptors (IL2Rα / β / γ), YT / STAT5-Luc reporter cells expressing endogenous IL2Rα were engineered to either lack or overexpress IL2Rα. Although the reporter cells express endogenous PD1, derivatives were generated in which PD1 was overexpressed to assess the effect of PD1 expression levels on anti-PD1-IL2Rα-IL2 potency.

[0208] RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium to prepare cell suspensions and antibody dilutions. The day before screening, engineered reporter YT / Stat5-Luc cells overexpressing or lacking IL2Rα and endogenously expressing or overexpressing PD1 were cultured at 3 × 10 5 On the day of the assay, reporter cells were diluted to 2.5 × 10 4Cells / well were plated in fresh assay medium in 96-well white flat-bottom plates and incubated with serially diluted (1:4) IL2-Fc (SEQ ID NO: 74), non-targeting IL2Rα-IL2, REGN10486, REGN10595, REGN10597, or isotype control in 11 titration ranges (200 nM to 191 fM). The 12th titration range contained no recombinant protein. After incubating the plates for 5 hours at 37°C / 5% CO2, 100 μL of ONE-Glo™ (Promega) reagent was added to the wells, cells were lysed, and luciferase activity was detected. Light emitted was measured in RLU using a multilabel plate reader Envision (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation on a 12-point dose-response curve using GraphPad Prism™ software. Fold induction was calculated using the following formula:

number

[0209] PD1 competition assay Anti-PD1 bivalent antibodies or isotype controls were added to engineered YT reporter cells prior to stimulation with either non-targeting IL2Rα-IL2 or anti-PD1-IL2Rα-IL2 fusion constructs. Competition between bivalent anti-PD1 and anti-PD1-IL2Rα-IL2 would blunt the ability of anti-PD1-IL2Rα-IL2 to target and thus activate PD-1-expressing STAT5-Luc reporter cells. PD1-overexpressing reporter cells are used to maximize targeting efficiency and allow best detection of anti-PD1 bivalent competition with anti-PD1-IL2Rα-IL2.

[0210] RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium to prepare cell suspensions and antibody dilutions. On the day before screening, YT / Stat5-Luc / hPD1 reporter cells with IL2Rα knockout or overexpression were cultured at 3 × 10 5On the day of the assay, reporter cells were diluted to 2.5 × 10 4 100μL / well were plated in assay medium into 96-well white flat-bottom plates and titrations of anti-PD1 bivalent or isotype control antibodies (5 steps, 1:10 dilutions from 500nM to 0.05nM, the 6th containing no recombinant protein) were added, followed by titrations of non-targeting IL2Rα-IL2, REGN10486, REGN10595, or REGN10597 (62.5nM to 59.6fM; 1:4 serial dilutions over 11 dilution ranges, the 12th containing no recombinant protein). Plates were incubated at 37°C / 5%CO2 for 4 hours, after which 100μL of ONE-Glo™ (Promega) reagent was added to the wells, cells were lysed, and luciferase activity was detected. Light emitted was measured in RLU using a multilabel plate reader Envision (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation on a 12-point dose-response curve using GraphPad Prism™ software. Fold induction was calculated using the following formula:

number

[0211] Primary T cell stimulation assay Primary T cells were stimulated by co-culture with mitomycin-treated Raji / CD80 KO / CD86 KO cells for 4 days. As Raji cells endogenously express CD20, CD20xCD3 bispecific antibody was added to activate primary T cells. Titrations of isotype control, IL2, anti-PD1, non-targeting IL2Rα-IL2 antibody (REGN9904) or anti-PD1-IL2Rα-IL2 antibodies (REGN10486, REGN10595 or REGN10597) were added during the co-culture and the effect on T cell activity was determined by measuring the release of IFN-γ in cell culture supernatants using a homogenous, no-wash AlphaLISA kit (Perkin Elmer).

[0212] Human peripheral blood mononuclear cells (PBMCs) were isolated from leukopacks of healthy donors using the EasySep Direct Human PBMC Isolation Kit (Stemcell) according to the manufacturer's recommended protocol. T cells were isolated from PBMCs using the EasySep Human T cell Isolation Kit (Stemcell) according to the manufacturer's protocol. Cells were spun down and resuspended in stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, 0.01 mM BME) and incubated at 1 × 10 cells. 5 Raji / CD80 KO / CD86 KO and Raji / CD80 KO / CD86 KO / hPD-L1 cells were stimulated with 20 μg / mL mitomycin C in primary stimulation medium at 10 × 10 cells / well. 6 The cells were treated with 5 × 10 cells / mL of IgG4-associated IgG4 antibodies at 37°C for 1 hour to stop cell proliferation. The cells were then washed three times with D-PBS containing 2% FBS and plated at 5 × 10 cells / mL per well. 4 Cells were then added. 0.5 nM of anti-CD3 x anti-CD20 bispecific antibody was then added in 1:6 serial dilutions ranging from 500 nM to 0.3 pM in nine steps, with the tenth dilution point containing only the bispecific antibody at 0.5 nM constant, in combination with titrations of anti-PD1, IL2, the combination of REGN2810 + IL2, non-targeting IL2Rα-IL2 antibody, anti-PD1-IL2Rα-IL2 (REGN10486, REGN10595 or REGN10597), or an isotype control antibody (isotype control 1 or isotype control 2). Plates were incubated for 96 h at 37 °C / 5% CO2. IFN-γ was quantified in cell culture supernatants using the AlphaLISA assay according to the manufacturer's protocol, using samples of known IFN-γ concentrations to extrapolate the pg / mL of IFN-γ in each sample well. Measurements were performed using a multilabel plate reader Envision (Perkin Elmer). 50Values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation on a 10-point dose-response curve, with the 10th dilution point containing a constant 0.5 nM bispecific antibody alone.

[0213] Summary of Results and Conclusions PD1 antagonist assay: The ability of anti-PD1-IL2Rα-IL2 fusion constructs to block PD1 / PD-L1-mediated inhibition of TCR signaling was assessed using an AP1 reporter cell-based bioassay. 293 / aCD3 / hPD-L1 cells were incubated with Jurkat / AP1-Luc / hPD1 reporter cells, and PD1 activation by PD-L1 reduced reporter activity. The ability of bivalent anti-PD1 antibodies or antibody-fused IL2Rα-IL2 proteins to inhibit PD1-PD-L1 interaction and rescue reporter activity was assessed. Engineered reporter cells were cultured in EC 50 and fold induction values ​​are summarized in Table 7.

[0214] No increase in luciferase activity was detected when reporter cells were treated with control proteins (isotype control Ab 1, isotype control Ab 2, or non-targeting IL2Rα-IL2). In contrast, incubation of reporter cells with the anti-PD1 antibody, REGN2810, or the corresponding anti-PD1-IL2Rα-IL2, REGN10486, enhanced luciferase activity (19.210-fold and 17.740-fold, respectively) with similar potency (3.668E-09 and 5.351E-09, respectively). On the other hand, the bivalent PD1 antibodies H4H29512 and H4H9048 did not enhance luciferase activity, and the corresponding anti-PD1-IL2R-IL2 proteins REGN10595 and REGN10597 induced low luciferase activity (2.725- and 3.038-fold, respectively) with weak potency (1.266E-08 and 1.264E-08, respectively).

[0215] IL2 reporter assay: The ability of anti-PD1-IL2Rα-IL2 fusion constructs to induce PD1-targeted IL-2 receptor signaling was assessed using a STAT5-reporter assay. YT / STAT5-Luc reporter cells expressing endogenous IL2Rα, lacking IL2Rα expression (KO cells), or overexpressing IL2Rα, and endogenous or overexpressed PD1, were co-incubated with isotype control, IL2-Fc, non-targeted IL2Rα-IL2, or anti-PD1-IL2Rα-IL2 (REGN10486, REGN10595, or REGN10597), and STAT5 reporter activity was assessed. Fold induction values ​​and EC 50 are summarized in Tables 8 and 9, respectively.

[0216] Compared to IL2, non-targeted IL2Rα-IL2 showed reduced induction of STAT5 reporter activity, regardless of IL2Rα expression. PD1-targeted molecules showed higher potency compared to non-targeted IL2Rα-IL2, regardless of IL2Rα expression. Furthermore, overexpressing PD1 on the reporter cells further increased the potency of anti-PD1-IL2Rα-IL2 toward a potency similar to IL2.

[0217] PD-1 competition assay: The ability of anti-PD1 bivalent mAbs to interfere with anti-PD1-IL2Rα-IL2 fusion constructs was assessed using a STAT5-reporter bioassay. YT / STAT5-Luc reporter cells overexpressing IL2Rα or lacking expression of IL2Rα and overexpressing PD1 were incubated with either 500 nM isotype control or anti-PD1 bivalent mAbs (REGN2810, Comp 1 or Comp 2) before incubation with a titration of non-targeting IL2Rα-IL2 or PD1-targeting anti-PD1-IL2Rα-IL2 constructs (REGN10486, REGN10595, or REGN10597). After incubation, STAT5 reporter activity was assessed. Competition between anti-PD1 bivalent mAbs and anti-PD1-IL2Rα-IL2 would result in loss of PD1 targeting by anti-PD1-IL2Rα-IL2, resulting in reduced reporter activity. EC 50The values ​​are summarized in Table 10.

[0218] In the absence of bivalent PD1 mAbs, PD1-targeted IL2Rα-IL2 (REGN10486, REGN10595, REGN10597) had greater potency compared to non-targeted IL2Rα-IL2 (REGN9904). The potency of REGN10486 was strongly reduced by incubating reporter cells with PD1 antibodies (REGN2810, Comp 1 or Comp 2), whereas the effect on potency of REGN10595 and REGN10597 was minimal regardless of IL2Rα expression. Addition of anti-PD1 (REGN2810, Comp 1 or Comp 2) did not affect the potency of non-targeted IL2Rα-IL2.

[0219] Primary T cell stimulation assay: The ability of anti-PD1-IL2Rα-IL-2 to enhance T cell stimulation was assessed in a functional primary T cell assay measuring IFN-γ cytokine production. T cells incubated with mitomycin C-treated Raji / CD80 KO / CD86 KO or Raji / CD80 KO / CD86 KO / hPD-L1 cells were treated with a fixed amount of 0.5 nM anti-CD3 × anti-CD20 bispecific antibody and a titration of isotype control, IL2, anti-PD1 bivalent antibody (REGN2810) alone or in combination with IL2, non-targeting IL2Rα-IL2, or anti-PD1-IL2Rα-IL2 (REGN10486, REGN10595, or REGN10597). Co-incubation of T cells with Raji cells for 4 days resulted in measurable IFN-γ release. Treatment of T cells with anti-PD1-IL2Rα-IL2, IL2, or the combination of IL2 and REGN2810 in the presence of Raji / CD80 KO / CD86 KO cells resulted in similar levels of IFNγ release, whereas non-targeted IL2Rα-IL2, isotype control antibody, and REGN2810 resulted in lower levels of IFNγ release. In the presence of RAJI / CD80 KO / CD86 KO cells expressing hPD-L1, overall IFNy release was reduced in isotype control treated samples compared to RAJI cells not expressing PD-L1. Treatment with non-targeted IL2Ra-IL2 and REGN2810 resulted in a similar increase in IFNy release compared to isotype control, whereas treatment with IL2, REGN10597, and REGN10595 resulted in even greater responses. The greatest responses were observed with REGN10486 or the combination of IL2 and REGN2810. EC across a range of antibody doses 50 and maximum interferon release values ​​are summarized in Table 11.

[0220] [Table 7]

[0221] [Table 8]

[0222] [Table 9]

[0223] [Table 10]

[0224] [Table 11] EXAMPLES

[0225] In vivo antitumor efficacy evaluation in PD1×LAG3 humanized mice Test 1: Human PD1×LAG3 knock-in mice (described in Burova E. et al., Mol Cancer Ther 2019(18)(11)2051-2062) were inoculated with 3×10 5 MC38 tumor cells were inoculated subcutaneously, and the average tumor size was 95 mm 3 Mice were then randomized on day 7, when tumor volume reached 100 mg / kg. Mice were then intraperitoneally administered isotype control Ab (0.33 mg / kg), isotype-IL2Rα-IL2 antibody (0.5 mg / kg) + anti-hPD1 antibody (0.33 mg / kg), or anti-hPD1-IL2Rα-IL2 (0.5 mg / kg) + isotype control Ab (0.33 mg / kg) every 6 weeks for a total of four doses. The mean tumor volume (mm 3 + SEM) was plotted (Figure 1A). Tumor size was calculated as v=ab^2 / 2, where a represents the longest tumor diameter and b is the perpendicular tumor diameter. Arrows in Figure 1A indicate days of treatment.

[0226] Kaplan-Meier survival curves for each treatment group were also plotted (Figure 1B). Loss of survival was determined when tumors showed deep ulceration or reached 20 mm in any dimension or 2250 mm in total volume. 3 Euthanasia was defined as when

[0227] Test 2: Human PD1×LAG3 knock-in mice were inoculated with 3×10 5 MC38 tumor cells were inoculated subcutaneously, and the average tumor size was 105 mm 3 Mice were then randomized on day 7, when tumor volume reached 100 mg / kg. Mice were then intraperitoneally injected with isotype (0.5 mg / kg) or three different clones of anti-hPD1-IL2Rα-IL2 (0.5 mg / kg) every two weeks for a total of four doses. The mean tumor volume (mm 3 + SEM) are shown (Figure 1C). Tumor size was calculated as v = ab^2 / 2, where a represents the longest tumor diameter and b is the perpendicular tumor diameter. Arrows in the figure indicate the days of treatment.

[0228] Kaplan-Meier survival curves for each treatment group were also plotted (Figure 1D). Loss of survival was determined when tumors showed deep ulceration or reached 20 mm in any dimension or 2250 mm in total volume. 3 Euthanasia was defined as when

[0229] Summary of Results and Conclusions In study 1, the anti-tumor efficacy of anti-hPD1-IL2Rα-IL2 was evaluated in comparison to the combination of the two individual components, isotype-IL2Rα-IL2 and an equimolar parental anti-hPD1 blocking antibody.

[0230] Although isotype-IL2Rα-IL2 + anti-hPD1 failed to confer effective tumor control at the doses tested, anti-hPD1-IL2Rα-IL2 + isotype treatment at the same molar dose was able to regress established tumors and resulted in long-term tumor-free survival in the majority of treated mice (Figure 1A-B). This result demonstrated that the anti-tumor efficacy of anti-hPD1-targeted IL2Rα-IL2 was superior to the combination of non-targeted IL2Rα-IL2 and parental anti-hPD1 antibodies.

[0231] In study 2, we compared the antitumor activity of three different clones of anti-hPD1-IL2Rα-IL2. One of the anti-hPD1 clones potently blocks, whereas the other two block hPD1 signaling minimally. All three clones of anti-hPD1-IL2Rα-IL2 molecules showed similarly potent antitumor efficacy and resulted in similar long-term tumor-free survival rates (Figure 1C-D). This result indicates that the robust antitumor efficacy of anti-hPD1-IL2Rα-IL2 therapy is primarily responsible for the anti-hPD1-targeted delivery of IL2Rα-IL2 rather than its blocking activity. It also suggests that the therapeutic efficacy may be further enhanced by combining anti-hPD1-IL2Rα-IL2 molecules with non-competitive PD1 blocking reagents. EXAMPLES

[0232] Ability of anti-PD1-IL2Rα-IL2 fusion constructs to bind recombinant monomeric IL2Rα, IL2Rβ, or IL2Rγ proteins in solution Human IL-2 receptor α binding Binding of human IL2Rα expressed with a C-terminal hexahistidine tag (hIL2Rα.6H, R&D, Cat. No. 10305-RL-050) to purified anti-PD1 antibodies with a C-terminal IL2Rα-IL2 fusion was measured using real-time surface plasmon resonance biosensor technology with a Biacore S-200 instrument. To prepare the hIgG capture surface, a CM5 Biacore sensor surface was derivatized with a mixture of monoclonal anti-human Fab antibodies (Cytiva, Cat. No. 28-9583-25) by amine coupling. All Biacore binding studies were performed at 25°C in a buffer containing 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). Anti-PD1-IL2Rα-IL2 constructs and controls were captured on this anti-hFab surface by injecting a 3 μg / mL solution at 8 μL / min for 1 min. A 1 μM hIL2Rα.6H solution prepared in HBS-EP running buffer was then injected over the capture antibody-IL2 fusion construct capture surface at a flow rate of 50 μL / min. Association of the IL2 fusion antibody was monitored for 1 min, followed by dissociation in HBS-EP running buffer for 1 min. Binding responses were measured at the end of the injection and are shown in Table 12. At the end of each cycle, the anti-PD1-IL2Rα-IL2 fusion construct capture surface was regenerated by injecting 10 mM glycine, pH 1.5 for 30 s.

[0233] Human IL-2 receptor β / γ binding Binding of human IL2 receptor beta (hIL2Rβ) expressed with a C-terminal myc-myc-hexahistidine tag (hIL2Rβ.mmH, REGN9169) or human IL2 receptor gamma (hIL2Rγ) expressed with a C-terminal myc-myc-hexahistidine tag (hIL2Rγ.mmH, REGN1183) to anti-PD1-IL2Rα-IL2 fusion constructs was measured using real-time surface plasmon resonance biosensor technology with a Biacore S-200 instrument. The CM5 Biacore sensor surface was derivatized by amine coupling with a mixture of monoclonal anti-human Fab antibodies (Cytiva, catalog no. 28-9583-25). All Biacore binding studies were performed at 25°C in a buffer containing 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.05% v / v Surfactant P20 (HBS-EP running buffer). All IL2 receptor components were prepared in the same buffer. Anti-PD1-IL2Rα-IL2 constructs and controls were captured onto this anti-hFab surface by injecting a 3μg / mL solution at 8μl / min for 1 min.

[0234] It has been reported in the literature (Liparoto et al., Biochemistry. 2002) that IL2Rγ has an affinity for IL2 that is dependent on IL2Rβ or IL2Rα / β. Thus, the addition of IL2Rβ allows the formation of a detectable ternary complex. A sequential binding experiment was designed to explore the binding of IL2Rγ in the presence of pre-bound IL2Rβ. The anti-PD1-IL2Rα-IL2 surface was injected with 1 μM IL2Rβ.mmH at 50 μl / min for 30 seconds, followed immediately by injection of a mixture of 1 μM IL2Rβ.mmh and 1 μM IL2Rγ.mmH for 1 minute. The binding signal at the end of each injection was recorded. The results are shown in Table 13. At the end of each cycle, the anti-PD1-IL2Rα-IL2 fusion construct capture surface was regenerated by injection of 10 mM glycine, pH 1.5 for 30 seconds.

[0235] To calculate the activity of the anti-PD1-IL2Rα-IL2 fusion construct, the theoretical maximum signal (TRmax) was first calculated based on the capture amount of the anti-PD1-IL2-IL2Rα fusion, the stoichiometry of the IL2 receptor interaction, and the molecular weight of the interacting proteins. The activity of the anti-PD1-IL2Rα-IL2 fusion construct was then expressed as a percentage of the calculated TRmax. Furthermore, the percentage of the TRmax results was normalized to the control antibodies (REGN1945 and REGN8512) using the following formula:

number

[0236] Results, Summary and Conclusions The anti-PD1-IL2-IL2Rα fusion construct showed significantly reduced binding to hIL2Rα compared to the non-attenuated (IL2 only) control.

[0237] The anti-PD1-IL2Rα-IL2 fusion construct demonstrated the ability to bind hIL2Rβ with a partially reduced binding signal compared to the non-attenuated (IL2 only) control. In the presence of bound hIL2Rβ, minimal binding of hIL2Rγ was observed compared to the non-attenuated IL2 control construct.

[0238] [Table 12]

[0239] [Table 13] EXAMPLES

[0240] Cell-binding characteristics of anti-PD1-IL2Rα-IL2 fusion constructs and parental mAbs by FACS binding assay To compare hPD1 binding of αhPD1-IL2Rα-IL2 protein with each parental anti-hPD1 antibody, YT / STAT5 luc / Cl4 cells expressing endogenous levels of hPD1 were washed twice with FACS wash (PBS+2%FBS) and resuspended at 1x10^6 cells / ml in FACS wash. Cells (100μl / well) were then plated in 96-well U-bottom plates. Cells were spun down (1200RPM, 5 minutes) and resuspended in 100μl / well of various antibody dilutions listed in the mAb clone ID. Antibody titrations were prepared from 20μg / ml, diluted 6-fold with FACS wash. Cells were incubated with antibodies for 30 minutes at 4°C. Cells were then washed twice with FACS wash to remove unbound antibody. Cells were then resuspended in 100μl / well of a 1:200 dilution of APC-anti-human Fcγ secondary antibody. The cells were incubated for 30 minutes at 4° C. The cells were then washed twice with FACS wash solution to remove unbound antibody. The cells were then resuspended in 160 μl / well of FACS wash solution and binding studies were performed using a BD FACSCanto™ Flow Cytometer.

[0241] Anti-hPD1-IL2Rα-IL2 protein and each of the anti-hPD1 parental antibodies bind to PD1+ cells to a similar extent (Figures 2A and 2B). Anti-hPD1-IL2Rα-IL2 protein binds with slightly lower affinity (approximately 2-fold) than the corresponding parental mAbs. EXAMPLES

[0242] Fab exchange and native mass spectrometry (MS) analysis of anti-PD1-IL2Rα-IL2 fusion constructs Fab exchange and native mass spectrometry (MS) analysis were used to determine the conformation of the PD-1-IL2Ra-IL2 fusion molecule. REGN10597, REGN8509, REGN2810 and REGN475 were each treated with peptide N-glycosidase F (PNGase F; 1 IUB milliunits per 10 μg protein) at 45° C. for 1 hour to completely remove glycan chains from their respective heavy chain constant regions. The deglycosylated protein samples were then mixed according to FIG. 3A and Table 14 below, and each mixture was incubated at 37° C. for 30 minutes in the presence of 2 mM DTT. The treated protein mixtures were then subjected to mass spectrometry-coupled native desalting size-exclusion chromatography analysis (SEC-MS) on a native LC-MS platform (see Provisional Patent No. 10724). Desalting SEC was performed using a BEH® SEC column (4.6 × 30 mm, 200 Å, 1.7 μm) with an isocratic flow of 150 mM ammonium acetate (pH 6.8) at a flow rate of 0.2 mL / min. Mass measurements were performed on a Thermo Q-Exactive UHMR mass spectrometer.

[0243] [Table 14]

[0244] When the disulfide bonds in the hinge region are disrupted, IgG4 molecules undergo rapid Fab exchange, the products of which can be monitored by native MS analysis. In control experiments with a mixture of two IgG4 antibodies (mixture 1) or a mixture of one IgG4 antibody + anti-hCD20-IL2 (mixture 2), Fab exchange was observed under partial reducing conditions and Fc exchange under FabRICATOR digestion conditions (Figure 3B). No Fab or Fc exchange products were observed under partial reducing conditions or FabRICATOR digestion conditions, respectively, for the mixture of PD1-IL2Rα-IL2 + IgG4 molecules (mixture 3), suggesting that PD1-IL2Rα-IL2 molecules exist primarily in an inactive form and that the strong interchain interactions between IL2Rα and IL2 prohibit Fab (or Fc) exchange (Figure 3B). In summary, Fab exchange and native MS analysis indicate that anti-PD1-IL2Rα-IL2 molecules exist primarily in a trans-sequestered conformation. EXAMPLES

[0245] Evaluation of the ability of anti-PD1-IL2Rα-IL2 constructs to enhance human primary T cell activation by a mixed lymphocyte reaction (MLR) assay A mixed lymphocyte reaction (MLR) assay was used to evaluate the ability of anti-PD1-IL2Ra-IL2 constructs to enhance human primary T cell activation. Allogeneic donor PBMCs stimulate T cells, resulting in proliferation and cytokine release. Addition of recombinant IL-2 further aids in T cell activation. Therefore, this assay was used to evaluate how PD1-targeted IL2Ra-IL2 affects T cell proliferation and release of IFNg, an indicator of T cell activation.

[0246] Isolation of human primary cells Human PBMCs were isolated from peripheral blood leukopacks of two healthy donors obtained from Precision for Medicine (Donor 1) or Stem Cell Technologies (Donor 2) using the EasySep™ Direct Human PBMC Isolation Kit following the manufacturer's recommended protocol. CD3+ T cells from Donor 2 were isolated from PBMCs using the EasySep™ Human CD3+ T Cell Isolation Kit from StemCell Technologies following the manufacturer's recommended protocol.

[0247] Assay procedure: Isolated CD3+ T cells from donor 2 were resuspended in primary culture medium (X Vivo 15 medium supplemented with 10% FBS, 10mM HEPES, 1mM sodium pyruvate, 1X non-essential amino acids, 0.01mM β-mercaptoethanol) at a concentration of 1x10^6 cells / ml. PBMCs from donor 1 (10x10^6 cells / ml) were treated with 50μg / ml mitomycin C diluted in primary culture medium for 1 hour at 37℃ / 5%CO2 (to stop cell proliferation). After washing three times with primary culture medium, PBMCs were resuspended and added to T cells from donor 2 to a final T cell to PBMC ratio of 1:3 (1x10^6 T cells + 3x10^6 PBMC per ml). After incubating the CD3+ T cell / PBMC mixture for 6 days, T cells were re-isolated using Miltenyi CD3+ Microbeads according to the manufacturer's instructions. Isolated cells were then rested in primary cell culture medium for 24 hours. 100,000 T cells were added to the wells of a round-bottom microtiter plate, and fresh mitomycin C-treated donor 1 PBMCs were added to the wells at 300,000 cells / well. Anti-PD1-IL2Rα-IL2 (REGN10597, REGN10595 or REGN10486), isotype-IL2Rα-IL2 control (REGN9903), anti-PD1 (REGN2810), recombinant IL2, or matched IgG4 and IgG4 isotype controls (REGN1945 and REGN7540, respectively) were titrated at 1:6 dilutions in nine steps ranging from 1000 nM to 0.595 pM, with the final tenth being no antibody (indicated as the lowest point on the curve). Each condition was performed in triplicate. After 72 h of incubation at 37°C / 5% CO2, the microtiter plates were centrifuged to pellet the cells and 50 μl of media supernatant was collected. 5 μl from the collected supernatant was tested in the Human IFNγAlphaLISA (PerkinElmer) assay according to the manufacturer's protocol. Measurements were performed using a multilabel plate reader Envision (PerkinElmer). Pelleted cells were resuspended in primary stimulation medium containing 3H-thymidine (1.25 mCi / ml) and incubated at 37°C / 5% CO2 for 6 hours.Plates were processed using a Filtermate Cell Harvester (PerkinElmer) and counted using a MicroBeta2 microplate counter (PerkinElmer). Values ​​were recorded as counts per minute (CPM). EC of antibodies. 50 Values ​​were determined from a four-parameter logistic equation on a 10-point dose-response curve using GraphPad™ software.

[0248] In the presence of allogeneic PBMCs, T cells treated with a dose titration of recombinant IL-2, anti-PD-1-IL2Rα-IL2 (REGN10597, REGN10595, REGN10486) or isotype-IL2Rα-IL2 control (REGN9903) resulted in a dose-dependent increase in IFNγ release and proliferation (Figure 4A, 4B and Table 15). Recombinant IL2 resulted in the highest maximum cytokine release and most potent proliferation, followed by the anti-PD1-IL2Rα-IL2 molecule. The isotype target IL2Rα-IL2 had reduced potency in cytokine release and proliferation compared to the PD-1 target. REGN2810 only slightly increased IFNγ in a dose-dependent manner, whereas matched IgG4 and IgG4s isotype controls (REGN1945 and REGN7540, respectively) had no effect on cytokine release or proliferation.

[0249] [Table 15] EXAMPLES

[0250] Comparison of anti-PD1-IL2Rα-IL2 constructs with isotype control IL2Rα-IL2 + anti-PD1 constructs in a Raji-based primary T cell stimulation assay The ability of anti-PD1-IL2Rα-IL2 constructs to enhance human primary T cell activation was assessed using a Raji cell-based primary T cell assay. T cells were stimulated to release cytokines by co-culture with Raji cells that have been engineered to knock out CD80 and CD86 and overexpress human PD-L1 (Raji / CD80 KO / CD86 KO / hPD-L1) in the presence of a CD3×CD20 bispecific antibody. The addition of recombinant IL-2 further aids in T cell activation. Therefore, this assay was used to assess how PD1-targeted IL2Rα-IL2 affects T cell release of IFNγ, an indicator of T cell activation.

[0251] Isolation of human primary cells: Human PBMCs were isolated from peripheral blood leukopacks of healthy donors obtained from Precision for Medicine using the EasySep™ Direct Human PBMC Isolation Kit following the manufacturer's recommended protocol. CD3+ T cells were isolated from PBMCs using the EasySep™ Human CD3+ T Cell Isolation Kit from StemCell Technologies following the manufacturer's recommended protocol.

[0252] Procedure for Raji-based T cell assay: T cells were spun down and resuspended in stimulation medium (X Vivo 15 medium supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 1x non-essential amino acids, 0.01 mM β-mercaptoethanol) at 1x10 cells. 5 Raji / CD80 KO / CD86 KO / hPD-L1 cells (1 × 10 cells / well) were plated in a 96-well round-bottom plate. 7 To stop cell proliferation, 5 × 10 cells / ml were treated with 20 mg / ml mitomycin C diluted in stimulation medium at 37°C / 5% CO2 for 1 h. Then, the cells were washed three times with D-PBS containing 2% FBS, resuspended in stimulation medium, and diluted to 5 × 10 cells / ml per well. 4cells were added. Then, 0.5 nM of anti-CD3 x anti-CD20 bispecific antibody (REGN1979) was added along with 20 nM of a fixed isotype control (REGN1945) or anti-PD1 (REGN2810). Titrations of either isotype control (REGN7540), isotype-IL2Rα-IL2 antibody (REGN9903), anti-PD1 antibody with VR of mAb9048 (REGN15187), anti-PD1-IL2Rα-IL2 (REGN10597), or equimolar titrations of REGN9903 + REGN15187 were added to the wells in nine 1:6 serial dilutions ranging from 500 nM to 0.3 pM per molecule, with the 10th dilution point containing only 0.5 nM fixed REGN1979 and 20 nM REGN2810 or REGN1945. Each condition was performed in duplicate. Plates were incubated for 96 h at 37°C / 5% CO2. IFNγ was quantified in cell culture supernatants using the AlphaLISA assay according to the manufacturer's protocol. Measurements were performed on a multilabel plate reader Envision (Perkin Elmer). EC 50 Values ​​were determined using GraphPad Prism™ software from a four-parameter logistic equation on a 10-point dose-response curve, with the 10th dilution point containing a constant 0.5 nM REGN1979 and 20 nM REGN2810 or REGN1945 alone.

[0253] Summary of Results T cells treated with dose titrations of isotype-IL2Rα-IL2 (REGN9903) or anti-PD1-IL2Rα-IL2 (REGN10597) in the presence of Raji / CD80 KO / CD86 KO / hPDL1, with or without REGN2810, resulted in a surprisingly significant dose-dependent increase in IFNγ release (Table 16 and Figures 5A, 5B). Anti-PD1-IL2Rα-IL2 molecules resulted in the highest maximum cytokine release, followed by isotype-IL2Rα-IL2 molecules alone or in combination with anti-PD1 antibody REGN15187. Matched IgG4 and IgG4s isotype controls (REGN1945 and REGN7540, respectively) did not affect IFNγ release.

[0254] [Table 16] EXAMPLES

[0255] In vivo antitumor efficacy and safety profile of anti-PD1-IL2Rα-IL2 fusion constructs In the first experiment, we evaluated the in vivo antitumor efficacy of single-agent REGN10597. Human PD1 × LAG3 knock-in mice (described in Burova E. et al., 2019) were treated with 3 × 10 5 MC38 tumor cells were inoculated subcutaneously, and the average tumor size was 105 mm 3 Mice were randomized on day 7, when tumor volume reached 100 mg / kg / day. Mice in each randomized group received intraperitoneal injections of the indicated molecules at a particular dose level once every two weeks for a total of four injections. The mean tumor volume (mm 3 +SD) were plotted (Figure 6A). Tumor size was calculated as v=a·b^2 / 2, where a represents the longest tumor diameter and b is the perpendicular tumor diameter. Arrows indicate days of treatment. Individual tumor growth curves and the frequency of mice with complete tumor rejection in each treatment group are shown (Figure 6B).

[0256] REGN10597 monotherapy at dose levels of 0.5 mg / kg or 1 mg / kg demonstrated superior antitumor activity compared with combination therapy of 1 mg / kg REGN9904 and 10 mg / kg REGN2810, resulting in a higher frequency of complete tumor regression in treated mice, with intermediate antitumor efficacy observed with 0.2 mg / kg REGN10597 treatment (Figures 6A, 6B).

[0257] In a second experiment, the in vivo activity and toxicity profile of REGN10597 was compared to that of comparator REGN13233, an anti-PD1-targeted IL2 mutein with inhibited binding to IL2Ra. Human PD1×LAG3 knock-in mice (described in Burova E. et al., 2019) were treated with 3×10 5 MC38 tumor cells were inoculated subcutaneously, and the average tumor size was 70 mm 3 Mice were randomized on day 7, when tumor volume reached 100 mg / kg. Mice in each randomized group received intraperitoneal injections of the indicated molecules at a specific dose level once every two weeks for a total of four injections. The mean tumor volume (mm 3 +SD (Fig. 6C), Kaplan-Meier survival curves (Fig. 6D), and percentage of body weight change (mean +SD) (Fig. 6E) were plotted. Arrows in Fig. 6A indicate days of treatment. The frequency of mice undergoing complete tumor rejection is shown for selected groups in (Fig. 6D). On day 13, blood was collected from all groups and analyzed by flow cytometry. Total white blood cell counts were shown (Fig. 6F).

[0258] Both REGN10597 and REGN13233 monotherapies demonstrated superior antitumor efficacy to the combination of REGN2810 and the respective non-targeted controls (REGN9904 for REGN10597 and REGN13234 for REGN13233), with the majority of treated mice in both groups showing complete tumor regression (Figures 6C, 6D).

[0259] However, unlike REGN10597, which did not cause obvious weight changes in treated mice, the same dose of REGN13233 treatment resulted in significant weight loss accompanied by a marked increase in circulating lymphocytes (Figure 6E). This difference in toxicity is likely due to the different IL-2 moieties of REGN10597 and REGN13233, although similar weight loss and increased white blood cell counts were observed in the REGN13234-treated group (Figure 6E, 6F). Further immunophenotypic analysis of white blood cells by multiparameter flow cytometry revealed that REGN10597 inhibited PD-1 + REGN13233 not only expanded these cells but also selectively expanded CD4 and CD8 T cells. + CD62L + Significant expansion of CD8 T cell and NK cell populations was also demonstrated.

[0260] These results suggest that although both REGN10597 and the comparator molecule REGN13233 showed robust antitumor efficacy, REGN10597 exerted a broader CD8 + Compared with REGN13233, which expanded T and NK cell populations, PD1 + It has been shown to have a better safety profile, likely due to its ability to more selectively expand T cells.

[0261] In the third experiment, the in vivo antitumor efficacy of REGN10597 was compared to that of aPD1-IL2-IL2Rα, a molecule that contains all the same moieties as REGN10597, but in which the IL2 and IL2Rα moieties are fused to each other in the reverse order. Human PD1×LAG3 knock-in mice (described in Burova E. et al., 2019) were treated with 3×10 5 MC38 tumor cells were inoculated subcutaneously, and the average tumor size was 120 mm 3 Mice were randomized on day 9, when tumor volume reached 100 mg / kg / day. Mice in each group then received weekly intraperitoneal injections of the indicated molecules at the specified dose levels for a total of two injections (Figure 6G). The mean tumor volume (mm 3+SD) were plotted. Tumor size was calculated as v=a·b^2 / 2, where a represents the longest tumor diameter and b is the perpendicular tumor diameter. Arrows in the figure indicate days of treatment (Figure 6H). Kaplan-Meier survival curves for each treatment group are also shown. Loss of survival was determined when tumors showed deep ulceration or reached 20 mm in any dimension or 2250 mm in total volume. 3 Euthanasia was defined as when the tumor rejection rate reached 0.05%. The frequency of mice undergoing complete tumor rejection is shown for selected groups.

[0262] Unlike REGN10597, which showed robust antitumor activity in vivo, the same or higher doses of aPD1-IL2-IL2Rα showed minimal tumor growth control, indicating that the correct order of IL2Rα-IL2 fusions in REGN10597 is critical for its potent antitumor activity in vivo (Figures 6G, 6H). EXAMPLES

[0263] Flow binding of Ab-IL2Ra-IL2 fusion molecules to the IL2 receptor Functional IL2 receptors are formed by differential assembly of IL2R subunits (IL2Ra=CD25, IL2Rb=CD122, IL2Rg=CD132), resulting in intermediate affinity IL2 receptors (IL2Rb / IL2Rg) and high affinity IL2 receptors (IL2Ra / IL2Rb / IL2Rg) expressed on immune cells (PMID:16293754). The binding ability of IL2 and IL2Ra-IL2 chimeric antibodies to cells expressing intermediate affinity IL2 receptors (IL2Rb / g, IL2Ra knockout) or high affinity receptors (IL2Rb / g and IL2Ra overexpression) was evaluated using flow cytometry.

[0264] Although YT cells endogenously express human PD1, overexpressing PD-1 cells (PD1 OE) were generated. To investigate the binding of human IL2 and human IL2Ra-IL2 chimeric antibodies (=primary antibodies) to the cells, YT / STAT5-Luc / PD1 OE reporter cells, which endogenously express all three IL2 receptor subunits, were genetically modified to express on the cell surface either a medium affinity IL2 receptor (CD25 KO) by knockout of the IL2Ra subunit, or a high affinity receptor (CD25 OE) by overexpression of IL2Ra. Binding of antibodies to these cells was detected by flow cytometry using fluorescently labeled secondary antibodies. Briefly, PD1 OE YT cells, CD25 KO or CD25 OE, were resuspended in staining buffer (2% FBS in PBS) and plated at 3 × 10 per well in a 96-well plate. 5 Cells were plated and incubated with 1:5 serially diluted primary antibodies [isotype-IL2 (REGN8512) or isotype-IL2Ra-IL2 (REGN9904)] for 30 min on ice. Final antibody concentrations ranged from 768 fM to 300 nM, and a no-antibody control labeled "secondary antibody only" was included. After incubation, samples were washed with ice-cold staining buffer and subsequently incubated with AF647-conjugated anti-human IgG antibody for 30 min on ice. Unbound secondary antibody was removed, and samples were washed once with ice-cold PBS, stained with viability dye, washed once with ice-cold staining buffer, fixed for 30 min at room temperature, washed with staining buffer, resuspended in staining buffer, and filtered before analysis on an iQue Plus flow cytometer to measure geometric mean fluorescence intensity (gMFI). The following formula was used to calculate maximum fold binding for secondary antibody alone:

number

[0265] Summary of Results Table 17 summarizes the maximum geometric MFI and fold induction values ​​of antibody-IL2 or antibody-IL2Ra-IL2 chimeric constructs binding to CD25 knockout or overexpressing cells. Dose-dependent binding of isotype IL2 molecules (REGN8512) and isotype IL2Ra-IL2 molecules (REGN9904) was observed on both engineered YT cells expressing either intermediate affinity IL2Rβ / γ receptors (CD25 KO) or high affinity IL2Rα / β / γ receptors (CD25 OE) (Figures 7A, 7B; Table 17). As expected, the maximum fold binding was greater for both constructs on CD25 OE cells. However, binding of Ab-IL2Ra-IL2 was greatly reduced compared to Ab-IL2 in both IL2Rα / β / γ- and IL2Rβ / γ-expressing cell lines, suggesting "masking" of IL-2 by the presence of IL2Ra in the fusion protein.

[0266] [Table 17] EXAMPLES

[0267] Bioassays to evaluate the potency of PD1-IL2R2-IL2 molecules Functional IL2 receptors are formed by differential assembly of IL2R subunits (IL2Ra=CD25, IL2Rb=CD122, IL2Rg=CD132), resulting in the intermediate affinity IL2 receptor (IL2Rβ / γ) and high affinity IL2 receptor (IL2Rα / β / γ) expressed on immune cells (PMID:16293754).

[0268] To evaluate the biological activity of IL2 or IL2Ra-IL2 or IL-2-IL2Ra chimeric antibodies, we established a cell-based reporter assay in which STAT5-driven luciferase expression was activated upon binding of IL2 to the intermediate or high affinity IL2 receptor on engineered YT / STAT5-Luc. YT cells endogenously express human PD1, but overexpressed PD-1 cells (PD1 OE) were generated. YT / STAT5-Luc / PD1 OE reporter cells, which endogenously express all three IL2 receptor subunits, were genetically modified to express either the intermediate affinity IL2 receptor (CD25 KO) by knockout of the IL2Ra subunit, or the high affinity receptor (CD25 OE) by overexpression of IL2Ra on the cell surface.

[0269] RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium to prepare cell suspensions and antibody dilutions. The day before screening, engineered YT / STAT5-Luc reporter cells (CD25 KO / PD1 OE and CD25 OE / PD1 OE) were cultured at 3 × 10 cells. 5 On the day of the assay, cells were spun down, resuspended in assay medium, and plated in 96-well white flat-bottom plates at 2.5 × 10 reporter cells. 4The antibodies were plated at 1000 / well and serially diluted (1:5) over an 11-step titration range (200 nM–21 fM) (Figures 1A, 1B, 1C, 1D) or serially diluted (1:4) over an 11-step titration range (250 nM–238 fM) (Figures 2A, 2B) with the 12th titration range incubated with isotype-IL2Ra-IL2 [REGN9903 or REGN9904], isotype-IL2-IL2Ra, isotype-IL2(3m) [REGN13234], anti-PD1(arm 9048)-target-IL2Ra-IL2 [REGN10597], anti-PD1(arm 9048)-target-IL2-IL2Ra or anti-PD1(arm 9048)-target-IL2(3m) [REGN13233] without recombinant protein. After incubating the plates at 37°C / 5% CO2 for 4 h 30 min (Figures 1A, 1B, 1C, 1D) or 4 h (Figures 2A, 2B), 100 mL of ONE-Glo™ (Promega) reagent was added to the wells to lyse the cells and detect luciferase activity. Light emitted was measured in RLU using a multilabel plate reader Envision (PerkinElmer). EC 50 Values ​​were determined from a four-parameter logistic equation on a 12-point dose-response curve using GraphPad Prism™ software. Fold induction was calculated using the following formula:

number

[0270] Summary of Results Isotype IL2Ra-IL2 (REGN9903), isotype-IL2-IL2Ra, anti-PD1 target-IL2Ra-IL2 (REGN10597) and anti-PD1 target-IL2-IL2Ra caused a dose-dependent increase in STAT5-driven reporter expression in both the absence (Figure 8A) or presence (Figure 8B) of IL2Ra expression on reporter cells (Table 18). PD-1 target molecules showed enhanced potency compared to isotype target molecules. IL2-IL2Ra chimeric molecules showed reduced potency compared to IL2Ra-IL2 chimeric molecules, regardless of the presence or absence of IL2Ra expression on reporter cells.

[0271] [Table 18]

[0272] Isotype IL2Ra-IL2 (REGN9903), isotype-IL2(3m) (REGN13234), anti-PD1 target-IL2Ra-IL2 (REGN10597) and anti-PD1 target-IL2(3m) (REGN13233) caused a dose-dependent increase in STAT5-driven reporter expression both in the absence (FIG. 9A) or presence (FIG. 9B) of IL2Ra expression on reporter cells (Table 19). PD1 targeting molecules showed similar potency in the absence (REGN13233: 53 pM, REGN10597: 84 pM) or presence (REGN13233: 46 pM, REGN10597: 37 pM) of IL2Ra expression on reporter cells. In the absence of PD1 targeting, the IL2Ra-IL2 chimeric molecule showed reduced potency compared to the IL2(3m) chimeric molecule.

[0273] [Table 19]

[0274] The present disclosure is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be within the scope of the appended claims.

Claims

1. A fusion protein comprising: (i) an antigen-binding portion that specifically binds to human programmed cell death protein 1 (PD-1), and (ii) an interleukin 2 (IL2) portion, The antigen-binding portion comprises an antibody that specifically binds to PD-1 or an antigen-binding fragment thereof, and three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained in the heavy-chain variable region (HCVR), and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained in the light-chain variable region (LCVR), The IL2 portion comprises (i) IL2 or a fragment thereof and (ii) interleukin 2 receptor α (IL2Rα) or a fragment thereof, and IL2 or a fragment thereof is connected to the C-terminus of IL2Rα or a fragment thereof via a first linker, a fusion protein.

2. The fusion protein according to claim 1, wherein the antibody or an antigen-binding fragment thereof that binds to human PD-1 is a human monoclonal antibody.

3. The fusion protein according to claim 1, wherein the antigen-binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively.

4. The fusion protein according to claim 1, wherein the antigen-binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 4, 6, 8, 12, 14, and 16, respectively.

5. The fusion protein according to claim 1, wherein the antigen-binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 24, 26, 28, 32, 14, and 35, respectively.

6. The fusion protein according to claim 1, wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 41 and the LCVR comprises the amino acid sequence of SEQ ID NO:

10.

7. The fusion protein according to claim 1, wherein the HCV R comprises the amino acid sequence of SEQ ID NO: 2 and the LCV R comprises the amino acid sequence of SEQ ID NO:

10.

8. The fusion protein according to claim 1, wherein the HCV R comprises the amino acid sequence of SEQ ID NO: 22 and the LCV R comprises the amino acid sequence of SEQ ID NO:

30.

9. The fusion protein according to claim 1, wherein the antigen-binding portion comprises a heavy chain constant region of SEQ ID NO: 55 and a light chain constant region of SEQ ID NO:

56.

10. The fusion protein according to claim 1, wherein the antigen-binding portion comprises a heavy chain / light chain sequence pair of SEQ ID NO: 61 / 62, 57 / 58, or 59 / 60.

11. The fusion protein according to claim 1, wherein the antigen-binding portion comprises a heavy chain / light chain sequence pair of SEQ ID NO: 61 / 62.

12. The fusion protein according to any one of claims 1 to 11, wherein the IL-2 or a fragment thereof is human IL-2 (hIL-2) or a fragment thereof.

13. The fusion protein according to any one of claims 1 to 11, wherein the IL-2Rα or a fragment thereof is human IL-2Rα (hIL-2Rα) or a fragment thereof.

14. The fusion protein according to any one of claims 1 to 11, wherein the IL-2 or a fragment thereof comprises the amino acid sequence of SEQ ID NO:

53.

15. The fusion protein according to any one of claims 1 to 11, wherein the IL-2Rα or a fragment thereof comprises the amino acid sequence of SEQ ID NO:

51.

16. The fusion protein according to any one of claims 1 to 11, wherein the antigen-binding portion comprises a heavy chain constant region connected to the HCV R, and the IL-2 portion is connected to the C-terminus of the heavy chain constant region of the antigen-binding portion via a second linker.

17. The fusion protein according to claim 16, wherein the first and / or second linker comprises one or more repeating amino acid sequences of GGGGS (SEQ ID NO: 67).

18. The fusion protein according to claim 16, wherein the first linker comprises the amino acid sequence of SEQ ID NO: 52, and the second linker comprises the amino acid sequence of SEQ ID NO:

50.

19. The fusion protein according to claim 16, wherein the IL2 portion comprises the amino acid sequence of SEQ ID NO:

54.

20. A fusion protein comprising: (i) a first polypeptide comprising a light chain variable region (LCVR) of an antibody; and (ii) a second polypeptide comprising (a) a heavy chain variable region (HCVR) of the antibody and (b) an IL2 portion, The antibody specifically binds to human programmed cell death protein 1 (PD-1) and comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) of the HCVR, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of the LCVR, The IL2 portion comprises (i) IL2 or a fragment thereof and (ii) IL2 receptor α (IL2Rα) or a fragment thereof, and IL2 or a fragment thereof is connected to the C-terminus of IL2R α or a fragment thereof via a first linker.

21. The fusion protein according to claim 20, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, respectively.

22. The fusion protein according to claim 20, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 4, 6, 8, 12, 14, and 16, respectively.

23. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the fusion proteins according to claim 20, each containing the amino acid sequence of SEQ ID NO: 24, 26, 28, 32, 14, and 35, respectively.

24. HCVr and LCvr are the fusion proteins according to claim 20, each containing the amino acid sequence of (i) SEQ ID NO: 41 and 10; (ii) SEQ ID NO: 2 and 10; or (iii) SEQ ID NO: 22 and 30, respectively.

25. The first polypeptide is the fusion protein according to claim 20, containing a light chain constant region linked to LCvr.

26. The light chain constant region is the fusion protein according to claim 25, containing the amino acid sequence of SEQ ID NO:

56.

27. The second polypeptide is the fusion protein according to any one of claims 20 to 26, containing a heavy chain constant region linked to HCVr, and the IL2 portion is linked to the C-terminus of the heavy chain constant region.

28. The heavy chain constant region is the fusion protein according to claim 27, containing the amino acid sequence of SEQ ID NO:

55.

29. (i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequence of SEQ ID NO: 43, 45, 47, 12, 14, and 16, and the first polypeptide contains the light chain amino acid sequence of SEQ ID NO: 62; (ii) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequence of SEQ ID NO: 4, 6, 8, 12, 14, and 16, and the first polypeptide contains the light chain amino acid sequence of SEQ ID NO: 58; or (iii) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 24, 26, 28, 32, 14, and 35, and the first polypeptide contains the light chain amino acid sequence of SEQ ID NO: 60, the fusion protein according to claim 27.

30. (i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 43, 45, 47, 12, 14, and 16, and the second polypeptide contains the heavy chain amino acid sequence of SEQ ID NO: 61; (ii) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 4, 6, 8, 12, 14, and 16, and the second polypeptide contains the heavy chain amino acid sequence of SEQ ID NO: 57; or (iii) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain the amino acid sequences of SEQ ID NOs: 24, 26, 28, 32, 14, and 35, and the second polypeptide contains the heavy chain amino acid sequence of SEQ ID NO: 59, the fusion protein according to claim 27.

31. The fusion protein according to claim 27, comprising a heavy chain / light chain sequence pair of SEQ ID NO: 61 / 62, 57 / 58, or 59 / 60.

32. The fusion protein according to claim 31, comprising a heavy chain / light chain sequence pair of SEQ ID NO: 61 / 62.

33. The IL2 portion is connected to the C-terminus of the heavy chain constant region via a second linker, the fusion protein according to claim 27.

34. IL2 or a fragment thereof is human IL2 (hIL2) or a fragment thereof, the fusion protein according to claim 33.

35. IL2Rα or a fragment thereof is human IL2Rα (hIL2Rα) or a fragment thereof, the fusion protein according to claim 33.

36. The IL2 or a fragment thereof is the fusion protein according to claim 33, which contains the amino acid sequence of SEQ ID NO:

53.

37. The IL2Rα or a fragment thereof is the fusion protein according to claim 33, which contains the amino acid sequence of SEQ ID NO:

51.

38. The first and / or second linker is the fusion protein according to claim 33, which contains one or more repeated amino acid sequences of GGGGS (SEQ ID NO: 67).

39. The first linker contains the amino acid sequence of SEQ ID NO: 52, and the second linker contains the amino acid sequence of SEQ ID NO:

50. The fusion protein according to claim 38.

40. The IL2 portion contains the amino acid sequence of SEQ ID NO:

54. The fusion protein according to claim 33.

41. (i) The first polypeptide contains the amino acid sequence of SEQ ID NO: 62, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 65; (ii) The first polypeptide contains the amino acid sequence of SEQ ID NO: 58, and the second polypeptide contains the amino acid sequence of SEQ ID NO: 64; or (iii) The first polypeptide contains the amino acid sequence of SEQ ID NO: 60, and the second polypeptide contains the amino acid sequence of SEQ ID NO:

63. The fusion protein according to claim 33.

42. The first polypeptide contains the amino acid sequence of SEQ ID NO: 62, and the second polypeptide contains the amino acid sequence of SEQ ID NO:

65. The fusion protein according to claim 33.

43. The first polypeptide contains the amino acid sequence of SEQ ID NO: 58, and the second polypeptide contains the amino acid sequence of SEQ ID NO:

64. The fusion protein according to claim 33.

44. The fusion protein according to claim 33, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 60 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

63.

45. The fusion protein according to claim 16, which forms a dimeric fusion protein comprising two monomers.

46. The fusion protein according to claim 45, wherein each monomer comprises a heavy chain constant region and the fusion protein dimerizes via the heavy chain constant region of the monomer.

47. The fusion protein according to claim 16, which does not cross-compete with semipilimab, pembrolizumab or nivolumab for binding to PD-1.

48. The fusion protein according to claim 16, which shows a decrease in activity to activate human IL2Rα / β / γ and IL2Rβ / γ complexes as compared to IL2.

49. The fusion protein according to claim 16, which shows an increase in activity to activate human IL2Rα as compared to non-target IL2Rα-IL2.

50. The fusion protein according to claim 16, which shows an increase in activity to stimulate T cells as measured by the level of IFN-γ release as compared to wild-type human IL2.

51. The fusion protein according to claim 16, which shows a reduced binding to IL2Rα, IL2Rβ and IL2Rγ.

52. A nucleic acid or a plurality of nucleic acids, comprising a nucleic acid molecule containing a polynucleotide sequence encoding the fusion protein according to claim 16.

53. A vector comprising the nucleic acid molecule according to claim 52.

54. A host cell comprising the vector according to claim 53.

55. A first vector comprising a nucleic acid molecule comprising a polynucleotide sequence encoding the first polypeptide of the fusion protein according to claim 33, and a second vector comprising a nucleic acid molecule comprising a polynucleotide sequence encoding the second polypeptide of the fusion protein according to claim 33. A host cell comprising the vector.

56. A method for producing the fusion protein according to claim 16, the method comprising culturing the host cell according to claim 54 under conditions that allow production of the fusion protein or fragment, and recovering the fusion protein or fragment so produced. A method comprising.

57. A pharmaceutical composition comprising the fusion protein according to claim 16.

58. A pharmaceutical composition for use in a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the fusion protein according to claim 16.

59. The cancer is selected from bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer, ovarian cancer, rectal cancer, skin cancer, squamous cell carcinoma, and uterine cancer. The pharmaceutical composition according to claim 58.

60. The method further comprises administering to the subject a second therapeutic agent or therapy. The pharmaceutical composition according to claim 58.

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