Uses of attenuated IL-2 immunoconjugates

Abstract

The present invention discloses a method for treating cancer in subjects by using an anti-hPD-1 antibody modified human interleukin-2 (hIL-2) immunoconjugate alone or in combination with an antagonistic anti-PD-1 antibody.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 612,007 filed December 19, 2023, the full text of which is incorporated herein by reference.

[0003] Sequence list

[0004] The present application comprises a sequence list, the full text of which is incorporated herein by reference, which is submitted electronically in XML format. The name of the XML copy created on December 17, 2024 is 102085.002049-BIO040-WO01_SequenceListing.xml and the size is 697 kilobytes.

[0005] Technology field

[0006] The present invention discloses a method for treating cancer in subjects using an anti-human PD-1 (hPD-1) antibody-attenuated human interleukin-2 (hIL-2) immunoconjugate alone or in combination with an antagonistic anti-PD-1 antibody. Background Technology

[0007] Over the past decade, immuno-oncology has brought groundbreaking benefits to cancer treatment. For instance, the approval of immunotherapies such as immune checkpoint inhibitors, adoptive cell therapy, and cancer vaccines has revolutionized approaches to cancer treatment. These advancements have transformed the Standard of Care (SOC) and improved survival rates across various tumor types. However, despite immune checkpoint inhibitors improving clinical outcomes in diverse tumor types, only a portion of patients exhibit a clinical response, and a significant number of these responders develop acquired resistance following the initial response.

[0008] For immune checkpoint inhibitors, such as PD-1 antagonists, to be effective, the patient's immune system must be normal and the number of immune cells must be adequate. Typically, non-responders who do not respond to PD-1 antagonists are characterized by low intratumoral T-cell infiltration and a poor T-cell proliferation response to PD-1 antagonism.

[0009] Human IL-2 (hIL-2) is a type 1, four-α-helical bundle, glycosylated cytokine produced by CD4+ T cells and CD8+ T cells. Autocrine and adrenal secretion IL-2 signaling occurs through the binding of high-affinity trimeric receptor complexes containing IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132), or medium-affinity dimeric receptor complexes containing IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-2 plays a dual and opposing role in that it can stimulate T cell proliferation to generate T cell effectors, T cell memory, and activated NK cells, as well as stimulate suppressive regulatory T cells to maintain immune homeostasis. Low-dose IL-2 primarily stimulates regulatory T cells as well as some T effectors and NK cells, whereas high-dose IL-2 broadly stimulates cytotoxic T cells, T effectors, NK cells, and regulatory T cells. However, the use of IL-2 in the treatment of autoimmune diseases and as cancer immunotherapy has been limited due to off-target effects and toxicity associated with IL-2 administration.

[0010] In this institution, as a method for treating cancer in subjects, this method is for subjects to

[0011] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0012] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ NO. 345; and

[0013] An anti-hPD-1 antibody that immunospecifically binds to human PD-1 (hPD-1), or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0014] (i) a heavy chain complementarity determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 containing the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 containing the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO. 423;

[0015] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0016] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0017] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411, or an antigen-binding fragment thereof

[0018] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0019] (B) Antagonistic anti-PD-1 antibody binding to PD-1 in the presence of an immune conjugate

[0020] A method is disclosed that includes the step of administering [something].

[0021] The present invention provides a method for treating renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors in subjects, wherein the present method comprises: to the subject

[0022] As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0023] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0024] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0025] (i) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 423;

[0026] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0027] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0028] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411, or an antigen-binding fragment thereof

[0029] anti-hPD-1 antibody modified hIL-2 immunoconjugate containing

[0030] A method comprising the step of administering [something] is disclosed.

[0031] At our clinic, for cancer treatment.

[0032] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0033] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ NO. 345; and

[0034] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0035] (i) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 423;

[0036] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0037] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0038] (iv) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO. 406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 408, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 409, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 411

[0039] An anti-hPD-1 antibody containing, or its antigen-binding fragment

[0040] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0041] (B) The use of an antagonistic anti-PD-1 antibody that binds to PD-1 in the presence of an immune conjugate is also disclosed.

[0042] For the treatment of renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or microsatellite instability-high tumors or DNA mismatch repair deficiency tumors,

[0043] As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0044] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ NO. 345; and

[0045] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0046] (i) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 423;

[0047] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0048] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0049] (iv) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO. 406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 408, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 409, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 411

[0050] The use of an anti-hPD-1 antibody comprising, or an anti-hPD-1 antibody modified hIL-2 immunoconjugate comprising its antigen-binding fragment, is also disclosed. Brief explanation of the drawing

[0051] In addition to the summary, the following detailed description is better understood when read together with the attached drawings. To illustrate the disclosed method and use, exemplary embodiments of the method and use are presented in the drawings; however, the method and use are not limited to the specific embodiments disclosed. Fig. 1a, Fig. 1b, Fig. 1c, Fig. 1d, Fig. 1e, Fig. 1f, Fig. 1g, and Fig. 1hThis illustrates an exemplary antibody-hIL-2 immunoconjugate as described in Example 1 of the present invention. An unattenuated human IL-2 cytokine (indicated by a gray square) was fused directly (df: direct fusion) or via an L6 linker (L6) to the N-terminus or C-terminus of both heavy chains or both kappa light chains of the antibody. FIGS. 2A, FIGS. 2B, FIGS. 2C, FIGS. 2D, FIGS. 2E, FIGS. 2F, FIGS. 2G, and FIGS. 2H Figure 1 illustrates an exemplary antibody-hIL-2 immunoconjugate having an hCD25 (1-164) extracellular domain designed to interfere with hIL-2 binding of the immunoconjugate to human IL-2Rα. For the N-terminal variant, the human CD25 / IL-2Rα extracellular domain (indicated by black triangles) was fused to an unattenuated hIL-2 cytokine (indicated by gray squares) via an L20 linker (light gray line). Subsequently, the unattenuated hIL-2 cytokine was fused directly (df) or fused to the antibody using an L6 linker. For the C-terminal variant, the hCD25 / IL-2Rα extracellular domain moiety was fused directly (df) or fused to the antibody using an L6 linker, followed by an L20 linker and an unattenuated hIL-2 cytokine. Fig. 3 Figure 1 illustrates an exemplary 1H3-hIgG1-L6-hIL-2 immunoconjugate containing a CD25 / IL-2Rα extracellular domain moiety. The hCD25 / IL-2Rα extracellular domain moiety was fused to 1H3-hIgG1-L6-hIL-2 via an L6 linker at the C-terminus of each heavy chain, followed by an L20 linker and an hIL-2 cytokine moiety (attenuated hIL-2) containing a substitution predicted to coordinate binding to CD122 / IL-2Rβ as described in Example 2. Figs. 4a, 4b, 4c, and Fig. 4dThis shows the experimental results analyzing the binding of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 to the human PD-1 receptor in Jurkat cells in the presence of saturated concentrations of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A (10 μM) prior to exposure to anti-hPD-1 antibodies. Fig. 5 Figure [Image] illustrates exemplary anti-hPD-1-attenuated hIL-2 immunoconjugates using an L6 linker (L6) (left) or as direct fusions (df) (right). Anti-hPD-1 antibodies containing hIgG4 or hIgG1 Fc domains, with or without L235E (LE) or L235A / G237A (LAGA) modifications in the Fc domain, were fused to hIL-2 cytokines attenuated at the C-terminus of the antibody heavy chain. Various substitutions were introduced into the hIL-2 cytokines to attenuate efficacy. Fig. 6a and Fig. 6b is anti-hPD-1 #1-mIgG2b-N297A in the presence of saturated concentration of anti-hPD-1-attenuated hIL-2 immunoconjugate (280 nM) Fig. 6a ) or anti-hPD-1 #2-mIgG2b-N297A( Fig. 6b ) shows the results of a competitive assay demonstrating that it binds to the anti-hPD-1 receptor in Jurkat cells. Fig. 7 This shows the results of a competitive assay demonstrating that anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-L6-hIgG4-hIL-2 (D20A / R38E), and A2-hIgG4-df-hIL-2 (D20A / R38E) do not inhibit the binding of human PD-L1 to the human PD-1 receptor using a PD-1 / PD-L1 blocking bioassay. Fig. 8This shows experimental results analyzing the effects of administering a vehicle, a surrogate anti-PD-1 antibody (anti-mPD-1 RMP1-14 mIgG2b-N297A and anti-mPD-1 RMP1-30 mIgG2b-N297A), and a surrogate anti-PD-1-attenuated hIL-2 immunoconjugate (anti-mPD-1 RMP1-14 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) or anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R)) on the growth of established subcutaneous MC38 synonymous tumors in C57BL / 6 mice, as described in Example 18. The test agent was administered intraperitoneally at a dose of 5 mg / kg twice a week for 4 weeks, starting on day 1. The dots on the graph represent the average tumor volume of an average of 10 mice per group. Fig. 9a, Fig. 9b, and Fig. 9c This shows the results of a study conducted to determine the efficacy of the surrogate anti-hPD-1-attenuated hIL-2 immunoconjugate anti-mPD-1 RMP1-30 mIgG2b-N297A-hIL-2 (F42K / Y45R / V69R) in an MC38 murine colorectal adenocarcinoma model. Fig. 9a Figure 1 shows the average subcutaneous tumor volume (mm³) measured every 3-4 days for 8 days following the first administration of the test agent (3 doses at 5 mg / kg on days 1, 4, and 8). The tumor growth curve represents the average of 15 animals per group. Fig. 9b This summarizes the results from the tumor immunophenotype analysis by flow cytometry on day 9, which includes the entire CD8 + Different CD8s as a fraction relative to the average absolute count of T cells + Shows the proportion of T cell subsets. Fig. 9c Figure 9 shows the results of the immunophenotyping analysis on day 9, which indicates CD8 after exposure to the surrogate immunoconjugate. + It was demonstrated that T effector memory cells significantly expanded, and regulatory T cells (cells / µL) in the tumor showed a decreasing trend. Fig. 10 As demonstrated by a significant decrease in body weight in the NCG-PBMC model, NOD- exposed to anti-hPD-1-attenuated hIL-2 immunoconjugates Prkdc em26Cd52 IL-2rg em26Cd22 This shows the experimental results analyzing the acceleration of graft-versus-host disease in / NjuCrl(NCG) mice. Fig. 11a and Fig. 11b NOD- processed as 2H7-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A) in the NCG-PBMC model Prkdc em26Cd52 IL-2rg em26Cd22 / NjuCrl(NCG) mouse CD8 + Effector memory T cells ( Fig. 11a ) and CD4 + Effector memory T cells ( Fig. 11b This shows the experimental results analyzing the dose-dependent expansion of )(cells / mL(blood)). Fig. 12 NOD- processed as 2H7-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A) in the NCG-PBMC model Prkdc em26Cd52 IL-2rg em26Cd22 This shows a decrease in regulatory T cells (cells / mL (blood)) in NjuCrl (NCG) mice. Fig. 13a and Fig. 13b is H7-632-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A)(designated as "H7-767")( Fig. 13b This shows that ) continues to bind to the human PD-1 receptor in Jurkat cells in the presence of saturated concentrations of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A (10 μM) before exposure. Fig. 14a and Fig. 14bis the binding of recombinant human PD-1 captured in H7-767 immobilized on the CM5 sensor chip, as evaluated by surface plasmon resonance (SPR), subsequently, ( Fig. 14a ) H7-767, Keytruda®, or Opdivo® or ( Fig. 14b This is a graph showing the combination of PD-L1 or PD-L2. Fig. 15 This demonstrates that H7-632-hIgG1-LAGA and H7-767 do not inhibit the binding of human PD-L1 to the human PD-1 receptor using an hPD-1 / hPD-L1 blocking bioassay. Figs. 16a, 16b, 16c and Fig. 16d This is a graph showing the binding of anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-df-hIL-2 (D20A / R38E), and A2-hIgG4-df-hIL-2 (D20A / R38E) to the human PD-1 receptor in Jurkat cells in the presence of saturation concentrations of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A (10 μM) prior to exposure to anti-hPD-1-attenuated hIL-2 immunoconjugates, as evaluated by flow cytometry. Fig. 17 This is a graph showing the binding of 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-df-hIL-2 (D20A / R38E), A2-hIgG4-df-hIL-2 (D20A / R38E) and the unrelated antibody control 1H3-hIgG4-df-hIL-2 (D20A / R38E) to HEK-293T cells recombinantly expressing synomolgus PD-1, as evaluated by flow cytometry. Figs. 18a, 18b, 18c and Fig. 18dThis shows the antagonist activity of anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-df-hIL-2 (D20A / R38E), and A2-hIgG4-df-hIL-2 (D20A / R38E) in the presence of anti-hPD-1 #1 or anti-hPD-1 #2. Fig. 18a, and Fig. 18b This shows the results from the titration of anti-hPD-1 #1 or anti-hPD-1 #2 in the presence of a fixed concentration of anti-hPD-1-attenuated hIL-2 immunoconjugate. Fig. 18c, and Fig. 18d is an anti-hPD-1-attenuated hIL-2 immunoconjugate at a fixed concentration of 100 nM anti-hPD-1 #1 ( Fig. 18c ) or 100 nM of anti-hPD-1 #2( Fig. 18d It shows the results from appropriate reverse experiments. Fig. 19 This shows the effects of administration of various test agents, including the surrogate anti-mouse PD-1 / attenuated IL-2 immunoconjugate, on the growth of established subcutaneous MC38 synonymous tumors in C57BL / 6 mice. Each growth curve represents the average tumor volume of 10 mice per treatment group. Fig. 20 This demonstrates the ability of MC38 tumor cells to grow in tumor-naive mice compared to mice from the MC38 primary tumor study shown in Fig. 19, which were previously administered anti-mPD-1-hIL-2 F42K / Y45R / V69R and showed long-term complete regression of the established primary tumor. In animals of both cohorts (10 mice per group), 5x10 were placed on the left flank, opposite the primary tumor site. 5Canine MC38 tumor cells were subcutaneously transplanted. Mice previously exposed to the surrogate agent anti-mPD-1-hIL-2 F42K / Y45R / V69R did not show tumor growth because they developed sustained immunity, whereas the corresponding naive mouse controls showed typical tumor growth on their flanks. Fig. 21 This shows the change in MC38 tumor volume over time in mice treated with anti-mouse PD-1 RMP1-14, anti-mouse PD-1 RMP1-30-hIL-2 F42K / Y45R / V69R, or a combination of anti-mouse PD-1 RMP1-14 and anti-mouse PD-1 RMP1-30-hIL-2 F42K / Y45R / V69R. Specific details for implementing the invention

[0052] Detailed description of exemplary embodiments

[0053] The disclosed methods and uses may be more easily understood by referring to the following detailed description taken in connection with the accompanying drawings, which form part of the present disclosure. The disclosed methods and uses are not limited to the specific methods and uses described and / or presented herein, and it should be understood that the terms used herein are merely for describing specific embodiments by example and are not intended to limit the claimed methods and uses.

[0054] Unless otherwise specifically stated, any description of possible mechanisms of action, modes of action, or reasons for improvement is merely illustrative, and the disclosed methods and uses should not be limited by the accuracy or inaccuracy of any of the aforementioned proposed mechanisms of action, modes of action, or reasons for improvement.

[0055] Throughout this specification, descriptions refer to treatment methods and uses. Where this disclosure describes or claims features or embodiments associated with a treatment method, such features or embodiments are equally applicable to a use. Similarly, where this disclosure describes or claims features or embodiments associated with a use, such features or embodiments are equally applicable to a treatment method. Uses included within the scope of this disclosure include, but are not limited to, Swiss-style uses, first pharmaceutical uses, second / or more pharmaceutical uses, and uses according to EPC2000.

[0056] Where a range of numerical values ​​is mentioned or established herein, said range includes its endpoint and all individual integers and fractions within that range, and also includes, respectively, a narrower range formed by all various possible combinations of said endpoint and internal integers and fractions, forming a subgroup of a larger group of values ​​within the mentioned range to the extent that said narrower ranges are each explicitly mentioned. Where a range of numerical values ​​is mentioned as being larger than the values ​​mentioned herein, the range is nevertheless finite and its upper limit is restricted to values ​​operable within the context of this disclosure. Where a range of numerical values ​​is mentioned as being smaller than the values ​​mentioned herein, the range is nevertheless restricted to non-zero values. Categories of methods and uses are not intended to be limited to specific values ​​mentioned when defining the range. All ranges are inclusive and combinable.

[0057] It should be understood that when a value is expressed as an approximation using the antecedent "approximately," the specific value constitutes another embodiment. References to specific numerical values ​​include at least that specific value unless the context clearly indicates otherwise. The term "approximately," used when referring to a numerical range, cutoff, or specific value, is used to indicate that the mentioned value may vary from the listed value by up to 10%. Accordingly, the term "approximately" is used to include variation of ± 10% or less, ± 5% or less, ± 1% or less, ± 0.5% or less, or ± 0.1% or less from the mentioned value.

[0058] For clarity, it should be understood that specific features of the disclosed method and use described herein in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed method and use described herein in the context of a single embodiment may be provided individually or in any sub-combination.

[0059] As used herein, the singular forms "han" ("a," "an") and "geu" include the plural forms.

[0060] Various terms relating to aspects of description are used throughout this specification and claims. Unless otherwise specified, such terms shall have their common meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0061] The term "comprising" is intended to include examples included in the terms "essentially composed of" and "composed of"; similarly, the term "essentially composed of" is intended to include examples included in the term "composed of".

[0062] The term "antibody molecule" is used in a broad sense and includes full-length immunoglobulin molecules and their antigen-binding fragments.

[0063] Immunoglobulins can be designated into five major classes, namely IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies of any vertebrate species can be designated into one of two distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0064] "Antigen-binding fragment" refers to a part of an immunoglobulin molecule (i.e., "its antigen-binding fragment") that retains the antigen-binding properties of a parent full-length antibody. Exemplary antigen-binding fragments may have heavy chain complementarity determining region (CDR) 1, 2, and / or 3; light chain CDR 1, 2, and / or 3; heavy chain variable region (VH); light chain variable region (VL); and combinations thereof. The antigen-binding fragments include a Fab fragment, which is a monovalent fragment composed of VL, VH, constant light chain (CL), and constant heavy chain 1 (CH1) domains; a F(ab)2 fragment, which is a divalent fragment comprising two Fab fragments connected by a disulfide bridge at a hinge region; an Fd fragment composed of VH and CH1 domains; an Fv fragment composed of VL and VH domains of a single arm of the antibody; and a domain antibody (dAb) fragment composed of a VH domain or a VL domain (reference [Ward et al[Nature 341:544-546, 1989]) is included. VH and VL domains are manipulated via a synthetic linker and linked to form pairs of VH / VL domains within a molecule, or between molecules where VH and VL domains are expressed by separate single-strand antibody constructs, to form various types of single-strand antibody designs that form monovalent antigen-binding sites, e.g., single-strand Fv (scFv) or diabodies, as described, for example, in International Publications Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO1992 / 01047. The antibody fragments are obtained using techniques widely known to those skilled in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0065] The phrase "binds immunospecifically" refers to the ability of the initiated antibody molecule to bind preferentially to its target (hPD-1 in the case of an anti-hPD-1 antibody molecule) without binding preferentially to other molecules in a sample containing a mixed population of molecules. There are substantially no other antibodies with different antigen specificities in antibody molecules that bind immunospecifically to hPD-1 (e.g., there are substantially no antibodies in anti-hPD-1 antibodies that bind specifically to antigens other than hPD-1). However, antibody molecules that bind immunospecifically to hPD-1 are Macaca facicularius ( Macaca fascicularis It may have cross-reactivity with other antigens, such as orthologs of hPD-1, including )(cynomolgus monkey) PD-1. The antibody molecules disclosed herein can immunospecifically bind to both naturally produced hPD-1 and PD-1 recombinantly produced in mammalian or prokaryotic cells.

[0066] The antibody variability region consists of four "framework" regions interposed with three "antigen binding sites." Antigen binding sites are defined using various terms: (i) the complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), are based on sequence variability (reference [Wu and Kabat J Exp Med 132:211-50, 1970]; [Kabat et al. , [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991]); (ii) The "hypervariable regions" (“HVR” or “HV”: “Hypervariable regions”), consisting of three (H1, H2, H3) in VH and three (L1, L2, L3) in VL, refer to structurally hypervariable antibody variable domain regions as defined by (Chothia) and (Lesk) (Reference [Chothia and Lesk Mol Biol 196:901-17, 1987]). The AbM definition of CDR is also widely used; This is a compromise between the (Kabat) and (Chothia) numbering systems and is called that because it was used in Oxford Molecular's AbM antibody modeling software (Reference [Rees, AR, Searle, SMJ, Henry, AH and Pedersen, JT (1996) In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172]). Another term is "IMGT-CDR" (Reference [Lefranc et al.,Includes Dev Comparat Immunol 27:55-77, 2003]) and "Specificity Determining Residue Usage" (SDRU) (Almagro Mol Recognit 17:132-43, 2004). The International ImMunoGeneTics (IMGT) database (www_imgt_org) provides standardized numbering and definitions of antigen binding sites. The correspondence between CDR, HV, and IMGT classifications is [Lefranc et al., It is described in Dev Comparat Immunol 27:55-77, 2003.

[0067] The "framework" or "framework sequence" is the remaining sequence of the variable region other than that defined as the antigen binding site. Since the antigen binding site can be defined by various terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.

[0068] "Human antibody," "fully human antibody," and similar terms refer to antibodies having heavy and light chain variable regions in which both the framework and the antigen-binding site are derived from sequences of human origin. If an antibody contains a constant region, the constant region is also derived from a sequence of human origin. A human antibody comprises a heavy chain and / or light chain variable region “derived” from a “sequence of human origin” when the variable region of the antibody is obtained from a system using a human germline immunoglobulin or rearranged immunoglobulin gene. The system comprises a library of human immunoglobulin genes displayed on phages and transgenic non-human animals, such as mice or chickens, having human immunoglobulin loci as described herein. The “human antibody” may contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequence, for example, due to naturally occurring somatic mutations or the introduction of intentional substitutions in the variable domain (framework and antigen binding site) or constant domain. Typically, the “human antibody” has an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and amino acid sequences coded by the human germline or rearranged immunoglobulin gene. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% are identical. In some cases, "human antibodies" are, for example, in the literature [Knappik et al., As described in [J Mol Biol 296:57-86, 2000], consensus framework sequences derived from human framework sequence analysis, or, for example, literature [Shi et al.,As described in [J Mol Biol 397:385-96, 2010] and International Patent Publication No. WO2009 / 085462, it may include synthetic HCDR3 introduced into a human immunoglobulin gene library displayed on a phage. Antibodies whose antigen-binding sites are derived from non-human species are not included in the definition of "human antibody".

[0069] Human antibodies are derived from human immunoglobulin sequences, but can be generated using systems such as phage displays that introduce synthetic CDRs and / or synthetic frameworks, for example, or antibodies that do not naturally exist in the in vivo human antibody germline repertoire can be generated through in vitro mutagenesis to improve antibody characteristics in variable or constant regions, or both.

[0070] "Recombinant antibody" comprises all antibodies produced, expressed, generated, or isolated by recombinant means: for example, antibodies isolated from animals that are transgenic or transchromosomal to the human immunoglobulin gene (e.g., mice) or hybridomas produced therefrom (described further below); antibodies isolated from host cells transformed to express antibodies; antibodies isolated from a library of recombinant, recombinant antibodies; and antibodies produced, expressed, generated, or isolated by any other means including splicing the human immunoglobulin gene sequence to another DNA sequence, or antibodies produced in vitro using Fab arm exchange.

[0071] "Monoclonal antibody" refers to a group of antibody molecules with a single molecular composition. The composition of a monoclonal antibody exhibits single-binding specificity and affinity for a specific epitope, or, in the case of bispecific monoclonal antibodies, bispecific specificity for two distinct epitopes. Thus, monoclonal antibodies refer to a group of antibodies having a single amino acid composition in each heavy chain and each light chain, excluding widely known modifications such as the removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody group. Monoclonal antibodies can be monospecific or multispecific, and can be monovalent, divalent, or multivalent. Bispecific antibodies are included within the term monoclonal antibody.

[0072] An "epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes generally consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of a moiety, such as amino acid or polysaccharide side chains, and may possess specific charge characteristics as well as specific three-dimensional structural features. Epitopes may consist of continuous and / or discontinuous amino acids that form structural spatial units. In the case of discontinuous epitopes, amino acids from different parts of the antigen's linear sequence become close in three-dimensional space through the folding of the protein molecule.

[0073] "Variant" refers to a polypeptide or polynucleotide that differs from the reference polypeptide or reference polynucleotide by one or more modifications, e.g., substitution, insertion, or deletion. As used herein, the term "mutation" is intended to mean one or more intentional substitutions made to a polypeptide or polynucleotide.

[0074] As used herein, "antagonistic anti-PD-1 antibody" refers to an antibody that binds to PD-1 and acts to at least partially inhibit the interaction between PD-1 and its ligands PD-L1 and / or PD-L2.

[0075] "Antagonistic anti-PD-1 antibody binding to PD-1 in the presence of an immunoconjugate" means that, for example, using a method as described in Protocol B described herein, at least a portion of the antagonistic anti-PD-1 antibody is detectably bound to PD-1 previously exposed to an immunoconjugate.

[0076] "Non-antagonist anti-hPD-1 antibody" refers to an anti-human PD-1 antibody that does not inhibit ligand-induced signaling mediated by the interaction between PD-1 and its ligands PD-L1 and / or PD-L2 when measured using a cell-based assay or SPR as described herein.

[0077] “Treat,” “treatment,” and similar terms refer to both therapeutic and prophylactic or preventive treatments, and include reducing the severity and / or frequency of symptoms, eliminating symptoms and / or the underlying cause of symptoms, reducing the frequency or likelihood of symptoms and / or their underlying cause, and improving or treating damage caused directly or indirectly by cancer. Treatment also includes extending the duration of survival compared to the expected survival rate of subjects not receiving treatment. Subjects to be treated include not only subjects with cancer, but also subjects at risk of developing cancer or subjects to be treated to prevent cancer.

[0078] As used herein, "administered to a subject" and similar terms refer to a procedure of injecting a disclosed immunoconjugate (monotherapy) or an immunoconjugate and an antagonistic anti-PD-1 antibody (combination therapy) into a subject so that target cells, tissues, or segments of the subject's body come into contact with the disclosed immunoconjugate or the immunoconjugate and antagonistic anti-PD-1 antibody.

[0079] The phrase “therapeutic effective dose” refers to an amount of an immunoconjugate (monotherapy) or an immunoconjugate and an antagonistic anti-PD-1 antibody (combination therapy) as described herein that is effective in achieving a specific biological or therapeutic outcome, e.g., but not limitingly, the biological or therapeutic outcome disclosed, described, or illustrated herein. The therapeutic effective dose may vary depending on factors such as, for example, the individual’s disease state, age, sex, and body weight, and the ability of the immunoconjugate (monotherapy) or the immunoconjugate and an antagonistic anti-PD-1 antibody (combination therapy) to elicit a desired response in the subject. Exemplary indicators of the therapeutic effective dose include, for example, improvement in the patient’s well-being, reduction of cancer symptoms, cessation or slowing of cancer progression, and / or absence of cancer symptoms.

[0080] As used herein, the term "subject" is intended to mean any animal, in particular, mammals. Accordingly, the method is most preferably used on humans, but is applicable to humans and non-human animals. "Subject" and "patient" are used interchangeably herein.

[0081] The disclosed modified hIL-2 protein is also referred to herein as "attenuated" hIL-2. As described herein, "reduced efficacy" of IL-2 activity and related terms such as, for example, "reduced efficacy" or "attenuation" of IL-2 activity refer to the EC of unmodified hIL-2 in IL-2-dependent assays. 50 EC compared to the value 50It refers to a decrease in efficacy of modified hIL-2 determined to be increased. As described herein, the decrease in efficacy of modified hIL-2 will occur in both high-affinity IL-2 receptors and medium-affinity IL-2 receptors. The IL-2-dependent assay for determining efficacy may be a engineered human erythroleukemia TF1 (TF1+IL-2Rβ) cell proliferation assay or a human natural killer NK-92 cell proliferation assay as described herein. In one embodiment, the IL-2-dependent assay for determining efficacy is a engineered human erythroleukemia TF1 (TF1+IL-2Rβ) cell proliferation assay. In another embodiment, the IL-2-dependent assay for determining efficacy is a human natural killer NK-92 cell proliferation assay. Other IL-2-dependent assays for determining efficacy may be the TF1+IL-2Rβ pSTAT5 assay or the human natural killer NK-92 pSTAT5 assay as described herein. The unmodified-hIL-2 may be a prokaryotic-expressed hIL-2, e.g., Proleukin® (having the natural human IL-2 amino acid sequence except for a C125S substitution to remove unbound cysteine, and not having normal human carbohydrate expression at residue T3), or the unmodified-hIL-2 may be an hIL-2 expressed in mammalian cell lines, such as CHO or HEK cell lines, having the amino acid sequence of SEQ ID NO. 345 or the amino acid sequence of SEQ ID NO. 345 including a C125S substitution.

[0082] Immunoconjugates and fusion proteins are used interchangeably in this institution.

[0083] Combination therapy

[0084] Standard cancer treatment may include administering PD-1 antagonist checkpoint inhibitors (e.g., antagonistic anti-PD-1 antibodies nivolumab, pembrolizumab, cemiflimab, dostarlimab, or retipanlimab) to antagonize PD-1 in tumor-resident T cells. For example, treatment with nivolumab or pembrolizumab may initially enhance the tumor response by effector T cells, but in many cases, this response diminishes over time as chronically stimulated tumor-resident T cells exhibit a "depletion" phenotype characterized by reduced proliferative capacity, reduced cytotoxic activity, and increased expression of immune checkpoint molecules. The immunoconjugates described herein do not interfere with antagonistic anti-PD-1 antibodies, such as nivolumab or pembrolizumab, binding to and antagonizing the PD-1 receptor. The mechanisms of action of the immunoconjugates and antagonistic anti-PD-1 antibodies described herein are different yet complementary. Specifically, the primary mechanism of action of PD-1 antagonists, such as antagonistic anti-PD-1 antibodies, is to block immunosuppressive signals transmitted through the interaction between PD-1 and PD-L1, thereby maintaining T cells in their activated state or restoring them to their activated state. In contrast, the mechanism of action of the immunoconjugates described herein is to induce T cell proliferation primarily through T cell-selective IL-2 signaling, and then promote activation only afterward. The different modes of action and the possibility that each agent can exhibit activity through simultaneous combination provide grounds for combining therapies to enhance clinical outcomes, for example, in cases of acquired resistance to PD-(L)1 blockade therapy. Therapies can be enhanced by clinically combining immunoconjugates initiated by these characteristics with antagonistic anti-PD-1 antibodies.For example, in a tumor that initially responded to a checkpoint inhibitor but subsequently developed T cell depletion, the tumor-resident T cells can be restimulated to acquire a non-depletion phenotype by administering the initiated immunoconjugate to deliver modified hIL-2 to PD-1-expressing cells, thereby promoting the T cells to respond to the checkpoint inhibitor again. In some embodiments, the antagonistic anti-PD-1 antibody and the immunoconjugate may be delivered jointly or sequentially as a single agent, as described herein, so that each agent can be administered at a concentration that optimizes the subject's anti-tumor response and for such a period.

[0085] In this institution, as a method for treating cancer in subjects, this method is for subjects to

[0086] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0087] A modified hIL-2 protein comprising a substitution at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ ID NO. 345 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0088] An anti-hPD-1 antibody that immunospecifically binds to human PD-1 (hPD-1), or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0089] (i) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to as "H7-632" in this application);

[0090] (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to as "2H7" in this application);

[0091] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5"); or

[0092] (iv) an anti-hPD-1 antibody or its antigen-binding fragment comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as "A2").

[0093] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0094] (B) Antagonistic anti-PD-1 antibody binding to PD-1 in the presence of an immune conjugate

[0095] A method is disclosed that includes the step of administering [something].

[0096] Suitable substitutions for amino acid position 20 of the modified hIL-2 portion of the immunoconjugate include, for example, any of the substitutions D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0097] Suitable substitutions for amino acid position 38 of the modified hIL-2 portion of the immunoconjugate include, for example, any of the substitutions R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0098] In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions may be combined with the R38E substitution.

[0099] Therefore, in this institution, as a method for treating cancer in a subject, this method is for the subject

[0100] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0101] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0102] An anti-hPD-1 antibody that immunospecifically binds to human PD-1 (hPD-1), or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0103] (i) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to as "H7-632" in this application);

[0104] (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to as "2H7" in this application);

[0105] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5"); or

[0106] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as "A2"), or an antigen-binding fragment thereof

[0107] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0108] (B) Antagonistic anti-PD-1 antibody binding to PD-1 in the presence of an immune conjugate

[0109] A method is disclosed that includes the step of administering [something].

[0110] The modified hIL-2 protein portion of the immunoconjugate may comprise any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein portion of the immunoconjugate may comprise any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 608, 611, 614, or 620. The modified hIL-2 protein portion of the immunoconjugate may comprise any one of the amino acid sequences of SEQ ID NOs 149, 307, 607-611, 614, 617, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 146.In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 620. Any one of the amino acid sequences of SEQ ID NO. 134-150, 307, 344, 607-611, 614, 617, or 620 of the modified hIL-2 protein may further comprise T3A substitution and / or C125A substitution. In some embodiments, any one of the amino acid sequences of SEQ ID NO. 134-150, 307, 344, 607-611, 614, 617, or 620 of the modified hIL-2 protein further comprises T3A substitution.In some embodiments, a modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, a modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

[0111] The modified hIL-2 protein portion of the immunoconjugate may include a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. The method disclosed herein for treating cancer in a subject to the subject

[0112] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0113] A modified hIL-2 protein comprising a D20A substitution at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0114] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0115] (i) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to as "H7-632" in this application);

[0116] (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to as "2H7" in this application);

[0117] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5"); or

[0118] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as "A2"), or an antigen-binding fragment thereof

[0119] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0120] (B) Antagonistic anti-PD-1 antibody binding to PD-1 in the presence of an immune conjugate

[0121] It may include a step of administering.

[0122] The modified hIL-2 protein portion of the immunoconjugate may additionally include a substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate includes T3A substitution, D20A substitution, and R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugate includes the amino acid sequence of SEQ ID NO. 216.

[0123] Alternatively, the modified hIL-2 protein portion of the immunoconjugate may further comprise a deletion at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises deletions of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 218.

[0124] The modified hIL-2 protein portion of the immunoconjugate may further comprise a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 215. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 217. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 219.

[0125] The modified hIL-2 protein portion of the immunoconjugate is, for example, EC in the hIL-2-dependent cell proliferation assay described herein. 50As quantified by value comparison, the modified hIL-2 may exhibit a reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a greater reduction in efficacy of about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2. A greater reduction in hIL-2 efficacy for the high-affinity hIL-2 receptor is possible and permissible for the modified hIL-2 protein described herein, but such reduction may not be quantified by the method described herein due to limitations of cell proliferation assay conditions.

[0126] Additionally, the modified hIL-2 protein portion of the immunoconjugate is, for example, EC in the hIL-2-dependent cell proliferation assay described herein. 50 As quantified by value comparison, the modified hIL-2 may exhibit a reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a reduction in efficacy of about 10,000-fold greater with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2.

[0127] The modified hIL-2 protein portion of the immunoconjugate is, for example, EC in the hIL-2-dependent cell proliferation assay described herein. 50As quantified by value comparison, it may exhibit up to approximately 10,000-fold reduction in efficacy for high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduction in efficacy for medium-affinity IL-2 receptors (hIL-2Rβγ) compared to unmodified hIL-2. The modified hIL-2 protein portion of the immunoconjugate may exhibit approximately 10,000-fold greater reduction in efficacy for high-affinity IL-2 receptors (hIL-2Rαβγ) and approximately 10,000-fold greater reduction in efficacy for medium-affinity IL-2 receptors (hIL-2Rβγ) compared to unmodified hIL-2.

[0128] The modified hIL-2 protein may be fused to the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment of the anti-hPD-1 antibody of the immunoconjugate. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate is fused directly to the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion by peptide bonding. For example, the modified hIL-2 protein portion of the immunoconjugate may be fused directly to the C-terminal amino acid residue of the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion by peptide bonding. In some embodiments, the modified hIL-2 protein is fused to the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion through a linker.

[0129] Fusion of an anti-hPD-1 antibody or its antigen-binding fragment portion of an immunoconjugate of a modified hIL-2 protein can construct the ability of the modified hIL-2 protein to activate an intermediate affinity IL-2 receptor. In some embodiments, the immunoconjugate can activate an intermediate affinity IL-2 receptor to a degree similar to that of wild-type hIL-2.

[0130] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to herein as "H7-632").

[0131] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to herein as "2H7").

[0132] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5").

[0133] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as “A2”).

[0134] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 417 (referred to herein as "H7-632").

[0135] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 385 (referred to herein as "2H7").

[0136] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 395 (referred to herein as "C51E6-5").

[0137] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 405 (referred to as "A2" in this application).

[0138] The anti-hPD-1 antibody of the immune conjugate or its antigen-binding fragment portion may include an IgG1 heavy chain constant region.

[0139] The anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion may have substitutions or deletions within the constant region to minimize Fc-mediated immune effector functions, e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by FcγRIIIA, antibody-dependent cellular phagocytosis (ADCP) mediated by FcγRI and FcγRIIA, and complement-dependent cytotoxicity (CDC). In some embodiments, the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion comprises an L235A substitution, wherein the amino acid numbering follows EU numbering. In some embodiments, the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion comprises a G237A substitution, wherein the amino acid numbering follows EU numbering. In some embodiments, the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion comprises L235A and G237A substitutions, wherein the amino acid numbering follows EU numbering.

[0140] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415 (referred to herein as "H7-632-hIgG1-LAGA").

[0141] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 424 and a light chain comprising the amino acid sequence of SEQ ID NO. 425 (referred to herein as "2H7-hIgG4").

[0142] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 426 and a light chain comprising the amino acid sequence of SEQ ID NO. 427 (referred to herein as "C51E6-5-hIgG4").

[0143] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 428 and a light chain comprising the amino acid sequence of SEQ ID NO. 429 (referred to herein as "A2-hIgG4").

[0144] Immunoconjugates may have one or more of the following characteristics:

[0145] · Characteristic of binding to PD-1 without inhibiting PD-L1 binding to PD-1;

[0146] · Standard therapeutic anti-PD-1 antibodies used in clinics (e.g., pembrolizumab (KEYTRUDA) ® ) or nivolumab (OPDIVO ® Characteristic of binding to PD-1 in the presence of );

[0147] · Highly selective for PD-1 and does not immune-specifically bind to other related B7 family members;

[0148] · Binding property of activated human T cells to PD-1 (EC in flow binding assay) 50 ~0.1-0.2 nM);

[0149] · The modified hIL-2 protein portion of the immunoconjugate exhibits a measurable reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2, as quantified, for example, by comparing EC50 values ​​in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a greater reduction in efficacy of about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2. A greater reduction in hIL-2 efficacy to high-affinity hIL-2 receptors is possible and permissible for the modified hIL-2 protein described herein, but such reduction may not be quantifiable by the method described herein due to limitations in cell proliferation assay conditions;

[0150] · The modified hIL-2 protein portion of the immunoconjugate exhibits a measurable reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2, as quantified, for example, by comparing EC50 values ​​in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a greater reduction in efficacy of about 10,000-fold with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2;

[0151] · Characteristics for constructing and expanding PD-1-expressing human memory T cell subsets in a GvHD animal model; and

[0152] · Characteristics of having minimal or no effect on body weight, blood chemistry, or hematological parameters after a single dose of 1 and 10 mg / kg in cynomolgus monkeys.

[0153] In some embodiments, the immunoconjugate comprises a modified hIL-2 protein comprising T3A substitution, R38E substitution, D20A substitution, and C125A substitution fused to the C-terminus of an anti-hPD-1 antibody heavy chain comprising a human IgG1 framework comprising L235A substitution and G237A substitution. In some embodiments, the immunoconjugate comprises a modified hIL-2 protein fusion comprising a light chain having the amino acid sequence of SEQ ID NO. 415 and a heavy chain having the amino acid sequence of SEQ ID NO. 532.

[0154] Suitable antagonistic anti-PD-1 antibodies include nivolumab (OPDIVO ® ), pembrolizumab (KEYTRUDA ® ), semiplimab (LIBTAYO®), dostarlimab (JEMPERLI), or retipanlimab (ZYNYZ TM Includes ). In some embodiments, the antagonistic anti-PD-1 antibody is nivolumab. In some embodiments, the antagonistic anti-PD-1 antibody is pembrolizumab. References to nivolumab, pembrolizumab, cemiplimab, dostarlimab, or retipanlimab are, in some cases, Opdivo ® , Keytruda ® It is intended to include any nivolumab-containing, pembrolizumab-containing, semiplimab-containing, dostarlimab-containing, or retipanlimab-containing product that has been approved by regulatory authorities as a biosimilar of LIBTAYO®, JEMPERLI, or ZYNYZ™.

[0155] The disclosed immunoconjugate can selectively deliver IL-2 signaling to PD-1-expressing T cells. The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate can be used exclusively to deliver modified hIL-2 to PD-1-expressing cells, for example, nivolumab (OPDIVO ® ) and pembrolizumab (KEYTRUDA ® It does not block PD-1 receptor function like classical anti-PD-1 inhibitor antibodies. The main mechanism of the immunoconjugate disclosed herein is mediated by the T cell-selective activation of IL-2. The human PD-1 receptor is primarily expressed in a small subset of T cells with potent tumor responsiveness. Without being bound by theory, it is considered that targeting the modified hIL-2 protein portion of the immunoconjugate to said T cell population can dramatically amplify anti-tumor immunity while reducing or minimizing off-target systemic IL-2-mediated toxicity mediated by the non-PD-1 expressing cell population.

[0156] Antagonistic anti-PD-1 antibody and an immunoconjugate may be administered to a subject together as a mixture, jointly as a single agent, or sequentially as a single agent in any order. In some embodiments, the antagonistic anti-PD-1 antibody and the immunoconjugate are administered to a subject together as a mixture. In some embodiments, the antagonistic anti-PD-1 antibody and the immunoconjugate are administered to a subject jointly as a single agent. In some embodiments, the antagonistic anti-PD-1 antibody and the immunoconjugate are administered sequentially as a single agent in any order. In some embodiments, the method may include the step of administering the immunoconjugate before administering the antagonistic anti-PD-1 antibody. In some embodiments, the method may include the step of administering the antagonistic anti-PD-1 antibody before administering the immunoconjugate. In some embodiments, the method may include the step of administering the immunoconjugate at substantially the same time as administering the antagonistic anti-PD-1 antibody. Immunoconjugates and antagonistic anti-PD-1 antibodies may be administered so that the second agent is administered while the first agent is still exhibiting a biological effect.

[0157] The immunoconjugate may be administered to subjects who begin to show signs of relapse / recurrence after a period of antagonistic PD-1 therapy. Accordingly, in some embodiments, the present method is performed on subjects who begin to show signs of relapse / recurrence after or during antagonistic PD-1 therapy.

[0158] Exemplary cancers that can be treated using the disclosed method include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, non-small cell lung carcinoma, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, melanoma, squamous cell carcinoma, bone cancer, and kidney cancer. In some embodiments, the disclosed method may be used to treat melanoma, Merkel cell carcinoma, non-small cell lung carcinoma, renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors.

[0159] Every aspect of the initiation and claimed combination therapy presented herein as a treatment method is equally applicable to the use of immunoconjugates and antagonistic anti-PD-1 antibodies, including, but not limited to, Swiss-type uses, first medicinal uses, second / additional medicinal uses, and uses under EPC2000. Accordingly, for example, herein, for the treatment of cancer,

[0160] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0161] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0162] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0163] (i) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 423;

[0164] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0165] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0166] (iv) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO. 406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 408, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 409, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 411

[0167] An anti-hPD-1 antibody containing, or its antigen-binding fragment

[0168] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0169] (B) The use of an antagonistic anti-PD-1 antibody that binds to PD-1 in the presence of an immune conjugate is disclosed.

[0170] monotherapy

[0171] The present invention provides a method for treating renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors in subjects, wherein the present method comprises: to the subject

[0172] As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0173] A modified hIL-2 protein comprising a substitution at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0174] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0175] (i) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to as "H7-632" in this application);

[0176] (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to as "2H7" in this application);

[0177] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5"); or

[0178] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as "A2"), or an antigen-binding fragment thereof

[0179] A method is disclosed comprising the step of administering an anti-hPD-1 antibody modified hIL-2 immunoconjugate containing [the component].

[0180] Suitable substitutions for amino acid position 20 of the modified hIL-2 portion of the immunoconjugate include, for example, any of the substitutions D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0181] Suitable substitutions for amino acid position 38 of the modified hIL-2 portion of the immunoconjugate include, for example, any of the substitutions R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0182] In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions may be combined with the R38E substitution.

[0183] Accordingly, the present invention provides a method for treating renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors in subjects, wherein the present method to the subject

[0184] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0185] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0186] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0187] (i) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to as "H7-632" in this application);

[0188] (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to as "2H7" in this application);

[0189] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5"); or

[0190] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as "A2"), or an antigen-binding fragment thereof

[0191] A method is disclosed comprising the step of administering an anti-hPD-1 antibody modified hIL-2 immunoconjugate containing [the component].

[0192] The modified hIL-2 protein portion of the immunoconjugate may comprise any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein portion of the immunoconjugate may comprise any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 608, 611, 614, or 620. The modified hIL-2 protein portion of the immunoconjugate may comprise any one of the amino acid sequences of SEQ ID NOs 149, 307, 607-611, 614, 617, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 146.In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 620. Any one of the amino acid sequences of SEQ ID NO. 134-150, 307, 344, 607-611, 614, 617, or 620 of the modified hIL-2 protein may further comprise T3A substitution and / or C125A substitution. In some embodiments, any one of the amino acid sequences of SEQ ID NO. 134-150, 307, 344, 607-611, 614, 617, or 620 of the modified hIL-2 protein further comprises T3A substitution.In some embodiments, a modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, a modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

[0193] The modified hIL-2 protein portion of the immunoconjugate may include a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. Accordingly, the present invention provides a method for treating renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors in a subject, wherein the present method comprises: to a subject

[0194] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0195] A modified hIL-2 protein comprising a D20A substitution at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0196] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0197] (i) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to as "H7-632" in this application);

[0198] (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to as "2H7" in this application);

[0199] (iii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5"); or

[0200] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as "A2"), or an antigen-binding fragment thereof

[0201] anti-hPD-1 antibody modified hIL-2 immunoconjugate containing

[0202] A method is disclosed that includes the step of administering [something].

[0203] The modified hIL-2 protein portion of the immunoconjugate may additionally include a substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate includes T3A substitution, D20A substitution, and R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugate includes the amino acid sequence of SEQ ID NO. 216.

[0204] Alternatively, the modified hIL-2 protein portion of the immunoconjugate may further comprise a deletion at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises deletions of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 218.

[0205] The modified hIL-2 protein portion of the immunoconjugate may further comprise a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 215. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 217. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO. 219.

[0206] The modified hIL-2 protein portion of the immunoconjugate is, for example, EC in the hIL-2-dependent cell proliferation assay described herein. 50As quantified by value comparison, the modified hIL-2 may exhibit a reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a greater reduction in efficacy of about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2. A greater reduction in hIL-2 efficacy for the high-affinity hIL-2 receptor is possible and permissible for the modified hIL-2 protein described herein, but such reduction may not be quantified by the method described herein due to limitations of cell proliferation assay conditions.

[0207] Additionally, the modified hIL-2 protein portion of the immunoconjugate is, for example, EC in the hIL-2-dependent cell proliferation assay described herein. 50 As quantified by value comparison, the modified hIL-2 may exhibit a reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a reduction in efficacy of about 10,000-fold greater with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2.

[0208] The modified hIL-2 protein portion of the immunoconjugate is, for example, EC in the hIL-2-dependent cell proliferation assay described herein. 50As quantified by value comparison, it may exhibit up to approximately 10,000-fold reduction in efficacy for high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduction in efficacy for medium-affinity IL-2 receptors (hIL-2Rβγ) compared to unmodified hIL-2. The modified hIL-2 protein portion of the immunoconjugate may exhibit approximately 10,000-fold greater reduction in efficacy for high-affinity IL-2 receptors (hIL-2Rαβγ) and approximately 10,000-fold greater reduction in efficacy for medium-affinity IL-2 receptors (hIL-2Rβγ) compared to unmodified hIL-2.

[0209] The modified hIL-2 protein may be fused to the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment of the anti-hPD-1 antibody of the immunoconjugate. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate is fused directly to the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion by peptide bonding. For example, the modified hIL-2 protein portion of the immunoconjugate may be fused directly to the C-terminal amino acid residue of the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion by peptide bonding. In some embodiments, the modified hIL-2 protein is fused to the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion through a linker.

[0210] Fusion of an anti-hPD-1 antibody or its antigen-binding fragment portion of an immunoconjugate of a modified hIL-2 protein can construct the ability of the modified hIL-2 protein to activate an intermediate affinity IL-2 receptor. In some embodiments, the immunoconjugate can activate an intermediate affinity IL-2 receptor to a degree similar to that of wild-type hIL-2.

[0211] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423 (referred to herein as "H7-632").

[0212] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 391 (referred to herein as "2H7").

[0213] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 401 (referred to herein as "C51E6-5").

[0214] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate comprises a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411 (referred to herein as “A2”).

[0215] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 417 (referred to herein as "H7-632").

[0216] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 385 (referred to herein as "2H7").

[0217] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 395 (referred to herein as "C51E6-5").

[0218] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 405 (referred to as "A2" in this application).

[0219] The anti-hPD-1 antibody of the immune conjugate or its antigen-binding fragment portion may include an IgG1 heavy chain constant region.

[0220] The anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion may have substitutions or deletions within the invariant region to minimize Fc-mediated immune effector functions, e.g., FcγRIIIA-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), FcγRI and FcγRIIA-dependent antibody-dependent phagocytosis (ADCP), and C1q-binding-mediated complement-dependent cytotoxicity (CDC). In some embodiments, the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion comprises an L235A substitution, wherein the amino acid numbering follows EU numbering. In some embodiments, the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion comprises a G237A substitution, wherein the amino acid numbering follows EU numbering. In some embodiments, the anti-hPD-1 antibody of the immunoconjugate or its antigen-binding fragment portion comprises L235A and G237A substitutions, wherein the amino acid numbering follows EU numbering.

[0221] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415 (referred to herein as "H7-632-hIgG1-LAGA").

[0222] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 424 and a light chain comprising the amino acid sequence of SEQ ID NO. 425 (referred to herein as "2H7-hIgG4").

[0223] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 426 and a light chain comprising the amino acid sequence of SEQ ID NO. 427 (referred to herein as "C51E6-5-hIgG4").

[0224] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 428 and a light chain comprising the amino acid sequence of SEQ ID NO. 429 (referred to herein as "A2-hIgG4").

[0225] Immunoconjugates may have one or more of the following characteristics:

[0226] · Characteristic of binding to PD-1 without inhibiting PD-L1 binding to PD-1;

[0227] · Standard therapeutic anti-PD-1 antibodies used in clinics (e.g., pembrolizumab (KEYTRUDA) ® ) or nivolumab (OPDIVO ® Characteristic of binding to PD-1 in the presence of );

[0228] · Highly selective for PD-1 and does not immune-specifically bind to other related B7 family members;

[0229] · Binding property of activated human T cells to PD-1 (EC in flow binding assay) 50 ~0.1-0.2 nM);

[0230] · The modified hIL-2 protein portion of the immunoconjugate exhibits a measurable reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2, as quantified, for example, by comparing EC50 values ​​in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a greater reduction in efficacy of about 10,000-fold with respect to the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to the unmodified hIL-2. A greater reduction in hIL-2 efficacy to high-affinity hIL-2 receptors is possible and permissible for the modified hIL-2 protein described herein, but such reduction may not be quantifiable by the method described herein due to limitations in cell proliferation assay conditions;

[0231] · The modified hIL-2 protein portion of the immunoconjugate exhibits a measurable reduction in efficacy of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2, as quantified, for example, by comparing EC50 values ​​in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate may exhibit a greater reduction in efficacy of about 10,000-fold with respect to the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to the unmodified hIL-2;

[0232] · Characteristics for constructing and expanding PD-1-expressing human memory T cell subsets in a GvHD animal model; and

[0233] · Characteristics of having minimal or no effect on body weight, blood chemistry, or hematological parameters after a single dose of 1 and 10 mg / kg in cynomolgus monkeys.

[0234] In some embodiments, the immunoconjugate comprises a modified hIL-2 protein comprising T3A substitution, R38E substitution, D20A substitution, and C125A substitution fused to the C-terminus of an anti-hPD-1 antibody heavy chain comprising a human IgG1 framework comprising L235A substitution and G237A substitution. In some embodiments, the immunoconjugate comprises a modified hIL-2 protein fusion comprising a light chain having the amino acid sequence of SEQ ID NO. 415 and a heavy chain having the amino acid sequence of SEQ ID NO. 532.

[0235] The disclosed immunoconjugate can selectively deliver IL-2 signaling to PD-1-expressing T cells. The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate can be used exclusively to deliver modified hIL-2 to PD-1-expressing cells, for example, nivolumab (OPDIVO ® ) and pembrolizumab (KEYTRUDA ® It does not block PD-1 receptor function like classical anti-PD-1 inhibitor antibodies. The main mechanism of the immunoconjugate disclosed herein is mediated by the T cell-selective activation of IL-2. The human PD-1 receptor is primarily expressed in a small subset of T cells with potent tumor responsiveness. Without being bound by theory, it is considered that targeting the modified hIL-2 protein portion of the immunoconjugate to said T cell population can dramatically amplify anti-tumor immunity while reducing or minimizing off-target systemic IL-2-mediated toxicity mediated by the non-PD-1 expressing cell population.

[0236] Each and every aspect of the initiation and claimed monotherapy presented herein as a treatment method is equally applicable to the use of immunoconjugates, including but not limited to Swiss-type uses, first medicinal uses, second / additional medicinal uses, and uses under EPC2000. Thus, for example, for the treatment of renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors,

[0237] As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0238] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; and

[0239] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0240] (i) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 423;

[0241] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0242] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0243] (iv) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO. 406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 408, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 409, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 411

[0244] An anti-hPD-1 antibody containing, or its antigen-binding fragment

[0245] The use of an anti-hPD-1 antibody modified hIL-2 immunoconjugate comprising [the following] is disclosed.

[0246] Examples

[0247] The following examples are provided to further describe some of the embodiments disclosed herein. These examples are not intended to limit the disclosed embodiments, but to explain them.

[0248] General method

[0249] Protocol A. Flow cytometry screening for the binding of anti-hPD-1 antibodies or anti-hPD-1 antibody-attenuated hIL-2 fusion agents to human PD-1

[0250] To test binding to hPD-1, the antibody and antibody-attenuated hIL-2 fusion protein were characterized in the full titration curve. Jurkat cell lines were transfected with a mammalian vector encoding amino acids 1-185 (SEQ No. 346) of human PD-1 to stably express parts of the extracellular and transmembrane domains of human PD-1, and binding of the anti-hPD-1 antibody was determined using said transfected cell lines. Jurkat + hPD-1 cells were washed and added to 96-well plates at a density of 100,000 cells per well in FACS buffer (PBS, 0.2% heat-inactivated fetal bovine serum). Cells were blocked with a 1:50 dilution of human FcR block (Miltenyi) for 10 minutes at 4°C and washed with FACS buffer.

[0251] Antibodies or antibody-attenuated hIL-2 immunoconjugates (fusion proteins) were serially diluted 6-fold in FACS buffer for an 8-point curve and added to human PD-1-expressing Jurkat cells in a 100 µL volume over ice for 1 hour. Cells were washed and resuspended in FACS buffer containing a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody. Cells were washed once more and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher), and analysis was performed by flow cytometry on a BD FACS Canto II, BD Celesta, or BD Fortessa (BD Biosciences) flow cytometer. Geometric mean fluorescent intensity (gMFI) was calculated using FlowJo software version 10. Half-maximum effective concentration (EC6) 50 The ) value was calculated from the gMFI of the allophycocyanin signal over titration concentrations using GraphPad Prism 7 software.

[0252] Protocol B. Flow cytometry competitive screening for the binding of anti-hPD-1 antibodies or anti-hPD-1 antibody-attenuated hIL-2 fusion agents to human PD-1

[0253] Antibodies and antibody-attenuated hIL-2 fusion proteins regarding the ability to bind to human PD-1 in the presence of saturated concentrations of anti-hPD-1 #1-mIgG2b-N297A (a sequence comprising the heavy and light chain variable region sequences (SEQNs 348 and 349) of nivolumab (clone 5C4) described in U.S. Patent Publication No. US 2009 / 0217401A1 formatted on a murine IgG2b-N297A background) or anti-hPD-1 #2-mIgG2b-N297A (a sequence comprising the heavy and light chain variable region sequences (SEQNs 350 and 351) of pembrolizumab (clone 109A-H / K09A-L-11) described in International Publication No. WO2008 / 156712A1 formatted on a murine IgG2b-N297A background) Tested.

[0254] Antibodies or antibody-attenuated hIL-2 fusion proteins were serially diluted 6-fold for an 8-point curve in the presence and absence of saturation amounts of 10 μM anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. Briefly, (as described in Protocol A above) stably expressing hPD-1 Jurkat cells were washed and resuspended in FACS buffer containing a 1:50 dilution of human FcR blocking reagent. Cells were incubated at 4°C for 10 minutes and then washed. Subsequently, cells were resuspended in a 100 μL volume containing anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N287A diluted to 10 μM in FACS buffer and incubated at 4°C for 1 hour. Cells were washed and incubated at 4°C for 1 hour with 6-fold serially diluted test antibodies or antibody-attenuated hIL-2 fusion proteins for an 8-point curve in a 100 µL volume. To detect the conjugated test anti-hPD-1 antibody or anti-hPD-1-attenuated hIL-2 fusion protein, cells were washed again and incubated on ice for 45 minutes with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody. Cells were washed and resuspended in FACS buffer containing a 1:1000 dilution of Cytox Green (Thermo Fisher). For comparison, Jurkat cells stably expressing human PD-1 were incubated with the appropriate test antibody or antibody-attenuated hIL-2 fusion protein alone (in the absence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A), followed by incubation with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc secondary.As a control group, variable regions of anti-hPD-1 #1 and anti-hPD-1 #2 were cloned into the hIgG4 framework and evaluated with and without the addition of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. Flow cytometry was performed on BD FACS Cantor II, BD Celesta, or BD Potessa (BD Biosciences) flow cytometers, and gMFI was calculated using FlowJo software version 10. EC. 50 The value was calculated from the gMFI of the allophycocyanin signal across titration concentrations using GraphPad Prism 7 software.

[0255] Protocol C. Cell-based screen for characterizing non-antagonist anti-hPD-1 antibodies or anti-hPD-1 antibody-attenuated hIL-2 fusions

[0256] Human PD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were characterized for their ability to block hPD-1 from binding to the ligand hPD-L1 (SEQ No. 584). Anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were characterized as antagonists or non-antagonists using an in vitro cell-based human PD-1 / PD-L1 blocking bioassay (Promega, catalog number J1255). This co-culture assay used two cell lines: FCγR11b artificial antigen presenting cell / Chinese ovary hamster K1 (aAPC / CHO-K1) and Jurkat effector cells. aAPC / CHO K1 cells stably express both human PD-L1 ligand and cell surface protein to activate the cognate T cell receptor (TCR), whereas Jurkat effector cells express hPD-1 and a luciferase reporter under the control of the Nuclear Factor of Activated T cells response element (NFAT-RE). When the cells are co-cultured in the presence of a non-antagonistic antibody, the hPD-1 / hPD-L1 interaction inhibits TCR signaling, and no luminescence is detected. In the presence of an antibody that antagonizes the interaction between hPD-L1 (SEQ No. 584) and hPD-1, the inhibitory signal is destroyed, and luminescence is detected.

[0257] Thawing and use assays were performed according to the manufacturer's instructions. Briefly, aAPC / CHO-K1 cells were thawed first and plated at 30,000 cells per well in flat-bottom 96-well plates in a 37°C 5% CO2 incubator for 18 hours. After the cells attached, the medium was removed, and 200 nM or 1000 nM test antibody or antibody-attenuated hIL-2 fusion protein was diluted in 440 µl of assay buffer (RPMI 1640 medium + 1% FBS) and added to the aAPC / CHO-K1 cells. Human IgG4 isotype control monoclonal antibodies (SEQ Nos. 585 and 586) targeting Keyhole Limpet Hemocyanin (KLH) clone C3 were used as negative controls. Jurkat effector cells expressing hPD-1 were added at a volume of 40 µl at a density of 24,000 cells per well. The final concentrations of the immobilized antibodies tested were 100 nM or 500 nM. In some examples, various concentrations of anti-hPD-11 or anti-hPD-1-attenuated hIL-2 fusion proteins were tested in this co-culture assay, where the highest concentration was a 5-fold titration series of 500 nM (Fig. 7).

[0258] The co-culture samples were incubated for an additional 18–20 hours in a 37°C 5% CO2 incubator. To read the luminescence signal, the plates were allowed to reach room temperature, and 80 µl of Bio-Glo™ reagent was added to each well. The plates were incubated in a dark room at room temperature for 15 minutes, and luminescence was read using a Victor X photometer (Perkin Elmer). The average relative luminescence unit (RLU) for each triple replicate was calculated and graphed using GraphPad Prism 7 software.

[0259] Protocol D. In vitro phosphorylated STAT5 assay to test for attenuation of hIL-2 variants

[0260] The level of attenuation of hIL-2 receptor activating activity of antibody-attenuated hIL-2 fusion proteins was characterized using the phosphorylated STAT5 assay. Variants were tested in both hIL-2 reactive human natural killer NK-92 cells and engineered human erythropoietic leukemia TF1 cells. While NK-92 cell lines naturally express the high-affinity hIL-2 receptor (IL-2Rαβγ) at physiological levels, TF1 cell lines naturally expressing IL-2Rγ (SEQ No. 352) were engineered to stably express human CD122 (IL-2Rβ) (SEQ No. 353) for the expression of the intermediate-affinity hIL-2 receptor complex (IL-2Rβγ). The TF1+IL-2Rβ stable cell lines do not express IL-2Rα (SEQ No. 354). Both NK-92 and TF1+IL-2Rβ cell lines were used to evaluate the attenuation level of IL-2 efficacy in the cell-based efficacy assay using a fixed-concentration screen and a full titration curve.

[0261] To perform a fixed-concentration screen, 100,000 NK-92 cells or TF1+IL-2Rβ cells were plated in 96 wells of 50 µl of fresh growth medium free of human IL-2 cytokines and incubated overnight in a 37°C CO2 incubator. After 15–16 hours, human IL-2 depleted cells were treated with 25.7 nM recombinant hIL-2 (referred to as rhIL-2) (SEQ No. 345) or a test antibody-attenuated hIL-2 fusion protein for the NK-92 cell assay, or with 33.3 nM hIL-2 or a test hIL-2 variant for the TF1+IL-2Rβ cell assay. Cells were incubated at 37°C in 5% CO2 for 10 minutes. Cells were fixed with Cytofix Buffer (BD Biosciences) at 37°C for 10 minutes, followed by treatment with Perm Buffer III (BD Biosciences) on ice for 30 minutes, and then permeated. Fixed and permeable cells were stained with Alexa Fluor-647 conjugated anti-Stat5 antibody (BD Biosciences) at a dose of 0.5 µL per sample for 45 minutes at room temperature in the dark, and hIL-2-dependent Stat5 phosphorylation was detected. Cells were washed, and the reagents were diluted in BD Pharmingen Buffer (BD Biosciences). Stained cells were acquired on a FACS-Celesta cytometer (BD Biosciences) and analyzed using FlowJo software version 10.7.2. The assay was performed on a cohort basis but normalized using rhIL-2 from each plate. The degree of attenuation of the selected antibody-attenuated hIL-2 fusion protein was evaluated on 8-point, 6-fold continuous titration curves ranging from 1200 nM to 7 pM in both NK-92 and TF1+IL-2Rβ cell lines. The procedure for the pStat5 curves was performed in the same manner as described above. EC50 Values ​​were calculated from geometric mean fluorescence intensity (gMFI) over titration concentrations using GraphPad Prism 7 software. Changes in active fold from rhIL-2 were the EC for the variant. 50 The value of hIL-2's EC 50 It was calculated by dividing by.

[0262] Protocol E. In vitro cell-based proliferation assay for testing the attenuation of antibody-attenuated hIL-2 fusion protein

[0263] Antibody-attenuated hIL-2 fusion proteins were also tested for attenuated hIL-2 activity in the hIL-2-dependent cell proliferation assay. 10,000 NK-92 cells (expressing high-affinity receptor hIL-2Rαβγ) or TF1+IL-2Rβ cells (expressing intermediate-affinity receptor hIL-2Rβγ) suspended in 50 µl of fresh growth medium free of hIL-2 cytokines were plated in each well of a 96-well U-bottom cell culture plate. An 8-point, 6-fold serial titration of antibody-attenuated hIL-2 fusion protein in fresh medium was diluted to a peak concentration of 996 nM and overlaid on the cells in the wells. Cells were incubated in a 37°C, 5% CO2 incubator for 3 days for TF1+IL-2Rβ cells or 4 days for NK-92 cells. To measure proliferation, Cell-Titer-Glo (Promega) was added to the wells, incubated at room temperature for 10 minutes, and luminescence was read for 0.1 seconds per well using a VictorX Multilabel Plate Reader (Perkin Elmer). EC 50 The values ​​were calculated from Relative Luminous Units (RLU) across titration concentrations using GraphPad Prism 7 software. The change in active factor from rhIL-2 is the EC for the variant. 50 The value of hIL-2's EC 50It was calculated by dividing by. Although the test method was performed on a cohort, the rhIL-2 EC of each plate 50 Normalized using the value.

[0264] Example 1: Optimization of antibody-attenuated hIL-2 fusion protein variants and determination of hIL-2 activity in their intermediate and high-affinity hIL-2 receptor complexes

[0265] To determine the optimal structure of the antibody-attenuated hIL-2 fusion protein, unattenuated hIL-2 was fused to an anti-DNase I antibody (clone 1H3) designated as 1H3-hIgG1 (SEQ No. 379, SEQ No. 374) in various ways to the antibody variable region as shown in FIG. 1. The variant included hIL-2 fused to the N-terminus of the human anti-DNase I antibody (clone 1H3) immunoglobulin hIgG1 heavy chain or human kappa light chain through a df represented by hIL-2 Nterm light chain direct fusion (df) (SEQ No. 379, SEQ No. 356) and hIL-2 Nterm heavy chain df (SEQ No. 358, SEQ No. 374), or through a 6-amino acid linker (L6) (SEQ No. 355) represented by hIL-2 Nterm light chain L6 fusion (SEQ No. 379, SEQ No. 357) and hIL-2 Nterm heavy chain L6 fusion (SEQ No. 359, SEQ No. 374). Variants in which the hIL-2 moiety is fused to the C-terminus of both the heavy and light chains via df or L6 were also generated and represented as hIL-2 Cterm heavy chain df (SEQ No. 360, SEQ No. 374), hIL-2 Cterm heavy chain L6 fusion (SEQ No. 361, SEQ No. 374), hIL-2 Cterm light chain df (SEQ No. 379, SEQ No. 362), and hIL-2 Cterm light chain L6 fusion (SEQ No. 379, SEQ No. 363). Additional variants were generated in which the CD25 / IL-2Rα extracellular domain (amino acid 1-164) (SEQ No. 126) was fused to the N-terminus or C-terminus of the heavy chain or kappa light chain to interfere with the binding of IL-2 to CD25 of the IL-2 receptor (Fig. 2).In the above composition, the human CD25 extracellular domain (amino acids 1-164) (SEQ No. 126) was fused to human IL-2 via a 20-amino acid linker (L20) (SEQ No. 364), and then subsequently fused directly to the 1H3-hIgG1 heavy chain or light chain at the N-terminus, or fused via an L6 linker (SEQ No. 355): hCD25-L20-hIL-2 N-term heavy chain df (SEQ No. 365, SEQ No. 374), hCD25-L20-hIL-2 N-term heavy chain L6 fusion (SEQ No. 366, SEQ No. 374), hCD25-L20-hIL-2 N-term light chain df (SEQ No. 379, SEQ No. 367), hCD25-L20-hIL-2 N-term light chain L6 fusion (SEQ No. 379, SEQ ID NO. 368). Finally, a set of final variants was produced in which the CD25 / IL-2Rα extracellular domain moiety (SEQ ID NO. 126) was fused to the C-terminus of the heavy chain and kappa light chain: hCD25-L20-hIL-2 Cterm heavy chain df (SEQ ID NO. 369, SEQ ID NO. 374), hCD25-L20-hIL-2 Cterm heavy chain L6 fusion (SEQ ID NO. 370, SEQ ID NO. 374), hCD25-L20-hIL-2 Cterm light chain df (SEQ ID NO. 379, SEQ ID NO. 371), and hCD25-L20-hIL-2 Cterm light chain L6 fusion (SEQ ID NO. 379, SEQ ID NO. 372). The antibody-hIL-2 fusion proteins were produced, expressed, and purified using standard techniques. The 16 N- or C-terminal and linker variants described above were evaluated in an in vitro cell-based phosphorylated STAT5 assay using an 8-point, 6-fold serial titration as described in Protocol D.

[0266] Table 1 shows the EC calculated across an 8-point, 6-fold continuous titration curve using geometric mean fluorescence intensity (gMFI) calculated with FlowJo version 10 software. 50This is a summary. As a measure of the level of attenuation relative to the activity of the rhIL-2 positive control, the fold change from rhIL-2 for each variant was also calculated. Some EC 50 The value could not be calculated with GraphPad Prism 7 software and was marked as Not Calculated (NC); however, there was no attenuation for the variant based on the dose titration curve.

[0267] In cell lines expressing high-affinity hIL-2 receptors (NK-92) or medium-affinity hIL-2 receptors (TF1+IL-2Rβ), IL-2 activity was not reduced upon fusion of the hIL-2 moiety to the N-terminus or C-terminus of the immunoglobulin heavy chain compared to rhIL-2. When compared to fusion between IL-2 and antibody components using a 6-amino acid linker (L6), there was no change in IL-2 activity upon direct fusion (df) of hIL-2 to the antibody component of the fusion protein. Similarly, when compared to rhIL-2, there was no change in IL-2 activity upon fusion of the IL-2 component to the heavy or light chain of the antibody component. For all N- or C-terminus and linker fusion protein variants in which the hCD25 / hIL-2Rα moiety was fused to hIL-2, it was predicted that the binding of the fusion protein to CD25 of the hIL-2 receptor in cells would be reduced. Experimentally, the above composition showed strongly attenuated hIL-2 activity (at least 45-fold) at high-affinity IL-2 receptors (NK-92) and attenuated activity 18-fold at intermediate hIL-2 receptors (TF1+IL-2Rβ).

[0268]

[0269]

[0270] Example 2: Production of antibody-attenuated hIL-2 fusion protein variants and determination of their binding kinetic properties to recombinant human CD25 and / or human CD122

[0271] Since hIL-2 activity was not reduced in various N-terminal or C-terminal immunoglobulin heavy chain fusion proteins, the hIL-2 Cterm heavy chain L6 fusion designated as "1H3-hIgG1-L6-hIL-2" (SEQ ID NOs 361, 374) was used as the base construct for antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety. Single, double, and / or multiple amino acid substitutions were introduced into selected residues of human IL-2 to investigate the role of said residues in the recognition of human CD25 / IL-2Rα and / or human CD122 / IL-2Rβ or CD132 / IL-2Rγ (human IL-2R subunits). Over 300 antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety were generated and evaluated in 6 rounds. The above variants were first screened in IL-2-dependent cell lines (NK-92 and TF1+IL-2Rβ) using a fixed-concentration flow-based phosphorylated STAT5 (pSTAT5) assay as well as dose-titration curves. Phosphorylated STAT5 is a downstream signal of IL-2 activity and was used as a snapshot measure of IL-2 efficacy. An IL-2-dependent cell proliferation assay was also performed to measure IL-2 activity for 3-4 days. Selection criteria for attenuated hIL-2 included (1) reduced IL-2 efficacy in both NK-92 and TF1+IL-2Rβ cell lines, and agonist activity greater than 50% for both cell lines; and (2) medium to high production yield.

[0272] A human anti-DNase I antibody-hIL-2 fusion protein was produced by fusing IL-2 or human IL-2 variants (SEQ Nos. 1-344, 377, 378, and 575) to the C-terminus of the heavy chain of a human anti-DNase I antibody (clone 1H3, having a human IgG1 isotype) via an L6 linker and combining it with the hIgG1 light chain (1H3-hKappa LC; SEQ No. 374) to produce a 1H3-hIgG1-L6-hIL-2 fusion protein (provided in Table 28). A mouse anti-yellow fever virus antibody-hIL-2 fusion protein was also produced by fusing a human IL-2 variant via an L6 linker to the C-terminus of the heavy chain of a mouse anti-yellow fever virus antibody (clone 2D12, possessing a mouse IgG1 isotype) containing a D265A substitution to reduce immune effector function, and combining this with the 2D12-mIgG1 light chain (2D12-mKappa LC; SEQ ID NO. 376) to produce the 2D12-mIgG1-D265A-L6-hIL-2 fusion protein (provided in Table 28). A portion of the mouse anti-yellow fever virus antibody-hIL-2 fusion protein was formatted into a human IgG1 constant region, produced using the same method as described above, and combined with the 2D12-hKappa light chain (2D12-hKappa LC; SEQ ID NO. 573). IL-2 amino acid substitution repetitions were performed in 6 rounds and designated as groups 1 to 6. Using standard techniques, 1H3-hIgG1-L6-hIL-2, 2D12-mIgG1-D265A-L6-hIL-2, and 2D12-hIgG1-L6-hIL-2 fusion proteins were produced, expressed, and Protein-A purified.

[0273] Group 1 included an initial series consisting only of 2D12-mIgG1-D265A-L6-hIL-2 or 2D12-hIgG1-L6-hIL-2 fusion proteins containing substitutions or combinations of substitutions among human IL-2 predicted to be involved in binding only to one of the IL-2 receptor subunits CD25 / IL-2Rα, CD122 / IL-2Rβ, or CD132 / IL-2Rγ. The fusion proteins of the above group included the following substitutions for IL-2 predicted to regulate binding to CD25 / IL-2Rα: F42K (SEQ No. 1), V69A (SEQ No. 2), V69E (SEQ No. 3), V69F (SEQ No. 4), V69G (SEQ No. 5), V69H (SEQ No. 6), V69I (SEQ No. 7), V69K (SEQ No. 8), V69L (SEQ No. 9), V69M (SEQ No. 10), V69Q (SEQ No. 11), V69S (SEQ No. 12), V69T (SEQ No. 13), V69W (SEQ No. 14), V69Y (SEQ No. 15), V69R (SEQ No. 581), (F42K / F44K)(SEQ No. 16), (F44K / Y45R)(Sequence No. 17), (F42K / V69R)(Sequence No. 18), (Y45R / V69R)(Sequence No. 19), (F42K / F44K / Y45R)(Sequence No. 20), (F42A / Y45A / L72G)(Sequence No. 574), (R38A / F42K / Y45R)(Sequence No. 21), (R38E / F42K / Y45R)(Sequence No. 22), (K43E / F42K / Y45R)(Sequence No. 23), (K43T / F42K / Y45R)(Sequence No. 24), (F42K / Y45R / E62A)(Sequence No. 25), (P65R / F42K / Y45R)(Sequence No 26), (P65S / F42K / Y45R)(Sequence No. 27), (V69A / F42K / Y45R)(Sequence No. 28), (V69D / F42K / Y45R)(Sequence No. 29), or (V69R / F42K / Y45R)(Sequence No. 30).The substitutions within the above group included the following substitutions predicted to coordinate binding to CD122 / IL-2Rβ: D20A (SEQ No. 31), D20N (SEQ No. 32), D20K (SEQ No. 33), N88A (SEQ No. 34), N88G (SEQ No. 35), N88H (SEQ No. 36), N88K (SEQ No. 37), (D20A / D84A)(SEQ No. 38), (D20A / E15A)(SEQ No. 39), (D20A / E95A)(SEQ No. 40), (D20A / N88A)(SEQ No. 41), (D20A / S87A)(SEQ No. 42), (D84A / N88A)(SEQ No. 43), (E15A / N88A)(SEQ No. 44), or (S87A / N88A) (SEQ No. 45). Group 1 also included the following substitutions for IL-2 predicted to modulate IL-2 binding to CD132 / IL-2-Rγ: Q126L (SEQ No. 377) or Q126E (SEQ No. 378). The IL-2 substitutions studied in Group 1 were not predicted to modulate binding to more than one of the IL-2 receptor subunits.

[0274] Group 2 included a series of 1H3-hIgG1-L6-hIL-2 fusion proteins containing one or more substitutions of human IL-2 predicted to be involved only in CD25 / IL-2Rα binding. The fusion proteins of the above group included the following substitutions for IL-2 predicted to regulate binding to CD25 / IL-2Rα: R38A (SEQ No. 46), R38D (SEQ No. 47), R38E (SEQ No. 48), R38Q (SEQ No. 49), F42R (SEQ No. 50), F42A (SEQ No. 51), F42D (SEQ No. 52), F42H (SEQ No. 53), K43A (SEQ No. 54), K43E (SEQ No. 55), K43Q (SEQ No. 56), Y45A (SEQ No. 57), Y45K (SEQ No. 58), Y45S (SEQ No. 59), Y45R (SEQ No. 60), E61A (SEQ No. 61), E61R (SEQ No. 62), E61K(SEQN 63), E62A(SEQN 64), E62R(SEQN 65), E62K(SEQN 66), E62Y(SEQN 67), E68Y(SEQN 68), E68A(SEQN 69), E68K(SEQN 70), E68R(SEQN 71), E68L(SEQN 72), L72Y(SEQN 73), L72R(SEQN 74), L72A(SEQN 75), L72D(SEQN 76), L72H(SEQN 77), L72F(SEQN 78), (R38D / E61R)(SEQN 79), (R38D / E61R / K43E)(SEQN 80), or (T3A / F42A / Y45A / L72G / C125A)(SEQ No. 81). Substitution T3A was introduced into the IL-2 amino acid sequence to remove a predicted O-linked glycosylation site on human IL-2 (see e.g., International Publication No. WO2012 / 107417), and substitution C125A was introduced into the IL-2 amino acid sequence to remove an unpaired cysteine ​​residue (see e.g., International Publication No. WO2018 / 184964).IL-2 substitutions studied in group 2 were predicted not to modulate IL-2 binding to CD132 / IL-2-Rγ, and the following substitutions were not predicted to modulate binding to more than 1 of the IL-2 receptor subunits.

[0275] Group 3 included a series of 1H3-hIgG1-L6-hIL-2 fusion proteins containing one or more substitutions of human IL-2 predicted to be involved only in CD122 / IL-2Rβ binding. The fusion proteins of the above group included the following substitutions for IL-2 predicted to regulate binding to CD122 / IL-2Rβ: E15A (SEQ No. 82), E15R (SEQ No. 83), E15K (SEQ No. 84), H16A (SEQ No. 85), H16Y (SEQ No. 86), H16E (SEQ No. 87), L19A (SEQ No. 88), D20I (SEQ No. 89), D20S (SEQ No. 90), D20H (SEQ No. 91), D20T (SEQ No. 92), D20W (SEQ No. 93), D20Y (SEQ No. 94), D20R (SEQ No. 95), D20F (SEQ No. 96), R81A (SEQ No. 97), D84A (SEQ No. 98), D84R (Sequence No. 99), D84K (Sequence No. 100), S87A (Sequence No. 101), N88Y (Sequence No. 102), N88D (Sequence No. 103), N88R (Sequence No. 104), N88E (Sequence No. 105), N88F (Sequence No. 106), N88I (Sequence No. 107), I92A (Sequence No. 108), I92Y (Sequence No. 109), I92S (Sequence No. 110), I92F (Sequence No. 111), I92R (Sequence No. 112), I92D (Sequence No. 113), I92E (Sequence No. 114), E95A (Sequence No. 115), E95R (Sequence No. 116), E95K (Sequence No. 117), (D20Y / H16E)(SEQ No. 118), (D20Y / H16A)(SEQ No. 119), (D20Y / H16Y)(SEQ No. 120), (D20Y / I92A)(SEQ No. 121), (D20Y / I92S)(SEQ No. 122), (D20Y / I92R)(SEQ No. 123), (D20Y / E95R)(SEQ No. 124), or (D20Y / E95A)(SEQ No. 125).

[0276] Group 4 included a series of fusion proteins containing a CD25 / IL-2Rα extracellular domain moiety (SEQ No. 126) containing one or more substitutions for residues predicted to be involved in binding to CD122 / IL-2Rβ, a 20-amino acid linker (L20) (SEQ No. 364), and 1H3-hIgG1-L6-hIL-2 HC fused to a human IL-2 variant. The fusion proteins of the above group included the following substitutions for IL-2, which are predicted to regulate binding to CD122 / IL-2Rβ: E15A (SEQ No. 82), D20I (SEQ No. 89), D20S (SEQ No. 90), D20H (SEQ No. 91), D20W (SEQ No. 93), D20Y (SEQ No. 94), D20R (SEQ No. 95), D20F (SEQ No. 96), D84K (SEQ No. 100), S87A (SEQ No. 101), N88Y (SEQ No. 102), N88D (SEQ No. 103), N88R (SEQ No. 104), N88E (SEQ No. 105), N88F (SEQ No. 106), N88I (SEQ No. 107), I92A (SEQ No. 108), E95A (sequence number 115), or E95K (sequence number 117). The antibody-attenuated hIL-2 fusion protein of the above group is represented as 1H3-hIgG1-L6-hCD25(1-164)-L20-hIL-2.

[0277] Group 5 included the 1H3-hIgG1-L6-hIL-2 series, which contained IL-2 substitution combinations predicted to be involved in the binding of IL-2 to CD25 / IL-2Rα and CD122 / IL-2Rβ or CD132 / IL-2Rγ. Additionally, some variants had the first three amino acids of the N-terminus of the hIL-2 moiety deleted (Δ1-3APT). The fusion proteins in Group 5 included the following substitutions for IL-2 predicted to regulate IL-2 binding to CD25 / IL-2Rα and CD122 / IL-2Rβ: (F42D / D20A)(SEQ No. 127), (F42R / D20A)(SEQ No. 128), (F42K / D20A)(SEQ No. 129), (F42A / D20A)(SEQ No. 130), (F42H / D20A)(SEQ No. 131), (Y45R / D20A)(SEQ No. 132), (Y45K / D20A)(SEQ No. 133), (R38N / D20A)(SEQ No. 134), (R38G / D20A)(SEQ No. 135), (R38H / D20A)(SEQ No. 136), (R38I / D20A)(Sequence No. 137), (R38L / D20A)(Sequence No. 138), (R38M / D20A)(Sequence No. 139), (R38F / D20A)(Sequence No. 140), (R38P / D20A)(Sequence No. 141), (R38S / D20A)(Sequence No. 142), (R38T / D20A)(Sequence No. 143), (R38W / D20A)(Sequence No. 144), (R38Y / D20A)(Sequence No. 145), (R38V / D20A)(Sequence No. 146), (R38A / D20A)(Sequence No. 147), (R38Q / D20A)(Sequence No. 148), (D20A / R38E)(Sequence No. 149), (R38D / D20A)(Sequence No. 150), (K43E / D20A)(Sequence No. 151), (E61A / D20A)(Sequence No. 152), (E62A / D20A)(Sequence No. 153), (E62Y / D20A)(Sequence No. 154), (L72D / D20A)(Sequence No. 155), (L72H / D20A)(Sequence No. 156), (L72R / D20A)(Sequence No. 157),(F42D / I92D)(SEQN 158), (F42R / I92D)(SEQN 159), (F42H / I92D)(SEQN 160), (F42A / I92D)(SEQN 161), (H16A / F42A)(SEQN 575), (K43E / I92D)(SEQN 162), (Y45R / I92D)(SEQN 163), (Y45K / I92D)(SEQN 164), (E62A / I92D)(SEQN 165), (E62Y / I92D)(SEQN 166), (L72D / I92D)(SEQN 167), (L72H / I92D)(SEQN 168), (L72R / I92D)(SEQ No. 169), (R38D / I92D)(SEQ No. 170), (R38E / I92D)(SEQ No. 171), (R38Q / I92D)(SEQ No. 172), (R38A / I92D)(SEQ No. 173), (R38E / N88R)(SEQ No. 174), (R38E / D84R)(SEQ No. 175), (R38E / D84K)(SEQ No. 176), (F42A / Y45R / D20A)(SEQ No. 177), (F42H / Y45R / D20A)(SEQ No. 178), (R38D / E61R / D20A)(SEQ No. 179), (R38E / E61R / D20A)(Sequence No. 180), (R38Q / E61R / D20A)(Sequence No. 181), (R38A / E61R / D20A)(Sequence No. 182), (R38A / D20A / E95A)(Sequence No. 183), (D20A / E95A / R38D)(Sequence No. 184), (D20A / E95A / R38E)(Sequence No. 185), (D20A / E95A / R38Q)(Sequence No. 186), (D20A / E95A / F42R)(Sequence No. 187), (D20A / E95A / F42A)(Sequence No. 188), (D20A / E95A / F42D)(Sequence No 189), (D20A / E95A / F42H)(Sequence No. 190), (D20A / E95A / F42K)(Sequence No. 191), (D20A / E95A / K43A)(Sequence No. 192), (D20A / E95A / K43E)(Sequence No. 193), (D20A / E95A / K43Q)(Sequence No. 194), (D20A / E95A / Y45A)(Sequence No. 195),(D20A / E95A / Y45K)(Sequence No. 196), (D20A / E95A / Y45S)(Sequence No. 197), (D20A / E95A / Y45R)(Sequence No. 198), (D20A / E95A / E61A)(Sequence No. 199), (D20A / E95A / E62A)(Sequence No. 200), (D20A / E95A / E62R)(Sequence No. 201), (D20A / E95A / E62K)(Sequence No. 202), (D20A / E95A / E62Y)(Sequence No. 203), (D20A / E95A / E68Y)(Sequence No. 204), (D20A / E95A / E68A)(Sequence No 205), (D20A / E95A / E68L)(Sequence No. 206), (D20A / E95A / L72Y)(Sequence No. 207), (D20A / E95A / L72R)(Sequence No. 208), (D20A / E95A / L72A)(Sequence No. 209), (D20A / E95A / L72D)(Sequence No. 210), (D20A / E95A / L72H)(Sequence No. 211), (D20A / E95A / L72F)(Sequence No. 212), (F42K / Y45R / D20A / S87A)(Sequence No. 213), (F42K / Y45R / D20A / E95A)(Sequence No. 214), (D20A / R38E / C125A)(SEQ No. 215), (T3A / D20A / R38E)(SEQ No. 216), (T3A / D20A / R38E / C125A)(SEQ No. 217), (Δ1-3APT / D20A / R38E)(SEQ No. 218), or (Δ1-3APT / D20A / R38E / C125A)(SEQ No. 219). The fusion proteins in Group 5 included the following substitutions for IL-2 predicted to regulate IL-2 binding to CD25 / IL-2Rα and CD132 / IL-2R: (R38E / Q22A)(SEQ No. 220), (R38E / T123A)(SEQ No. 221), (R38E / I129A)(SEQ No. 222), (R38E / S130A)(SEQ No. 223), (R38E / Q126A)(SEQ No. 224), (R38E / Q126D)(SEQ No. 225), (R38E / Q126V)(SEQ No. 226), (R38E / Q22A / S130A)(SEQ No. 227),(F42K / Y45R / Q126D)(SEQ No. 228), or (D20A / E95A / Q126D)(SEQ No. 229). Mutations to the hIL-2 sequence of group 5 antibody-attenuated hIL-2 fusion proteins numbered according to the IL-2 sequence are listed in SEQ Nos. 127-229 and 575.

[0278] Group 6 included a series of 1H3-hIgG1-L6-hIL-2 fusion proteins containing human IL-2 substitution combinations predicted to be involved in the binding of IL-2 to CD25 / IL-2Rα and CD122 / IL-2Rβ, but not to CD132 / IL-2Rγ. The fusion proteins in Group 6 included the following combinations of substitutions in IL-2 predicted to regulate IL-2 binding to CD25 / IL-2Rα and CD122 / IL-2Rβ: (D20A / E61R)(SEQ No. 230), (D20A / E61N)(SEQ No. 231), (D20A / E61D)(SEQ No. 232), (D20A / E61Q)(SEQ No. 233), (D20A / E61G)(SEQ No. 234), (D20A / E61H)(SEQ No. 235), (D20A / E61I)(SEQ No. 236), (D20A / E61L)(SEQ No. 237), (D20A / E61K)(SEQ No. 238), (D20A / E61M)(SEQ No. 239), (D20A / E61F)(Sequence No. 240), (D20A / E61P)(Sequence No. 241), (D20A / E61S)(Sequence No. 242), (D20A / E61T)(Sequence No. 243), (D20A / E61W)(Sequence No. 244), (D20A / E61Y)(Sequence No. 245), (D20A / E61V)(Sequence No. 246), (D20A / F42N)(Sequence No. 247), (D20A / F42Q)(Sequence No. 248), (D20A / F42E)(Sequence No. 249), (D20A / F42G)(Sequence No. 250), (D20A / F42I)(Sequence No. 251), (D20A / F42L)(Sequence No. 252), (D20A / F42M)(Sequence No. 253), (D20A / F42P)(Sequence No. 254), (D20A / F42S)(Sequence No. 255), (D20A / F42T)(Sequence No. 256), (D20A / F42W)(Sequence No. 257), (D20A / F42Y)(Sequence No. 258), (D20A / F42V)(Sequence No. 259), (D20A / Y45A)(Sequence No. 260), (D20A / Y45N)(Sequence No. 261), (D20A / Y45D)(Sequence No. 262),(D20A / Y45Q)(SEQ No. 263), (D20A / Y45E)(SEQ No. 264), (D20A / Y45G)(SEQ No. 265), (D20A / Y45H)(SEQ No. 266), (D20A / Y45I)(SEQ No. 267), (D20A / Y45L)(SEQ No. 268), (D20A / Y45M)(SEQ No. 269), (D20A / Y45F)(SEQ No. 270), (D20A / Y45P)(SEQ No. 271), (D20A / Y45S)(SEQ No. 272), (D20A / Y45T)(SEQ No. 273), (D20A / Y45W)(SEQ No. 274), (D20A / Y45V)(Sequence No. 275), (I92D / F42N)(Sequence No. 276), (I92D / F42Q)(Sequence No. 277), (I92D / F42E)(Sequence No. 278), (I92D / F42G)(Sequence No. 279), (I92D / F42I)(Sequence No. 280), (I92D / F42L)(Sequence No. 281), (I92D / F42K)(Sequence No. 282), (I92D / F42M)(Sequence No. 283), (I92D / F42P)(Sequence No. 284), (I92D / F42S)(Sequence No. 285), (I92D / F42T)(Sequence No. 286), (I92D / F42W)(Sequence No. 287), (I92D / F42Y)(Sequence No. 288), (I92D / F42V)(Sequence No. 289), (I92D / Y45A)(Sequence No. 290), (I92D / Y45N)(Sequence No. 291), (I92D / Y45D)(Sequence No. 292), (I92D / Y45Q)(Sequence No. 293), (I92D / Y45E)(Sequence No. 294), (I92D / Y45G)(Sequence No. 295), (I92D / Y45H)(Sequence No. 296), (I92D / Y45I)(Sequence No. 297), (I92D / Y45L)(Sequence No. 298), (I92D / Y45M)(Sequence No. 299), (I92D / Y45F)(Sequence No. 300), (I92D / Y45P)(Sequence No. 301), (I92D / Y45S)(Sequence No. 302), (I92D / Y45T)(Sequence No. 303), (I92D / Y45W)(Sequence No. 304), (I92D / Y45V)(Sequence No. 305),(R38E / D20H)(Sequence No. 306), (R38E / D20S)(Sequence No. 307), (F42A / N88R)(Sequence No. 308), (F42A / N88D)(Sequence No. 309), (R38E / D84A)(Sequence No. 310), (R38E / D84N)(Sequence No. 311), (R38E / D84Q)(Sequence No. 312), (R38E / D84E)(Sequence No. 313), (R38E / D84G)(Sequence No. 314), (R38E / D84H)(Sequence No. 315), (R38E / D84I)(Sequence No. 316), (R38E / D84L)(Sequence No. 317), (R38E / D84M)(Sequence No. 318), (R38E / D84F)(Sequence No. 319), (R38E / D84P)(Sequence No. 320), (R38E / D84S)(Sequence No. 321), (R38E / D84T)(Sequence No. 322), (R38E / D84W)(Sequence No. 323), (R38E / D84Y)(Sequence No. 324), (R38E / D84V)(Sequence No. 325), (R38E / I92A)(Sequence No. 326), (R38E / I92R)(Sequence No. 327), (R38E / I92N)(Sequence No. 328), (R38E / I92Q)(Sequence No. 329), (R38E / I92E)(Sequence No. 330), (R38E / I92G)(Sequence No. 331), (R38E / I92H)(Sequence No. 332), (R38E / I92L)(Sequence No. 333), (R38E / I92K)(Sequence No. 334), (R38E / I92M)(Sequence No. 335), (R38E / I92F)(Sequence No. 336), (R38E / I92P)(Sequence No. 337), (R38E / I92S)(Sequence No. 338), (R38E / I92T)(Sequence No. 339), (R38E / I92W)(Sequence No. 340), (R38E / I92Y)(Sequence No. 341), (R38E / I92V)(SEQ No. 342), (R38E / H16E)(SEQ No. 343), or (R38K / D20A)(SEQ No. 344). Mutations to the hIL-2 sequence of group 6 antibody-attenuated hIL-2 fusion proteins numbered according to the IL-2 sequence are listed in SEQ Nos. 230–344.

[0279] The binding kinetics of some purified 1H3-hIgG1-L6-hIL-2 variant proteins to individual recombinant human CD25 and human CD122 were determined using bio-layer interferometry (BLI). Briefly, binding experiments were performed at 25°C using an Octet Red96 instrument (Pall ForteBio). The extracellular domains of human CD25 and human CD122 tagged with C-terminal poly-histidine were captured by an anti-His2 sensor (Pall ForteBio). The receptor-loaded sensor was dipped into seven consecutive 3-fold dilutions of each 1H3-hIgG-L6-hIL-2 variant, starting at a peak concentration of 300 nM. The 1H3-hIgG1-L6-hIL-2 fusion protein was diluted in assay buffer consisting of phosphate-buffered saline (PBMS) supplemented with 0.1% BSA and 0.02% Tween-20 (pH 7.2). The loaded sensor was regenerated using 10 mM glycine buffer (pH 1.7). The kinetic constants were calculated using a monovalent binding model.

[0280] Table 2 shows the association constants (k) of 74 immunoglobulin-hIL-2 fusion protein variants bound to recombinant human CD25 or recombinant human CD122. on ), dissociation constant(k off ), and equilibrium constant (K D It recorded ).

[0281]

[0282]

[0283]

[0284]

[0285] Table 3 shows the assembly of 74 1H3-hIgG1-L6-hIL-2 fusion proteins bound to recombinant human CD122 (kon ) constant, dissociation(k off ) constant, and equilibrium constant (K D It recorded ).

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293] Example 3: Attenuation test for high-affinity and medium-affinity hIL-2 receptors using a fixed-concentration cell-based efficacy pSTAT5 screen

[0294] As described in Protocol D, the attenuation of the antibody-attenuated hIL-2 fusion protein described in Example 2 was tested on a fixed-concentration pSTAT5 screen using NK-92 (high-affinity hIL-2 receptor expression) and TF1+IL-2Rβ (medium-affinity hIL-2 receptor expression) cell lines. Tables 4-8 list the multiples of change in the geometric mean fluorescence intensity (gMFI) of the antibody-attenuated hIL-2 fusion protein from wild-type rhIL-2, a free cytokine measuring the reduction in IL-2 activity. For the fixed-concentration screen, the multiple of change was calculated by dividing the gMFI of rhIL-2 by the gMFI of the variant. For experiments using the full titration curve, the multiple of change from rhIL-2 was the EC₀ for rhIL-2. 50 The value of the variant's EC 50Calculated by dividing by. The change multiple was rounded to the nearest integer. The reduced gMFI in both NK-92 and TF1+IL-2Rβ cell lines compared to the gMFI derived from rhIL-2 indicates that IL-2 activity was attenuated in both high-affinity and medium-affinity receptors. The Group 1 variants described in Example 2 were not tested in the fixed-concentration cell-based efficacy pSTAT5 screen.

[0295] Each tested variant was also evaluated for IL-2 agonist activity and characterized as a full or partial IL-2 agonist, or as having no IL-2 activity (inactive). Dose titration curves of 1H3-hIgG1-L6-hIL-2 fusion proteins reaching the maximum gMFI level observed in rhIL-2 positive controls were considered as antibody-attenuated hIL-2 fusion proteins with full agonist activity. Partial agonist activity was calculated as the percentage (%) of full activity using the rhIL-2 maximum gMFI as 100%. Antibody-attenuated hIL-2 fusion proteins with less than 10% of the rhIL-2 maximum gMFI at a peak concentration of 1200 nM were considered to have no agonist activity (inactive). Some EC 50 The values ​​and attenuation levels could not be accurately calculated using GraphPad Prism 7 software because the activity did not reach its maximum value, so the above values ​​are estimates.

[0296] While some single residue substitutions attenuated IL-2 activity in high-affinity cell lines (NK-92), it was demonstrated through pSTAT5 fixed concentration results that substitution combinations that coordinated binding to both the alpha and beta chains, or both the alpha and gamma chains, were required to significantly attenuate IL-2 activity in high-affinity IL-2 receptors (attenuated by more than 20-fold from recombinant hIL-2).

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] Example 4: Attenuation test of IL-2 fusion proteins for each high-affinity and medium-affinity hIL-2 receptor using a cell-based efficacy pSTAT5 dose titration screen

[0310] As described in Protocol D, the attenuation of the selected antibody-attenuated hIL-2 fusion protein (1H3-hIgG1-L6-hIL-2 fusion protein from groups 2-6) described in Example 2 was tested on the pSTAT5 titration curve using NK-92 and TF1+IL-2Rβ cell lines.

[0311] After generating a 4-parameter logistic curve using the gMFI of the Alexa Fluor 647 pSTAT5-positive signal, subsequently, EC using GraphPad Prism 7 software 50 The values ​​were calculated. As attenuation measures, the above values ​​were compared with a recombinant hIL-2 (rhIL-2) control. Table 9-13 summarizes the multiples of activity change from rhIL-2 calculated using the gMFI of the Alexa Fluorine 647 signal.

[0312] An increase in the change factor from rhIL-2 indicated the degree of attenuation of hIL-2 activity. Each antibody-attenuated hIL-2 fusion protein tested in the pSTAT5 titration curve was also evaluated for agonist activity and characterized as fully active, partially active, or inactive (inactive). Dose titration curves of antibody-attenuated hIL-2 fusion proteins that reached the maximum gMFI level as rhIL-2 were considered variants with full agonist activity. Partial agonist activity was calculated as described in Example 3. Inactive antibody-attenuated hIL-2 fusion proteins were classified as having less than 10% activity compared to rhIL-2. Since full 4 pharmaliter logistic curves were not generated, some change factors from rhIL-2 could not be accurately calculated using GraphPad Prism 7 software (indicated as not calculated or "NC"), and therefore, the above values ​​are estimates (in Table 9-13 a (commented out as ). However, the above variant showed a attenuation of more than 10,000 times from rhIL-2 in the graph (data not presented). This is indicated in Table 9-13 as ">10,000 in graph; NC".

[0313] Results similar to those presented in Example 3 were demonstrated through the whole-titration pSTAT5 curves, showing that substitutions coordinating binding to both the alpha and beta chains significantly attenuated IL-2 activity in high-affinity IL-2 receptors compared to single substitutions binding only to the alpha or beta chains. The whole-titration pSTAT5 assay was also able to distinguish variants with inactive substitutions from highly attenuated variants. Finally, a comparison of dose-titration curves showed the level of attenuation more accurately compared to the fixed-concentration assay.

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] Example 5: Attenuation test for high-affinity and medium-affinity hIL-2 receptors using a cell-based proliferation assay

[0324] As described in Protocol E, the IL-2 activity attenuation of antibody-attenuated hIL-2 fusion proteins (2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, and 1H3-hIgG1-L6-hIL-2 fusion proteins) from groups 1-6 of Example 2 was tested in a proliferation assay in both NK-92 and TF1+IL-2Rβ cell lines. The results of the assay are provided in Tables 14-19.

[0325] The selected 1H3-hIgG1-L6-hIL-2 fusion protein, which showed significant attenuation in the pSTAT5 titration curve from Example 4, was tested in the cell-based proliferation assay. pSTAT5 is a downstream readout of IL-2 activity, and only a 10-minute stimulation is required for the assay, which may be a small snapshot of IL-2-dependent activity. For the proliferation assay, cells were incubated for 3-4 days with 2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, 1H3-hIgG1-L6-hIL-2 fusion protein, or a recombinant hIL-2 control to provide a more physiologically relevant readout of IL-2-dependent activity in vivo. Other 2D12-mIgG1-D265A-L6-hIL-2 and 2D12-hIgG1-L6-hIL-2 fusion proteins that were generated but not tested in the pSTAT5 assay were tested for IL-2-dependent activity using this proliferation assay.

[0326] Similar to cell-based pSTAT5 dose titration experiments, EC from relative luminescence units (RLU) instead of gMFI 50 Determine the calculated value, and EC 50 Once this was calculated, the results were analyzed in the same manner as in Example 4. Similar to the results confirmed in Example 4, it was demonstrated through the proliferation curve that some substitutions coordinating binding to both the alpha and beta chains significantly attenuated IL-2 activity at high-affinity receptors compared to single substitutions binding only to the alpha or beta chains. The selected 1H3-hIgG1-L6-hIL-2 fusion protein was also tested for proliferation in TF1+IL-2Rβ cell lines, which demonstrated that some of the same substitutions significantly attenuated IL-2 activity at medium-affinity receptors.

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] Example 6: Generation of anti-hPD-1 antibody

[0341] Various different anti-hPD-1 antibodies with desired characteristics were generated using various approaches.

[0342] In one approach, an anti-hPD-1 human monoclonal antibody was generated using transgenic chickens (OmniChicken™) expressing human antibody genes (human light chain (VLCL or VKCK) and human VH) and a chicken heavy chain constant region (Reference [Ching et al. [ , mAbs 2018]). Transgenic chickens were immunized with 100 µg of Fc-tagged human PD-1 protein (huPD-1-Fc) (SEQ No. 380) every 14 days for 14 weeks. In another approach, transgenic chickens were genetically immunized six times with DNA encoding human PD-1 (SEQ No. 347), followed by a final boost with 100 µg of huPD-1-Fc (SEQ No. 380). Serum immune responses of each animal were monitored by ELISA for biotinylated human PD-1 on streptavidin-coated plates.

[0343] Splenocytes were isolated from each immunized animal and tested for positive antibody clones using the Gel Encapsulated Microenvironment (GEM) assay (Mettler Izquierdo, S., Varela, S., Park, M., Collarini, EJ, Lu, D., Pramanick, S., Rucker, J., Lopalco, L., Etches, R., & Harriman, W. (2016). High-efficiency antibody discovery achieved with multiplexed microscopy. Microscopy (Oxford, England) , 65 Screening was performed on human PD-1 labeled beads as described in [4], 341-352. Positive clones were sequenced, the variable regions of the heavy and light chains were cloned, assembled into single-chain variable fragments, and fused to the hinge and Fc regions of immunoglobulins (ScFv-Fc). The unique scFv-Fc fusion proteins were transiently expressed in Expi293 cells, and the supernatants were tested for binding activity by ELISA on plates coated with huPD-1-Fc (SEQ No. 380) or synomolgus-PD-1-Fc (SEQ No. 381). Using the above method, a total of 102 unique anti-human PD-1 variable heavy chain and variable light chain pairs were identified. 2H7-hIgG4 (sequence numbers 382-391, 424, and 425) and A2-hIgG4 (sequence numbers 402-411, 428, and 429) were some of the antibodies identified in the above approach.

[0344] Through a different approach, an anti-hPD-1 antibody labeled C51E6-hIgG4 was identified, germline optimized to become the antibody designated as C51E6-5-hIgG4 (sequence numbers 392-401, 426, 427), humanized, and further sequence optimized to become the antibody designated as Abz1mod-hIgG4 (sequence numbers 449, 450).

[0345] The anti-PD-1 variable region sequence was expressed as a human IgG4 kappa antibody, and its ability to bind to PD-1-expressing cells was evaluated using flow cytometry as described in General Methods Protocol A. The antibodies to be tested were first screened for binding to human PD-1 using a Jurkat cell line (Jurkat + hPD-1 cell line) expressing recombinant human PD-1. After serially diluting the antibodies starting from a peak concentration of 280 nM, detection was performed by adding an allophycocyanin-conjugated anti-human IgG secondary antibody to the cells. Of the 92 hits, 79 of the test anti-PD-1 antibodies had an EC <30 nM (according to flow cytometry). 50Binding was shown. 2H7-hIgG4 (SEQN NOs 382-391, 424, and 425), C51E6-5-hIgG4 (SEQN NOs 392-401, 426, and 427), A2-hIgG4 (SEQN NOs 402-411, 428, and 429), OMC.1.B6-hIgG4 (SEQN NOs 438 and 439), OMC.1.D6-hIgG4 (SEQN NOs 442 and 443), OMC.2.C6-hIgG4 (SEQN NOs 440 and 441), 1H9-hIgG4 (SEQN NOs 576 and 525), 1D5-hIgG4 (SEQN NOs 577 and 527), and 2A3.H7-hIgG4 (SEQNs 424 and 523) was part of a group of antibodies identified as exhibiting intermediate to high-affinity binding to hPD-1 using Jurkat cell lines expressing human PD-1 (SEQN 346). In multiple experiments, the EC10 of binding to recombinant hPD-1-expressing Jurkat cells by flow cytometry... 50 The calculated values ​​were 0.1–0.3 nM for 2H7-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4. EC of binding to hPD-1-expressing Jurkat cells by flow 50 The calculated values ​​were 2-4 nM for C51E6-5-hIgG4 and 3-16 nM for A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4. Since antibody titrations of 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, 1H9-hIgG4, 1D5-hIgG4, 2A3.H7-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4 did not bind to parental Jurkat cell lines that do not express hPD-1 (data not presented), the binding was specific to hPD-1.

[0346] Example 7: Characterization of anti-hPD-1 antibody binding in the presence of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A antibodies

[0347] As described in the general method protocol B, 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 were evaluated for binding competition to hPD-1 in the presence of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A.

[0348] As a control, Opdivo® (nivolumab) was titrated in the presence of anti-hPD-1 #1-mIgG2b-N297A at a saturation concentration of 10 μM (Fig. 4a). When compared to the dose titration curve of Opdivo® in the absence of the anti-hPD-1 #1-mIgG2b-N297A competitor, the dose titration curve in the presence of the anti-hPD-1 #1-mIgG2b-N297A competitor was significantly reduced (the dose titration curve shifted 100 to 1000-fold to the right of the graph). When anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A was added at a saturation concentration (10 μM) prior to exposure with 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4, the binding of 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 to hPD-1 was not discarded, as shown by a shift of less than 10-fold in Figures 4b-4d, which implies that 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 in the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A This suggests that there was no competition for binding to PD-1.

[0349] Example 8: Characterization of a non-antagonist hPD-1 antibody

[0350] Anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 were tested for PD-1 antagonist activity using an in vitro cell-based human PD-1 / PD-L1 blocking bioassay as described in General Methods Protocol C. All antibodies except A2-hIgG4 were tested at a final concentration of 200 nM. A2-hIgG4 was tested at a final concentration of 500 nM.

[0351] The anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, OMC.2.C6-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4 all showed an average luminescence level of 3000 relative luminescence units (RLU), and showed RLU similar to the negative control KLH-C3-hIgG4 (data not presented), so none of the above antibodies demonstrated hPD-1 antagonist activity. In contrast, anti-hPD-1 #1, a known hPD-1 antagonist that blocks the binding of hPD-L1 (sequence number 584) to hPD-1, showed luminescence of over 14,000 RLU (data not presented).

[0352] Example 9: Anti-hPD-1-attenuated hIL-2 fusion protein binds to Jurkat cells expressing human PD-1.

[0353] To construct various antibody and antibody-attenuated hIL-2 fusion protein expression vectors, corresponding polynucleotide coding sequences of antibodies, cytokines, cytokine receptors, and linkers were generated and cloned into expression vectors. After transiently expressing the antibodies or antibody fusion proteins in Human Embryonic Kidney (HEK) 293 cells, they were purified by affinity chromatography using protein A or protein G Sepharose. The purified proteins were concentrated, and protein concentrations were determined after exchanging the buffer with phosphate-buffered saline or phosphate-buffered saline containing 100 mM L-arginine and 10 mM L-histidine using ultracentrifugation filtration.

[0354] In some approaches, 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 harboring the S228P hinge stabilization mutation were fused directly to hIL-2 (df) or fused to hIL-2 at the C-terminus of the immunoglobulin heavy chain using an L6 linker. Examples of the above anti-PD-1-attenuated hIL-2 fusion proteins are summarized in Figure 5. Various constructs were produced through substitutions in hIL-2 that attenuate hIL-2 activity, as described in Example 2. The anti-hPD-1-attenuated hIL-2 fusion proteins listed in Table 20 were tested for binding to hPD-1 using an hPD-1-expressing Jurkat cell line as described in General Methods Protocol A. The variable region of 2H7-hIgG4 (sequence numbers 384 and 385) was further optimized, and the isotype was converted to human IgG1 having the effector function null substitution L235A / G237A (LAGA, as described in WO1998 / 006248) to become H7-632-hIgG1-LAGA (sequence numbers 414 and 415). Optimized H7-632-hIgG1-LAGA was also directly fused (df) to a variant of hIL-2 with reduced hIL-2 activity (hIL-2 T3A / D20A / R38E / C125A; SEQ ID NO. 217) to become H7-767 (SEQ ID NOs. 412-413, 415-423, 532), and both H7-632-hIgG1-LAGA and H7-767 were tested for binding to hPD-1 (Table 20). EC 50 The values ​​were calculated from the geometric mean fluorescence intensity (gMFI) across titration concentrations using GraphPad Prism 7 software.

[0355] The generation of anti-hPD-1-attenuated hIL-2 fusion proteins did not reduce binding to hPD-1, and the anti-hPD-1-attenuated hIL-2 fusion proteins were still able to bind to Jurkat cells expressing human PD-1. The EC of the tested anti-hPD-1-attenuated hIL-2 fusion proteins compared to each anti-hPD-1 antibody lacking the attenuated hIL-2 moiety. 50 The calculated values ​​are summarized in Table 20.

[0356]

[0357] EC of anti-hPD-1-hIL-2 fusion protein on Jurkat+ hPD-1 cells compared to anti-hPD-1 antibody without attenuated hIL-2 moiety 50 As demonstrated by the fact that binding increased by less than twofold, the addition of an attenuated hIL-2 moiety to the anti-hPD-1 antibody did not abolish binding to human PD-1.

[0358] Example 10: Anti-hPD-1-attenuated hIL-2 fusion protein binds to hPD-1 in the presence of anti-hPD-1 #1 and anti-hPD-1 #2 antibodies.

[0359] As described in General Methods Protocol B and Example 7, the binding of the anti-hPD-1-attenuated hIL-2 fusion protein to the hPD-1 receptor in the presence of anti-hPD-1 #1 and anti-hPD-1 #2 was tested. A reverse experiment was also performed to investigate the binding of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A to hPD-1 in the presence of saturated concentrations of the test antibody-attenuated hIL-2 fusion protein. In the above format, hPD-1 expressing Jurkat cells were plated at 100,000 cells per well in FACS buffer, blocked with an anti-human FcγR blocking agent (Miltenyi) for 10 minutes at 4°C, and then washed. Test antibodies-attenuated hIL-2 fusion proteins 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-L6-hIL-2 (D20A / R38E), A2-hIgG4-df-hIL-2 (D20A / R38E), H7-767, and isotype control anti-DNase 1H3-hIgG4-df-hIL-2 (D20A / R38E) were diluted to a final concentration of 280 nM in 100 µl of FACS buffer and incubated on ice with Jurkat cells expressing hPD-1 cells for 1 hour. Cells were washed and resuspended on ice for 1 hour in FACS buffer containing 6-fold serial titrations of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A, starting at a maximum concentration of 50 nM. Cells were washed and resuspended on ice for 45 minutes in a 1:100 dilution of phycoerythrin-conjugated anti-mouse IgG light chain kappa monoclonal antibody. Cells were washed again and resuspended in FACS buffer containing a 1:1000 dilution of Citox Green (Thermo Fisher).Analysis was performed by flow cytometry using BD FACS Kanto II (BD Biosciences), and gMFI was calculated using FlowJo software version 10. EC. 50 The value was calculated from the gMFI of the phycoerythrin signal over titration concentrations using GraphPad Prism 7 software.

[0360] Similar to the results described in Example 7, the addition of attenuated hIL-2 to the anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 did not reduce the ability of the anti-hPD-1 protein to bind to human PD-1 in the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A (Figs. 16b-16d). H7-767 was also tested in this competitive assay, and Fig. 13b shows that H7-767 continues to bind to the hPD-1 receptor in the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. In contrast, the binding of the positive control anti-hPD-1 #1 was significantly reduced in the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A (Fig. 16a, 13b).

[0361] Figures 6a and 6b show that, in the case of a reverse competition assay, anti-hPD-1 #1-mIgG2b-N297A (Figure 6a) and anti-hPD-1 #2-mIgG2b-N297A (Figure 6b) can bind to hPD-1 in Jurkat cells even in the presence of saturated (280 nM) anti-hPD-1-attenuated hIL-2 fusion proteins 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-df-hIL-2 (D20A / R38E) or A2-hIgG4-df-hIL-2 (D20A / R38E). The binding curve using the saturated anti-hPD-1-attenuated hIL-2 fusion protein prior to exposure to the anti-hPD-1 fusion protein overlapped with the binding curve of anti-hPD-1 #1-mIgG2b-N297A (non-competitive) or anti-hPD-1 #2-mIgG2b-N297A (non-competitive). The binding curve also overlapped with the saturated negative control fusion protein that did not bind to hPD-1, 1H3-hIgG4-df-hIL-2 (D20A / R38E).

[0362] Example 11: Anti-hPD-1-attenuated hIL-2 fusion protein binds to recombinantly expressed synomolgus PD-1.

[0363] Binding of the anti-hPD-1-attenuated hIL-2 fusion protein to cynomolgus PD-1 was tested using human embryonic kidney 293 cell lines (HEK-293T) expressing SV40 large T cell antigen, transiently transfected to recombinantly express cynomolgus PD-1 in flow cytometry. For each transfection reaction, 2 million HEK-293T cells were transfected with 2 µg of pCMV6-hygro-HA-cyno-PD-1(1-185) (SEQ No. 448), a mammalian vector containing the extracellular domain of cynomolgus PD-1 tagged with human influenza hemagglutinin and a sequence encoding hygromycin resistance. Transfection was performed by electroporation. Transfected cells were blocked with a human FcγR blocking agent and stained with an appropriate amount of anti-hPD-1-attenuated hIL-2 fusion protein. Additionally, phycoerythrin-conjugated anti-hemagglutinin clone 15B12 was added to cells to stain transfected cells, and allophycocyanin-conjugated anti-human IgG Fc secondary clone HP6017 (BioLegend catalog number 409306) was added to cells to stain the conjugated antibody. Cells were analyzed in a BD Canto II, and live transfected (hemagglutinin-positive) cells were gated using FlowJo software version 10, and the gMFI of the allophycocyanin signal was calculated. EC 50 The value was calculated from gMFI over titration concentrations using GraphPad Prism 7 software.

[0364] The anti-hPD-1-attenuated hIL-2 fusion protein bound to synomolgus PD-1-expressing HEK-293T cells in a manner similar to the binding profile observed in human PD-1-expressing Jurkat T cells (Fig. 17). EC for binding to synomolgus PD-1-expressing HEK-293T cells 50The EC was 5 nM for 2H7-hIgG4-df-hIL-2 (D20A / R38E), 6 nM for C51E6-5-hIgG4-df-hIL-2 (D20A / R38E), and 11 nM for A2-hIgG4-df-hIL-2 (D20A / R38E). Anti-hPD-1 #1 and anti-hPD-1 #2, formatted as competitor anti-hPD-1-attenuated hIL-2 fusion proteins, also had ECs of 9 nM and 2 nM, respectively. 50 It bound to Synomolgus PD-1 with a value, suggesting that the binding to Synomolgus PD-1 was not canceled even when a weakened hIL-2 moiety was added to the anti-hPD-1 antibody.

[0365] Example 12: Anti-hPD-1-attenuated hIL-2 fusion protein binds to activated primary human and synomolgus PD-1.

[0366] The binding of anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins to activated primary T cells expressing hPD-1 was investigated by flow cytometry. To test whether 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 bind to natural hPD-1, cryopreserved human peripheral blood mononuclear cells (PBMCs) were thawed and activated with 50 ng / mL phorbol 12-myristate 13-acetate (PMA) and 1 μg / mL ionomycin to upregulate the hPD-1 receptor. Activated PBMCs were collected and blocked with a 1:50 dilution of human FcγR blocking reagent (Miltenyi) for 10 minutes at 4°C, and then stained with appropriate concentrations of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, anti-hPD-1 #1, and isotype controls. Subsequently, cells were stained with a 1:20 dilution of allophycocyanin-conjugated anti-human IgG Fc to detect the bound antibodies. To differentiate immune subsets, a cocktail of anti-human CD3, anti-CD4, and anti-CD8 antibodies containing surface markers was used. Additionally, sample fractions were investigated for cellular expression of hPD-1, hCD25, hCD122, and hCD132. Cells were analyzed on BD Potesa (BD Biosciences), and after gating T cell subsets using FlowJo software version 10, the gMFI of the allophycocyanin signal was calculated. EC 50 Values ​​were calculated from gMFI over titration concentrations using GraphPad Prism 7 software. To test the binding of the anti-hPD-1-hIL-2 fusion protein, cryopreserved CD3+ T cells were activated with PMA / ionomycin, and binding by flow cytometry was performed as described above.

[0367] The binding of human PD-1 antibody-attenuated hIL-2 fusion protein to activated synomolgus T cells was also tested using flow cytometry. Synomolgus PBMCs were activated with 0.081 μM PMA and 1.34 μM ionomycin. After 24 hours, cells were stained using the same procedure as described above for the binding of human PD-1 to primary cells, except that a synomolgus cross-reactive marker was used. Live, CD3 + CD4 + or CD3 + CD8 + After gating T cells, the gMFI of allophycocyanin signaling was calculated. EC 50 Values ​​were calculated from gMFI across titration concentrations of anti-hPD-1 antibody or hPD-1 antibody-attenuated hIL-2 fusion protein using GraphPad Prism 7 software.

[0368] In some of the variants tested, the weakened hIL-2 also included substitutions T3A and C125A, which provide a site for O-linked glycosylation and substitute free cysteine ​​residues, respectively.

[0369] After PMA and ionomycin activation, CD4 + 40-50% of T cells are PD-1 + On the other hand, CD8 + 30-40% of T cells are PD-1 + was (data not presented). According to flow cytometry, activated human CD3 + CD4 + EC regarding binding to T cells 50 The calculated values ​​were 0.1–0.7 nM for 2H7-hIgG4, 12 nM for C51E6-5-hIgG4, 30 nM for A2-hIgG4, and 0.04 nM for 2H7-hIgG4-df-hIL-2(T3A / D20A / R38E / C125A). Activated human CD3+ EC for binding to CD8+ T cells 50 For 2H7-hIgG4, it was 0.1-0.8 nM, for C51E6-5-hIgG4, 16 nM, for A2-hIgG4, 22 nM, and for 2H7-hIgG4-df-hIL-2(T3A / D20A / R38E / C125A), 0.03 nM. Activated human CD3 + CD4 + EC regarding binding to T cells 50 is 0.19 nM, and activated human CD3 + CD8 + T cell levels were 0.12 nM for H7-767. Activated synomolgus CD3 + EC for binding to CD4+ T cells 50 It was 0.09 nM for 2H7-hIgG4 and 0.04 nM for 2H7-hIgG4-df-hIL-2(T3A / D20A / R38E / C125A). Activated synomologous CD3 + EC for binding to CD8+ T cells 50 It was 0.08 nM for 2H7-hIgG4 and 0.03 nM for 2H7-hIgG4-df-hIL-2(T3A / D20A / R38E / C125A). Activated synomologous CD3 + CD4 + EC regarding binding to T cells 50 is 0.26 nM, and activated synomologous CD3 + CD8 + The T cell level was 0.24 nM for H7-767. These data represent the calculated EC10 for binding to activated hPD-1 when the hPD-1 antibody was converted to the anti-hPD-1-attenuated hIL-2 fusion protein. 50 It showed that the value remained similar to the calculated EC50 value of the hPD-1 naked antibody binding to hPD-1.

[0370] H7-767 and H7-632-hIgG1-LAGA anti-PD-1 naked antibodies against primary inactivated human CD4 by flow cytometry + and CD8 + Binding to T cells was tested. Frozen human CD3 + T was thawed, and flow cytometry was performed as described above. H7-767 and H7-632-hIgG1-LAGA anti-PD-1 naked antibodies, both inactivated human CD4 + and CD8 + It did not bind to T cells (data not presented).

[0371] Example 13: Quantification of binding of anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to recombinant human or synomolous PD-1 by surface plasmon resonance (SPR)

[0372] The binding affinities of the anti-hPD-1 antibody and the anti-hPD-1-attenuated hIL-2 fusion protein were determined by performing surface plasmon resonance coupling analysis using a high-throughput SPR Carterra® LSA™. The antibodies were diluted to 2 or 10 µg / mL in 10 mM sodium acetate containing 0.01% Tween-20 at pH 4.5, coupled to an HC30M (Carterra Bio) chip using the sulfo-N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylamino)propyl carbodiimide (sulfo-NHS / EDC) coupling chemistry, and blocked with ethanolamine. The binding kinetics were determined for commercially available recombinant His-tagged human PD-1 and His-tagged synomolgus PD-1 (Acro Biosystems) using a non-regenerative kinetic coupling process.

[0373] Anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were expressed as modified human IgG1 or modified IgG4 isotypes with a kappa light chain framework. Additional substitutions L235E or L235A / G237A (described in LAGA, International Publication No. WO1998 / 006248) (numbered according to the EU numbering scheme) were introduced into the Fc domain to abolish the effector function of the immunoglobulin components.

[0374] Association constants (k) of binding of various anti-hPD-1 antibodies and anti-hPD-1 antibody-attenuated hIL-2 fusion proteins to recombinant human or synomolgus PD-1 protein from titration curves and Katerra kinetic properties software a ), dissociation constant(k d ), and equilibrium constant (K D ) was determined. The generated maximum possible SPR signal (R max ) and the residual standard deviation (Res SD) were also calculated. The results from the kinetic properties screen are summarized in Table 21 and showed that the addition of the attenuated hIL-2 moiety to the anti-hPD-1 antibody did not modulate PD-1 antibody binding to human PD-1 or synomolous PD-1 antigens. In a separate experiment, H7-632-hIgG1-LAGA (SEQ Nos. 414 and 415) was measured by SPR, and the steady-state equilibrium dissociation constant (K D ) is 1.23 x 10 -9 It was M, and H7-767's K D is 1.93 x 10 -9 It was M.

[0375]

[0376]

[0377] Example 14: Determination by Surface Plasmon Resonance (SPR) whether anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein compete with anti-hPD-1 #1 and anti-hPD-1 #2 for binding to PD-1

[0378] The sandwich method was used to test whether anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins competed with each other. Antibodies and corresponding antibody-IL-2 cytokine-fusion proteins were immobilized on an HC30M chip using the amine coupling chemistry described in Example 13. According to the kinetic analysis described in Example 13, 80 nM human PD-1 (Acro Biosystems, catalog number PD-1-H5221-100 µg) was injected into the entire array. Competing anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins (analytes) were diluted to 30 µg / mL, then injected into the array, and binding parameters were evaluated using SPR. Both anti-hPD-1 and anti-hPD-1-hIL-2 fusion proteins were evaluated in duplexes. Some tested variants had a modified human IgG1 or IgG4 kappa light chain framework with additional L235E or L235A / G237A (LAGA) substitutions that discard immunoglobulin effector function.

[0379] Screening of anti-hPD-1 or anti-hPD-1-attenuated hIL-2 fusion protein pairs identified two bins as presented in Table 22. Antibodies and fusion proteins from Group 1 were able to bind to hPD-1 in the presence of all antibodies and fusion proteins from Group 2, but competed with all members of the same group. Antibodies and fusion proteins from Group 2 were able to bind to hPD-1 in the presence of all antibodies and fusion proteins from Group 1, but competed with all members of the same group. None of the anti-hPD-1s listed in Group 1 of Table 22 competed with Keytruda® and Opdivo®.

[0380]

[0381] Example 15: Antagonism of anti-hPD-1-attenuated hIL-2 fusion protein to hPD-1 in the presence of anti-hPD-1 #1 and anti-hPD-1 #2

[0382] Anti-hPD-1-attenuated hIL-2 fusion proteins were tested for antagonism against hPD-1. Characterization of the anti-hPD-1-attenuated hIL-2 fusion proteins was performed according to General Methods Protocol C. Figure 7 illustrates these results. Compared to the PD-1 antagonists Keytruda® or Opdivo®, 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-L6-hIL-2 (D20A / R38E), and A2-hIgG4-df-hIL-2 (D20A / R38E) were non-antagonistic to human PD-1, as evidenced by low levels of detectable luminescence. H7-632-hIgG1-LAGA and H7-767 were also tested for antagonist activity as described in General Protocol C. Figure 15 illustrates that H7-632-hIgG1-LAGA and H7-767 do not block hPD-L1 (sequence number 584) from interacting with the hPD-1 receptor.

[0383] For a competitive assay using the cell-based co-culture assay described in General Protocol C, several modifications were performed. An anti-hPD-1-attenuated hIL-2 fusion protein sample was diluted to a fixed concentration of 400 nM, and 20 µL was added to 20 µL of titrated anti-hPD-1 #1 or anti-hPD-1 #2. 40 µL of the mixture was added to CHO cells. 40 (40) µL of Jurkat PD-1 effector cells were overlaid onto the mixture of CHO cells and anti-hPD-1-attenuated hIL-2 fusion protein. In this competitive assay, a final concentration of saturated anti-hPD-1-attenuated hIL-2 fusion protein at 100 nM was tested in combination with titrated anti-hPD-1 #1 or anti-hPD-1 #2. The remaining assays were performed as described in General Protocol C. Figures 18a and 18b show that the addition of 100 nM anti-hPD-1-attenuated hIL-2 fusion protein did not compete with the blocking of binding of titrated anti-hPD-1 #1 to hPD-L1 (SEQN 584). The dose titration curve of anti-hPD-1 #1 remained unchanged from the curve without the competing antibody, which suggests that the presence of anti-hPD-1-attenuated hIL-2 fusion protein did not compete with the function of anti-hPD-1 #1 even at high concentrations. In the presence of 100 nM 2H7-hIgG4-df-hIL-2 (D20A / R38E) and 100 nM C51E6-5-hIgG4-L6-hIL-2 (D20A / R38E), the luminescence (RLU) of anti-hPD-1 #2 decreased by 35% at higher concentrations of anti-hPD-1 #2 (Fig. 18b), but it is unclear whether this decrease is significant due to the degree of standard deviation.

[0384] In the reverse experiment, anti-hPD-1 #1 or anti-hPD-1 #2 was diluted to a concentration of 400 nM, and 20 µL was combined with 20 µL of titrated anti-hPD-1-attenuated hIL-2 fusion protein. The anti-hPD-1-attenuated hIL-2 fusion protein was serially titrated, and 40 µL of the mixture was added to CHO cells and then overlaid onto 40 µL of Jurkat PD-1 effector cells. The remaining assays were performed as described in General Protocol C. Figures 18c and 18d show that the ability of the anti-hPD-1-attenuated hIL-2 fusion protein to act as an antagonist is not impaired upon the addition of 100 nM anti-hPD-1 #1 (Figure 18c) or 100 nM anti-hPD-1 #2 (Figure 18d). The flat curve observed above 18,000 relative luminescent units (RLU) indicated that there was no competition for antagonist activity, and that the tested anti-hPD-1-attenuated hIL-2 fusion protein could continue to exhibit antagonist function even in the presence of anti-hPD-1 #1 or anti-hPD-1 #2.

[0385] Example 16: Anti-hPD-1-attenuated hIL-2 fusion protein test for attenuation to high-affinity and medium-affinity hIL-2 receptors using a cell-based proliferation assay

[0386] As described in General Protocol E, anti-hPD-1-attenuated hIL-2 fusion proteins were evaluated for the level of attenuation of hIL-2 activity using a cell proliferation assay on NK-92 and TF1+IL-2Rβ cell lines. To demonstrate the effect of the untargeted attenuated hIL-2 fusion proteins, control fusion proteins included fusion proteins that either directly fused to hIL-2 or introduced an anti-DNase I antibody (designated 1H3) along with a human IgG4 or human IgG1 backbone fused using a linker (SEQ No. 355). The hIL-2 sequences of the above constructs included substitutions for attenuated hIL-2 activity as described in Example 2. Full, partial agonist IL-2 activity, or the absence of agonist IL-2 activity (inactivity) were also evaluated similarly to Example 3. Some of the tested variants were expressed in modified human IgG1 or IgG4 isotypes containing additional L235E or L235A / G237A (LAGA) substitutions in the Fc region to discard immunoglobulin effector function, along with the kappa light chain. In some antibody-cytokine fusion proteins, the hIL-2 cytokine was fused to the C-terminus of the light chain (LC fusions).

[0387] EC for each antibody-cytokine fusion protein 50 The calculated value was determined from the relative luminescence unit (RLU), and the change multiple EC was compared with recombinant human IL-2 (rhIL-2). 50...was calculated. The change multiples from rhIL-2 and agonist activity are summarized in Table 23. Agonist activity was measured as full, partial, or inactive, determined by the peak luminescence of the antibody-attenuated hIL-2 fusion protein compared to the peak luminescence of rhIL-2. Dose titration curves of antibody-attenuated hIL-2 fusion proteins that reached the same peak luminescence as rhIL-2 were considered to be variants with full activity. Partial activity was calculated as a percentage (%) relative to full activity, using the rhIL-2 peak luminescence as 100%. The peak RLU of antibody-attenuated hIL-2 fusion proteins with a rhIL-2 peak RLU of less than 10% at the peak concentration of 1200 nM was considered to have no agonist activity or to be inactive. For some variants, EC was... because peak luminescence was not reached 50 Only the values ​​were estimated, and these are in Table 23 a It is commented out.

[0388]

[0389]

[0390]

[0391]

[0392]

[0393] Example 17: IL-2 activity structure of anti-hPD-1-attenuated hIL-2 fusion protein in cell lines expressing intermediate affinity hIL-2 receptor and hPD-1

[0394] Anti-hPD-1-attenuated hIL-2 fusion proteins were evaluated for hIL-2 activity using targeted cell lines expressing hPD-1. Briefly, TF1+IL-2Rβ cell lines were modified via lentiviral transduction as described in General Methods Protocol D to express the hPD-1 receptor (SEQ No. 580). hPD-1 expressing TF1+IL-2Rβ cells were detected using flow cytometry with a Brilliant Blue 515 conjugated hPD-1 antibody (BD Biosciences Catalog No. 565936). Cells with low hPD-1 expression (10 in Brilliant Blue 515 fluorescence) 3 Classification was performed based on (intensity below). The pool was classified two more times to collect cells with hPD-1 expression levels close to those in activated primary cells. The cell line (TF1+IL-2Rβ+hPD-1) was expanded and frozen in aliquots for a cell-based proliferation assay. The proliferation assay was performed as described in General Methods Protocol E, with an incubation period of 3 days. Some of the tested variants had modified human IgG1 or IgG4 kappa light chain frameworks with additional L235E or L235A / G237A (LAGA) substitutions to discard the effector function of immunoglobulins.

[0395] Table 24 summarizes the results of the proliferation assay in targeted TF1+IL-2Rβ+hPD-1 cell lines. Agonistic activity was measured as full, partial, or inactive, determined by the peak luminescence of the antibody-attenuated hIL-2 fusion protein compared to the peak luminescence of rhIL-2. Dose titration curves of antibody-attenuated hIL-2 fusion proteins that reached the same peak luminescence as rhIL-2 were considered variants with full activity. Partial activity was calculated as a percentage (%) relative to full activity, using the rhIL-2 peak luminescence as 100%. The peak RLU of antibody-attenuated hIL-2 fusion proteins with a rhIL-2 peak RLU of less than 10% at a peak concentration of 1200 nM was considered to have no agonist activity or be inactive. For some variants, EC was calculated because they did not reach the full curve. 50 The values ​​were merely estimates. Many examples of anti-hPD-1-hIL-2 fusion proteins with attenuated hIL-2 constructed hIL-2 activity in targeted cell lines, whereas the non-targeted antibody control (indicated as 1H3) failed to construct hIL-2 activity. The complete construct was presented as a decrease in the change factor from rhIL-2 to a value of 0 or 1.

[0396]

[0397]

[0398]

[0399] Example 18: Evaluation of anti-hPD-1-attenuated hIL-2 fusion proteins that block or do not block mouse PD-L1 in vivo murine colorectal adenocarcinoma (MC38) model

[0400] Since there is no acceptable model in primates to investigate the in vivo efficacy of tumor therapeutic agents, a surrogate anti-mPD-1-attenuated hIL-2 fusion protein was produced and tested in a synonymous murine tumor model. The MC38 colon adenocarcinoma model is commonly used to test the efficacy of immuno-oncology therapeutic agents. To investigate the in vivo effects of the anti-PD-1-attenuated hIL-2 fusion protein, surrogate anti-mouse PD-1 antibodies designated as RMP1-14 (known to block mouse PD-L1 binding) and RMP1-30 (described as a mouse PD-L1 non-blocker) were fused to attenuated hIL-2 at the C-terminus of the mouse IgG2b-N297 heavy chain and tested in the MC38 colon adenocarcinoma model. hIL-2 moiety was tested in IL-2-dependent mouse T lymphoblast cell lines (CTLL-2) and included substitutions F42K, Y45R, and V69R, which were proven to be attenuated for mouse IL-2 activity. Although human IL-2 can stimulate mouse T cell proliferation at similar concentrations, the same substitutions that attenuate activity in human IL-2-dependent cell lines do not attenuate activity in CTLL-2 cell lines (data not presented). Therefore, since the F42K / Y45R / V69R substitution showed attenuated IL-2 activity in mouse cell lines, the said substitution was used for hIL-2 as a surrogate. (Reference [Matsumoto K et al.Sequences containing the heavy and light chain variable region sequences of the anti-mouse PD-1 antibodies RMP1-14 and RMP1-30 (as described in [J Immunol. 2004 Feb 15;172(4):2530-41]) were also formatted on a murine IgG2b-N297A background to generate anti-mPD-1 RMP1-14 mIgG2b-N297A (SEQ NOs 564 and 566) and anti-mPD-1 RMP1-30 mIgG2b-N297A (SEQ NOs 567 and 568). Mouse IgG2b isotypes with N297A substitutions are murine equivalents of the Fc isotypes that discard Fc immune effector function. Using standard techniques, surrogate antibodies and antibody-attenuated hIL-2 fusion proteins were produced, expressed, and purified into Protein-A.

[0401] In the above murine tumor model, 5x10 10-week-old female C57BL / 6NCrl(Charles River) mice 5 Canine MC38 colorectal carcinoma cells were injected into the right flank. When the tumor reached 80-120 mm³, mice were classified into cohorts (10 mice / group) and treatment was started on day 1 of the study. Anti-mPD-1 RMP1-14 mIgG2b-N297A, anti-mPD-1 RMP1-30 mIgG2b-N297A, anti-mPD-1 RMP1-14 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) (SEQ Nos. 565 and 566), and anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) (SEQ Nos. 568 and 569) were administered intraperitoneally at 5 mg / kg twice weekly for 4 weeks, along with a vehicle control (phosphate-buffered saline). Tumor size during the study period was [calculated using] formula (w 2 Measurements were taken twice a week with calipers using x L) / 2, where w = width and L = length). The study endpoint was when the tumor volume reached 1,000 mm³ or survival at 50 days, whichever came first.

[0402] Figure 8 demonstrates that while administration of anti-mPD-1 RMP1-14-mIgG2b-N297A or anti-mPD-1 RMP1-30-mIgG2b-N297A antibodies alone did not significantly promote efficacy compared to vehicle control treatment, administration of anti-mPD-1 RMP1-14 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) or anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) anti-PD-1-attenuated hIL-2 fusion proteins was associated with 90% and 100% complete tumor regression, respectively. The above data demonstrate that the anti-tumor efficacy mediated by the anti-mPD-1-hIL-2 (F42K / Y45R / V69R) fusion protein does not require PD-1 checkpoint blockade, and that the efficacy depends on hIL-2 activity. The data further demonstrate that antibody-mediated targeting of PD-1-expressing T cells is sufficient to promote potent anti-tumor efficacy in the MC38 tumor model.

[0403] Example 19: Substitute anti-hPD-1-attenuated hIL-2 fusion protein expands effector memory CD8+ T cells in an in vivo murine colon adenocarcinoma model.

[0404] To understand the mechanism of action of the surrogate anti-hPD-1-attenuated hIL-2 fusion protein in vivo, in vivo experiments similar to Example 18 were performed after three administrations, followed by immunophenotyping of T cell populations generated in tumors, blood, spleen, and lymph nodes. 5 x 10 oz of 10-week-old female C57BL / 6NCrl (Charles River) mice were injected into the right flank. 5Canine murine MC38 colon adenocarcinoma tumor cells were subcutaneously implanted, and tumor growth was monitored. For the study, animals with tumors of 150–260 mm³ were divided into four groups of 10 mice per group. Twenty-one days after transplantation, on days 1, 4, and 8, 0.2 mL / volume of phosphate-buffered saline (PBS) was administered to the vehicle control group, and 5 mg / kg anti-KLH-C3-mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R), 5 mg / kg anti-mPD-1 RMP1-30 mIgG2b-N297A, or 5 mg / kg anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) was administered intraperitoneally. On day 9, tumors, spleens, and inguinal lymph nodes were harvested from all mice and processed into single-cell suspensions for subsequent analysis by flow cytometry.

[0405] Figure 9a charts tumor volume growth (mm³) over 9 days following the first administration on day 1, where each point represents the average of 10 mice. Up to day 8, anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) showed a reduction in tumor volume compared to other treatment groups. Figure 9b shows various CD8s in the tumors of each treatment group. + This summarizes the contribution of a T cell subset, where T-centered memory is phenotyped CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 - CD103 - It was analyzed to be, and the T effector memory is CD45 + CD3 + CD4 - CD8 + CD44 + CD127+ CD69 - CD103 - CD62L - was, and the T resident memory is CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 + CD103 + was, and CD44 - CD62L - T cells are CD45 + CD3 + CD4 - CD8 + CD44 + CD62L - It was, and the T Naive is CD45 + CD3 + CD4 - CD8 + CD44 - CD62L + It was. Compared to other treatment groups, in mice treated with anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R), CD8 + The T effector memory subset was expanded (indicated by the light gray slice increase in Fig. 9b). This was also illustrated in Fig. 9c as the absolute count (cells / µL) within the MC38 dissected tumor. Additionally, CD45 within the tumor + CD3 + CD4 + CD8 - CD25 + FoxP3 + The absolute count (cells / µL) of regulatory T cells defined as expressing the marker decreased.

[0406] CD8 + The expansion of T effector memory cells and the reduction of regulatory T cells are associated with effective immunotherapy in both mice and humans.

[0407] Example 20: Anti-hPD-1-attenuated hIL-2 fusion protein is active in an in vivo NCG-PBMC model.

[0408] NOD- deficient in functional T, B, and NK cells Prkdc em26Cd52 IL-2rg em26Cd22 Transplanting human immune cells into / NjuCrl(NCG) mice has been a valuable tool for evaluating the efficacy of therapies hypothesized to stimulate human T cells. In the aforementioned model, if the therapies activate human T cells, the T cells will expand, and graft-versus-host disease (GvHD) will accelerate.

[0409] Three independent donors for human peripheral mononuclear cell (hPBMC) transplantation were evaluated for 4 weeks for transplant kinetics as well as the expression of human PD-1 and human IL-2 receptors in T cells. Among the three donors tested, the donor who induced the most T cells via the median window for GvHD was selected. 1.5 x 10⁶ 7Cans of hPBMC were injected intravenously into NCG mice and divided into 8 groups of 8 to 16 mice each. On days 7, 10, and 14, mice were given three doses of 2H7-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A)(SEQN 471, 425)(2.5 mg / kg, 5 mg / kg, or 10 mg / kg), 1H3-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A)(SEQN 546, 374)(5 mg / kg or 10 mg / kg), 1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A)(SEQN 563, 374)(10 mg / kg), or 2H7-hIgG1-LAGA-df-hIL-2(T3A / R38E / I92K / C125A)(SEQ ID NOs 474, 425)(5 mg / kg) was injected intraperitoneally. The anti-DNase fusion protein as wild-type hIL-2 (1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A)) and the anti-DNase fusion protein with an attenuated hIL-2 moiety (1H3-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A)) were both used as non-targeted antibody controls. The 1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A) fusion protein did not alter the hIL-2 moiety, but included T3A and C125A substitutions to remove the O-linked glycosylation site (e.g., see International Publication No. WO2012 / 107417) and unpaired cysteine ​​residue (e.g., see International Publication No. WO2018 / 184964) predicted in human IL-2, respectively. The substitutions did not demonstrate reduced hIL-2 efficacy in the clinic. On day 21, blood, spleen, and lungs were harvested, whereby the blood and spleen were processed for immunophenotyping by flow cytometry, and the weight of the lungs was measured.

[0410] After 21 days, immunophenotyping was performed on the blood and spleen of the animals using flow cytometry. Table 25 summarizes the markers used to distinguish human T cell populations for subsequent analysis.

[0411]

[0412] Body weight was measured for 21 days and normalized to the body weight on day 1 for each individual animal as an evaluation of graft-versus-host disease (GvHD) as shown in Fig. 10. Acceleration of GvHD was observed in mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A). Additionally, small body weight loss was observed in mice treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), and in mice treated with 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / R38E / I92K / C125A). Weight loss was observed in 1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A), but it did not persist.

[0413] Flow cytometry analysis revealed a correlation with the observed acceleration of graft-versus-host disease (GvHD). Flow cytometry analysis of peripheral blood using phenotypic markers to differentiate human T cell subsets provided in Table 25 showed that CD3 in mice treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), and in mice treated with 10 mg / kg of 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A), + , CD4 + , and CD8 + It was found that T cell subset expansion occurred only minimally (CD3 +(Quantified as a 10 to 50-fold change from the vehicle control for T cells). Additionally, CD3 + , CD4 + , and CD8 + T cell subsets expanded significantly in the peripheral blood of mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) (the number of changes from the vehicle control was CD3 + In the case of T cells, it was more than 50-fold). Table 26 summarizes the expanded human T cell subsets.

[0414]

[0415] CD3 between treatment groups + , CD4 + , and CD8 + In addition to evaluating T cells, CD4 + and CD8 + Memory and naive subsets for T cell subsets were also evaluated. CD4 + and CD8 + Phenotypic markers used to distinguish naive, effector, effector memory, and central memory T cells for both groups are summarized in Table 25. There were no changes in naive, effector, or central memory T cells between treatment groups (data not presented). However, in mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), CD4 in peripheral blood + and CD8 + Effector memory (EM) T cells were shown to have expanded significantly, where the average number of cells per milliliter was CD8 + In the case of T cells, it was over 5 million, and CD4 +For T cells, the count exceeded 50 million (Figs. 11a and 11b). The box-and-whisker plots were plotted with the 1st and 3rd quartiles marked in boxes, the horizontal line representing the median, and the line representing the minimum and maximum points. For animals treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), as well as those treated with 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A), the median CD8 count was defined as the average cell count per million, ranging from 1 million to 5 million per milliliter. + It showed effector memory (EM) T cell expansion. In mice treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), CD4 + Intermediate CD4 count of 6 to 13 million per milliliter for T cells + It showed effector memory (EM) T cell expansion.

[0416] IL-2 has been described as stimulating NK cells and regulatory T cells (Tregs) in addition to stimulating effector T cells; since Tregs express high levels of CD25 and NK cells express CD122, these immune cell types were also evaluated. Figure 12 illustrates that animals treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) at the highest dose of 10 mg / kg did not expand human regulatory T cells; instead, the proportion of regulatory T cells in the animals' peripheral blood was found to be the lowest (defined as phenotypic analysis in Table 25). A dose-dependent decrease in human regulatory T cells occurred, and human CD3 Tregs +Compared to a vehicle control group with an average T cell count of 1.6%, 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) was compared to human CD3 T cells, which are Tregs. + The average T cell count was 0.16%. Compared to the vehicle control group, there was no change in the percentage (%) of human NK cells (phenotypes defined in Table 25) in peripheral blood across all treatment groups (data not presented).

[0417] Example 21: Nonclinical safety profile of anti-hPD-1-attenuated hIL-2 fusion protein

[0418] Synomolgus monkeys were previously used to evaluate the toxicity of unmodified IL-2. Lethal results were observed in synomolgus monkeys even at exogenous recombinant IL-2 doses as low as 50 µg / kg / day. As it was confirmed by flow cytometry that H7-767 binds to cynomolgus monkey hPD-1 in primary activated PBMCs (Example 12), a single-dose study for preliminary safety evaluation was performed using both H7-767 and a variant of H7-767 (H7-02-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) (SEQ ID NOs. 582 and 583). H7-02-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) was administered to eight monkeys by 15-minute IV infusion at a dose of 1 mg / kg (four animals) or 10 mg / kg (four animals). Sampling was performed up to 360 hours after infusion. No adverse effects, total toxicity, body weight A decrease or mortality was observed (data not presented). Subsequent single-dose studies using H7-767 were conducted at higher doses of 5 mg / kg and 50 mg / kg, similar to the primary study, and samples were taken at time points up to 360 hours after infusion. Again, no adverse effects, total toxicity, body weight loss, or mortality were observed (data not presented).

[0419] Example 22: Attenuation of IL-2 activity of modified hIL-2 protein

[0420] The IL-2 activity attenuation of modified hIL-2 proteins, including substitutions at amino acid position 20 (D20) and at amino acid position 38 (R38), was tested in the proliferation assay in both NK-92 and TF1+IL-2Rβ cell lines as described in Example 5 above. The modified hIL-2 proteins were grouped into seven groups (1 to 7) based on the maximum agonist activity and attenuation levels of the modified hIL-2 proteins for both intermediate and high-affinity receptors relative to unmodified recombinant hIL-2 (Table 27). The criteria used to group the modified hIL-2 proteins were as follows:

[0421] · Group 1: Variants that exhibit maximum attenuation (i.e., >10,000-fold) and at least about 80% activity for medium-affinity receptors, as well as high attenuation and at least about 70% activity for high-affinity receptors.

[0422] · Group 2: Variants showing at least about 70% activity and >1,000-fold attenuation for medium-affinity receptors, and about 20% to about 30% activity for high-affinity receptors.

[0423] · Group 3: Variants showing about 50% to about 70% activity and >1,000-fold attenuation for medium-affinity receptors, and about 20% activity for high-affinity receptors.

[0424] · Group 4: Variants showing at least about 70% activity toward medium-affinity receptors but only >500-fold attenuation, and about 50% activity toward high-affinity receptors.

[0425] · Group 5: Variants showing at least about 70% activity for both receptors, but >10 to >300 attenuation in descending order for the intermediate affinity receptor and 70 to 1500 attenuation in descending order for the high affinity receptor.

[0426] · Group 6: A variant showing only about 30% activity and >2,500-fold attenuation for intermediate affinity receptors, and no activity for high affinity receptors.

[0427] · Group 7: Variants that showed no activity toward both intermediate affinity receptors and high affinity receptors.

[0428]

[0429]

[0430] Example 23: Activity of surrogate fusion protein in a murine MC38 colorectal tumor model

[0431] 5x10 on the right flank of a 10-week-old female C57BL / 6NCrl mouse 5 Canine syngeneic MC38 colorectal carcinoma cells were injected. When the tumor reached 80–120 mm³, mice were classified into cohorts (10 mice / group), and treatment was initiated on Day 1 of the study. Starting on Day 1, all agents except hIL-2 were administered intraperitoneally at a dose of 5 mg / kg twice weekly for 4 weeks. hIL-2 was administered intraperitoneally at a dose of 36,000 international units once daily from Days 1 to 5. Tumor size was measured twice weekly using calipers during the study period. The study endpoint was reached at a tumor volume of 1,000 mm³, or at either Day 50 survival or Day 70 progression-free survival, whichever came first.

[0432] All test agents, including antibody molecules and antibody-hIL-2 fusion proteins, were generated using a mouse IgG2b Fc region having a single N297A amino acid substitution at position 297, which prevents glycosylation of the Fc region and significantly reduces Fc region-mediated immune effector function, thereby hindering cell depletion in vivo. Anti-mPD-1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (reference [Matsumoto, J Immunol 172: 2530-2541, 2004]). Anti-mPD-1 RMP1-14-hIL-2 F42K / Y45R / V69R is a bifunctional fusion protein composed of a monoclonal RMP1-14 antibody antagonist of the mouse PD-1 receptor fused to the C-terminus of hIL-2 F42K / Y45R / V69R (SEQ No. 621), a reduced-efficacy IL-2 variant, via a flexible 6-amino acid glycine / serine linker. The molecule was designed to directly target the reduced-efficacy hIL-2 variant to PD-1-expressing T cells in vivo in mice. Anti-KLH-hIL-2 F42K, Y45R, V69R is a control fusion protein composed of an isotype control monoclonal antibody that recognizes a non-mammalian antigen (keyhole limpet hemocyanin, KLH) fused to the C-terminus of hIL-2 F42K, Y45R, V69R, an IL-2 variant with reduced efficacy, via a flexible 6-amino acid glycine / serine linker.

[0433] The results are presented in Figure 19. The MC38 colorectal tumor model responds particularly well to antibody-mediated PD-1 receptor inhibition. While growing tumors in vehicle-treated mice rapidly reached the study endpoint, 50% of the mice treated with anti-mPD-1 RMP1-14 experienced complete tumor regression. In contrast, 100% of the mice treated with the anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, V69R fusion protein experienced sustained and long-term tumor regression. Mice treated with various combinations of individual components of the anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, V69R fusion protein, including anti-mPD-1 RMP1-14 combined with hIL-2 free cytokines (administered at doses and regimens equivalent to therapeutic doses in humans) or anti-mPD-1 RMP1-14 combined with untargeted anti-KLH-hIL-2 F42K, Y45R, V69R fusion proteins, failed to replicate the efficacy observed with anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, V69R. The above data demonstrate that targeting reduced-efficacy hIL-2 to PD-1-expressing cells significantly improves anti-tumor efficacy compared to anti-PD-1 receptor antagonists, and that the activity of the fusion protein is not due to the additive effect of the individual components of the molecule.

[0434] Example 24: Evaluation of protective anti-tumor immunity induced by surrogates anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, V69R in an MC38 colorectal tumor model

[0435] A second tumor challenge was performed without any additional drug therapy on mice that showed complete tumor regression in the primary tumor study described in Example 23 and survived until day 50. For the tumor re-challenge, 5 x 10 were placed on the left flank of the mice, opposite the location of the primary tumor. 5 Canine MC38 tumor cells were transplanted. As a control group, MC38 tumor cells were also transplanted into 10 age-matched tumor naive mice.

[0436] Figure 20 shows that following treatment with anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, and V69R, all mice that showed complete tumor regression in the preceding primary tumor study and survived until day 50 were completely protected from secondary tumor development. In contrast, in all tumor-naive mice transplanted with MC38 tumor cells, tumors began to develop and rapidly reached the study endpoint of a tumor volume of 100 mm³. The development of protective anti-tumor immunity without continued drug therapy suggests that anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, and V69R induced an anti-tumor memory T cell response.

[0437] Example 25: Evaluation of the suitability of a combination therapy using surrogate anti-mPD-1 RMP1-30-hIL-2 F42K, Y45R, V69R and anti-mPD-1 RMP1-14 antibodies in a mouse MC38 colorectal tumor model

[0438] To evaluate the suitability of the disclosed immunoconjugate for use in combination therapy with an antagonistic anti-PD-1 antibody, a surrogate antagonist anti-mouse PD-1 antibody and a surrogate immunoconjugate were used, as the antagonistic anti-human PD-1 antibody does not bind to mouse PD-1 and the modified IL-2 protein does not act on the mouse IL-2 receptor. 5 x 10⁶ 10⁶ female C57BL / 6NCrl mice were tested. 5Canine synonyms for MC38 colorectal carcinoma cells were injected into the right flank. When the tumor reached 80–120 mm³, mice were classified into cohorts (10 mice / group), and treatment was initiated on Day 1 of the study. Starting on Day 1, all agents were administered intraperitoneally once a week for 4 weeks. Anti-mPD-1 RMP1-30-hIL-2 F42K, Y45R, and V69R were administered at 1 mg / kg, and anti-mPD-1 RMP1-14 was administered at 2 mg / kg. Tumor size was measured with calipers twice a week during the study period. The endpoint was reached when the average tumor weight in the vehicle-treated group reached 1,500 mm³ or on Day 45, whichever came first. Sampling for the endpoint was performed over two days due to the large number of mice to be sampled.

[0439] Both test agents were generated using a mouse IgG2b Fc region having a single N297A amino acid substitution at position 297, which prevents glycosylation of the Fc region and significantly reduces any Fc region-mediated immune effector function, thereby hindering cell depletion in vivo. Anti-mPD-1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (reference [Matsumoto, J Immunol 172: 2530-2541, 2004]). Anti-mPD-1 RMP1-30-hIL-2 F42K / Y45R / V69R is a bifunctional fusion protein composed of a monoclonal RMP1-30 antibody non-antagonist of the mouse PD-1 receptor fused to the C-terminus of hIL-2 F42K / Y45R / V69R (SEQ No. 621), an IL-2 variant with reduced efficacy against the mouse IL-2 receptor, via a flexible 6-amino acid glycine / serine linker. The molecule was designed to directly target the reduced-efficacy hIL-2 variant to PD-1-expressing T cells in vivo in mice.

[0440] The results are presented in Figure 21. Tumors grown in vehicle-treated mice reached the study endpoint on day 25. In contrast, partial tumor regression was observed in mice treated with anti-mPD-1 RMP1-14 or anti-mPD-1 RMP1-30-hIL-2 F42K / Y45R / V69R, and superior results were observed in the anti-mPD-1 RMP1-30-hIL-2 F42K / Y45R / V69R group. The most effective reduction in tumor volume was observed in the combination group. The data demonstrated that neither antibody interfered with the PD-1 binding ability of the other antibody, and that higher efficacy could be achieved when administered in combination compared to when each agent was administered alone.

[0441] The data presented herein, combined with the data demonstrating that the disclosed immunoconjugate binds to PD-1 in the presence of an antagonistic anti-PD-1 antibody, and the data demonstrating that the disclosed immunoconjugate is designed to deliver a modified hIL-2 protein to the IL-2 receptor rather than antagonizing PD-1, demonstrate that the disclosed immunoconjugate and the antagonistic anti-PD-1 antibody are effective for use as a combination therapy to reduce tumor volume.

[0442] Example 26: Combination therapy with an anti-human PD-1 (hPD-1) antibody-modified human interleukin-2 (hIL-2) immunoconjugate and an antagonistic anti-PD-1 antibody

[0443] The data from Example 25 above demonstrate that superior therapeutic efficacy can be achieved by combining an anti-hPD-1 antibody-modified hIL-2 immunoconjugate with an antagonistic anti-PD-1 antibody. A single-dose study for preliminary safety evaluation will be performed in cynomolgus monkeys using 1) the initiated immunoconjugate in the presence and absence of nivolumab; and 2) the initiated immunoconjugate in the presence and absence of pembrolizumab. Sampling will be performed at time points up to 360 hours after infusion. Adverse events, total toxicity, body weight loss, or lethality will be evaluated. Subsequent studies will be performed using higher doses of the immunoconjugate and / or the antagonistic anti-PD-1 antibody.

[0444] Those skilled in the art will understand that numerous changes and variations may be made to the preferred embodiments disclosed herein, and that such changes and variations may be made without departing from the spirit of the invention. Accordingly, the appended claims are intended to encompass all such equivalent variations within the true spirit and scope of the invention.

[0445] The full text of each patent, patent application, and publication disclosed in or described herein is incorporated herein by reference.

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[0540] The following list of embodiments is intended to replace, or rather supplement, the preceding description.

[0541] Embodiment 1. A method for treating cancer in a subject, wherein the present method is to a subject

[0542] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0543] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ NO. 345; and

[0544] An anti-hPD-1 antibody that immunospecifically binds to human PD-1 (hPD-1), or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0545] (i) heavy chain complementarity determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 420, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 421, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 423;

[0546] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0547] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0548] (iv) an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411, or an antigen-binding fragment thereof

[0549] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0550] (B) Antagonistic anti-PD-1 antibody binding to PD-1 in the presence of an immune conjugate

[0551] A method comprising the step of administering

[0552] Embodiment 2. The method of Embodiment 1, wherein the antagonistic anti-PD-1 antibody is nivolumab, pembrolizumab, cemiflimab, dostarlimab, or retipanlimab.

[0553] Embodiment 3. The method of Embodiment 1 or 2, wherein the modified hIL-2 protein comprises any one of the amino acid sequences of SEQ ID NOs 149, 307, 607-611, 614, 617, or 620.

[0554] Embodiment 4. A method in which, in any one of the above embodiments, the modified hIL-2 protein comprises a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0555] Embodiment 5. The method of Embodiment 4, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 149.

[0556] Embodiment 6. A method in which, in any one of the above embodiments, the modified hIL-2 protein additionally comprises a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0557] Embodiment 7. The method of Embodiment 6, wherein the substitution of amino acid position 3 of the modified hIL-2 protein is T3A.

[0558] Embodiment 8. The method of Embodiment 7, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 216.

[0559] Embodiment 9. The method of Embodiment 6, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 218.

[0560] Embodiment 10. A method in which, in any one of the above embodiments, the modified hIL-2 protein additionally comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0561] Embodiment 11. The method of Embodiment 10, wherein the substitution at amino acid position 125 is C125A.

[0562] Embodiment 12. The method of Embodiment 11, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 215, 217, or 219.

[0563] Embodiment 13. The method of Embodiment 12, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 217.

[0564] Embodiment 14. A method in which, in any one of the above embodiments, the modified hIL-2 protein is fused to the anti-hPD-1 antibody of the immune conjugate or the portion thereof containing the antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

[0565] Embodiment 15. A method in which, in any one of the above embodiments, the modified hIL-2 protein is directly fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by a peptide bond.

[0566] Embodiment 16. The method of Embodiment 15, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by a peptide bond.

[0567] Embodiment 17. A method in any one of embodiments 1-14 in which the modified hIL-2 protein is fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of an immune conjugate through a linker.

[0568] Embodiment 18. In any one of the above embodiments, the portion of the anti-hPD-1 antibody or its antigen-binding fragment of the immune conjugate

[0569] a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 417;

[0570] b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 385;

[0571] c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 395; or

[0572] d) A method comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 405.

[0573] Embodiment 19. A method in which, in any one of the above embodiments, the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises an IgG1 heavy chain constant region.

[0574] Embodiment 20. The method of Embodiment 19, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises L235A substitution and G237A substitution according to EU numbering.

[0575] Embodiment 21. In any one of the above embodiments, the portion of the anti-hPD-1 antibody or its antigen-binding fragment of the immune conjugate

[0576] a) a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415;

[0577] b) a heavy chain comprising the amino acid sequence of SEQ ID NO. 424 and a light chain comprising the amino acid sequence of SEQ ID NO. 425;

[0578] c) a heavy chain comprising the amino acid sequence of SEQ ID NO. 426 and a light chain comprising the amino acid sequence of SEQ ID NO. 427; or

[0579] d) A method comprising a heavy chain having the amino acid sequence of SEQ ID NO. 428 and a light chain having the amino acid sequence of SEQ ID NO. 429.

[0580] Embodiment 22. The method of Embodiment 21, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415.

[0581] Embodiment 23. In any one of the above embodiments, the immune conjugate

[0582] A light chain comprising the amino acid sequence of SEQ ID NO. 415; and

[0583] A method comprising a heavy chain modified hIL-2 protein fusion comprising the amino acid sequence of SEQ ID NO. 532.

[0584] Embodiment 24. A method in which, in any one of the above embodiments, an antagonistic anti-PD-1 antibody and an immunoconjugate are administered to a subject together as a mixture, together as a single agent, or sequentially as a single agent in any order.

[0585] Embodiment 25. In Embodiment 24,

[0586] A step of administering an immunoconjugate before administering an antagonistic anti-PD-1 antibody;

[0587] A step of administering an antagonistic anti-PD-1 antibody before administering an immune conjugate; or

[0588] A method comprising the step of administering an immune conjugate at substantially the same time as administering an antagonistic anti-PD-1 antibody.

[0589] Embodiment 26. A method in which, in any one of the above embodiments, the cancer is melanoma, Merkel cell carcinoma, non-small cell lung carcinoma, renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, or a high-frequency microsatellite instability tumor or a DNA mismatch repair deficiency tumor.

[0590] Embodiment 27. A method for treating renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors in a subject, wherein the present method comprises: to a subject

[0591] As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0592] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ NO. 345; and

[0593] An anti-hPD-1 antibody that immunospecifically binds to human PD-1 (hPD-1), or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0594] (i) heavy chain complementarity determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 420, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 421, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 423;

[0595] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0596] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0597] (iv) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO. 406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 408, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 409, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 411

[0598] An anti-hPD-1 antibody containing, or its antigen-binding fragment

[0599] anti-hPD-1 antibody modified hIL-2 immunoconjugate containing

[0600] A method comprising the step of administering

[0601] Embodiment 28. The method of Embodiment 27, wherein the modified hIL-2 protein comprises any one of the amino acid sequences of SEQ ID NOs 149, 307, 607-611, 614, 617, or 620.

[0602] Embodiment 29. The method of Embodiment 27 or 28, wherein the modified hIL-2 protein comprises a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0603] Embodiment 30. The method of Embodiment 29, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 149.

[0604] Embodiment 31. A method in which, in any one of embodiments 27-30, the modified hIL-2 protein additionally comprises a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0605] Embodiment 32. The method of Embodiment 31, wherein the substitution of amino acid position 3 of the modified hIL-2 protein is T3A.

[0606] Embodiment 33. The method of Embodiment 32, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 216.

[0607] Embodiment 34. The method of Embodiment 31, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 218.

[0608] Embodiment 35. A method in which, in any one of embodiments 27-34, the modified hIL-2 protein additionally comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345.

[0609] Embodiment 36. The method of Embodiment 35, wherein the substitution at amino acid position 125 is C125A.

[0610] Embodiment 37. The method of Embodiment 36, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 215, 217, or 219.

[0611] Embodiment 38. The method of Embodiment 37, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 217.

[0612] Embodiment 39. A method in which, in any one of embodiments 27-38, the modified hIL-2 protein is fused to the anti-hPD-1 antibody of an immune conjugate or the portion thereof containing the antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

[0613] Embodiment 40. A method in any one of embodiments 27-39 in which the modified hIL-2 protein is directly fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by a peptide bond.

[0614] Embodiment 41. The method of Embodiment 40, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by a peptide bond.

[0615] Embodiment 42. A method in which, in any one of embodiments 27-39, the modified hIL-2 protein is fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of an immune conjugate through a linker.

[0616] Embodiment 43. In any one of embodiments 27-42, the portion of the anti-hPD-1 antibody or its antigen-binding fragment of the immune conjugate

[0617] a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 417;

[0618] b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 385;

[0619] c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 395; or

[0620] d) A method comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 405.

[0621] Embodiment 44. A method in any one of embodiments 27-43, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises an IgG1 heavy chain constant region.

[0622] Embodiment 45. The method of Embodiment 44, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises L235A substitution and G237A substitution according to EU numbering.

[0623] Embodiment 46. In any one of embodiments 27-45, the portion of the anti-hPD-1 antibody or its antigen-binding fragment of the immune conjugate

[0624] a) a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415;

[0625] b) a heavy chain comprising the amino acid sequence of SEQ ID NO. 424 and a light chain comprising the amino acid sequence of SEQ ID NO. 425;

[0626] c) a heavy chain comprising the amino acid sequence of SEQ ID NO. 426 and a light chain comprising the amino acid sequence of SEQ ID NO. 427; or

[0627] d) A method comprising a heavy chain having the amino acid sequence of SEQ ID NO. 428 and a light chain having the amino acid sequence of SEQ ID NO. 429.

[0628] Embodiment 47. The method of Embodiment 46, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415.

[0629] Embodiment 48. In any one of embodiments 27-47, the immune conjugate

[0630] A light chain comprising the amino acid sequence of SEQ ID NO. 415; and

[0631] A method comprising a heavy chain modified hIL-2 protein fusion comprising the amino acid sequence of SEQ ID NO. 532.

[0632] Embodiment 49. For cancer treatment,

[0633] (A) As an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate,

[0634] A modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ NO. 345; and

[0635] As an anti-hPD-1 antibody that immunospecifically binds to hPD-1, or as its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is

[0636] (i) heavy chain complementarity determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO. 418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 420, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 421, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 423;

[0637] (ii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 391;

[0638] (iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or

[0639] (iv) heavy chain CDR1 containing the amino acid sequence of SEQ ID NO. 406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO. 407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 408, light chain CDR1 containing the amino acid sequence of SEQ ID NO. 409, light chain CDR2 containing the amino acid sequence of SEQ ID NO. 410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO. 411

[0640] An anti-hPD-1 antibody containing, or its antigen-binding fragment

[0641] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising; and

[0642] (B) Use of an antagonistic anti-PD-1 antibody that binds to PD-1 in the presence of an immune conjugate.

Claims

Claim 1 As a method for treating cancer in a subject, (A) an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate comprising a modified hIL-2 protein comprising a substitution of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ ID NO. 345 and a substitution of R38E at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; An anti-hPD-1 antibody that is immune-specifically bound to human PD-1 (hPD-1), or an antigen-binding fragment thereof, comprising: (i) a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423; (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and an amino acid sequence of SEQ ID NO. 391 (iii) a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 396, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 397, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 398, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 399, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 400, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 401;or (iv) an anti-hPD-1 antibody modified hIL-2 immunoconjugate comprising an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411, or an antigen-binding fragment thereof; and (B) a step comprising administering an antagonistic anti-PD-1 antibody that binds to PD-1 to a subject in the presence of the immunoconjugate.; Claim 2 In claim 1, the antagonistic anti-PD-1 antibody is nivolumab, pembrolizumab, cemiflimab, dostarlimab, or retipanlimab. Claim 3 The method of claim 1 or 2, wherein the modified hIL-2 protein comprises any one of the amino acid sequences of SEQ ID NOs 149, 307, 607-611, 614, 617, or 620. Claim 4 A method according to any one of claims 1 to 3, wherein the modified hIL-2 protein comprises a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO.

345. Claim 5 A method according to claim 4, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

149. Claim 6 A method according to any one of claims 1 to 5, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO.

345. Claim 7 In claim 6, the method in which the substitution at amino acid position 3 of the modified hIL-2 protein is T3A. Claim 8 A method according to claim 7, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

216. Claim 9 A method according to claim 6, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

218. Claim 10 A method according to any one of claims 1 to 9, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO.

345. Claim 11 In paragraph 10, the method in which the substitution at amino acid position 125 is C125A. Claim 12 The method of claim 11, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 215, 217, or 219. Claim 13 A method according to claim 12, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

217. Claim 14 A method according to any one of claims 1 to 13, wherein the modified hIL-2 protein is fused to the anti-hPD-1 antibody of the immune conjugate or the portion thereof of the antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. Claim 15 A method according to any one of claims 1 to 14, wherein the modified hIL-2 protein is directly fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by peptide bonding. Claim 16 In paragraph 15, the modified hIL-2 protein is fused directly to the C-terminal amino acid residue of the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by a peptide bond. Claim 17 A method according to any one of claims 1 to 14, wherein the modified hIL-2 protein is fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of an immune conjugate through a linker. Claim 18 A method according to any one of claims 1 to 17, wherein the anti-hPD-1 antibody or the antigen-binding fragment portion of the immune conjugate comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 417; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 385; c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 395; or d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO.

405. Claim 19 A method according to any one of claims 1 to 18, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises an IgG1 heavy chain constant region. Claim 20 A method according to claim 19, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises L235A substitution and G237A substitution according to EU numbering. Claim 21 A method according to any one of claims 1 to 20, wherein the portion of the anti-hPD-1 antibody or its antigen-binding fragment of the immune conjugate comprises: a) a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415; b) a heavy chain comprising the amino acid sequence of SEQ ID NO. 424 and a light chain comprising the amino acid sequence of SEQ ID NO. 425; c) a heavy chain comprising the amino acid sequence of SEQ ID NO. 426 and a light chain comprising the amino acid sequence of SEQ ID NO. 427; or d) a heavy chain comprising the amino acid sequence of SEQ ID NO. 428 and a light chain comprising the amino acid sequence of SEQ ID NO.

429. Claim 22 A method according to claim 21, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO.

415. Claim 23 A method according to any one of claims 1 to 22, wherein the immunoconjugate comprises a light chain having the amino acid sequence of SEQ ID NO. 415; and a heavy chain modified hIL-2 protein fusion having the amino acid sequence of SEQ ID NO.

532. Claim 24 A method according to any one of claims 1 to 23, wherein the antagonistic anti-PD-1 antibody and the immunoconjugate are administered to a subject together as a mixture, together as a single agent, or sequentially as a single agent in any order. Claim 25 The method of claim 24 comprises the step of administering an immunoconjugate before administering an antagonistic anti-PD-1 antibody; the step of administering an antagonistic anti-PD-1 antibody before administering the immunoconjugate; or the step of administering the immunoconjugate at substantially the same time as administering the antagonistic anti-PD-1 antibody. Claim 26 A method according to any one of claims 1 to 25, wherein the cancer is melanoma, Merkel cell carcinoma, non-small cell lung carcinoma, renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, or high-frequency microsatellite instability tumor or DNA mismatch repair deficiency tumor. Claim 27 A method for treating renal cell carcinoma, triple-negative breast cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma, hepatocellular carcinoma, or high-frequency microsatellite instability tumors or DNA mismatch repair deficiency tumors in subjects, comprising: an anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate comprising a modified hIL-2 protein comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ ID NO. 345 and an R38E substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; An anti-hPD-1 antibody that is immune-specifically bound to human PD-1 (hPD-1), or an antigen-binding fragment thereof, comprising: (i) a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 423; (ii) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 390, and an amino acid sequence of SEQ ID NO. 391 Light chain CDR3 containing;(iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401; or (iv) a method comprising the step of administering to a subject an anti-hPD-1 antibody modified hIL-2 immunoconjugate comprising an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411, or an antigen-binding fragment thereof.; Claim 28 The method of claim 27, wherein the modified hIL-2 protein comprises any one of the amino acid sequences of SEQ ID NOs 149, 307, 607-611, 614, 617, or 620. Claim 29 A method according to claim 27 or 28, wherein the modified hIL-2 protein comprises a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345 and an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO.

345. Claim 30 A method according to claim 29, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

149. Claim 31 A method according to any one of claims 27 to 30, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO.

345. Claim 32 In paragraph 31, the method in which the substitution at amino acid position 3 of the modified hIL-2 protein is T3A. Claim 33 A method according to claim 32, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

216. Claim 34 A method according to claim 31, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

218. Claim 35 A method according to any one of claims 27 to 34, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO.

345. Claim 36 In paragraph 35, the method in which the substitution at amino acid position 125 is C125A. Claim 37 A method according to claim 36, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO. 215, 217, or 219. Claim 38 In claim 37, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO.

217. Claim 39 A method according to any one of claims 27 to 38, wherein the modified hIL-2 protein is fused to the anti-hPD-1 antibody of the immune conjugate or the portion thereof of the antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. Claim 40 A method according to any one of claims 27 to 39, wherein the modified hIL-2 protein is directly fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by peptide bonding. Claim 41 In paragraph 40, the modified hIL-2 protein is fused directly to the C-terminal amino acid residue of the anti-hPD-1 antibody or its antigen-binding fragment portion of the immune conjugate by a peptide bond. Claim 42 A method according to any one of claims 27 to 39, wherein the modified hIL-2 protein is fused to the anti-hPD-1 antibody or its antigen-binding fragment portion of an immune conjugate through a linker. Claim 43 A method according to any one of claims 27 to 42, wherein the anti-hPD-1 antibody or the antigen-binding fragment portion of the immunoconjugate comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 417; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 385; c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 395; or d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO.

405. Claim 44 A method according to any one of claims 27 to 43, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises an IgG1 heavy chain constant region. Claim 45 A method according to claim 44, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises L235A substitution and G237A substitution according to EU numbering. Claim 46 A method according to any one of claims 27 to 45, wherein the portion of the anti-hPD-1 antibody or its antigen-binding fragment of the immune conjugate comprises: a) a heavy chain comprising the amino acid sequence of SEQ ID NO. 414 and a light chain comprising the amino acid sequence of SEQ ID NO. 415; b) a heavy chain comprising the amino acid sequence of SEQ ID NO. 424 and a light chain comprising the amino acid sequence of SEQ ID NO. 425; c) a heavy chain comprising the amino acid sequence of SEQ ID NO. 426 and a light chain comprising the amino acid sequence of SEQ ID NO. 427; or d) a heavy chain comprising the amino acid sequence of SEQ ID NO. 428 and a light chain comprising the amino acid sequence of SEQ ID NO.

429. Claim 47 A method according to claim 46, wherein the anti-hPD-1 antibody of the immune conjugate or the antigen-binding fragment portion thereof comprises a heavy chain having the amino acid sequence of SEQ ID NO. 414 and a light chain having the amino acid sequence of SEQ ID NO.

415. Claim 48 A method according to any one of claims 27 to 47, wherein the immunoconjugate comprises a light chain having the amino acid sequence of SEQ ID NO. 415; and a heavy chain modified hIL-2 protein fusion having the amino acid sequence of SEQ ID NO.

532. Claim 49 (A) An anti-human PD-1 (hPD-1) antibody modified human interleukin-2 (hIL-2) immunoconjugate comprising a modified hIL-2 protein comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20 relative to the unmodified human interleukin-2 (hIL-2) amino acid sequence of SEQ ID NO. 345 and an R38E substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO. 345; An anti-hPD-1 antibody that binds immunospecifically to hPD-1, or an antigen-binding fragment thereof, comprising: (i) a heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO. 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO. 423; (ii) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO. 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO. 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO. 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO. 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO. 390, and a light chain comprising the amino acid sequence of SEQ ID NO. 391 CDR3;(iii) heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 396, heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 397, heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 398, light chain CDR1 having the amino acid sequence of SEQ ID NO. 399, light chain CDR2 having the amino acid sequence of SEQ ID NO. 400, and light chain CDR3 having the amino acid sequence of SEQ ID NO. 401;or (iv) an anti-hPD-1 antibody modified hIL-2 immunoconjugate comprising an anti-hPD-1 antibody comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO. 406, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO. 407, a heavy chain CDR3 having the amino acid sequence of SEQ ID NO. 408, a light chain CDR1 having the amino acid sequence of SEQ ID NO. 409, a light chain CDR2 having the amino acid sequence of SEQ ID NO. 410, and a light chain CDR3 having the amino acid sequence of SEQ ID NO. 411, or an antigen-binding fragment thereof; and (B) use of an antagonistic anti-PD-1 antibody that binds to PD-1 in the presence of an immunoconjugate for the treatment of cancer.;

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