Uses of attenuated IL-2 immunoconjugates

Abstract

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

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 612,007, filed December 19, 2023, the disclosure of which is hereby incorporated by reference in its entirety. sequence list

[0002] This application contains a sequence list that has been submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy was created on December 17, 2024, named 102085.002049-BIO040-WO01_SequenceListing.xml, and is 697 kilobytes in size. Technical Field

[0003] This article discloses a method for treating cancer in subjects with either 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

[0004] Over the past decade, immuno-oncology therapies have achieved remarkable efficacy in treating cancer. The approval of immunotherapies such as immune checkpoint inhibitors, adoptive cell therapy, and cancer vaccines has revolutionized cancer treatment. These developments have altered the standard of care (SOC) and improved survival rates for several tumor types. However, while immune checkpoint inhibitors have improved clinical outcomes across multiple tumor types, only a fraction of patients exhibit clinical response, and a large number of responders develop acquired resistance after the initial response.

[0005] The efficacy of immune checkpoint inhibitors, such as PD-1 antagonists, requires patients to have a fully functional immune system and a sufficient number of immune cells. Patients who do not respond to PD-1 antagonists typically exhibit low tumor T-cell infiltration and poor proliferative T-cell responses to PD-1 antagonism.

[0006] Human IL-2 (hIL-2) is a type I four-alpha helix, a glycosylated cytokine produced by CD4+ T cells and CD8+ T cells. Autocrine and paracrine IL-2 signaling occurs via conjugation of a high-affinity trimeric receptor complex containing IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132), or a medium-affinity dimeric receptor complex containing IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-2 exhibits dual, pleiotropic effects, stimulating both T cell proliferation to produce T cell effectors, T cell memory, and activated NK cells, and 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, while 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 a cancer immunotherapy is limited by off-target effects and toxicity associated with IL-2 administration. Summary of the Invention

[0007] This article discloses a method for treating cancer in a subject, the method comprising administering the following to the subject:

[0008] (A) An immunoconjugate modified with an anti-human PD-1 (hPD-1) antibody, said immunoconjugate comprising:

[0009] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0010] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0011] (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO: 421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO: 422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO: 423;

[0012] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0013] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0014] (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; and

[0015] (B) Anti-PD-1 antibody that antagonizes PD-1 in the presence of the said immunoconjugate.

[0016] This article discloses a method for treating subjects with renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or tumors with high microsatellite instability or DNA mismatch repair defects, the method comprising administering the following to the subjects:

[0017] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0018] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0019] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0020] (i) Heavy chain 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;

[0021] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0022] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0023] (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.

[0024] This article also discloses the uses of the following items:

[0025] (A) An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0026] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0027] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0028] (i) Heavy chain 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;

[0029] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0030] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0031] (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; and

[0032] (B) Anti-PD-1 antibodies that antagonize PD-1 in the presence of the aforementioned immunoconjugate

[0033] It is used to treat cancer.

[0034] The use of an anti-hPD-1 antibody-modified hIL-2 immunoconjugate is also disclosed, the immunoconjugate comprising:

[0035] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0036] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0037] (i) Heavy chain 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;

[0038] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0039] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0040] (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.

[0041] It is used to treat renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or tumors with high microsatellite instability or those with DNA mismatch repair defects. Attached Figure Description

[0042] The invention and the following detailed description will be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of the disclosed methods and uses are shown in the drawings for the purpose of illustrating the methods and uses; however, the methods and uses are not limited to the specific embodiments disclosed. In the drawings:

[0043] Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H illustrate exemplary antibody-hIL-2 immunoconjugates as described in Example 1 of this document. Unattenuated human IL-2 cytokines (gray rectangles) are fused directly (df) or via an L6 linker (L6) to the N-terminus or C-terminus of either the heavy chain or the two κ light chains of the antibody.

[0044] Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H illustrate exemplary antibody-hIL-2 immunoconjugates having an hCD25(1-164) extracellular domain designed to interfere with the binding of hIL-2 to human IL-2Rα. For the N-terminal variant, the human CD25 / IL-2Rα extracellular domain (black triangle) is fused to unattenuated hIL-2 cytokines (gray rectangles) via an L20 linker (light gray line). The unattenuated hIL-2 cytokines are then fused directly to the antibody (df) or via an L6 linker. For the C-terminal variant, the hCD25 / IL-2Rα extracellular domain is partially fused directly to the antibody (df) or via an L6 linker, followed by an L20 linker and unattenuated hIL-2 cytokines.

[0045] Figure 3 An exemplary 1H3-hIgG1-L6-hIL-2 immunoconjugate containing the CD25 / IL-2Rα extracellular domain portion is shown. The hCD25 / IL-2Rα extracellular domain portion is fused to 1H3-hIgG1-L6-hIL-2 at the C-terminus of each heavy chain via an L6 linker, followed by an L20 linker and a substituted hIL-2 cytokine portion (attenuated hIL-2) as described in Example 2, which is intended to regulate binding to CD122 / IL-2Rβ.

[0046] Figure 4A , Figure 4B , Figure 4C and Figure 4DThe results show the experimental results of analyzing the binding of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 to human PD-1 receptor on 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.

[0047] Figure 5 Exemplary anti-hPD-1-attenuated hIL-2 immunoconjugates with an L6 linker (L6) (left) or direct fusion (df) (right) are shown. An anti-hPD-1 antibody containing an hIgG4 or hIgG1 Fc domain (with or without L235E (LE) or L235A / G237A (LAGA) modification in the Fc domain) is fused to an attenuated hIL-2 cytokine at the C-terminus of the antibody heavy chain. Various substitutions are introduced into the hIL-2 cytokine to enhance attenuation potency.

[0048] Figure 6A and Figure 6B The results of the competitive assay are shown, indicating that in the presence of a saturated concentration of anti-hPD-1-attenuated hIL-2 immunoconjugate (280 nM), anti-hPD-1 #1-mIgG2b-N297A ( Figure 6A ) or anti-hPD-1 #2-mIgG2b-N297A ( Figure 6B It binds to the anti-hPD-1 receptor on Jurkat cells.

[0049] Figure 7 Results of the competitive assay are shown, demonstrating that the anti-hPD-1-attenuation 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 when using the PD-1 / PD-L1 blocking bioassay.

[0050] Figure 8The results of experiments analyzing the effects of administration of the mediator, alternative anti-PD-1 antibodies (anti-mPD-1 RMP1-14 mIgG2b-N297A and anti-mPD-1 RMP1-30 mIgG2b-N297A), and alternative anti-PD-1-attenuated hIL-2 immunoconjugates (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 homologous tumors in C57BL / 6 mice are presented as described in Example 18. The test agents were administered intraperitoneally at a dose of 5 mg / kg twice weekly for 4 weeks, starting on day 1. The dots in the graph represent the average tumor volume of 10 mice in each group.

[0051] Figure 9A , Figure 9B and Figure 9C The results of a study conducted to determine the efficacy of an alternative anti-hPD-1-attenuating hIL-2 immunoconjugate, anti-mPD-1RMP1-30 mIgG2b-N297A-hIL-2 (F42K / Y45R / V69R), in an MC38 mouse colon adenocarcinoma model are presented. Figure 9A The average subcutaneous tumor volume (mm²) measured every 3–4 days over 8 days following the first administration of the test drug (administered three times at 5 mg / kg on days 1, 4, and 8) is depicted. 3 The tumor growth curves represent an average of 15 animals per group. Figure 9B The results of tumor immunophenotyping by flow cytometry on day 9 were summarized, showing different CD8... + T cell subsets account for a significant portion of total CD8+. + The proportion of the mean absolute count of T cells. Figure 9C The immunophenotyping results on day 9 were presented, indicating that CD8+ decreased after exposure to the alternative immunoconjugate. + T effector memory was significantly amplified, and the number of regulatory T cells (cells / μL) in tumors tended to decrease.

[0052] Figure 10 The analysis of NOD-Prkdc exposed to hIL-2 immunoconjugates with anti-hPD-1 attenuation is shown. em26Cd52 IL-2rg em26Cd22 Experimental results demonstrating accelerated graft-versus-host disease in NjuCrl (NCG) mice, as evidenced by significant weight loss in the NCG-PBMC model.

[0053] Figure 11A and Figure 11BThe analysis shows NOD-Prkdc treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) in the NCG-PBMC model. em26Cd52 IL-2rg em26Cd22 / NjuCrl (NCG) mouse blood CD8 + Effector memory T cells ( Figure 11A ) and CD4 + Effector memory T cells ( Figure 11B Experimental results of dose-dependent expansion of cells / mL.

[0054] Figure 12 This illustrates NOD-Prkdc treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) in the NCG-PBMC model. em26Cd52 IL-2rg em26Cd22 / NjuCrl (NCG) mice showed a decrease in the number of regulatory T cells per mL of blood.

[0055] Figure 13A and Figure 13B This indicates that, 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, H7-632–hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) (named "H7-767") Figure 13B It continues to bind to the human PD-1 receptor on Jurkat cells.

[0056] Figure 14A and Figure 14B This demonstrates the binding of recombinant human PD-1 captured by the H7-767 immobilized to the CM5 sensor chip, as evaluated by surface plasmon resonance (SPR). (The following is...) Figure 14A H7-767, KEYTRUDA® or OPDIVO® or ( Figure 14B A diagram showing the combination of PD-L1 or PD-L2.

[0057] Figure 15 The results indicate that, using the hPD-1 / hPD-L1 blocking bioassay, H7-632-hIgG1-LAGA and H7-767 do not inhibit the binding of human PD-L1 to the human PD-1 receptor.

[0058] Figure 16A , Figure 16B , Figure 16C and Figure 16DThis is a graph showing the binding of the anti-hPD-1-degraded 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 human PD-1 receptors on 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 the anti-hPD-1-degraded hIL-2 immunoconjugates, as assessed by flow cytometry.

[0059] Figure 17 The figure shows 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 recombinant HEK-293T cells expressing cynomolgus monkey PD-1, as assessed by flow cytometry.

[0060] Figure 18A , Figure 18B , Figure 18C and Figure 18D The antagonistic activity of the 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 was demonstrated. Figure 18A and Figure 18B The titration results 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 are shown. Figure 18C and Figure 18D This demonstrates the use of a fixed concentration of 100 nM antihPD-1 #1 ( Figure 18C ) or 100 nM antihPD-1 #2 ( Figure 18D The results of the opposite experiment on the titration of hIL-2 immunoconjugates that counteract hPD-1-attenuation.

[0061] Figure 19The effects of administration of various test agents (including alternative anti-mouse PD-1 / attenuated IL-2 immunoconjugates) on the growth of established subcutaneous MC38 homologous tumors in C57BL / 6 mice are shown. Each growth curve represents the mean tumor volume of ten mice in each treatment group.

[0062] Figure 20 It shows the relationship with Figure 19 The demonstrated ability of MC38 tumor cells to grow in tumor-naïve mice, compared to mice in a primary tumor study previously treated with anti-mPD-1-hIL-2 F42K / Y45R / V69R, and the established primary tumors showed complete long-term regression. Animals from both groups (n=10 per group) underwent subcutaneous implantation of 5 × 10⁶ MC38 tumor cells in the left ventricular region contralateral to the primary tumor site. 5 MC38 tumor cells. Mice previously exposed to the alternative anti-mPD-1-hIL-2 F42K / Y45R / V69R did not show tumor growth because they had developed sustained immunity, while the corresponding original mouse controls showed typical tumor growth on their flanks.

[0063] Figure 21 The changes 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 are shown. Detailed Implementation

[0064] The disclosed methods and uses can be more readily understood by referring to the following detailed description of the accompanying drawings, which form a part of this disclosure. It should be understood that the disclosed methods and uses are not limited to the specific methods and uses described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed methods and uses.

[0065] Unless otherwise specifically stated, any description of possible mechanisms or modes of action or reasons for improvement is intended to be illustrative only, and the disclosed methods and uses are not subject to the correctness or incorrectness of any such suggested mechanisms or modes of action or reasons for improvement.

[0066] In this document, the description relates to treatment methods and uses. Where features or embodiments associated with a treatment method are described or claimed in this disclosure, such features or embodiments also apply to the use. Similarly, where features or embodiments associated with a use are described or claimed in this disclosure, such features or embodiments also apply to the treatment method. Uses included within the scope of this disclosure include, but are not limited to, Swiss-style use, first medical use, second / additional medical use, and use according to EPC2000.

[0067] When a numerical range is enumerated or established herein, the range includes its endpoints and all individual integers and fractions within the range, and also includes each of the various possible combinations of endpoints and internal integers and fractions of a subgroup forming a larger group of values ​​within the range, to the same extent as each narrower range is explicitly enumerated. Where a numerical range is stated herein as being greater than the stated value, the range remains finite, and its upper limit is defined by values ​​operable within the context of this disclosure. Where a numerical range is stated herein as being less than the stated value, the lower limit of the range remains defined by non-zero values. When defining a range, it is not intended to limit the scope of methods and uses to the specific values ​​enumerated. All ranges are inclusive and composable.

[0068] Similarly, when a value is expressed as an approximation using the antecedent "about," it will be understood that the specific value forms another embodiment. Unless the context explicitly specifies otherwise, references to a specific numerical value include at least that specific value. The term "about" is used when referring to a range of numbers, a threshold, or a specific value to indicate that the listed value may differ from the stated value by up to 10%. Thus, the term "about" is used to cover variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the specified value.

[0069] It should be understood that, for clarity, certain features of the disclosed methods and uses described herein in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed methods and uses described in the context of a single embodiment may also be provided individually or in any sub-combination.

[0070] As used herein, the singular forms “a”, “an”, and “the” include the plural.

[0071] Various terms are used throughout the specification and claims in connection with the described aspects. Unless otherwise stated, such terms have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0072] The term “comprising” is intended to include instances covered by the terms “substantially consisting of” and “consisting of”; similarly, the term “substantially consisting of” is intended to include instances covered by the term “consisting of”.

[0073] The term "antibody molecule" is used in a broad sense and includes both full-length immunoglobulin molecules and their antigen-binding fragments.

[0074] Immunoglobulins can be classified into five major classes based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be designated as one of two distinct types (i.e., kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain.

[0075] "Antigen-binding fragment" refers to the portion of an immunoglobulin molecule that retains the antigen-binding properties of the full-length parental antibody (i.e., "its antigen-binding fragment"). Exemplary antigen-binding fragments may have: heavy chain complementarity-determining regions (CDRs) 1, 2, and / or 3; light chain CDRs 1, 2, and / or 3; a heavy chain variable region (VH); a light chain variable region (VL); and combinations thereof. Antigen-binding fragments include: Fab fragments, which are monovalent fragments composed of VL, VH, constant light chain (CL), and constant heavy chain 1 (CH1) domains; F(ab)2 fragments, which are divalent fragments comprising two Fab fragments linked by disulfide bridges at the hinge region; Fd fragments composed of VH and CH1 domains; Fv fragments composed of VL and VH domains of a single arm of the antibody; and domain antibody (dAb) fragments composed of either a VH domain or a VL domain (Ward et al., Nature 341:544-546, 1989). VH and VL domains can be engineered and linked together via synthetic linkers to form various types of single-chain antibody designs, wherein when the VH and VL domains are expressed by separate single-chain antibody constructs, in these cases, the VH / VL domains pair intramolecularly or intermolecularly to form monovalent antigen-binding sites, such as single-chain Fv (scFv) or biantibodies, as described, for example, in International Patent Publications WO1998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO1992 / 01047. These antibody fragments are obtained using techniques well known to those skilled in the art and are screened for practicality in the same manner as full-length antibodies.

[0076] The phrase "immunospecific binding" refers to the ability of a disclosed antibody molecule to preferentially bind to its target (hPD-1 in the case of anti-hPD-1 antibody molecules) rather than to other molecules in a sample containing a mixed molecular population. Immunospecific binding hPD-1 antibody molecules are substantially free of other antibodies with different antigen specificities (e.g., anti-hPD-1 antibodies are substantially free of antibodies that specifically bind to antigens other than hPD-1). However, immunospecific binding hPD-1 antibody molecules can exhibit cross-reactivity with other antigens, such as orthologs of hPD-1, including cynomolgus monkey (Macaca fascicularis) PD-1. The antibody molecules disclosed herein are capable of immunospecific binding to both naturally occurring hPD-1 and recombinant PD-1 produced in mammalian or prokaryotic cells.

[0077] The antibody variable region consists of four "framework" regions that are interrupted by three "antigen binding sites". Antigen binding sites are defined using various terms: (i) complementarity-determining regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3), based on sequence variability (Wu and Kabat, *Journal of Experimental Medicine* 132:211-50, 1970; Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, NIH, Bethesda, Maryland, 1991); and (ii) hypervariable regions ("HVR" or "HV"), three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3), referring to regions of antibody variable regions that are hypervariable in structures as defined by Chothia and Lesk (Chothia and Lesk, *Molecular Biology* 196:901-17). 1987). The AbM definition of CDR is also widely used; it is a compromise between the Kabat and Chothia numbering schemes, and is so named because it is used by Oxford Molecular's AbM antibody modeling software (Rees, AR, Searle, SMJ, Henry, AH and Pedersen, JT (1996) Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, 141–172). Other terms include "IMGT-CDR" (Lefranc et al., Developmental and Comparative Immunology 27:55-77, 2003) and "Specific Determining Residue Use" (SDRU) (Almagro, Molecular Recognition 17:132-43, 2004). The International Immunogenetics (IMGT) database (www.imgt.org) provides standardized numbers and definitions for antigen-binding sites. The correspondence between CDR, HV, and IMGT descriptions is described in Lefranc et al., Developmental and Comparative Immunology 27:55-77, 2003.

[0078] A "frame" or "frame sequence" is the remaining sequence of the variable region excluding the sequence defined as the antigen-binding site. Because antigen-binding sites can be defined using various terms as described above, the exact amino acid sequence of the framework depends on how the antigen-binding site is defined.

[0079] The terms "human antibody," "fully human antibody," and similar terms refer to antibodies with variable regions of both heavy and light chains, wherein both the framework and antigen-binding site are derived from human sequences. If the antibody contains a constant region, that constant region is also derived from a human sequence. Human antibodies contain variable regions of both heavy and / or light chains, and if the variable regions of the antibody are obtained from a system using human germline immunoglobulin or rearranged immunoglobulin genes, the variable regions are "derived" from human sequences. Such systems include phage-displayed human immunoglobulin gene libraries and transgenic nonhuman animals, such as mice or chickens, carrying human immunoglobulin loci as described herein. "Human antibodies" may contain amino acid differences compared to human germline or rearranged immunoglobulin sequences due to, for example, naturally occurring somatic mutations or intentional substitutions introduced into the variable domains (framework and antigen-binding sites) or constant domains. Typically, a "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to the sequence encoded by human germline or rearranged immunoglobulin genes. In some cases, a "human antibody" may contain a common frame sequence derived from human frame sequence analysis, as described by Knappik et al., *Journal of Molecular Biology* 296:57-86, 2000, or contain synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library, as described, for example, by Shi et al., *Journal of Molecular Biology* 397:385-96, 2010, and International Patent Publication No. WO2009 / 085462. Antibodies with antigen-binding sites derived from non-human species are not included in the definition of a "human antibody."

[0080] Although human antibodies are derived from human immunoglobulin sequences, they can be generated using systems such as phage display that incorporates synthetic CDRs and / or synthetic frameworks, or they can be mutagenized in vitro to improve the antibody properties of variable or constant regions or both, thereby generating antibodies that are not naturally present in human antibody strains in vivo.

[0081] "Recombinant antibodies" include all antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as: antibodies isolated from transgenic or transchromatic animals (e.g., mice) of the human immunoglobulin gene or hybridomas prepared therefrom (described further below); antibodies isolated from host cells converted to express antibodies; antibodies isolated from recombinant combined antibody libraries; and antibodies prepared, expressed, generated, or isolated by any other means involving splicing the human immunoglobulin gene sequence into other DNA sequences, or antibodies generated in vitro using Fab arm exchange.

[0082] "Monoclonal antibody" refers to a group of antibody molecules consisting of a single molecule. A monoclonal antibody composition exhibits single binding specificity and affinity for a specific epitope, or, in the case of a bispecific monoclonal antibody, dual binding specificity for two different epitopes. Therefore, a monoclonal antibody is a group of antibodies having a single amino acid composition in each heavy chain and each light chain, except for possible well-known alterations (such as the removal of a C-terminal lysine from the antibody heavy chain). Monoclonal antibodies may exhibit heterogeneous glycosylation within the antibody group. Monoclonal antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. The term monoclonal antibody includes bispecific antibodies.

[0083] An epitope is the part of an antigen that specifically binds to an antibody. Epitopes are typically composed of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups, such as amino acids or polysaccharide side chains, and can possess specific three-dimensional structural and charge properties. Epitopes can be composed of continuous and / or discontinuous amino acids forming conformational spatial units. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are very close in three-dimensional space through protein folding.

[0084] A “variant” is a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide through one or more modifications (e.g., substitution, insertion, or deletion). As used herein, the term “mutation” is intended to refer to one or more intentional substitutions of a polypeptide or polynucleotide.

[0085] As used herein, “antagonistic anti-PD-1 antibody” refers to an antibody that binds to PD-1 and is used to at least partially inhibit the interaction between PD-1 and its ligands PD-L1 and / or PD-L2.

[0086] "An antagonistic anti-PD-1 antibody that binds to PD-1 in the presence of an immunoconjugate" means that at least a portion of the antagonistic anti-PD-1 antibody can be detectably bound to PD-1 that has been previously exposed to an immunoconjugate, for example, using the method described in Protocol B as described herein.

[0087] "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, as measured using cell-based assays or SPR as described herein.

[0088] "Treatment" and similar terms refer to therapeutic treatments and preventative or preventative measures, including reducing the severity and / or frequency of symptoms, eliminating symptoms and / or their underlying causes, reducing the frequency or likelihood of symptoms and / or their underlying causes, and improving or remedying damage caused directly or indirectly by cancer. Treatment also includes extending survival compared to the expected survival of untreated subjects. Subjects to be treated include those with cancer, those susceptible to cancer, or those seeking cancer prevention.

[0089] As used herein, “administer to a subject” and similar terms refer to a procedure of injecting the disclosed immunoconjugate (monotherapy) or immunoconjugate and antagonistic anti-PD-1 antibody (combination therapy) into a subject so that the subject’s target cells, tissues or body segments come into contact with the disclosed immunoconjugate or immunoconjugate and antagonistic anti-PD-1 antibody.

[0090] The phrase “therapeutic effective dose” refers to the amount of an immunodrug (monotherapy) or an immunodrug combined with an antagonistic anti-PD-1 antibody (combination therapy) as described herein that effectively achieves a specific biological or therapeutic outcome (such as, but not limited to, the biological or therapeutic outcomes disclosed, described, or exemplified herein). Therapeutic effective doses can vary depending on factors such as an individual’s disease state, age, sex, and weight, and the ability of the immunodrug (monotherapy) or the immunodrug combined with an antagonistic anti-PD-1 antibody (combination therapy) to elicit a desired response in the subject. Exemplary measures of therapeutic effectiveness include, for example, improvement in patient health, reduction in cancer symptoms, cessation or slowing of cancer symptom progression, and / or disappearance of cancer symptoms.

[0091] As used herein, the term "subject" is intended to refer to any animal, particularly a mammal. Therefore, the methods described are applicable to both humans and non-human animals, but are most preferably used for humans. "Subject" and "patient" are used interchangeably herein.

[0092] The modified hIL-2 protein disclosed herein is also referred to herein as “attenuated” hIL-2. As described herein, the term “potency reduction” and related terms such as “potency reduction” or “attenuation” of IL-2 activity refer to a reduction in the potency of the modified hIL-2, as demonstrated by EC2 in IL-2-dependent assays. 50 The value relative to the EC of unmodified hIL-2 50The efficacy of modified hIL-2 against high-affinity and intermediate-affinity IL-2 receptors is determined by increasing the value. As described herein, the efficacy of modified hIL-2 against high-affinity and intermediate-affinity IL-2 receptors is reduced. IL-2-dependent assays for determining efficacy may be engineered human erythroleukemia TF1 (TF1+IL-2Rβ) or human natural killer NK-92 cell proliferation assays as described herein. In one embodiment, the IL-2-dependent assay for determining efficacy is an 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 also be TF1+IL-2Rβ or human natural killer NK-92 pSTAT5 assays as described herein. Unmodified hIL-2 can be prokaryotically expressed hIL-2, such as Proleukin® (which has the native human IL-2 amino acid sequence except for the C125S substitution used to remove unbound cysteine ​​and does not have normal human carbohydrate expression at residue T3), or unmodified hIL-2 can be hIL-2 with the amino acid sequence of SEQ ID NO: 345 or hIL-2 with the amino acid sequence of SEQ ID NO: 345 and the C125S substitution, which is expressed in mammalian cell lines such as CHO or HEK cell lines.

[0093] Immunoconjugates and fusion proteins are used interchangeably in this article.

[0094] Combination therapy

[0095] Standard care cancer treatment may include administration of PD-1 antagonist checkpoint inhibitors (such as antagonistic anti-PD-1 antibodies nivolumab, pembrolizumab, cemiplimab, dostarlimab, or retifanlimab) to antagonize PD-1 in tumor-resident T cells. For example, treatment with nivolumab or pemblimab may initially enhance the effector T cell response to the tumor, but in many cases, these responses diminish over time as chronically stimulated tumor-resident T cells develop an “exhausted” phenotype characterized by reduced proliferative potential, decreased cytotoxic activity, and increased expression of immune checkpoint molecules. The immunoconjugates described herein do not prevent antagonistic anti-PD-1 antibodies such as nivolumab or pemblimab from 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 and complementary. Specifically, the primary mechanism of action of PD-1 antagonists (such as antagonistic anti-PD-1 antibodies) is to block immunosuppressive signals delivered via the interaction of PD-1 and PD-L1, thereby maintaining T cells in their activated state or restoring them to their activated state. On the other hand, the mechanism of action of the immunoconjugates described herein is achieved through T cell-selective IL-2 signaling, which primarily induces T cell proliferation and subsequently promotes activation. The different modes of action and the potential for each agent to combine and simultaneously possess activity provide a theoretical basis for combination therapies to enhance clinical outcomes in cases of acquired resistance to, for example, PD-(L)1 blockade therapy. This property enables clinical combinations of the disclosed immunoconjugates with antagonistic anti-PD-1 antibodies, thereby enhancing the therapy. For example, in tumors that initially respond to checkpoint inhibitors but subsequently exhibit T cell exhaustion, administration of the disclosed immunoconjugate (which delivers modified hIL-2 to PD-1-expressing cells) can restimulate tumor-resident T cells to present a non-exhausted phenotype and can induce T cells to become responsive to checkpoint inhibitors again. In some embodiments, antagonistic anti-PD-1 antibodies and immunoconjugates may be delivered simultaneously or sequentially as single agents as described herein, such that each agent can be administered at concentrations and durations that optimize the subject's antitumor response.

[0096] This article discloses a method for treating cancer in a subject, the method comprising administering the following to the subject:

[0097] (A) An immunoconjugate modified with an anti-human PD-1 (hPD-1) antibody, said immunoconjugate comprising:

[0098] A modified hIL-2 protein, wherein the modified hIL-2 protein comprises 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

[0099] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0100] (i) Heavy chain 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 (referred to herein as "H7-632");

[0101] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7");

[0102] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401 (referred to herein as "C51E6-5"); or

[0103] (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 (referred to herein as "A2"); and

[0104] (B) Anti-PD-1 antibody that antagonizes PD-1 in the presence of the said immunoconjugate.

[0105] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, suitable substitutions at amino acid position 20 of the modified hIL-2 moiety of the immunoconjugate include, for example, any substitutions of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E.

[0106] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, suitable substitutions at amino acid position 38 of the modified hIL-2 moiety of the immunoconjugate include, for example, any substitutions of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K.

[0107] 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.

[0108] Therefore, this article discloses a method for treating cancer in a subject, the method comprising administering the following to the subject:

[0109] (A) An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0110] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0111] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0112] (i) Heavy chain 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 (referred to herein as "H7-632");

[0113] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7");

[0114] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401 (referred to herein as "C51E6-5"); or

[0115] (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 (referred to herein as "A2"); and

[0116] (B) Anti-PD-1 antibody that antagonizes PD-1 in the presence of the said immunoconjugate.

[0117] The modified hIL-2 protein moiety of the immunoconjugate may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein moiety of the immunoconjugate may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 608, 611, 614, or 620. The modified hIL-2 protein moiety of the immunoconjugate may contain the amino acid sequence of any one of SEQ ID NO: 149, 307, 607-611, 614, 617, or 620. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein contains 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. The modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 may further comprise a T3A substitution and / or a C125A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0118] The modified hIL-2 protein moiety of the immunoconjugate may contain a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, and may contain an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345. The disclosed method of treating a subject's cancer may comprise administering the following to the subject:

[0119] (A) An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0120] A modified hIL-2 protein, wherein the modified hIL-2 protein comprises, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, a D20A substitution at amino acid position 20 and an R38E substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345; and

[0121] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0122] (i) Heavy chain 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 (referred to herein as "H7-632");

[0123] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7");

[0124] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401 (referred to herein as "C51E6-5"); or

[0125] (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 (referred to herein as "A2"); and

[0126] (B) Anti-PD-1 antibody that antagonizes PD-1 in the presence of the said immunoconjugate.

[0127] Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein moiety of the immunoconjugate may further include a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein moiety of the immunoconjugate comprises the amino acid sequence of SEQ ID NO: 216.

[0128] Alternatively, the modified hIL-2 protein moiety 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 moiety of the immunoconjugate comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein moiety of the immunoconjugate comprises the amino acid sequence of SEQ ID NO: 218.

[0129] Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein moiety of the immunoconjugate may further include a deletion or substitution at amino acid position 125. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes 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.

[0130] Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of approximately 10,000-fold or more compared to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such a reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

[0131] Furthermore, compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) by 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency of more than 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) compared to the unmodified hIL-2.

[0132] Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit up to approximately 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ), for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction of more than 10,000-fold in potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and a reduction of more than 10,000-fold in potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ).

[0133] The modified hIL-2 protein can be fused to the anti-hPD-1 antibody or its antigen-binding fragment of an immunoconjugate at the N-terminus, C-terminus, N-terminus, C-terminus of the antibody light chain, N-terminus of the antibody heavy chain, or C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate is fused directly to the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate via a peptide bond. The modified hIL-2 protein moiety of the immunoconjugate can, for example, be fused directly to the C-terminal amino acid residue of the anti-hPD-1 antibody or its antigen-binding fragment moiety of the immunoconjugate via a peptide bond. In some embodiments, the modified hIL-2 protein is fused to the anti-hPD-1 antibody or its antigen-binding fragment moiety of the immunoconjugate via a linker.

[0134] The fusion of modified hIL-2 protein with an anti-hPD-1 antibody or its antigen-binding fragment portion of an immunoconjugate can rescue the ability of the modified hIL-2 protein to activate intermediate-affinity IL-2 receptors. In some embodiments, the immunoconjugate can activate intermediate-affinity IL-2 receptors to a degree comparable to wild-type hIL-2 activation of intermediate-affinity IL-2 receptors.

[0135] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a heavy chain 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 (referred to herein as "H7-632").

[0136] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7").

[0137] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a heavy chain CDR1 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 (referred to herein as "C51E6-5").

[0138] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 410, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 411 (referred to herein as “A2”).

[0139] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and a light chain variable region containing the amino acid sequence of SEQ ID NO:417 (referred to herein as "H7-632").

[0140] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and a light chain variable region containing the amino acid sequence of SEQ ID NO:385 (referred to herein as "2H7").

[0141] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and a light chain variable region containing the amino acid sequence of SEQ ID NO:395 (referred to herein as "C51E6-5").

[0142] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate 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 herein as "A2").

[0143] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may contain the constant region of the IgG1 heavy chain.

[0144] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may have substitutions or deletions in the constant region to minimize Fc-mediated immune effector functions, such as 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 or its antigen-binding fragment portion of the immunoconjugate contains an L235A substitution, wherein the amino acid numbering is according to EU numbers. In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate contains a G237A substitution, wherein the amino acid numbering is according to EU numbers. In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate contains both L235A and G237A substitutions, wherein the amino acid numbering is according to EU numbers.

[0145] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:414 and a light chain containing the amino acid sequence of SEQ ID NO:415 (referred to herein as "H7-632-hIgG1-LAGA").

[0146] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:424 and a light chain containing the amino acid sequence of SEQ ID NO:425 (referred to herein as "2H7-hIgG4").

[0147] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:426 and a light chain containing the amino acid sequence of SEQ ID NO:427 (referred to herein as "C51E6-5-hIgG4").

[0148] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:428 and a light chain containing the amino acid sequence of SEQ ID NO:429 (referred to herein as "A2-hIgG4").

[0149] Immunoconjugates may have one or more of the following properties:

[0150] ● It binds to PD-1, but does not inhibit the binding of PD-L1 to PD-1;

[0151] ● The presence of standard care anti-PD-1 antibodies used in clinical practice (e.g., pembrolizumab (KEYTRUDA)) ® ) or nivolumab (OPDIVO) ® In the case of binding with PD-1;

[0152] ● It exhibits high selectivity for PD-1 and does not specifically bind to other related B7 family members;

[0153] ● Binds to PD-1 on activated human T cells (EC1 in flow cytometry) 50 (~0.1-0.2 nM).

[0154] ● Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate exhibits a measurable reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) by 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of approximately 10,000-fold or more compared to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such a reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

[0155] ● Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate exhibits a measurable reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) by 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency of more than 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) compared to the unmodified hIL-2;

[0156] ●Rescue and expand PD-1-expressing human memory T cell subsets in a GvHD animal model; and

[0157] ● After a single dose of 1 and 10 mg / kg to cynomolgus monkeys, there were minimal or no effects on body weight, blood chemistry, or hematological parameters.

[0158] In some embodiments, the immunoconjugate comprises a modified hIL-2 protein having T3A, R38E, D20A, and C125A substitutions, fused to the C-terminus of an anti-hPD-1 antibody heavy chain comprising a human IgG1 framework having L235A and G237A substitutions. In some embodiments, the immunoconjugate comprises a fusion of an hIL-2 protein modified with a light chain comprising the amino acid sequence of SEQ ID NO: 415 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 532.

[0159] Suitable antagonistic anti-PD-1 antibodies include nivolumab (OPDIVO). ® ), Pembrolizumab (KEYTRUDA) ® ), cimiprimab (LIBTAYO®), dotalimab (JEMPERLI), or rivetimab (ZYNYZ) TM 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, cimiprimab, dotalimab, or revilimab are intended to include any product containing nivolumab, pembrolizumab, cimiprimab, dotalimab, or revilimab that is approved by a regulatory agency and, where applicable, is an OPDIVO. ® KEYTRUDA ® LIBTAYO®, JEMPERLI, or ZYNYZ TM Biosimilars.

[0160] The disclosed immunoconjugates selectively deliver IL-2 signaling to PD-1-expressing T cells. The anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate can be used solely to deliver modified hIL-2 to PD-1-expressing cells without blocking PD-1 receptor function, as nivolumab (OPDIVO2). ® ) and pembrolizumab (KEYTRUDA) ® Similar to classic anti-PD-1 inhibitor antibodies, the immunoconjugates disclosed in this paper achieve their main mechanism of action through the T-cell selective activity of IL-2. The human PD-1 receptor is primarily expressed on a small subset of T cells with potent tumor reactivity. Without being bound by theory, it is believed that targeting this T-cell population with the modified hIL-2 protein of the immunoconjugate can significantly enhance anti-tumor immunity while reducing or minimizing off-target systemic IL-2-mediated toxicity mediated by cell populations lacking PD-1 expression.

[0161] The antagonistic anti-PD-1 antibody and the immunodrug conjugate can be administered to the subject together in a mixture, simultaneously as a single agent, or sequentially as a single agent in any order. In some embodiments, the antagonistic anti-PD-1 antibody and the immunodrug conjugate are administered to the subject together in a mixture. In some embodiments, the antagonistic anti-PD-1 antibody and the immunodrug conjugate are administered to the subject simultaneously as a single agent. In some embodiments, the antagonistic anti-PD-1 antibody and the immunodrug conjugate are administered to the subject sequentially as a single agent in any order. In some embodiments, the method may include administering the immunodrug conjugate prior to administering the antagonistic anti-PD-1 antibody. In some embodiments, the method may include administering the antagonistic anti-PD-1 antibody prior to administering the immunodrug conjugate. In some embodiments, the method may include administering the immunodrug conjugate substantially simultaneously with the administration of the antagonistic anti-PD-1 antibody. The immunodrug conjugate and the antagonistic anti-PD-1 antibody can be administered such that the second agent is administered while the first agent is still showing a biological effect.

[0162] The immunoconjugate can be administered to subjects who begin to show signs of relapse / recurrence after a period of antagonistic PD-1 therapy. Therefore, in some embodiments, the method is performed on subjects who begin to show signs of relapse / recurrence after or during antagonistic PD-1 therapy.

[0163] Exemplary cancers that can be treated using the disclosed methods include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, leukemia, skin cancer, melanoma, squamous cell carcinoma, bone cancer, and kidney cancer. In some embodiments, the disclosed methods can be used to treat melanoma, Merkel cell carcinoma, non-small cell lung cancer, renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, hepatocellular carcinoma, or tumors with high microsatellite instability or those with DNA mismatch repair defects.

[0164] Each aspect of the disclosed and claimed combination therapies presented herein as treatment methods also applies to the use of the immunoconjugate and the antagonistic anti-PD-1 antibody, including but not limited to Swiss-style use, primary medical use, secondary / additional medical use, and use according to EPC2000. Thus, for example, the following uses are disclosed herein:

[0165] (A) An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0166] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0167] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0168] (i) Heavy chain 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;

[0169] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0170] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0171] (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; and

[0172] (B) Anti-PD-1 antibodies that antagonize PD-1 in the presence of the aforementioned immunoconjugate

[0173] It is used to treat cancer.

[0174] Monotherapy

[0175] This article discloses a method for treating a subject with renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or a tumor with high microsatellite instability or a tumor with DNA mismatch repair deficiency, the method comprising administering the following to the subject:

[0176] An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0177] A modified hIL-2 protein, wherein the modified hIL-2 protein comprises 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

[0178] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0179] (i) Heavy chain 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 (referred to herein as "H7-632");

[0180] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7");

[0181] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401 (referred to herein as "C51E6-5"); or

[0182] (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 (referred to herein as “A2”).

[0183] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, suitable substitutions at amino acid position 20 of the modified hIL-2 moiety of the immunoconjugate include, for example, any substitutions of D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E.

[0184] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, suitable substitutions at amino acid position 38 of the modified hIL-2 moiety of the immunoconjugate include, for example, any substitutions of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K.

[0185] 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.

[0186] Therefore, this article discloses a method for treating a subject with renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or a tumor with high microsatellite instability or a tumor with DNA mismatch repair deficiency, the method comprising administering the following to the subject:

[0187] (A) An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0188] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0189] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0190] (i) Heavy chain 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 (referred to herein as "H7-632");

[0191] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7");

[0192] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401 (referred to herein as "C51E6-5"); or

[0193] (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 (referred to herein as “A2”).

[0194] The modified hIL-2 protein moiety of the immunoconjugate may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein moiety of the immunoconjugate may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 608, 611, 614, or 620. The modified hIL-2 protein moiety of the immunoconjugate may contain the amino acid sequence of any one of SEQ ID NO: 149, 307, 607-611, 614, 617, or 620. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein contains 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. The modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 may further comprise a T3A substitution and / or a C125A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0195] The modified hIL-2 protein moiety of the immunoconjugate may contain a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, and may contain an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345. Therefore, this document discloses a method for treating a subject with renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or tumors with high microsatellite instability or DNA mismatch repair deficiency, the method comprising administering the following to the subject:

[0196] (A) An anti-hPD-1 antibody-modified hIL-2 immunoconjugate, said immunoconjugate comprising:

[0197] A modified hIL-2 protein, wherein the modified hIL-2 protein comprises, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, a D20A substitution at amino acid position 20 and an R38E substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345; and

[0198] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0199] (i) Heavy chain 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 (referred to herein as "H7-632");

[0200] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391 (referred to herein as "2H7");

[0201] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401 (referred to herein as "C51E6-5"); or

[0202] (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 (referred to herein as “A2”).

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

[0204] Alternatively, the modified hIL-2 protein moiety 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 moiety of the immunoconjugate comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein moiety of the immunoconjugate comprises the amino acid sequence of SEQ ID NO: 218.

[0205] Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein moiety of the immunoconjugate may further include a deletion or substitution at amino acid position 125. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes 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] Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of approximately 10,000-fold or more compared to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such a reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

[0207] Furthermore, compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) by 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency of more than 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) compared to the unmodified hIL-2.

[0208] Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit up to approximately 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ), for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction of more than 10,000-fold in potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and a reduction of more than 10,000-fold in potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ).

[0209] The modified hIL-2 protein can be fused to the anti-hPD-1 antibody or its antigen-binding fragment of an immunoconjugate at the N-terminus, C-terminus, N-terminus, C-terminus of the antibody light chain, N-terminus of the antibody heavy chain, or C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate is fused directly to the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate via a peptide bond. The modified hIL-2 protein moiety of the immunoconjugate can, for example, be fused directly to the C-terminal amino acid residue of the anti-hPD-1 antibody or its antigen-binding fragment moiety of the immunoconjugate via a peptide bond. In some embodiments, the modified hIL-2 protein is fused to the anti-hPD-1 antibody or its antigen-binding fragment moiety of the immunoconjugate via a linker.

[0210] The fusion of modified hIL-2 protein with an anti-hPD-1 antibody or its antigen-binding fragment portion of an immunoconjugate can rescue the ability of the modified hIL-2 protein to activate intermediate-affinity IL-2 receptors. In some embodiments, the immunoconjugate can activate intermediate-affinity IL-2 receptors to a degree comparable to wild-type hIL-2 activation of intermediate-affinity IL-2 receptors.

[0211] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a heavy chain 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 (referred to herein as "H7-632").

[0212] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: 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 CDR3 comprising 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 immunoconjugate comprises: a heavy chain CDR1 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 (referred to herein as "C51E6-5").

[0214] In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 406, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 407, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 408, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 409, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 410, and a light chain CDR3 comprising 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 immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and a light chain variable region containing 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 immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and a light chain variable region containing 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 immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and a light chain variable region containing 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 immunoconjugate 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 herein as "A2").

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

[0220] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may have substitutions or deletions in the constant region to minimize Fc-mediated immune effector functions, such as 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 or its antigen-binding fragment portion of the immunoconjugate contains an L235A substitution, wherein the amino acid numbering is according to EU numbers. In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate contains a G237A substitution, wherein the amino acid numbering is according to EU numbers. In some embodiments, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate contains both L235A and G237A substitutions, wherein the amino acid numbering is according to EU numbers.

[0221] The anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:414 and a light chain containing 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 immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:424 and a light chain containing 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 immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:426 and a light chain containing 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 immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:428 and a light chain containing 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 properties:

[0226] ● It binds to PD-1, but does not inhibit the binding of PD-L1 to PD-1;

[0227] ● The presence of standard care anti-PD-1 antibodies used in clinical practice (e.g., pembrolizumab (KEYTRUDA)) ® ) or nivolumab (OPDIVO) ® In the case of binding with PD-1;

[0228] ● It exhibits high selectivity for PD-1 and does not specifically bind to other related B7 family members;

[0229] ● Binds to PD-1 on activated human T cells (EC1 in flow cytometry) 50 (~0.1-0.2 nM).

[0230] ● Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate exhibits a measurable reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) by 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of approximately 10,000-fold or more compared to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such a reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

[0231] ● Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate exhibits a measurable reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) by 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, for example, as compared to EC in the hIL-2-dependent cell proliferation assay described herein. 50 The values ​​are quantified. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency of more than 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) compared to the unmodified hIL-2;

[0232] ●Rescue and expand PD-1-expressing human memory T cell subsets in a GvHD animal model; and

[0233] ● After a single dose of 1 and 10 mg / kg to cynomolgus monkeys, there were minimal or no effects on body weight, blood chemistry, or hematological parameters.

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

[0235] The disclosed immunoconjugates selectively deliver IL-2 signaling to PD-1-expressing T cells. The anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate can be used solely to deliver modified hIL-2 to PD-1-expressing cells without blocking PD-1 receptor function, as nivolumab (OPDIVO2). ® ) and pembrolizumab (KEYTRUDA) ® Similar to classic anti-PD-1 inhibitor antibodies, the immunoconjugates disclosed in this paper achieve their main mechanism of action through the T-cell selective activity of IL-2. The human PD-1 receptor is primarily expressed on a small subset of T cells with potent tumor reactivity. Without being bound by theory, it is believed that targeting this T-cell population with the modified hIL-2 protein of the immunoconjugate can significantly enhance anti-tumor immunity while reducing or minimizing off-target systemic IL-2-mediated toxicity mediated by cell populations lacking PD-1 expression.

[0236] Each aspect of the disclosed and claimed monotherapy presented herein as a treatment method also applies to the use of the immunoconjugate, including but not limited to Swiss-style use, first medical use, second / additional medical use, and use according to EPC2000. Thus, for example, this document discloses the use of an anti-hPD-1 antibody-modified hIL-2 immunoconjugate comprising:

[0237] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0238] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0239] (i) Heavy chain 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;

[0240] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0241] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0242] (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.

[0243] It is used to treat renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or tumors with high microsatellite instability or those with DNA mismatch repair defects.

[0244] Example

[0245] The following examples are provided to further describe some embodiments of the embodiments disclosed herein. These examples are intended to illustrate, and not limit, the disclosed embodiments.

[0246] General Method

[0247] Option A. Flow cytometry screening of human PD-1 binding to anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusion product.

[0248] To test binding to hPD-1, the antibody and antibody-attenuated hIL-2 fusion protein were characterized in complete titration curves. Jurkat cell lines were transfected with a mammalian vector encoding amino acids 1-185 (SEQ ID NO: 346) of human PD-1 to stably express the extracellular domain and part of the transmembrane domain of human PD-1, and this transfected cell line was used to determine the binding of the anti-hPD-1 antibody. Jurkat+ hPD-1 cells were washed and added at 100,000 cells per well to FACS buffer (PBS, 0.2% heat-inactivated fetal bovine serum) in 96-well plates. Cells were blocked for 10 min at 4°C with a 1:50 dilution of human FcR blocking agent (Miltenyi) and washed with FACS buffer.

[0249] The antibody or antibody-attenuated hIL-2 immunoconjugate (fusion protein) was serially diluted six-fold in FACS buffer to obtain an 8-point curve, and added to Jurkat cells expressing human PD-1 at a volume of 100 µL on 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 again and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific), and flow cytometry was performed on a BD FACS Canto II, BD Celesta, or BD Fortessa (BD Biosciences). Geometric mean fluorescence intensity (gMFI) was calculated using FlowJo software version 10. The half-maximum effective concentration (EC50) was calculated using GraphPad Prism 7 software based on the gMFI of the allophycocyanin signal across titration concentrations. 50 )value.

[0250] Option B. Competitive screening by flow cytometry of anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusion with human PD-1 binding.

[0251] The ability of the antibody and antibody-attenuated hIL-2 fusion protein to bind human PD-1 was tested in the presence of saturated concentrations of anti-hPD-1 #1-mIgG2b-N297A (containing the heavy and light chain variable region sequences of nivolumab (clone 5C4), formatted on a mouse IgG2b-N297A background as described in US Patent Publication No. US 2009 / 0217401A1) (SEQ ID NO: 348 and 349) or anti-hPD-1 #2-mIgG2b-N297A (containing the heavy and light chain variable region sequences of pembrolizumab (clone 109A-H / K09A-L-11), formatted on a mouse IgG2b-N297A background as described in International Publication No. WO2008 / 156712A1) (SEQ ID NO: 350 and 351).

[0252] The antibody or antibody-attenuated hIL-2 fusion protein was serially diluted six-fold to obtain an 8-point titration profile, with or without a saturation level of 10 μM anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. Briefly, Jurkat cells stably expressing hPD-1 were washed (as described in Protocol A above) and resuspended in FACS buffer containing a 1:50 dilution of human FcR blocking agent. Cells were incubated at 4°C for 10 minutes and washed. Cells were then resuspended in a 100 µL volume with 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 one hour. Cells were washed and incubated at 4°C for one hour with either the test antibody or the antibody-attenuated hIL-2 fusion protein serially diluted six-fold in 100 μL volumes to obtain an 8-point curve. To detect binding of the 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 then washed and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific). For comparison, Jurkat cells stably expressing human PD-1 were incubated only with titrated test antibodies or antibody-degraded hIL-2 fusion proteins (without anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A) and subsequently with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc secondary. As controls, the 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 BDCanto II, BD Celesta, or BD Fortessa (BD Biosciences), and gMFI was calculated using FlowJo software version 10. EC was calculated using GraphPad Prism 7 software based on gMFI of allophycocyanin signals across titration concentrations. 50 value.

[0253] Option C. Cell-based screening of non-antagonist anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusions.

[0254] Characterizing the ability of human PD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to block the binding of hPD-1 to the ligand hPD-L1 (SEQ ID NO: 584). The anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein 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 utilized two cell lines: FCγR11b artificial antigen-presenting cells / Chinese ovarian hamster K1 (aAPC / CHO-K1) and Jurkat effector cells. aAPC / CHO K1 cells stably expressed both the human PD-L1 ligand and cell surface protein to activate the homologous T cell receptor (TCR), while Jurkat effector cells expressed hPD-1 and a luciferase reporter gene under the control of the activated nuclear factor response element (NFAT-RE) of T cells. When these cells were co-cultured in the presence of a non-antagonistic antibody, the hPD-1 / hPD-L1 interaction inhibited TCR signaling, and no luminescence was detected. In the presence of an antibody that antagonized the interaction between hPD-1 and hPD-L1 (SEQ ID NO: 584), the inhibitory signaling was disrupted, and luminescence was detected.

[0255] Thawing and assaying were performed according to the manufacturer's instructions. In short, aAPC / CHO-K1 cells were first thawed and seeded at 30,000 cells per well in a 5% CO2 incubator at 37°C for 18 hours on flat-bottomed 96-well plates. After cell adhesion, the culture medium was removed, and 200 nM or 1000 nM of the test antibody or antibody-degraded hIL-2 fusion protein was diluted in 40 μL of assay buffer (RPMI 1640 medium + 1% FBS) and added to the aAPC / CHO-K1 cells. A human IgG4 isotype control monoclonal antibody targeting keyforamina hemocyanin (KLH) clone C3 (SEQ ID NO: 585 and 586) was used as a negative control. Jurkat effector cells expressing hPD-1 were added to a 40 μL volume at 24,000 cells per well. The final concentration of the fixed antibody tested was 100 nM or 500 nM. In some instances, this co-culture assay tested a series of concentrations of hIL-2 fusion protein against hPD-11 or hPD-1-attenuation, with the highest concentration in the five-fold titration series being 500 nM. Figure 7 ).

[0256] Co-culturing assays were performed at 37°C in a 5% CO2 incubator for an additional 18–20 hours. To read the luminescence signal, the plate was brought to room temperature, and 80 μL of Bio-Glo™ reagent was added to each well. The plate was incubated in the dark at room temperature for 15 minutes, and the luminescence was read on a Victor X-ray spectrophotometer (PerkinElmer). The average of every three relative luminescence units (RLU) was calculated, and the results were plotted using GraphPad Prism 7 software.

[0257] Protocol D. In vitro phosphorylated STAT5 assay to test hIL-2 variant attenuation

[0258] Phosphorylated STAT5 was used to determine the attenuation level of hIL-2 receptor activation activity of the antibody-attenuated hIL-2 fusion protein. Variants were tested in both hIL-2-responsive human natural killer (NK-92) cells and engineered human erythroleukemia (TF1) cells. The NK-92 cell line naturally expresses a high-affinity hIL-2 receptor (IL-2Rαβγ) at physiological levels, while the TF1 cell line, which naturally expresses IL-2Rγ (SEQ ID NO: 352), was engineered to stably express human CD122 (IL-2Rβ) (SEQ ID NO: 353) to express the intermediate-affinity hIL-2 receptor complex (IL-2Rβγ). This stable TF1+IL-2Rβ cell line does not express IL-2Rα (SEQ ID NO: 354). Both the NK-92 and TF1+IL-2Rβ cell lines were used to assess the attenuation level of IL-2 potency in these cell-based potency assays, such as fixed-concentration screening and complete titration curves.

[0259] For fixed concentration screening, 100,000 NK-92 cells or TF1+IL-2Rβ cells were plated into 96 wells of 50 μL of fresh growth medium lacking human IL-2 cytokines and incubated overnight at 37°C in a CO2 incubator. After 15–16 hours, human IL-2-starved cells were treated with 25.7 nM recombinant hIL-2 (denoted as rhIL-2) (SEQ ID NO: 345) or a test antibody-degraded hIL-2 fusion protein for NK-92 cell assays, or with 33.3 nM hIL-2 or a test hIL-2 variant for TF1+IL-2Rβ cell assays. 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 and then permeabilized on ice with Perm buffer III (BD Biosciences) for 30 minutes. hIL-2-dependent Stat5 phosphorylation was detected after staining fixed and permeabilized cells for 45 min at room temperature in the dark with an Alexa Fluor-647-conjugated anti-Stat5 antibody (BD Biosciences) at 0.5 μL / sample. Cells were washed and the reagent was diluted in BD Pharmingen buffer (BD Biosciences). Stained cells were acquired on a FACS-Celesta hematology counter (BD Biosciences) and analyzed using FlowJo software version 10.7.2. Assays were performed in groups, but each plate was normalized using rhIL-2. The degree of decay of the selected antibody-decaying hIL-2 fusion protein was assessed in 8-point, 6-fold sequential titration curves in the range of 1200 nM to 7 pM on both the NK-92 cell line and the TF1+IL-2Rβ cell line. The pStat5 curve procedure was performed in the same manner as described above. EC was calculated based on the geometric mean fluorescence intensity (gMFI) across titration concentrations using GraphPad Prism 7 software. 50 Value. By changing the EC variant 50 Value divided by hIL-2 EC 50 To calculate the fold change in rhIL-2 activity.

[0260] Protocol E. In vitro cell proliferation assay to test the attenuation of antibody-attenuated hIL-2 fusion protein.

[0261] The attenuated hIL-2 activity of the antibody-attenuated hIL-2 fusion protein was also tested in an 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 without hIL-2 cytokines were seeded in each well of a 96-well U-bottom cell culture plate. An eight-point, 6-fold serial titration of the antibody-attenuated hIL-2 fusion protein at a maximum concentration of 996 nM was diluted in fresh medium and applied to the cells in the wells. Cells were incubated at 37°C in a 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 the luminescence of each well was read for 0.1 seconds using a VictorX multi-tag reader (PerkinElmer). EC was calculated using GraphPad Prism 7 software based on relative luminescence units (RLU) across titration concentrations. 50 Value. By changing the EC variant 50 Value divided by hIL-2 EC 50 To calculate the fold change in rhIL-2 activity. Assays were performed in a cohort, but using rhIL-2 ECGs. 50 The values ​​are normalized for each board.

[0262] Example 1: Optimization of antibody-attenuated hIL-2 fusion protein variants and determination of their hIL-2 activity on intermediate-affinity and high-affinity hIL-2 receptor complexes.

[0263] To determine the optimal structure of the antibody-attenuated hIL-2 fusion protein, unattenuated hIL-2 was fused with an anti-DNase I antibody (clone 1H3) named 1H3-hIgG1 (SEQ ID NO: 379, SEQ ID NO: 374) in the variable region of the antibody in various ways as shown in Figure 1. Variants include hIL-2 fused at the N-terminus of the human anti-DNase I antibody (clone 1H3) immunoglobulin hIgG1 heavy chain or human κ light chain via a direct fusion (df) of hIL-2 N-terminal light chain df (SEQ ID NO: 379, SEQ ID NO: 356) or hIL-2 N-terminal heavy chain df (SEQ ID NO: 358, SEQ ID NO: 374) or a six-amino acid linker (L6) (SEQ ID NO: 355) of hIL-2 N-terminal light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 357) and hIL-2 N-terminal heavy chain L6 fusion (SEQ ID NO: 359, SEQ ID NO: 374). Variations were also generated in which the hIL-2 moiety is fused to the C-terminus of both the heavy and light chains via df or L6, and are represented as hIL-2 C-terminal heavy chain df (SEQ ID NO: 360, SEQ ID NO: 374), hIL-2 C-terminal heavy chain L6 fusion (SEQ ID NO: 361, SEQ ID: 374), hIL-2 C-terminal light chain df (SEQ ID NO: 379, SEQ ID NO: 362), and hIL-2 C-terminal light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 363). Further variations were generated in which the CD25 / IL-2Rα extracellular domain (amino acids 1-164) (SEQ ID NO: 126) is fused to the N-terminus or C-terminus of the heavy or κ light chain to interfere with the binding of IL-2 to CD25 of the IL-2 receptor (Figure 2).In these constructs, the extracellular domain of human CD25 (amino acids 1-164) (SEQ ID NO: 126) is fused to human IL-2 via a 20-amino acid linker (L20) (SEQ ID NO: 364), and then directly fused to the N-terminus or fused to the 1H3-hIgG1 heavy or light chain via an L6 linker (SEQ ID NO: 355): hCD25-L20-hIL-2 N-terminal heavy chain df (SEQ ID NO: 365, SEQ ID NO: 374), hCD25-L20-hIL-2 N-terminal heavy chain L6 fusion (SEQ ID NO: 366, SEQ ID NO: 374), hCD25-L20-hIL-2 N-terminal light chain df (SEQ ID NO: 379, SEQ ID NO: 367), hCD25-L20-hIL-2 N-terminal light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 367). 368). Finally, a final set of variants were generated in which the extracellular domain portion of CD25 / IL-2Rα (SEQ ID NO: 126) was fused to the C-terminus of the heavy chain and κ light chain: hCD25-L20-hIL-2 C-terminal heavy chain df (SEQ ID NO: 369, SEQ ID NO: 374), hCD25-L20-hIL-2 C-terminal heavy chain L6 fusion (SEQ ID NO: 370, SEQ ID NO: 374), hCD25-L20-hIL-2 C-terminal light chain df (SEQ ID NO: 379, SEQ ID NO: 371), and hCD25-L20-hIL-2 C-terminal light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 372). These antibody-hIL-2 fusion proteins were generated, expressed, and purified using standard techniques. The aforementioned 16 N-terminal or C-terminal and adapter variants were evaluated in an in vitro cell-based phosphorylated STAT5 assay using an 8-point, 6-fold serial titration as described in Protocol D.

[0264] Table 1 summarizes the EC values ​​calculated using the geometric mean fluorescence intensity (gMFI) from FlowJo version 10 software on an 8-point, 6-fold sequential titration curve. 50 The fold change of each variant relative to rhIL-2 was also calculated as a measure of the attenuation level compared to the activity of the rhIL-2 positive control. Some EC 50 The values ​​could not be calculated using GraphPad Prism 7 software and were marked as not calculated (NC); however, based on the dose titration curves, these variants showed no decay.

[0265] Compared to rhIL-2 expressing either a high-affinity hIL-2 receptor (NK-92) or a moderate-affinity hIL-2 receptor (TF1+IL-2Rβ) on cell lines, fusion of the hIL-2 moiety with the N-terminus or C-terminus of the immunoglobulin heavy chain did not result in decreased IL-2 activity. Direct fusion (df) of hIL-2 with the antibody component of the fusion protein did not lead to changes in IL-2 activity compared to fusion using a six-amino acid linker (L6) between IL-2 and the antibody component. Similarly, fusion of the IL-2 component with either the heavy or light chain of the antibody component did not lead to changes in IL-2 activity compared to rhIL-2. All N-terminal or C-terminal and linker fusion protein variants in which the hCD25 / hIL-2Rα moiety is fused to hIL-2 are expected to exhibit reduced binding of the fusion protein to the CD25 of the hIL-2 receptor on cells. In experiments, these constructs exhibited strongly attenuated hIL-2 activity (at least 45-fold attenuation) against the high-affinity IL-2 receptor (NK-92) and 18-fold attenuation against the intermediate-affinity hIL-2 receptor (TF1+IL-2Rβ).

[0266]

[0267]

[0268] Example 2: Generation of antibody-attenuated hIL-2 fusion protein variants and determination of their binding kinetics with recombinant human CD25 and / or human CD122.

[0269] Since hIL-2 activity is not reduced in various N-terminal or C-terminal immunoglobulin heavy chain fusion proteins, the hIL-2 C-terminal heavy chain L6 fusion protein named "1H3-hIgG1-L6-hIL-2" (SEQ ID NO: 361, 374) was used as the basic construct for antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety. To investigate the role of these residues in recognizing human CD25 / IL-2Rα and / or human CD122 / IL-2Rβ or CD132 / IL-2Rγ (human IL-2R subunits), single, two, and / or multiple amino acid substitutions were introduced into selected residues of human IL-2. More than three hundred antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety were generated and evaluated in six rounds. These variants were first screened on IL-2-dependent cell lines (NK-92 and TF1+IL-2Rβ) using a fixed-concentration, flow-based phosphorylated STAT5 (pSTAT5) assay in dose-titer curves. Phosphorylated STAT5 is a downstream signal of IL-2 activity and was used as a snapshot measure of IL-2 potency. IL-2-dependent cell proliferation assays were also performed to measure IL-2 activity over a 3–4 day timeframe. Selection criteria for attenuated hIL-2 included: (1) reduced IL-2 potency in both the NK-92 and TF1+IL-2Rβ cell lines, with agonist activity greater than 50% in both cell lines; and (2) moderate to high yield.

[0270] Human anti-DNase I antibody-hIL-2 fusion protein is produced by fusing human IL-2 or human IL-2 variants (SEQ ID NO: 1-344, 377, 378 and 575) with 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. The fusion protein is then combined with the hIgG1 light chain (1H3-hkappa LC; SEQ ID NO: 374) to produce the 1H3-hIgG1-L6-hIL-2 fusion protein (provided in Table 28). Mouse anti-yellow fever virus antibody-hIL-2 fusion proteins are also produced by fusing a human IL-2 variant with the C-terminus of a mouse anti-yellow fever virus antibody (clone 2D12, having a mouse IgG1 isotype) heavy chain, with D265A substitution to replace the reduced immune effector function, via an L6 linker. This fusion protein is then combined with a 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). Some of these mouse anti-yellow fever virus antibody-hIL-2 fusion proteins are formatted into the human IgG1 constant region and produced in the same manner as described above (i.e., combined with a 2D12-hKappa light chain (2D12-hKappa LC; SEQ ID NO: 573)). IL-2 amino acid substitutions are repeated six times, designated as groups 1 through 6. Standard techniques were used to generate and express fusion proteins of 1H3-hIgG1-L6-hIL-2, 2D12-mIgG1-D265A-L6-hIL-2, and 2D12-hIgG1-L6-hIL-2, and to purify protein A.

[0271] Group 1 contains only the initial series of 2D12-mIgG1-D265A-L6-hIL-2 or 2D12-hIgG1-L6-hIL-2 fusion proteins, which contain human IL-2 and are expected to involve binding only to one of the IL-2 receptor subunits CD25 / IL-2Rα, CD122 / IL-2Rβ or CD132 / IL-2Rγ, or a combination of substitutions. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD25 / IL-2Rα: F42K (SEQ ID NO: 1), V69A (SEQ ID NO: 2), V69E (SEQ ID NO: 3), V69F (SEQ ID NO: 4), V69G (SEQ ID NO: 5), V69H (SEQ ID NO: 6), V69I (SEQ ID NO: 7), V69K (SEQ ID NO: 8), V69L (SEQ ID NO: 9), V69M (SEQ ID NO: 10), V69Q (SEQ ID NO: 11), V69S (SEQ ID NO: 12), V69T (SEQ ID NO: 13), V69W (SEQ ID NO: 14), V69Y (SEQ ID NO: 15), V69R (SEQ ID NO: 581), (F42K / F44K) (SEQ ID NO: 581). 16), (F44K / Y45R) (SEQ ID NO: 17), (F42K / V69R) (SEQ ID NO: 18), (Y45R / V69R) (SEQ ID NO: 19), (F42K / F44K / Y45R) (SEQ ID NO: 20), (F42A / Y45A / L72G) (SEQ ID NO: 574), (R38A / F42K / Y45R) (SEQ ID NO: 21), (R38E / F42K / Y45R) (SEQ ID NO: 22), (K43E / F42K / Y45R) (SEQ ID NO: 23), (K43T / F42K / Y45R) (SEQ ID NO: 24), (F42K / Y45R / E62A) (SEQ ID NO: 25), (P65R / F42K / Y45R) (SEQ ID NO: 26), (P65S / F42K / Y45R) (SEQ ID NO: 27), (V69A / F42K / Y45R) (SEQ ID NO: 28), (V69D / F42K / Y45R) (SEQ ID NO: 29) or (V69R / F42K / Y45R) (SEQ IDNO: 30).The substitutions in this group include the following substitutions that are expected to regulate binding to CD122 / IL-2Rβ: D20A (SEQ ID NO: 31), D20N (SEQ ID NO: 32), D20K (SEQ ID NO: 33), N88A (SEQ ID NO: 34), N88G (SEQ ID NO: 35), N88H (SEQ ID NO: 36), N88K (SEQ ID NO: 37), (D20A / D84A) (SEQ ID NO: 38), (D20A / E15A) (SEQ ID NO: 39), (D20A / E95A) (SEQ ID NO: 40), (D20A / N88A) (SEQ ID NO: 41), (D20A / S87A) (SEQ ID NO: 42), (D84A / N88A) (SEQ ID NO: 43), (E15A / N88A) (SEQ ID NO: 44). 44) or (S87A / N88A) (SEQ ID NO: 45). Group 1 also includes the following IL-2 substitutions that are expected to regulate the binding of IL-2 to CD132 / IL-2-Rγ: Q126L (SEQ ID NO: 377) or Q126E (SEQ ID NO: 378). The IL-2 substitutions studied in Group 1 are not expected to regulate binding to more than one IL-2 receptor subunit.

[0272] Group 2 contains a series of 1H3-hIgG1-L6-hIL-2 fusion proteins, which contain one or more substitutions in human IL-2 that are expected to involve only CD25 / IL-2Rα binding. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD25 / IL-2Rα: R38A (SEQ ID NO: 46), R38D (SEQ ID NO: 47), R38E (SEQ ID NO: 48), R38Q (SEQ ID NO: 49), F42R (SEQ ID NO: 50), F42A (SEQ ID NO: 51), F42D (SEQ ID NO: 52), F42H (SEQ ID NO: 53), K43A (SEQ ID NO: 54), K43E (SEQ ID NO: 55), K43Q (SEQ ID NO: 56), Y45A (SEQ ID NO: 57), Y45K (SEQ ID NO: 58), Y45S (SEQ ID NO: 59), Y45R (SEQ ID NO: 60), E61A (SEQ ID NO: 61), E61R (SEQ ID NO: 60), and E61A (SEQ ID NO: 61). 62), E61K (SEQ ID NO: 63), E62A (SEQ ID NO: 64), E62R (SEQ ID NO: 65), E62K (SEQ ID NO: 66), E62Y (SEQ ID NO: 67), E68Y (SEQ ID NO: 68), E68A (SEQ ID NO: 69), E68K (SEQ ID NO: 70), E68R (SEQ ID NO: 71), E68L (SEQ ID NO: 72), L72Y (SEQ ID NO: 73), L72R (SEQ ID NO: 74), L72A (SEQ ID NO: 75), L72D (SEQ ID NO: 76), L72H (SEQ ID NO: 77), L72F (SEQ ID NO:78), (R38D / E61R) (SEQ ID NO: 79), (R38D / E61R / K43E) (SEQ ID NO: 80) or (T3A / F42A / Y45A / L72G / C125A) (SEQ ID NO: 81). The substitution T3A is introduced into the IL-2 amino acid sequence to remove the predicted O-linked glycosylation site on human IL-2 (see, for example, International Publication WO2012 / 107417), and the substitution C125A is introduced into the IL-2 amino acid sequence to remove unpaired cysteine ​​residues (see, for example, International Publication WO2018 / 184964).The IL-2 substitutions studied in Group 2 are not expected to regulate the binding of IL-2 to CD132 / IL-2-Rγ, nor are they expected to regulate binding to more than one IL-2 receptor subunit.

[0273] Group 3 contains a series of 1H3-hIgG1-L6-hIL-2 fusion proteins, which contain one or more substitutions in human IL-2 that are expected to involve only CD122 / IL-2Rβ binding.The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD122 / IL-2Rβ: E15A (SEQ ID NO: 82), E15R (SEQ ID NO: 83), E15K (SEQ ID NO: 84), H16A (SEQ ID NO: 85), H16Y (SEQ ID NO: 86), H16E (SEQ ID NO: 87), L19A (SEQ ID NO: 88), D20I (SEQ ID NO: 89), D20S (SEQ ID NO: 90), D20H (SEQ ID NO: 91), D20T (SEQ ID NO: 92), D20W (SEQ ID NO: 93), D20Y (SEQ ID NO: 94), D20R (SEQ ID NO: 95), D20F (SEQ ID NO: 96), R81A (SEQ ID NO: 97), D84A (SEQ ID NO: 98), and D20S (SEQ ID NO: 99). 98), D84R (SEQ ID NO: 99), D84K (SEQ ID NO: 100), S87A (SEQ ID NO: 101), N88Y (SEQ ID NO: 102), N88D (SEQ ID NO: 103), N88R (SEQ ID NO: 104), N88E (SEQ ID NO: 105), N88F (SEQ ID NO: 106), N88I (SEQ ID NO: 107), I92A (SEQ ID NO: 108), I92Y (SEQ ID NO: 109), I92S (SEQ ID NO: 110), I92F (SEQ ID NO: 111), I92R (SEQ ID NO: 112), I92D (SEQ ID NO: 113), I92E (SEQ ID NO: 114), E95A (SEQ ID NO: 115), E95R (SEQ ID NO: 116), E95K (SEQ ID NO: 117), (D20Y / H16E) (SEQ ID NO: 118), (D20Y / H16A) (SEQ ID NO: 119), (D20Y / H16Y) (SEQ ID NO: 120), (D20Y / I92A) (SEQ ID NO: 121), (D20Y / I92S) (SEQ ID NO: 122), (D20Y / I92R) (SEQ ID NO: 123), (D20Y / E95R) (SEQ ID NO: 124) or (D20Y / E95A) (SEQ ID NO: 125).

[0274] Group 4 contains a series of fusion proteins containing 1H3-hIgG1-L6-hIL-2 HC fused to the extracellular domain portion of CD25 / IL-2Rα (SEQ ID NO: 126), a 20-amino acid linker (L20) (SEQ ID NO: 364), and a human IL-2 variant containing one or more residues substituted that are expected to be involved in binding to CD122 / IL-2Rβ. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD122 / IL-2Rβ: E15A (SEQ ID NO: 82), D20I (SEQ ID NO: 89), D20S (SEQ ID NO: 90), D20H (SEQ ID NO: 91), D20W (SEQ ID NO: 93), D20Y (SEQ ID NO: 94), D20R (SEQ ID NO: 95), D20F (SEQ ID NO: 96), D84K (SEQ ID NO: 100), S87A (SEQ ID NO: 101), N88Y (SEQ ID NO: 102), N88D (SEQ ID NO: 103), N88R (SEQ ID NO: 104), N88E (SEQ ID NO: 105), N88F (SEQ ID NO: 106), N88I (SEQ ID NO: 89), D20S (SEQ ID NO: 90), D20H (SEQ ID NO: 91), D20W (SEQ ID NO: 93), D20Y (SEQ ID NO: 94), D20R (SEQ ID NO: 95), D20F (SEQ ID NO: 96), D84K (SEQ ID NO: 100), S87A (SEQ ID NO: 101), N88Y (SEQ ID NO: 102), N88D (SEQ ID NO: 103), N88R (SEQ ID NO: 104), N88E (SEQ ID NO: 105), N88F (SEQ ID NO: 106), N88I (SEQ ID NO: 107), and D88S (SEQ ID NO: 108). 107), I92A (SEQ ID NO: 108), E95A (SEQ ID NO: 115), or E95K (SEQ ID NO: 117). The antibody-degraded hIL-2 fusion protein in this group is represented as 1H3-hIgG1-L6-hCD25(1-164)-L20-hIL-2.

[0275] Group 5 contains a series of 1H3-hIgG1-L6-hIL-2 variants, which include IL-2 with substitution combinations expected to involve IL-2 binding to CD25 / IL-2Rα and CD122 / IL-2Rβ or CD132 / IL-2Rγ. Additionally, some variants lack the first three amino acids at the N-terminus of the hIL-2 moiety (Δ1-3APT). The fusion proteins in group 5 include the following IL-2 substitutions expected to regulate the binding of IL-2 to CD25 / IL-2Rα and CD122 / IL-2Rβ: (F42D / D20A) (SEQ ID NO: 127), (F42R / D20A) (SEQ ID NO: 128), (F42K / D20A) (SEQ ID NO: 129), (F42A / D20A) (SEQ ID NO: 130), (F42H / D20A) (SEQ ID NO: 131), (Y45R / D20A) (SEQ ID NO: 132), (Y45K / D20A) (SEQ ID NO: 133), (R38N / D20A) (SEQ ID NO: 134), (R38G / D20A) (SEQ ID NO: 135), (R38H / D20A) (SEQ ID NO: 136). 136), (R38I / D20A) (SEQ ID NO: 137), (R38L / D20A) (SEQ ID NO: 138), (R38M / D20A) (SEQ ID NO: 139), (R38F / D20A) (SEQ ID NO: 140), (R38P / D20A) (SEQ ID NO: 141), (R38S / D20A) (SEQ ID NO: 142), (R38T / D20A) (SEQ ID NO: 143), (R38W / D20A) (SEQ ID NO: 144), (R38Y / D20A) (SEQ ID NO: 145), (R38V / D20A) (SEQ ID NO:146), (R38A / D20A) (SEQ ID NO: 147), (R38Q / D20A) (SEQ ID NO: 148), (D20A / R38E) (SEQ ID NO: 149), (R38D / D20A) (SEQ ID NO: 150), (K43E / D20A) (SEQ ID NO: 151), (E61A / D20A) (SEQ ID NO: 152), (E62A / D20A) (SEQ ID NO: 153), (E62Y / D20A) (SEQ ID NO:154), (L72D / D20A) (SEQ ID NO:155)、(L72H / D20A)(SEQ ID NO: 156)、(L72R / D20A)(SEQID NO: 157)、(F42D / I92D)(SEQ ID NO: 158)、(F42R / I92D)(SEQ ID NO: 159)、(F42H / I92D)(SEQ ID NO: 160)、(F42A / I92D)(SEQ ID NO: 161)、(H16A / F42A)(SEQ ID NO:575)、(K43E / I92D)(SEQ ID NO: 162)、(Y45R / I92D)(SEQ ID NO: 163)、(Y45K / I92D)(SEQID NO: 164)、(E62A / I92D)(SEQ ID NO: 165)、(E62Y / I92D)(SEQ ID NO: 166)、(L72D / I92D)(SEQ ID NO: 167)、(L72H / I92D)(SEQ ID NO: 168)、(L72R / I92D)(SEQ ID NO:169)、(R38D / I92D)(SEQ ID NO: 170)、(R38E / I92D)(SEQ ID NO: 171)、(R38Q / I92D)(SEQID NO: 172)、(R38A / I92D)(SEQ ID NO: 173)、(R38E / N88R)(SEQ ID NO: 174)、(R38E / D84R)(SEQ ID NO: 175)、(R38E / D84K)(SEQ ID NO: 176)、(F42A / Y45R / D20A)(SEQ ID NO:177)、(F42H / Y45R / D20A)(SEQ ID NO: 178)、(R38D / E61R / D20A)(SEQ ID NO: 179)、(R38E / E61R / D20A)(SEQ ID NO: 180)、(R38Q / E61R / D20A)(SEQ ID NO: 181)、(R38A / E61R / D20A)(SEQ ID NO: 182)、(R38A / D20A / E95A)(SEQ ID NO: 183)、(D20A / E95A / R38D)(SEQ ID NO:184)、(D20A / E95A / R38E)(SEQ ID NO: 185)、(D20A / E95A / R38Q)(SEQ ID NO: 186)、(D20A / E95A / F42R)(SEQ ID NO: 187)、(D20A / E95A / F42A)(SEQID NO: 188)、(D20A / E95A / F42D)(SEQ ID NO: 189)、(D20A / E95A / F42H)(SEQ ID NO: 190)、(D20A / E95A / F42K)(SEQID NO: 191)、(D20A / E95A / K43A)(SEQ ID NO: 192)、(D20A / E95A / K43E)(SEQ ID NO:193)、(D20A / E95A / K43Q)(SEQ ID NO: 194)、(D20A / E95A / Y45A)(SEQ ID NO: 195)、(D20A / E95A / Y45K)(SEQ ID NO: 196)、(D20A / E95A / Y45S)(SEQ ID NO: 197)、(D20A / E95A / Y45R)(SEQ ID NO: 198)、(D20A / E95A / E61A)(SEQ ID NO: 199)、(D20A / E95A / E62A)(SEQ ID NO:200)、(D20A / E95A / E62R)(SEQ ID NO: 201)、(D20A / E95A / E62K)(SEQ ID NO: 202)、(D20A / E95A / E62Y)(SEQ ID NO: 203)、(D20A / E95A / E68Y)(SEQ ID NO: 204)、(D20A / E95A / E68A)(SEQ ID NO: 205)、(D20A / E95A / E68L)(SEQ ID NO: 206)、(D20A / E95A / L72Y)(SEQ ID NO:207)、(D20A / E95A / L72R)(SEQ ID NO: 208)、(D20A / E95A / L72A)(SEQ ID NO: 209)、(D20A / E95A / L72D)(SEQ ID NO: 210)、(D20A / E95A / L72H)(SEQ ID NO: 211)、(D20A / E95A / L72F)(SEQ ID NO: 212)、(F42K / Y45R / D20A / S87A)(SEQ ID NO: 213)、(F42K / Y45R / D20A / E95A)(SEQ ID NO: 214)、(D20A / R38E / C125A)(SEQ ID NO: 215)、(T3A / D20A / R38E)(SEQ ID NO:216)、(T3A / D20A / R38E / C125A)(SEQ ID NO:217), (Δ1-3APT / D20A / R38E) (SEQ ID NO: 218) or (Δ1-3APT / D20A / R38E / C125A) (SEQ ID NO: 219). The fusion proteins in group 5 include the following IL-2 substitutions expected to regulate the binding of IL-2 to CD25 / IL-2Rα and CD132 / IL-2R: (R38E / Q22A) (SEQ ID NO: 220), (R38E / T123A) (SEQ ID NO: 221), (R38E / I129A) (SEQ ID NO: 222), (R38E / S130A) (SEQ ID NO: 223), (R38E / Q126A) (SEQ ID NO: 224), (R38E / Q126D) (SEQ ID NO: 225), (R38E / Q126V) (SEQ ID NO: 226), (R38E / Q22A / S130A) (SEQ ID NO: 227), (F42K / Y45R / Q126D) (SEQ ID NO: 228). 228) or (D20A / E95A / Q126D) (SEQ ID NO: 229). SEQ ID NO: 127-229 and 575 list the group 5 antibody-attenuated hIL-2 fusion proteins based on mutations in the hIL-2 sequence of the IL-2 sequence.

[0276] Group 6 contains a series of 1H3-hIgG1-L6-hIL-2 fusion proteins, which contain human IL-2 with expected combinations of substitutions involving IL-2 binding to CD25 / IL-2Rα and CD122 / IL-2Rβ but not to CD132 / IL-2Rγ. The fusion proteins in group 6 include the following substitution combinations that are expected to regulate the binding of IL-2 to CD25 / IL-2Rα and CD122 / IL-2Rβ: (D20A / E61R) (SEQ ID NO: 230), (D20A / E61N) (SEQ ID NO: 231), (D20A / E61D) (SEQ ID NO: 232), (D20A / E61Q) (SEQ ID NO: 233), (D20A / E61G) (SEQ ID NO: 234), (D20A / E61H) (SEQ ID NO: 235), (D20A / E61I) (SEQ ID NO: 236), (D20A / E61L) (SEQ ID NO: 237), (D20A / E61K) (SEQ ID NO: 238), (D20A / E61M) (SEQ ID NO: 239). 239), (D20A / E61F) (SEQ ID NO: 240), (D20A / E61P) (SEQ ID NO: 241), (D20A / E61S) (SEQ ID NO: 242), (D20A / E61T) (SEQ ID NO: 243), (D20A / E61W) (SEQ ID NO: 244), (D20A / E61Y) (SEQ ID NO: 245), (D20A / E61V) (SEQ ID NO: 246), (D20A / F42N) (SEQ ID NO: 247), (D20A / F42Q) (SEQ ID NO: 248), (D20A / F42E) (SEQ ID NO: 249), (D20A F42G) (SEQ ID NO:250), (D20A / F42I) (SEQ ID NO: 251), (D20A / F42L) (SEQ ID NO: 252), (D20A / F42M) (SEQ ID NO: 253), (D20A / F42P) (SEQ ID NO: 254), (D20A / F42S) (SEQ ID NO: 255), (D20A / F42T) (SEQ ID NO: 256), (D20A / F42W) (SEQ ID NO: 257), (D20A / F42Y) (SEQ ID NO: 258), (D20A / F42V) (SEQ ID NO:259)、(D20A / Y45A)(SEQ ID NO: 260)、(D20A / Y45N)(SEQID NO: 261)、(D20A / Y45D)(SEQ ID NO: 262)、(D20A / Y45Q)(SEQ ID NO: 263)、(D20A / Y45E)(SEQ ID NO: 264)、(D20A / Y45G)(SEQ ID NO: 265)、(D20A / Y45H)(SEQ ID NO:266)、(D20A / Y45I)(SEQ ID NO: 267)、(D20A / Y45L)(SEQ ID NO: 268)、(D20A / Y45M)(SEQID NO: 269)、(D20A / Y45F)(SEQ ID NO: 270)、(D20A / Y45P)(SEQ ID NO: 271)、(D20A / Y45S)(SEQ ID NO: 272)、(D20A / Y45T)(SEQ ID NO: 273)、(D20A / Y45W)(SEQ ID NO:274)、(D20A / Y45V)(SEQ ID NO: 275)、(I92D / F42N)(SEQ ID NO: 276)、(I92D / F42Q)(SEQID NO: 277)、(I92D / F42E)(SEQ ID NO: 278)、(I92D / F42G)(SEQ ID NO: 279)、(I92D / F42I)(SEQ ID NO: 280)、(I92D / F42L)(SEQ ID NO: 281)、(I92D / F42K)(SEQ ID NO:282)、(I92D / F42M)(SEQ ID NO: 283)、(I92D / F42P)(SEQ ID NO: 284)、(I92D / F42S)(SEQID NO: 285)、(I92D / F42T)(SEQ ID NO: 286)、(I92D / F42W)(SEQ ID NO: 287)、(I92D / F42Y)(SEQ ID NO: 288)、(I92D / F42V)(SEQ ID NO: 289)、(I92D / Y45A)(SEQ ID NO:290)、(I92D / Y45N)(SEQ ID NO: 291)、(I92D / Y45D)(SEQ ID NO: 292)、(I92D / Y45Q)(SEQID NO: 293)、(I92D / Y45E)(SEQ ID NO: 294)、(I92D / Y45G)(SEQ ID NO:295)、(I92D / Y45H)(SEQ ID NO: 296)、(I92D / Y45I)(SEQ ID NO: 297)、(I92D / Y45L)(SEQ ID NO:298)、(I92D / Y45M)(SEQ ID NO: 299)、(I92D / Y45F)(SEQ ID NO: 300)、(I92D / Y45P)(SEQID NO: 301)、(I92D / Y45S)(SEQ ID NO: 302)、(I92D / Y45T)(SEQ ID NO: 303)、(I92D / Y45W)(SEQ ID NO: 304)、(I92D / Y45V)(SEQ ID NO: 305)、(R38E / D20H)(SEQ ID NO:306)、(R38E / D20S)(SEQ ID NO: 307)、(F42A / N88R)(SEQ ID NO: 308)、(F42A / N88D)(SEQID NO: 309)、(R38E / D84A)(SEQ ID NO: 310)、(R38E / D84N)(SEQ ID NO: 311)、(R38E / D84Q)(SEQ ID NO: 312)、(R38E / D84E)(SEQ ID NO: 313)、(R38E / D84G)(SEQ ID NO:314)、(R38E / D84H)(SEQ ID NO: 315)、(R38E / D84I)(SEQ ID NO: 316)、(R38E / D84L)(SEQID NO: 317)、(R38E / D84M)(SEQ ID NO: 318)、(R38E / D84F)(SEQ ID NO: 319)、(R38E / D84P)(SEQ ID NO: 320)、(R38E / D84S)(SEQ ID NO: 321)、(R38E / D84T)(SEQ ID NO:322)、(R38E / D84W)(SEQ ID NO: 323)、(R38E / D84Y)(SEQ ID NO: 324)、(R38E / D84V)(SEQID NO: 325)、(R38E / I92A)(SEQ ID NO: 326)、(R38E / I92R)(SEQ ID NO: 327)、(R38E / I92N)(SEQ ID NO: 328)、(R38E / I92Q)(SEQ ID NO: 329)、(R38E / I92E)(SEQ ID NO:330)、(R38E / I92G)(SEQ ID NO:331), (R38E / I92H) (SEQ ID NO: 332), (R38E / I92L) (SEQ ID NO: 333), (R38E / I92K) (SEQ ID NO: 334), (R38E / I92M) (SEQ ID NO: 335), (R38E / I92F) (SEQ ID NO: 336), (R38E / I92P) (SEQ ID NO: 337), (R38E / I92S) (SEQ ID NO: 338), (R38E / I92T) (SEQ ID NO: 339), (R38E / I92W) (SEQ ID NO: 340), (R38E / I92Y) (SEQ ID NO: 341), (R38E / I92V) (SEQ ID NO: 342), (R38E / H16E) (SEQ ID NO: (343) or (R38K / D20A) (SEQ ID NO: 344). SEQ ID NO: 230-344 lists the group 6 antibody-attenuated hIL-2 fusion proteins numbered according to mutations in the hIL-2 sequence of the IL-2 sequence.

[0277] Binding kinetics of several purified 1H3-hIgG1-L6-hIL-2 variants of individual recombinant human CD25 and human CD122 were determined using biolayer interferometry (BLI). Briefly, binding experiments were performed at 25 °C using an Octet Red96 instrument (Pall ForteBio). C-terminal multihistidine-tagged extracellular domains of human CD25 and human CD122 were captured onto an anti-His2 sensor (Pall ForteBio). Starting at a maximum concentration of 300 nM, the receptor-loaded sensor was immersed in seven consecutive 3-fold dilutions for each 1H3-hIgG-L6-hIL-2 variant. The 1H3-hIgG1-L6-hIL-2 fusion protein was diluted in an 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). Kinetic constants were calculated using a monovalent binding model.

[0278] Table 2 records the association constants (k) of 74 immunoglobulin-hIL-2 fusion protein variants that bind to recombinant human CD25 or recombinant human CD122. on ), dissociation constant (k) off ) and equilibrium constant (K D ).

[0279] Table 2. Binding kinetics of 1H3-hIgG-L6-hIL-2 fusion protein obtained by Octet BLI with recombinant human CD25 or CD122

[0280]

[0281]

[0282]

[0283]

[0284] Table 3 records the association constants (k) of 74 1H3-hIgG1-L6-hIL-2 fusion proteins that bind to recombinant human CD122. on ), dissociation constant (k) off ) and equilibrium constant (K D ).

[0285]

[0286]

[0287]

[0288]

[0289]

[0290] Example 3: Screening for attenuation of high-affinity and intermediate-affinity hIL-2 receptors using fixed concentrations of cell-based potency pSTAT5

[0291] As described in Protocol D, the attenuation of the antibody-attenuated hIL-2 fusion protein described in Example 2 was tested in a fixed-concentration pSTAT5 screening using NK-92 (expressing high-affinity hIL-2 receptor) and TF1+IL-2Rβ (expressing medium-affinity hIL-2 receptor) cell lines. Tables 4-8 list the fold change in geometric mean fluorescence intensity (gMFI) of the antibody-attenuated hIL-2 fusion protein from free cytokine wild-type rhIL-2, i.e., the measurement of reduced IL-2 activity. For fixed-concentration screening, the fold change was calculated by dividing the gMFI of rhIL-2 by the gMFI of the variant. For experiments with complete titration curves, the fold change was calculated by dividing the EC5 of rhIL-2 by the EC5 of the variant. 50 Value divided by the variant's EC 50The fold change relative to rhIL-2 was calculated. The fold change was rounded to the nearest integer. The decrease in gMFI in both the NK-92 and TF1+IL-2Rβ cell lines, compared to gMFI produced by rhIL-2, indicates a decline in IL-2 activity at high and intermediate affinity receptors. The Group 1 variant described in Example 2 was not tested in the pSTAT5 efficacy screening based on fixed cell concentrations.

[0292] The IL-2 agonist activity of each variant tested was also evaluated and characterized as a complete or partial IL-2 agonist, or as having no IL-2 activity (inactive). A dose-titer curve of the 1H3-hIgG1-L6-hIL-2 fusion protein reaching the maximum gMFI level shown by the rhIL-2 positive control was considered an antibody-attenuated hIL-2 fusion protein with complete agonist activity. The percentage of partial agonist activity to complete activity was calculated using the maximum gMFI of rhIL-2 as 100%. Antibody-attenuated hIL-2 fusion proteins with less than 10% of the maximum gMFI of rhIL-2 at the highest concentration of 1200 nM were considered to have no agonist activity (inactive). The GraphPad Prism 7 software could not accurately calculate certain EC values. 50 The values ​​and decay levels are estimates because the activity has not reached its maximum value.

[0293] pSTAT5 fixed concentration results demonstrated that although some single residue substitutions attenuated IL-2 activity on the high-affinity cell line (NK-92), a combination of substitutions that modulate the combination of both α and β chains or both α and γ chains was required to significantly attenuate IL-2 activity on the high-affinity IL-2 receptor (the attenuation of recombinant hIL-2 was more than 20-fold).

[0294] Table 4. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from Group 2

[0295]

[0296]

[0297] Table 5. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 3.

[0298]

[0299]

[0300]

[0301] Table 6. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 4

[0302]

[0303]

[0304] Table 7. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 5

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] Table 8. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 6

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] Example 4: Screening for attenuation of IL-2 fusion proteins in each of the high-affinity and intermediate-affinity hIL-2 receptors using cell-based efficacy pSTAT5 dose titration.

[0317] 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 in pSTAT5 titration curves using NK-92 and TF1+IL-2Rβ cell lines as described in Protocol D.

[0318] The four-parameter logic curve was generated using gMFI with a positive pSTAT5 signal from the Alexa Fluor 647, and then EC was calculated using GraphPad Prism 7 software. 50 Values. These values ​​were compared to a recombinant hIL-2 (rhIL-2) control as measurements of attenuation. Table 9-13 summarizes the fold changes in rhIL-2 activity calculated using gMFI with the Alexa Fluor 647 signal.

[0319] An increase in the fold change relative to rhIL-2 indicates the degree of decay in hIL-2 activity. The agonistic activity of each antibody-decaying hIL-2 fusion protein tested in pSTAT5 titration profiles was also evaluated and characterized as complete, partial, or inactive (inactive). Antibody-decaying hIL-2 fusion proteins that reached maximum gMFI levels, similar to rhIL-2, were considered variants with complete agonist activity. Partial agonist activity was calculated as described in Example 3. Inactive antibody-decaying hIL-2 fusion proteins were classified as having less than 10% activity compared to rhIL-2. Certain fold changes relative to rhIL-2 could not be accurately calculated using GraphPad Prism 7 software (indicated as not calculated or “NC”) because a complete four-parameter logistic curve was not generated, and therefore these values ​​are estimates (labeled in Tables 9-13). a However, on the graph, these variants attenuated by more than 10,000 times relative to rhIL-2 (data not shown). This is indicated in Tables 9-13 as ">10,000; NC on the graph".

[0320] The complete titration of pSTAT5 yielded results similar to those presented in Example 3, where substitutions regulating binding to both the α and β chains significantly attenuated IL-2 activity on the high-affinity IL-2 receptor compared to single substitutions binding only the α or β chain. Furthermore, the complete titration of pSTAT5 also distinguished between variants with substitutions leading to inactivation and those with high attenuation. Finally, the comparison of dose-titer curves demonstrated a more precise attenuation level than fixed-concentration assays.

[0321] Table 9. Fold change relative to rhIL-2 and agonistic activity on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 2

[0322]

[0323]

[0324] Table 10. Fold change relative to rhIL-2 and agonistic activity on the 1H3-hIgG1-L6-hIL-2 fusion protein variant from group 3

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334] Example 5: Testing the attenuation of high-affinity and intermediate-affinity hIL-2 receptors using cell-based proliferation assays

[0335] As described in Protocol E, the attenuation of IL-2 activity 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 proliferation assays of both the NK-92 and TF1+IL-2Rβ cell lines. The results are provided in Tables 14-19.

[0336] In this cell-based proliferation assay, selected 1H3-hIgG1-L6-hIL-2 fusion proteins from Example 4, exhibiting significant attenuation in the pSTAT5 titration curve, were tested. pSTAT5 is a downstream reading of IL-2 activity, and the assay requires only 10 minutes of stimulation, potentially providing a 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 proteins, or a recombinant hIL-2 control, to provide a more physiologically relevant reading of in vivo IL-2-dependent activity. The IL-2-dependent activity of other 2D12-mIgG1-D265A-L6-hIL-2 and 2D12-hIgG1-L6-hIL-2 fusion proteins generated but not tested in the pSTAT5 assay was also determined using this proliferation assay.

[0337] Similar to cell-based pSTAT5 dose titration experiments, the calculated EC is determined based on relative luminescence units (RLU) rather than gMFI. 50 And once EC is calculated 50 The results analysis was performed in the same manner as in Example 4. Similar to the results identified in Example 4, the proliferation curves demonstrated that some substitutions regulating binding to both the α and β chains significantly attenuated IL-2 activity on high-affinity receptors compared to single substitutions that bound only the α or β chain. The proliferation of these selected 1H3-hIgG1-L6-hIL-2 fusion proteins in the TF1+IL-2Rβ cell line was also tested, demonstrating that some of these identical substitutions substantially attenuated IL-2 activity on intermediate-affinity receptors.

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353] Example 6: Production of anti-hPD-1 antibodies

[0354] Several methods were used to generate a variety of different anti-hPD-1 antibodies with the desired properties.

[0355] In one approach, transgenic chickens (OmniChicken™) expressing human antibody genes (human light chain (VLCL or VKCK) and human VH) and the constant region of the chicken heavy chain were used to generate anti-hPD-1 human monoclonal antibodies (Ching et al., mAbs 2018). The transgenic chickens were immunized every 14 days with 100 µg of Fc-labeled human PD-1 protein (huPD-1-Fc) (SEQ ID NO: 380) for 14 weeks. In another approach, the transgenic chickens were genetically immunized six times with DNA encoding human PD-1 (SEQ ID NO: 347), followed by a final boost with 100 µg of huPD-1-Fc (SEQ ID NO: 380). Serum immune responses in each animal were monitored by an ELISA against biotinylated human PD-1 on streptavidin-coated plates.

[0356] Spleen cells were isolated from each immunized animal and tested for positive antibody clones using gel encapsulation microenvironment (GEM) assays (e.g., 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, 65(4), 341-352)) and screened for human PD-1-tagged beads. Positive clones were sequenced, and the variable regions of the heavy and light chains were cloned, assembled into single-chain variable fragments, and fused into the hinge and Fc regions of immunoglobulins (ScFv-Fc). These unique scFv-Fc fusion proteins were transiently expressed in Expi293 cells, and their binding activity was tested by ELISA on supernatants coated with either huPD-1-Fc (SEQ ID NO: 380) or cynomolgus monkey PD-1-Fc (SEQ ID NO: 381). A total of 102 unique anti-human PD-1 variable weight and variable light pairings were identified using this method. 2H7-hIgG4 (SEQ ID NO: 382-391, 424, and 425) and A2-hIgG4 (SEQ ID NO: 402-411, 428, and 429) are antibodies identified using this method.

[0357] Other methods enabled the identification of an anti-hPD-1 antibody denoted as C51E6-hIgG4, which was germline optimized to become an antibody named C51E6-5-hIgG4 (SEQ ID NO: 392-401, 426, 427), and then humanized and further sequence optimized to become an antibody named Abz1mod-hIgG4 (SEQ ID NO: 449, 450).

[0358] As described in General Method Protocol A, antibodies expressing the anti-PD-1 variable region sequence were used to assess their binding ability to PD-1-expressing cells using flow cytometry. First, test antibodies were screened for binding to human PD-1 using a Jurkat cell line expressing recombinant human PD-1 (Jurkat + hPD-1 cell line). Antibodies were serially diluted starting at a maximum concentration of 280 nM, and then allophycocyanin-conjugated anti-human IgG secondary antibody was added to the cells for detection. Of 92 hits, 79 of the tested anti-PD-1 antibodies showed EC50 values. 50 Binding (by flow cytometry) < 30 nM. 2H7-hIgG4 (SEQ ID NO: 382-391, 424 and 425), C51E6-5-hIgG4 (SEQ ID NO: 392-401, 426 and 427), A2-hIgG4 (SEQ ID NO: 402-411, 428 and 429), OMC.1.B6-hIgG4 (SEQ ID NO: 438 and 439), OMC.1.D6-hIgG4 (SEQ ID NO: 442 and 443), OMC.2.C6-hIgG4 (SEQ ID NO: 440 and 441), 1H9-hIgG4 (SEQ ID NO: 576 and 525), 1D5-hIgG4 (SEQ ID NO: 577 and 527), and 2A3.H7-hIgG4 (SEQ ID NO: 577 and 527). Antibodies 424 and 523 are a group of antibodies identified as having moderate to high affinity for hPD-1 using Jurkat cell lines (SEQ ID NO: 346) expressing human PD-1. EC50 binding to recombinant hPD-1-expressing Jurkat cells was calculated by flow cytometry in multiple experiments for 2H7-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4. 50 The concentration was 0.1–0.3 nM. EC20 binding to Jurkat cells expressing hPD-1 was calculated using flow computing. 50The binding intensity was 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. Binding was specific to hPD-1 because antibodies against 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 did not express hPD-1 (data not shown).

[0359] 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

[0360] As described in General Method Protocol B, in the presence of anti-hPD-1 #1-mIgG2b-N297A and anti-hPD-1 #2-mIgG2b-N297A, the binding competition of 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 with hPD-1 was evaluated.

[0361] As a control, OPDIVO® (nivolumab) was titrated in the presence of a saturated concentration of 10 μM anti-hPD-1 #1-mIgG2b-N297A. Figure 4A Compared to the dose-titer profile of OPDIVO® without an anti-hPD-1 #1-mIgG2b-N297A competitor, the dose-titer profile in the presence of the anti-hPD-1 #1-mIgG2b-N297A competitor was significantly reduced (the dose-titer profile shifted 100-fold to 1000-fold to the right of the graph). Adding a saturated concentration (10 μM) of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A before exposure to 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 did not eliminate the binding of 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 to hPD-1, as... Figure 4B-4D The shift of less than 10-fold indicates that 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 do not competitively bind to PD-1 in the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A.

[0362] Example 8: Characterization of non-antagonist hPD-1 antibody

[0363] As described in General Method Protocol C, the PD-1 antagonist activity of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 was tested using an in vitro cell-based human PD-1 / PD-L1 blocking bioassay. 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.

[0364] None of 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 exhibited hPD-1 antagonist activity, as they all showed a luminescence level of an average of 3000 relative luminescent units (RLU) and exhibited an RLU similar to that of the negative control KLH-C3-hIgG4 (data not shown). In contrast, anti-hPD-1 #1, a known hPD-1 antagonist that blocks the binding of hPD-L1 (SEQ ID NO: 584) to hPD-1, exhibited a luminescence level exceeding 14,000 RLU (data not shown).

[0365] Example 9: hIL-2 fusion protein against hPD-1 attenuation binds to Jurkat cells expressing human PD-1

[0366] To construct expression vectors for various antibodies and antibody-attenuated hIL-2 fusion proteins, corresponding polynucleotides encoding antibody, cytokine, cytokine receptor, and adaptor sequences were generated and cloned into expression vectors. Antibodies or antibody fusion proteins were transiently expressed in human embryonic kidney (HEK) 293 cells and then purified using affinity chromatography with protein A or protein G agarose. The purified proteins were concentrated using ultracentrifugation and buffer exchanged to phosphate-buffered saline or phosphate-buffered saline containing 100 mM L-arginine and 10 mM L-histidine, after which protein concentrations were determined.

[0367] In some methods, 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 carrying the S228P hinge stable mutation are directly fused with hIL-2 (df) or fused with hIL-2 at the C-terminus of the immunoglobulin heavy chain using the L6 linker. Figure 5This paper summarizes the descriptions of these anti-PD-1-attenuation hIL-2 fusion proteins. As described in Example 2, various constructs were generated by substituting hIL-2 to attenuate hIL-2 activity. As described in General Method Protocol A, the binding of the anti-PD-1-attenuation hIL-2 fusion proteins listed in Table 20 to hPD-1 was tested using a Jurkat cell line expressing hPD-1. The variable region of 2H7-hIgG4 (SEQ ID NO: 384 and 385) was further optimized, and the isotype was converted to human IgG1 with zero-substitution L235A / G237A (LAGA, as described in WO1998 / 006248) to become H7-632-hIgG1-LAGA (SEQ ID NO: 414 and 415). The optimized H7-632-hIgG1-LAGA was also directly fused (df) with an hIL-2 variant (hIL-2 T3A / D20A / R38E / C125A; SEQ ID NO: 217) with attenuated hIL-2 activity to become H7-767 (SEQ ID NO: 412-413, 415-423, 532), and the binding of both H7-632-hIgG1-LAGA and H7-767 to hPD-1 was tested (Table 20). EC was calculated based on the geometric mean fluorescence intensity (gMFI) across titration concentrations using GraphPad Prism 7 software. 50 value.

[0368] The production of the anti-hPD-1-attenuated hIL-2 fusion protein did not reduce binding to hPD-1, and the anti-hPD-1-attenuated hIL-2 fusion protein was still able to bind to Jurkat cells expressing human PD-1. Table 20 summarizes the calculated EC50 values ​​of the tested anti-hPD-1-attenuated hIL-2 fusion protein compared to the corresponding anti-hPD-1 antibody without the attenuated hIL-2 moiety. 50 .

[0369]

[0370] Compared to anti-hPD-1 antibodies without the attenuated hIL-2 moiety, the anti-hPD-1-hIL-2 fusion protein showed better adhesion to ECGs of Jurkat+ hPD-1 cells. 50 The binding increase was less than 2-fold, which demonstrates that adding the attenuated hIL-2 moiety to the anti-hPD-1 antibody does not eliminate the binding to human PD-1.

[0371] 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.

[0372] As described in General Method Protocol B and Example 7, the binding of the anti-hPD-1-attenuated hIL-2 fusion protein to the hPD-1 receptor was tested in the presence of anti-hPD-1 #1 and anti-hPD-1 #2. The reverse experiment was also performed, in which the binding of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A to hPD-1 was examined in the presence of saturated concentrations of the test antibody-attenuated hIL-2 fusion protein. In this manner, Jurkat cells expressing hPD-1 were seeded at 100,000 cells per well in FACS buffer, blocked with anti-human FcγR blocking agent (Mitenia Biosciences) at 4°C for 10 min, and washed. The tested antibody-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 the isotype control anti-DNase 1H3-hIgG4-df-hIL-2 (D20A / R38E) were diluted to a final concentration of 280 nM in 100 µL FACS buffer and incubated on ice with Jurkat cells expressing hPD-1 for 1 hour. Cells were washed and resuspended on ice in FACS buffer for 1 hour, containing six-fold sequential titrations of either anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A, starting at a maximum concentration of 50 nM. Cells were then washed and resuspended on ice in a 1:100 dilution of phycoerythrin-conjugated anti-mouse IgG light chain κ monoclonal antibody for 45 minutes. Cells were washed again and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific). Flow cytometry analysis was performed using BD FACS Canto II (BD Biosciences), and gMFI was calculated using FlowJo software version 10. EC was calculated using GraphPad Prism 7 software based on gMFI of the phycoerythrin signal across titration concentrations. 50 value.

[0373] In the presence of anti-hPD-1 #1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A, adding attenuated hIL-2 to anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 did not weaken the ability of anti-hPD-1 protein to bind to human PD-1, similar to the results described in Example 7. Figure 16B-16DH7-767 was also tested in this competitive assay, and Figure 13B This demonstrates that H7-767 continues to bind to the hPD-1 receptor in the presence of either anti-hPD-1-#1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A. Conversely, in the presence of either anti-hPD-1-#1-mIgG2b-N297A or anti-hPD-1 #2-mIgG2b-N297A, the binding of the positive control anti-hPD-1 #1 is significantly reduced. Figure 16A ,13A).

[0374] Figure 6A and Figure 6B This indicates that, for the reverse competition assay, 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), anti-hPD-1 #1-mIgG2b-N297A ( Figure 6A ) and anti-hPD-1 #2-mIgG2b-N297A ( Figure 6B These proteins were still able to bind to hPD-1 on Jurkat cells. Prior to exposure to the anti-hPD-1 fusion protein, the binding curves of these saturated anti-hPD-1-attenuated hIL-2 fusion proteins overlapped with the binding curves of anti-hPD-1 #1-mIgG2b-N297A (non-competitive) or anti-hPD-1 #2-mIgG2b-N297A (non-competitive). The binding curves also overlapped with the saturated negative control fusion protein 1H3-hIgG4-df-hIL-2 (D20A / R38E), which did not bind to hPD-1.

[0375] Example 11: Anti-hPD-1 attenuation hIL-2 fusion protein binds to recombinantly expressed cynomolgus monkey PD-1

[0376] The binding of the anti-hPD-1-attenuated hIL-2 fusion protein to cynomolgus monkey PD-1 was tested by flow cytometry using a human embryonic kidney 293 cell line expressing the SV40 large T cell antigen (HEK-293T). This cell line was transiently transfected to recombinantly express cynomolgus monkey PD-1. For each transfection reaction, 2 million HEK-293T cells were transfected with 2 µg pCMV6-hygro-HA-cyno-PD-1 (1-185) (SEQ ID NO: 448), a mammalian vector containing a cynomolgus monkey PD-1 extracellular domain labeled with human influenza hemagglutinin and a sequence encoding hygromycin resistance. Transfection was performed by electroporation. Transfected cells were blocked with human FcγR blocking agent and stained with a titration of the anti-hPD-1-attenuated hIL-2 fusion protein. In addition, phycoerythrin-conjugated antihemagglutinin clone 15B12 was added to cells for staining transfected cells, and allophycocyanin-conjugated anti-human IgG Fc secondary clone HP6017 (BioLegend, catalog number 409306) was added to cells for staining with bound antibodies. Cells were analyzed on BD Canto II, and live, transfected (hemagglutinin-positive) cells were selected using FlowJo software version 10, and the gMFI of the allophycocyanin signal was calculated. EC was calculated based on the gMFI across titration concentrations using GraphPad Prism 7 software. 50 value.

[0377] The hIL-2 fusion protein, which is anti-hPD-1 attenuation, binds to HEK-293T cells expressing cynomolgus monkey PD-1 in a similar manner to that observed on Jurkat T cells expressing human PD-1. Figure 17 EC binding to HEK-293T cells expressing cynomolgus monkey PD-1. 50 The EC50 values ​​were 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 the comparative anti-hPD-1-attenuated hIL-2 fusion protein) also showed EC50 values ​​of 9 nM and 2 nM, respectively. 50 The value of binding to cynomolgus monkey PD-1 indicates that adding the attenuated hIL-2 moiety to the anti-hPD-1 antibody did not eliminate the binding to cynomolgus monkey PD-1.

[0378] Example 12: Anti-hPD-1 attenuation hIL-2 fusion protein binds to activated primary human and cynomolgus monkey PD-1

[0379] The binding of anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to activated primary T cells expressing hPD-1 was examined by flow cytometry. To test whether 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 binds to native 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 iomycin to upregulate the hPD-1 receptor. Activated PBMCs were collected, blocked with a 1:50 dilution of human FcγR blocking agent (Mitenia Biotech) at 4°C for 10 min, and stained with titrated concentrations of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, anti-hPD-1 #1, and isotype controls. Cells were then stained with 1:20 diluted allophycocyanin-conjugated anti-human IgG Fc to detect bound antibodies. To characterize immune subsets, a mixture of surface markers including anti-human CD3, anti-CD4, and anti-CD8 antibodies was used. Additionally, cellular expression of hPD-1, hCD25, hCD122, and hCD132 in sample fractions was examined. Cells were analyzed on BD Fortessa (BD Biosciences), and T cell subsets were gated using FlowJo software version 10, followed by calculation of the gMFI of the allophycocyanin signal. EC was calculated based on the gMFI across titration concentrations using GraphPadPrism 7 software. 50 Value. To test the binding of the anti-hPD-1-hIL-2 fusion protein, cryopreserved CD3+ T cells were activated with PMA / ionomycin and subjected to flow cytometry in the same manner as described above.

[0380] The binding of the human PD-1 antibody-attenuated hIL-2 fusion protein to activated cynomolgus T cells was also tested using flow cytometry. Cynomolgus PBMCs were activated with a mixture of 0.081 µM PMA and 1.34 µM iomycin. After 24 hours, cells were stained using the same procedure as for the human PD-1-bound primary cells described above, except for the use of cynomolgus cross-reactivity markers. FlowJo software version 10 was used to select active CD3+ cells. + CD4 + or CD3 + CD8 + T cells were then used, and the gMFI of the allophycocyanin signal was calculated. EC was calculated using GraphPad Prism 7 software based on the gMFI of anti-hPD-1 antibody or hPD-1 antibody-attenuated hIL-2 fusion protein across titrated concentrations. 50 value.

[0381] In some tested variants, the decayed hIL-2 also includes substitutions for T3A and C125A, which remove O-linked glycosylation sites and replace free cysteine ​​residues, respectively.

[0382] After activation by PMA and ionomycin, 40-50% of CD4+ + T cells are PD-1 + 30-40% of CD8 + T cells are PD-1 + (Data not shown). Flow cytometry calculations of the ratio of activated human CD3... + CD4 + T-cell-bound EC 50 For 2H7-hIgG4, it is 0.1-0.7 nM; for C51E6-5-hIgG4, it is 12 nM; for A2-hIgG4, it is 30 nM; and for 2H7-hIgG4-df-hIL-2, it is 0.04 nM (T3A / D20A / R38E / C125A). This is in contrast to activated human CD3. + CD8+ T cell-binding EC 50 For 2H7-hIgG4, it is 0.1-0.8 nM; for C51E6-5-hIgG4, it is 16 nM; for A2-hIgG4, it is 22 nM; and for 2H7-hIgG4-df-hIL-2, it is 0.03 nM (T3A / D20A / R38E / C125A). For H7-767, it is related to activated human CD3. + CD4 + T-cell-bound EC 50 It is 0.19 nM, and activated human CD3 + CD8 + T cell count was 0.12 nM. This was consistent with activated CD3+ in cynomolgus monkeys. + CD4+ T cell-binding EC 50 The value was 0.09 nM for 2H7-hIgG4 and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A / D20A / R38E / C125A). This was compared with activated cynomolgus monkey CD3. + CD8+ T cell-binding EC 50 For 2H7-hIgG4, it was 0.08 nM, and for 2H7-hIgG4-df-hIL-2, it was 0.03 nM (T3A / D20A / R38E / C125A). For H7-767, it was associated with activated cynomolgus monkey CD3. + CD4 + T-cell-bound EC 50The value is 0.26 nM, and the activated CD3 of the cynomolgus monkey is... + CD8 + The T cell count was 0.24 nM. This data demonstrates that when the hPD-1 antibody was converted to an anti-hPD-1-attenuated hIL-2 fusion protein, the calculated EC50 binding to activated hPD-1 increased. 50 The value remains the same as the calculated EC50 of the naked hPD-1 antibody binding to hPD-1. 50 The values ​​are similar.

[0383] Flow cytometry was used to detect the interaction between H7-767 and H7-632-hIgG1-LAGA anti-PD-1 naked antibodies and primary inactivated human CD4. + and CD8 + T-cell binding. Frozen human CD3... + T was thawed and flow cytometry was performed as described above. Neither the H7-767 nor the H7-632-hIgG1-LAGA naked anti-PD-1 antibody bound to inactive human CD4. + and CD8 + T cells (data not shown).

[0384] Example 13: Quantification of the binding of anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to recombinant human or cynomolgus monkey PD-1 by surface plasmon resonance (SPR).

[0385] Surface plasmon resonance binding assays were performed using high-throughput SPR Carterra® LSA™ to determine the binding affinity of the anti-hPD-1 antibody and the anti-hPD-1-attenuated hIL-2 fusion protein. The proteins were diluted to 2 or 10 µg / mL in 10 mM sodium acetate (pH 4.5) containing 0.01% Tween-20 and coupled to an HC30M (Carterra Bio) chip using a sulfonyl-N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylamino)propylcarbodiimide (sulfonyl-NHS / EDC) coupling chemistry, followed by blocking with ethanolamine. Binding kinetics with commercially available recombinant His-labeled human PD-1 and His-labeled cynomolgus monkey PD-1 (Acro Biosystems) were determined using a non-regenerative kinetic coupling process.

[0386] Anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein were expressed using modified human IgG1 or modified IgG4 isotype with a κ light chain framework. Additional substitutions, L235E or L235A / G237A (LAGA, e.g., International Publication No. WO1998 / 006248) (based on the EU numbering system), were introduced into the Fc region to eliminate the effector function of the immunoglobulin component.

[0387] The binding constant (k) of various anti-hPD-1 antibodies and anti-hPD-1 antibody-attenuated hIL-2 fusion proteins that bind to recombinant human or cynomolgus monkey PD-1 protein. a ), dissociation constant (k) d ) and equilibrium constant (K D The value was determined by titration curves and Carterra kinetics software. The maximum feasible SPR signal (Rmax) was also calculated. max The results of the kinetic screening are summarized in Table 21, and it is shown that adding the attenuated hIL-2 moiety to the anti-hPD-1 antibody does not modulate the binding of the PD-1 antibody to human PD-1 or cynomolgus monkey PD-1 antigen. In separate experiments, H7-632-hIgG1-LAGA (SEQ ID NO: 414 and 415) were measured by SPR, and their steady-state equilibrium dissociation constant (K0) was also measured. D The value is 1.23 × 10 -9 M, and H7-767's K D =1.93 × 10 -9 M.

[0388]

[0389]

[0390] Example 14: Determining 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 by surface plasmon resonance (SPR).

[0391] The competition between anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins was determined using a sandwich assay. Antibodies and corresponding antibody-IL-2 cytokine fusion proteins were immobilized onto an HC30M chip using the amine coupling chemistry described in Example 13. Following the kinetic analysis described in Example 13, 80 nM human PD-1 (Bepsys, catalog PD-1-H5221-100ug) was injected into the entire array. The competitive anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins (analytes) were diluted to 30 μg / mL and subsequently injected into the array, and binding parameters were assessed using SPR. The assessment of all anti-hPD-1 and anti-hPD-1-hIL-2 fusion proteins was repeated. Some test variants had a modified human IgG1 or IgG4 light chain framework with additional L235E or L235A / G237A (LAGA) substitutions to eliminate the effector function of the immunoglobulins.

[0392] Screening for paired anti-hPD-1 or anti-hPD-1-attenuated hIL-2 fusion proteins allows for the identification of two bins, as shown in Table 22. In the presence of all antibodies and fusion proteins from Group 2, antibodies and fusion proteins from Group 1 are able to bind hPD-1 but compete with all members of the same group. In the presence of all antibodies and fusion proteins from Group 1, antibodies and fusion proteins from Group 2 are able to bind hPD-1 but compete with all members of the same group. The anti-hPD-1 antibodies listed in Group 1 of Table 22 do not compete with KEYTRUDA® and OPDIVO®.

[0393] Table 22. Groups 1 and 2 in the loading screening of anti-hPD-1 and anti-hPD-1-attenuation hIL-2 fusion proteins by SPR

[0394]

[0395]

[0396] Example 15: Antagonistic effect of hPD-1-attenuating hIL-2 fusion protein against hPD-1 in the presence of anti-hPD-1 #1 and anti-hPD-1 #2

[0397] The antagonistic effect of the hPD-1-attenuation-resistant hIL-2 fusion protein on hPD-1 was tested. The hPD-1-attenuation-resistant hIL-2 fusion protein was characterized according to general method protocol C. Figure 7 These results are presented. When compared to 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) are non-antagonistic to human PD-1, as demonstrated by low levels of detectable luminescence. H7-632-hIgG1-LAGA and H7-767 were also tested for antagonistic activity as described in General Protocol C. Figure 15 This indicates that H7-632-hIgG1-LAGA and H7-767 do not block the interaction between hPD-L1 (SEQ ID NO: 584) and the hPD-1 receptor.

[0398] For the competitive assay using the cell-based co-culture assay described in General Protocol C, some modifications were made. The 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. Forty (40) μL of Jurkat PD-1 effector cells were then placed over the CHO cells and the mixture of anti-hPD-1-attenuated hIL-2 fusion protein. In this competitive assay, the final concentration of saturated 100 nM anti-hPD-1-attenuated hIL-2 fusion protein was tested in combination with titrated anti-hPD-1 #1 or anti-hPD-1 #2. The remainder of the assay was performed as described in General Protocol C. Figure 18A and Figure 18B This indicates that the addition of 100 nM anti-hPD-1-attenuated hIL-2 fusion protein does not compete with the binding of anti-hPD-1 #1 to hPD-L1 (SEQ ID NO: 584) during titration blocking. The dose-titer curve of anti-hPD-1 #1 remained unchanged compared to the curve without competing antibodies, indicating that even at high concentrations, the presence of anti-hPD-1-attenuated hIL-2 fusion protein does not compete with the function of anti-hPD-1 #1. In the presence of 100 nM 2H7-hIgG4-df-hIL-2 (D20A / R38E) and 100 nM C51E6-5-hIgG4-L6-hIL-2 (D20A / R38E), anti-hPD-1 #2 showed a 35% decrease in luminescence (RLU) at higher concentrations of anti-hPD-1 #2. Figure 18B However, due to the extent of the standard deviation, it is unclear whether this reduction is significant.

[0399] In the opposite 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 titrated sequentially, and 40 μL of the mixture was added to CHO cells, followed by coverage with 40 μL of jurkat PD-1 effector cells. The remainder of the assay was performed as described in General Protocol C. Figure 18C and Figure 18D This indicates that adding 100 nM antihPD-1 #1 ( Figure 18C ) or 100nM antihPD-1 #2 ( Figure 18DThe ability of the anti-hPD-1-attenuation hIL-2 fusion protein to act as an antagonist is not diminished. The observed flat curves with relative luminescent units (RLU) above 18,000 indicate no competition for antagonist activity, and the tested anti-hPD-1-attenuation hIL-2 fusion protein still exhibits antagonistic function even in the presence of anti-hPD-1 #1 or anti-hPD-1 #2.

[0400] Example 16: Testing the attenuation of anti-hPD-1-attenuation hIL-2 fusion protein on high-affinity and intermediate-affinity hIL-2 receptors using cell-based proliferation assays

[0401] As described in General Protocol E, cell proliferation assays on NK-92 and TF1+IL-2Rβ cell lines were used to assess the attenuation level of hIL-2 activity of the anti-hPD-1-attenuated hIL-2 fusion protein. Control fusion proteins included those incorporating an anti-DNase I antibody (designated 1H3) having a human IgG4 or human IgG1 backbone directly fused to hIL-2 or having a linker (SEQ ID NO: 355) to demonstrate the effectiveness of the non-targeted attenuated hIL-2 fusion protein. As described in Example 2, the hIL-2 sequence of these constructs contained substitutions for attenuated hIL-2 activity. Similar to Example 3, complete, partial, or non-agonistic IL-2 activity (inactivity) was also evaluated. Some tested variants were expressed on modified human IgG1 or IgG4 isoforms with a κ light chain, the isoforms having additional L235E or L235A / G237A (LAGA) substitutions in the Fc region to eliminate immunoglobulin effector function. In some antibody-cytokine fusion proteins, hIL-2 cytokine is fused to the C-terminus of the light chain (LC fusion).

[0402] EC calculation for each antibody-cytokine fusion protein 50 EC was determined by relative luminescent units (RLU) and calculated when compared with recombinant human IL-2 (rhIL-2). 50 The fold change relative to rhIL-2 is summarized in Table 23. The fold change and agonistic activity relative to rhIL-2 are determined by comparing the maximum luminescence of the antibody-attenuated hIL-2 fusion protein with that of rhIL-2. Antibody-attenuated hIL-2 fusion proteins whose dose-titer profiles reach the same maximum luminescence as rhIL-2 are considered variants with full activity. The percentage of partial activity relative to full activity is calculated using the maximum luminescence of rhIL-2 as 100%. At the highest concentration of 1200 nM, a maximum RLU of the antibody-attenuated hIL-2 fusion protein less than 10% of the maximum RLU of rhIL-2 is considered to have no agonistic activity or be inactive. See Table 23 for details.a As noted, for some variants, EC 50 The value is only an estimate because the maximum luminosity was not reached.

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409] Example 17: Rescuing the IL-2 activity of an anti-hPD-1-attenuated hIL-2 fusion protein in cell lines expressing intermediate affinity hIL-2 receptor and hPD-1

[0410] The rescue of hIL-2 activity by an anti-hPD-1-attenuated hIL-2 fusion protein was evaluated using a targeted cell line expressing hPD-1. Briefly, the TF1+IL-2Rβ cell line described in Universal Method Protocol D was modified to express the hPD-1 receptor (SEQ ID NO: 580) via lentiviral transduction. Flow cytometry was used to detect hPD-1-expressing TF1+IL-2Rβ cells using an hPD-1 antibody incorporating Brilliant Blue 515 (BD Biosciences, catalog number 565936). Cells with low hPD-1 expression (less than 10⁻⁶ on the Brilliant Blue 515 fluorophore) were sorted. 3 (Intensity). The collection was sorted twice more to collect cells with near-hPD-1 expression levels on activated primary cells. The cell line (TF1+IL-2Rβ+hPD-1) was expanded and frozen in aliquots for cell-based proliferation assays. The proliferation assay was performed according to General Method Protocol E, with an incubation period of 3 days. Some tested variants have a modified human IgG1 or IgG4 light chain framework with additional L235E or L235A / G237A (LAGA) substitutions to eliminate the effector function of the immunoglobulin.

[0411] Table 24 summarizes the results of proliferation assays targeting TF1+IL-2Rβ+hPD-1 cell lines. The agonist activity (full, partial, or inactive) was determined by comparing the maximum luminescence of the antibody-attenuated hIL-2 fusion protein with that of rhIL-2. Antibody-attenuated hIL-2 fusion proteins whose dose-titer profiles reached the same maximum luminescence as rhIL-2 were considered variants with full activity. The percentage of partial activity to full activity was calculated using the maximum luminescence of rhIL-2 as 100%. At the highest concentration of 1200 nM, a maximum RLU of the antibody-attenuated hIL-2 fusion protein less than 10% of the maximum RLU of rhIL-2 was considered to have no agonist activity or be inactive. For some variants, EC 50 The values ​​are estimates only, as the full curve was not reached. Numerous examples of anti-hPD-1-hIL-2 fusion proteins with attenuated hIL-2 showed that hIL-2 activity was rescued on the targeted cell lines, while the non-targeted antibody control (denoted as 1H3) showed that hIL-2 activity was not rescued. A fold decrease in change relative to rhIL-2 to a value of 0 or 1 indicates complete rescue.

[0412] Table 24. Folds of change relative to rhIL-2 and agonistic activity of the antibody-hIL-2 fusion protein on the TF1+IL-2Rβ+hPD-1 cell line (a human PD-1 expression cell line with intermediate affinity for IL-2R).

[0413]

[0414]

[0415]

[0416]

[0417] Example 18: Evaluation of alternative anti-hPD-1-attenuation hIL-2 fusion proteins with or without blocking mouse PD-L1 in an in vivo mouse colon adenocarcinoma (MC38) model.

[0418] Because there is no universally accepted model for exploring the in vivo efficacy of tumor therapy in primates, alternative anti-mPD-1-attenuated hIL-2 fusion proteins were developed and tested in a syngeneic mouse tumor model. This MC38 colon adenocarcinoma model is commonly used to test the efficacy of immuno-oncology therapies. To investigate the in vivo effects of the anti-PD-1-attenuated hIL-2 fusion protein, alternative anti-mouse PD-1 antibodies named RMP1-14 (known for blocking mouse PD-L1 binding) and RMP1-30 (described as a non-blocker of mouse PD-L1) were fused to the C-terminus of the mouse IgG2b-N297A heavy chain with attenuated hIL-2 and tested in the MC38 colon adenocarcinoma model. The hIL-2 moiety includes substitutions for F42K, Y45R, and V69R, which were tested in the IL-2-dependent mouse T-lymphoblastic cell line (CTLL-2) and demonstrated to attenuate mouse IL-2 activity. Human IL-2 can stimulate the proliferation of mouse T cells at similar concentrations; however, the same substitutions that attenuate the activity of human IL-2-dependent cell lines do not attenuate the activity of CTLL-2 cell lines (data not shown). Therefore, F42K / Y45R / V69R substitutions are used as alternatives in hIL-2 because they exhibit attenuated IL-2 activity in mouse cell lines. Sequences containing the heavy and light chain variable regions of anti-mouse PD-1 antibodies RMP1-14 and RMP1-30 (as described in Matsumoto K et al., *Journal of Immunology*, Feb. 15, 2004; 172(4):2530-41) were also formed on a mouse IgG2b-N297A background to produce anti-mPD-1 RMP1-14 mIgG2b-N297A (SEQ ID NO: 564 and 566) and anti-mPD-1 RMP1-30 mIgG2b-N297A (SEQ ID NO: 567 and 568). The mouse IgG2b isotype with N297A substitution is the mouse equivalent of the Fc isotype with Fc immune effector function eliminated. Standard techniques were used to generate, express, and purify the alternative antibodies and antibody-attenuated hIL-2 fusion proteins, followed by protein A purification.

[0419] In this mouse tumor model, 5 × 10 5 MC38 colorectal cancer cells were injected into the right ventral region of ten-week-old female C57BL / 6NCrl (Charles River) mice. When the tumor reached 80-120 mm... 3Mice were randomly assigned to groups (n=10 per group) and treatment began 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 ID NO: 565 and 566) and anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) (SEQ ID NO: 568 and 569) were administered intraperitoneally at a dose of 5 mg / kg, along with a mediator control (phosphate-buffered saline), twice weekly for 4 weeks. During the study, the formula (w) was used. 2 The tumor size was measured twice weekly using calipers, where w = width and L = length. The study endpoint was 1000 mm. 3 The tumor volume or survival on day 50, whichever comes first, will be used as the criterion.

[0420] Figure 8 The results showed that while administration of anti-mPD-1 RMP1-14-mIgG2b-N297A or anti-mPD-1 RMP1-30-mIgG2b-N297A antibodies alone did not significantly improve efficacy compared to treatment with the mediator control, administration of the 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 protein was associated with 90% and 100% complete tumor regression, respectively. These 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 this efficacy depends on hIL-2 activity. The data further demonstrate that, in the MC38 tumor model, antibody-mediated targeting of PD-1-expressing T cells is sufficient to promote potent anti-tumor efficacy.

[0421] Example 19: Expansion of effector memory CD8+ T cells in an in vivo mouse colon adenocarcinoma model by replacing hIL-2 fusion protein with anti-hPD-1 attenuation.

[0422] To understand the mechanism of action of the alternative anti-hPD-1-attenuation hIL-2 fusion protein in vivo, in vivo experiments similar to those in Example 18 were performed, followed by immunophenotypic analysis of the resulting T cell populations in tumors, blood, spleen, and lymph nodes after three doses. 5 × 10 5MC38 colon adenocarcinoma cells were subcutaneously implanted into the right ventral region of ten-week-old female C57BL / 6NCrl (Chasliva) mice, and tumor growth was monitored. Tumors were grown to 150-260 mm in diameter. 3 The animals were divided into four groups of 10 mice each for the study. Twenty-one days after implantation, on days 1, 4, and 8, the animals were intraperitoneally administered 0.2 mL / dose of phosphate-buffered saline (PBS) as a mediator control, 5 mg / kg of anti-KLH-C3-mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R), 5 mg / kg of anti-mPD-1 RMP1-30 mIgG2b-N297A, or 5 mg / kg of anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R). On day 9, tumors, spleens, and inguinal lymph nodes were collected from all mice and processed into single-cell suspensions for subsequent flow cytometry analysis.

[0423] Figure 9A The tumor volume growth (mm) over 9 days from the first dose on day 1 was plotted. 3 (), where each point represents the average of 10 mice. By day 8, tumor volume was reduced in the anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) group compared to other treatment groups. Figure 9B A summary of various CD8 values ​​in each processing group. + The contribution of T cell subsets to tumors, among which the T central memory phenotype is CD45. + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 - CD103 - The T-effect sub-memory is CD45. + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 - CD103 - CD62L - T-resident memory is CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69+ CD103 + CD44 - CD62L - T cells are CD45 + CD3 + CD4 - CD8 + CD44 + CD62L - And T was originally CD45 + CD3 + CD4 - CD8 + CD44 - CD62L + Compared with other treatment groups, in mice treated with anti-mPD-1 RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R), CD8 + T-effect subpopulation amplification, such as Figure 9B The increase in the light gray portion is indicated by this. This is also... Figure 9C The absolute count (cells / μL) within the tumor after MC38 cleavage is used to illustrate this. Furthermore, within the tumor, expression of CD45 is defined as... + CD3 + CD4 + CD8 - CD25 + FoxP3 + The absolute count (cells / μL) of regulatory T cells, a marker of the disease, is reduced.

[0424] CD8 + The expansion of T effector memory and the reduction of regulatory T cells are associated with effective immunotherapy in mice and humans.

[0425] Example 20: In the NCG-PBMC model, the hIL-2 fusion protein resistant to hPD-1 attenuation is active in vivo.

[0426] Transplanting human immune cells into NOD-Prkdc cells lacking functional T, B, and NK cells em26Cd52 IL-2rg em26Cd22 The / NjuCrl (NCG) mouse model has become a valuable tool for evaluating the efficacy of hypothetical therapies that stimulate human T cells. In this model, if a therapeutic agent activates human T cells, it leads to T cell proliferation and accelerated graft-versus-host disease (GvHD).

[0427] The transplantation kinetics and expression of human PD-1 and human IL-2 receptors on T cells were evaluated in three independent donors for human peripheral mononuclear cell (hPBMC) transplantation over a 4-week period. Among the three tested donors, those that induced the most T cells and had the GvHD intermediate window were selected. 1.5 × 10⁻⁶ 7 hPBMCs were intravenously injected into NCG mice and divided into 8 groups of 8-16 mice each. Mice were intraperitoneally injected on days 7, 10, and 14 with three doses of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) (SEQ ID NO: 471, 425) (2.5 mg / kg, 5 mg / kg, or 10 mg / kg), 1H3-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) (SEQ ID NO: 546, 374) (5 mg / kg or 10 mg / kg), 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A) (SEQ ID NO: 563, 374) (10 mg / kg), or 2H7-hIgG1-LAGA-df-hIL-2 (T3A / R38E / I92K / C125A) (SEQ ID NO: 10 mg / kg). NO: 474, 425 (5 mg / kg). Anti-DNase fusion proteins of wild-type hIL-2 (1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A)) and those with a degraded hIL-2 moiety (1H3-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A)) were used as non-targeted antibody controls. Although the 1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A) fusion protein did not exhibit changes in the hIL-2 moiety with reduced hIL-2 activity, it did contain T3A and C125A substitutions to remove the predicted O-linked glycosylation site on human IL-2 (see, for example, International Publication No. WO2012 / 107417) and unpaired cysteine ​​residues (see, for example, International Publication No. WO2018 / 184964), respectively. These replacements did not clinically demonstrate a decrease in hIL-2 potency. On day 21, blood, spleen, and lungs were collected, with the blood and spleen processed for flow cytometry immunophenotypic analysis, and the lungs weighed.

[0428] Twenty-one days later, flow cytometry immunophenotyping was performed on the animals' blood and spleen. Table 25 summarizes the biomarkers used to characterize the human T cell population for subsequent analysis.

[0429] Table 25. Phenotypic markers defining human T cell subsets in NCG-PBMC mice

[0430]

[0431] like Figure 10 As shown, the body weight of each individual animal was measured at 21 days and normalized relative to day 1 as an assessment of graft-versus-host disease (GvHD). Accelerated GvHD was observed in mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A). Slight weight loss was also observed in mice treated with 2.5 mg / kg, 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). Although weight loss was observed in mice treated with 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A), this did not persist.

[0432] Flow cytometry analysis was associated with an accelerated observed graft-versus-host disease (GvHD). Using phenotypic markers for human T cell subset profiling provided in Table 25, flow cytometry analysis of peripheral blood showed that 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), CD3... + CD4 + and CD8 + T cell subsets showed only slight expansion (e.g., through CD3) + The change in T cells relative to the drug control was quantified as a fold increase of 10 to 50 times. Furthermore, in the peripheral blood of mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), CD3... + CD4 + and CD8 + T cell subsets have greatly expanded (for CD3) + T cells (with a fold increase of more than 50-fold relative to the drug control). Table 26 summarizes the expanded human T cell subsets.

[0433]

[0434] In addition to assessing CD3 levels between treatment groups +CD4 + and CD8 + In addition to T cells, CD4 was also assessed. + and CD8 + T cell subsets: memory and primary subsets. Table 25 summarizes the subsets used to characterize CD4. + and CD8 + Phenotypic markers of primary, effector, effector memory, and central memory T cells. No changes were observed in primary, effector, or central memory T cells between treatment groups (data not shown). However, CD4+ levels were significantly increased in peripheral blood of mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A). + and CD8 + Effector memory (EM) T cells greatly expanded, CD8 + The average number of T cells per milliliter is greater than 5 million, and CD4 + T cells greater than 50 million ( Figure 11A and 11B The box-and-whisker plot is drawn using boxes around the first and third quartiles, with the horizontal line representing the median and the lines indicating 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) and 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A), CD8... + Effector memory (EM) T cells showed moderate expansion, defined as an average cell count between 1 million and 5 million per milliliter. In mice treated with 2.5 mg / kg and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), for CD4... + T cells, CD4 + Effector memory (EM) T cells showed moderate expansion at a rate of 6 million to 13 million per milliliter.

[0435] In addition to stimulating effector T cells, IL-2 has also been described as stimulating NK cells and regulatory T cells (Tregs), and these immune cell types were also evaluated because Tregs express high levels of CD25 and NK cells express CD122. Figure 12This indicates that animals treated with the highest dose of 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) did not expand human regulatory T cells, but instead exhibited the lowest percentage of regulatory T cells in their peripheral blood (as defined by phenotype in Table 25). A dose-dependent reduction in human regulatory T cells was observed, and this was associated with an average of 1.6% human CD3+. + Compared to a control group where T cells act as a mediator for Tregs, 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) had an average of 0.16% human CD3+. + The T cells were Tregs. Compared with the drug control, the percentage of human NK cells in peripheral blood (phenotype as defined in Table 25) did not change in any of the treatment groups (data not shown).

[0436] Example 21: Non-clinical safety of hIL-2 fusion protein against hPD-1 attenuation

[0437] Cynomolgus monkeys have previously been used to evaluate the toxicity of unmodified IL-2. Lethality of exogenous recombinant IL-2 at doses as low as 50 μg / kg / day has been observed in cynomolgus monkeys. Since the binding of H7-767 to cynomolgus monkey hPD-1 on primary activated PBMCs was confirmed by flow cytometry (Example 12), single-dose studies were conducted to preliminarily assess the safety of variants of H7-767 (H7-02-hIgG1-LAGA-df-hIL-2) (T3A / D20A / R38E / C125A) (SEQ ID NO: 582 and 583) and H7-767. H7-02-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) was delivered to eight monkeys via intravenous infusion over 15 minutes at doses of 1 mg / kg (4 animals) or 10 mg / kg (4 animals). Sampling was performed at time points up to 360 hours post-infusion. No adverse reactions, total toxicity, weight loss, or lethality were observed (data not shown). Similar to the first study, subsequent single-dose studies using H7-767 were conducted at higher doses of 5 mg / kg and 50 mg / kg, with sampling at time points up to 360 hours post-infusion. Again, no adverse reactions, total toxicity, weight loss, or lethality were observed (data not shown).

[0438] Example 22: Attenuation of IL-2 activity in modified hIL-2 protein

[0439] As described in Example 5 above, the attenuation of IL-2 activity in modified hIL-2 proteins containing substitutions at amino acid position 20 (D20) and amino acid position 38 (R38) was tested in proliferation assays of both the NK-92 and TF1+IL-2Rβ cell lines. Based on the maximum agonist activity of the modified hIL-2 proteins and the attenuation level of their potency against intermediate and high affinity receptors relative to unmodified recombinant hIL-2 (Table 27), the modified hIL-2 proteins were divided into 7 groups (1 to 7). The criteria used for grouping the modified hIL-2 proteins were:

[0440] ●Group 1: Variants with the highest attenuation (i.e., >10,000-fold) and at least about 80% activity to intermediate affinity receptors, but also with high attenuation and at least about 70% activity to high affinity receptors.

[0441] ●Group 2: Variants with at least about 70% activity and > 1,000-fold attenuation to intermediate affinity receptors and about 20% to about 30% activity to high affinity receptors.

[0442] ●Group 3: Variants with approximately 50% to 70% activity and > 1,000-fold attenuation to intermediate affinity receptors and approximately 20% activity to high affinity receptors.

[0443] ●Group 4: Variants with at least about 70% activity against intermediate affinity receptors but only > 500-fold attenuation and about 50% activity against high affinity receptors.

[0444] ●Group 5: Variants that have at least about 70% activity against both receptors but exhibit a decrease in activity of >10 to >300 times against intermediate affinity receptors and a decrease in activity of 70 to 1500 times against high affinity receptors.

[0445] ●Group 6: Variants with only about 30% activity and >2,500-fold attenuation to intermediate affinity receptors and no activity to high affinity receptors.

[0446] ●Group 7: Variants that are inactive against both intermediate-affinity and high-affinity receptors.

[0447] Table 27. Fold change relative to rhIL-2 and agonistic activity of modified hIL-2 protein in cell-based proliferation assays, including substitutions at amino acid position 20 (D20) and amino acid position 38 (R38).

[0448]

[0449]

[0450] Example 23: Activity of Alternative Fusion Proteins in a Mouse MC38 Colorectal Tumor Model

[0451] 5 × 10 5 Syngeneic MC38 colorectal cancer cells were injected into the right ventral region of ten-week-old female C57BL / 6NCrl mice. When the tumor reached 80-120 mm... 3 Mice were randomly assigned to groups (n=10 per group) and treatment began on day 1 of the study. From day 1, all drugs except hIL-2 were administered intraperitoneally at 5 mg / kg twice weekly for 4 weeks. From days 1-5, hIL-2 was administered intraperitoneally at 36,000 IU once daily. Tumor size was measured twice weekly using calipers during the study. The study endpoint was 1000 mm. 3 The criterion is determined by the tumor volume, survival on day 50, or progression-free survival on day 70, whichever comes first.

[0452] All test agents, including antibody molecules and antibody-hIL-2 fusion proteins, were produced using the mouse IgG2b Fc region with a single N297A amino acid substitution at position 297. This substitution prevents glycosylation of the Fc region and significantly reduces the function of any Fc region-mediated immune effectors, thereby preventing cellular exhaustion in vivo. Anti-mPD-1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (Matsumoto, *Journal of Immunology* 172: 2530-2541, 2004). Anti-mPD-1RMP1-14-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 at its C-terminus to a reduced-potency IL-2 variant, hIL-2 F42K / Y45R / V69R (SEQ ID NO: 621), via a flexible six-amino acid glycine / serine linker. This molecule was engineered to directly target PD-1-expressing T cells in mice with a reduced-potency hIL-2 variant. Anti-KLH-hIL-2 F42K, Y45R, V69R is a control fusion protein composed of an isotype control monoclonal antibody that recognizes a non-mammal antigen (keyfora hemocyanin, KLH), fused to a reduced-potency IL-2 variant, hIL-2F42K, Y45R, V69R, via a flexible six-amino acid glycine / serine linker at its C-terminus.

[0453] The result is Figure 19The MC38 colorectal tumor model was particularly sensitive to antibody-mediated PD-1 receptor inhibition. Although tumors rapidly reached the study endpoint in mice treated with the mediated agent, 50% of mice treated with anti-mPD-1 RMP1-14 experienced complete tumor regression. In contrast, 100% of mice treated with the anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, V69R fusion protein experienced durable long-term tumor regression. Mice treated with various combinations of the components of the anti-mPD-1 RMP1-14-hIL-2F42K, Y45R, V69R fusion protein, including combinations of anti-mPD-1 RMP1-14 with free hIL-2 cytokines (administered at doses and regimens comparable to human therapeutic doses) or combinations of anti-mPD-1 RMP1-14 with the non-targeted anti-KLH-hIL-2 F42K, Y45R, V69R fusion protein, did not replicate the efficacy observed with anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, V69R. These data demonstrate that targeting PD-1-expressing cells with reduced hIL-2 potency significantly enhances antitumor efficacy compared to anti-PD-1 receptor antagonists, and that the activity of the fusion protein is not due to an additive effect of the individual components of the molecule.

[0454] Example 24: Evaluation of protective antitumor immunity induced by alternative anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, and V69R in an MC38 colorectal tumor model

[0455] Mice that experienced complete tumor regression and survived to day 50 in the primary tumor study described in Example 23 underwent secondary tumor challenge without any additional drug treatment. For tumor re-attack, 5 × 10 5 MC38 tumor cells were implanted into the left ventral region of mice contralateral to the primary tumor site. As a control group, 10 age-matched mice with unknown tumors were also implanted with MC38 tumor cells.

[0456] Figure 20 The results showed that all mice that had previously experienced complete tumor regression in the primary tumor study and survived to day 50 after treatment with anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, and V69R were protected from secondary tumor development. In contrast, all tumor-unaware mice implanted with MC38 tumor cells continued to develop tumors, which rapidly reached 100 mm. 3Tumor volume was the endpoint of the study. In the absence of sustained drug therapy, the development of protective antitumor immunity demonstrated that anti-mPD-1 RMP1-14-hIL-2 F42K, Y45R, and V69R induced antitumor memory T cell responses.

[0457] Example 25: Evaluation of compatibility with combination therapy with alternatives to anti-mPD-1 RMP1-30-hIL-2F42K, Y45R, V69R and anti-mPD-1 RMP1-14 antibodies in a mouse MC38 colorectal tumor model

[0458] To evaluate the compatibility of the disclosed immunoconjugate with combination therapy against antagonistic anti-PD-1 antibodies, alternative antagonistic anti-mouse PD-1 antibodies and alternative immunoconjugates were used because the antagonistic anti-human PD-1 antibody does not bind to mouse PD-1, and the modified IL-2 protein does not function against the mouse IL-2 receptor. 5 × 10 5 Syngeneic MC38 colorectal cancer cells were injected into the right ventral region of ten-week-old female C57BL / 6NCrl mice. When the tumor reached 80-120 mm... 3 Mice were randomly assigned to groups (n=10 per group) and treatment began on day 1 of the study. All agents were administered intraperitoneally once weekly for four weeks, starting on day 1. Anti-mPD-1 RMP1-30-hIL-2 F42K, Y45R, and V69R were administered at 1 mg / kg, and anti-mPD-1 RMP1-14 at 2 mg / kg. Tumor size was measured twice weekly using calipers during the study. The study endpoint was a mean tumor weight of 1500 mm³ in the mediated control group, or 45 days of study, whichever came first. Due to the need for sampling a large number of mice, endpoint sampling was conducted over two days.

[0459] Both test agents were produced using the mouse IgG2b Fc region with a single N297A amino acid substitution at position 297. This substitution prevents glycosylation of the Fc region and significantly reduces the function of any Fc region-mediated immune effectors, thereby preventing cellular exhaustion in vivo. Anti-mPD-1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (Matsumoto, *Journal of Immunology* 172: 2530-2541, 2004). Anti-mPD-1 RMP1-30-hIL-2 F42K / Y45R / V69R is a bifunctional fusion protein consisting of a non-antagonist of the mouse PD-1 receptor monoclonal RMP1-30 antibody, fused at its C-terminus to the IL-2 variant hIL-2F42K / Y45R / V69R (SEQ ID NO: 621), which has reduced potency against the mouse IL-2 receptor, via a flexible hexa-amino acid glycine / serine linker. This molecule was designed to directly target PD-1-expressing T cells in mice with a reduced-potency hIL-2 variant.

[0460] The result is Figure 21 The tumors growing in mice treated with the catalytic agent reached the study endpoint on day 25. In contrast, mice treated with either anti-mPD-1 RMP1-14 or anti-mPD-1 RMP1-30-hIL-2 F42K / Y45R / V69R experienced partial tumor regression, with the anti-mPD-1 RMP1-30-hIL-2 F42K / Y45R / V69R group showing better results. The combination group achieved the most effective tumor volume reduction. This data demonstrates that neither antibody interferes with the ability of the other to bind to PD-1, and that when administered in combination, greater efficacy can be achieved than when each agent is administered alone.

[0461] This data, combined with the data presented herein demonstrating that the disclosed immunoconjugate binds to PD-1 in the presence of an antagonistic anti-PD-1 antibody, and the data presented herein demonstrating that the disclosed immunoconjugate is designed to deliver modified hIL-2 protein to the IL-2 receptor rather than antagonizing PD-1, suggests that the disclosed immunoconjugate and the antagonistic anti-PD-1 antibody can be effectively used as a combination therapy to reduce tumor volume.

[0462] Example 26: Combination therapy of anti-human PD-1 (hPD-1) antibody-modified human interleukin-2 (hIL-2) immunoconjugate and antagonistic anti-PD-1 antibody

[0463] The data in Example 25 above demonstrate that combining an anti-hPD-1 antibody-modified hIL-2 immunoconjugate with an antagonistic anti-PD-1 antibody can achieve better therapeutic efficacy. Single-dose studies for preliminary safety assessment will be conducted in cynomolgus monkeys using the following: 1) the disclosed immunoconjugate with and without nivolumab; and 2) the disclosed immunoconjugate with and without pembrolizumab. Samples will be taken at time points up to 360 hours post-infusion. Adverse reactions, total toxicity, weight loss, and lethality will be assessed. Further studies will be conducted using higher doses of the immunoconjugate and / or the antagonistic anti-PD-1 antibody.

[0464] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments disclosed herein, and that these changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent changes that fall within the true spirit and scope of the invention.

[0465] The disclosure of each patent, patent application, and publication cited or described in this document is hereby incorporated in its entirety by reference.

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[0583] Example

[0584] The following list of examples is intended to supplement, rather than replace or supersede, the previous descriptions.

[0585] Example 1. A method for treating cancer in a subject, the method comprising administering the following to the subject:

[0586] (A) An immunoconjugate modified with an anti-human PD-1 (hPD-1) antibody, said immunoconjugate comprising:

[0587] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0588] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0589] (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO: 421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO: 422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO: 423;

[0590] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0591] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0592] (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; and

[0593] (B) Anti-PD-1 antibody that antagonizes PD-1 in the presence of the said immunoconjugate.

[0594] Example 2. The method according to Example 1, wherein the antagonistic anti-PD-1 antibody is nivolumab, pembrolizumab, cimiprimab, dotalimab, or revivalimab.

[0595] Example 3. The method according to Example 1 or 2, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 149, 307, 607-611, 614, 617 or 620.

[0596] Example 4. The method according to any one of the preceding examples, wherein the modified hIL-2 protein contains a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, and contains an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0597] Example 5. The method according to Example 4, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149.

[0598] Example 6. The method according to any one of the foregoing examples, 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.

[0599] Example 7. According to the method of Example 6, wherein the substitution at position 3 of the modified hIL-2 protein is T3A.

[0600] Example 8. The method according to Example 7, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 216.

[0601] Example 9. The method according to Example 6, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.

[0602] Example 10. The method according to any one of the preceding examples, 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.

[0603] Example 11. The method according to Example 10, wherein the substitution at position 125 of the amino acid is C125A.

[0604] Example 12. The method according to Example 11, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

[0605] Example 13. The method according to Example 12, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217.

[0606] Example 14. According to any one of the preceding examples, the modified hIL-2 protein is fused with the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate 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.

[0607] Example 15. The method according to any one of the preceding examples, wherein the modified hIL-2 protein is directly fused to the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate via a peptide bond.

[0608] Example 16. According to the method of Example 15, wherein 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 immunoconjugate via a peptide bond.

[0609] Example 17. The method according to any one of Examples 1 to 14, wherein the modified hIL-2 protein is fused via a linker to the anti-hPD-1 antibody or its antigen-binding fragment thereof of the immunoconjugate.

[0610] Example 18. The method according to any one of the foregoing examples, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises:

[0611] a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 416 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 417;

[0612] b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 384 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 385;

[0613] c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 394 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 395; or

[0614] d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 404 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 405.

[0615] Example 19. The method according to any one of the preceding examples, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises the IgG1 heavy chain constant region.

[0616] Example 20. The method according to Example 19, wherein, according to EU designation, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises L235A substitution and G237A substitution.

[0617] Example 21. The method according to any one of the foregoing embodiments, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises:

[0618] a) The heavy chain containing the amino acid sequence of SEQ ID NO: 414 and the light chain containing the amino acid sequence of SEQ ID NO: 415;

[0619] b) The heavy chain containing the amino acid sequence of SEQ ID NO: 424 and the light chain containing the amino acid sequence of SEQ ID NO: 425;

[0620] c) The heavy chain containing the amino acid sequence of SEQ ID NO: 426 and the light chain containing the amino acid sequence of SEQ ID NO: 427; or

[0621] d) The heavy chain containing the amino acid sequence of SEQ ID NO: 428 and the light chain containing the amino acid sequence of SEQ ID NO: 429.

[0622] Example 22. According to the method of Example 21, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 414 and the light chain comprises the amino acid sequence of SEQ ID NO: 415.

[0623] Example 23. The method according to any one of the foregoing embodiments, wherein the immunoconjugate comprises:

[0624] A light chain comprising the amino acid sequence of SEQ ID NO: 415; and

[0625] A heavy chain modified hIL-2 protein fusion containing the amino acid sequence of SEQ ID NO: 532.

[0626] Example 24. The method according to any one of the preceding examples, wherein the antagonistic anti-PD-1 antibody and the immunoconjugate are administered to the subject together in a mixture, as a single agent simultaneously or as a single agent in any order.

[0627] Example 25. The method according to Example 24, comprising:

[0628] The immunoconjugate was administered prior to the administration of the antagonistic anti-PD-1 antibody;

[0629] Administer the antagonistic anti-PD-1 antibody prior to the administration of the immunoconjugate; or

[0630] Essentially, the immunoconjugate is administered simultaneously with the administration of the antagonistic anti-PD-1 antibody.

[0631] Example 26. The method according to any one of the foregoing examples, wherein the cancer is melanoma, Merkel cell carcinoma, non-small cell lung cancer, renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, hepatocellular carcinoma, or a tumor with high microsatellite instability or a tumor with DNA mismatch repair defects.

[0632] Example 27. A method for treating a subject with renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or a tumor with high microsatellite instability or a tumor with DNA mismatch repair deficiency, the method comprising administering to the subject the following:

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

[0634] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0635] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

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

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

[0638] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing 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] Example 28. The method according to Example 27, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 149, 307, 607-611, 614, 617 or 620.

[0641] Example 29. The method according to Example 27 or 28, wherein the modified hIL-2 protein contains a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, and contains an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0642] Example 30. The method according to Example 29, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149.

[0643] Example 31. The method according to any one of Examples 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.

[0644] Example 32. The method according to Example 31, wherein the substitution at amino acid position 3 of the modified hIL-2 protein is T3A.

[0645] Example 33. The method according to Example 32, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 216.

[0646] Example 34. The method according to Example 31, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.

[0647] Example 35. The method according to any one of Examples 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.

[0648] Example 36. The method according to Example 35, wherein the substitution at position 125 of the amino acid is C125A.

[0649] Example 37. The method according to Example 36, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

[0650] Example 38. The method according to Example 37, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217.

[0651] Example 39. The method according to any one of Examples 27 to 38, wherein the modified hIL-2 protein is fused with the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate 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.

[0652] Example 40. The method according to any one of Examples 27 to 39, wherein the modified hIL-2 protein is fused directly to the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate via a peptide bond.

[0653] Example 41. According to the method of Example 40, wherein 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 immunoconjugate via a peptide bond.

[0654] Example 42. The method according to any one of Examples 27 to 39, wherein the modified hIL-2 protein is fused via a linker to the anti-hPD-1 antibody or its antigen-binding fragment thereof of the immunoconjugate.

[0655] Example 43. The method according to any one of Examples 27 to 42, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises:

[0656] a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 416 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 417;

[0657] b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 384 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 385;

[0658] c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 394 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 395; or

[0659] d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 404 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 405.

[0660] Example 44. The method according to any one of Examples 27 to 43, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises the IgG1 heavy chain constant region.

[0661] Example 45. The method according to Example 44, wherein, according to EU designation, the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises L235A substitution and G237A substitution.

[0662] Example 46. The method according to any one of Examples 27 to 45, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises:

[0663] a) The heavy chain containing the amino acid sequence of SEQ ID NO: 414 and the light chain containing the amino acid sequence of SEQ ID NO: 415;

[0664] b) The heavy chain containing the amino acid sequence of SEQ ID NO: 424 and the light chain containing the amino acid sequence of SEQ ID NO: 425;

[0665] c) The heavy chain containing the amino acid sequence of SEQ ID NO: 426 and the light chain containing the amino acid sequence of SEQ ID NO: 427; or

[0666] d) The heavy chain containing the amino acid sequence of SEQ ID NO: 428 and the light chain containing the amino acid sequence of SEQ ID NO: 429.

[0667] Example 47. The method according to Example 46, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 414, and the light chain comprising the amino acid sequence of SEQ ID NO: 415.

[0668] Example 48. The method according to any one of Examples 27 to 47, wherein the immunoconjugate comprises:

[0669] A light chain comprising the amino acid sequence of SEQ ID NO: 415; and

[0670] A heavy chain modified hIL-2 protein fusion containing the amino acid sequence of SEQ ID NO: 532.

[0671] Example 49. Uses of the following items:

[0672] (A) An anti-hPD-1 antibody-modified human interleukin-2 (hIL-2) immunoconjugate, said immunoconjugate comprising:

[0673] A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and

[0674] An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises:

[0675] (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO: 421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO: 422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO: 423;

[0676] (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391;

[0677] (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or

[0678] (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; and

[0679] (B) Anti-PD-1 antibodies that antagonize PD-1 in the presence of the aforementioned immunoconjugate

[0680] It is used to treat cancer.

Claims

1. A method of treating cancer in a subject, the method comprising administering the following to the subject: (A) An immunoconjugate modified with an anti-human PD-1 (hPD-1) antibody, said immunoconjugate comprising: A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises: (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO: 421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO: 422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO: 423; (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391; (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or (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; and (B) Anti-PD-1 antibody that antagonizes PD-1 in the presence of the said immunoconjugate.

2. The method according to claim 1, wherein the antagonistic anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, dostarlimab, or retifanlimab.

3. The method according to claim 1 or 2, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 149, 307, 607-611, 614, 617 or 620.

4. The method according to any one of the preceding claims, wherein the modified hIL-2 protein contains a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, and contains an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345.

5. The method of claim 4, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

149.

6. The method according to any one of the preceding claims, 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.

7. The method according to claim 6, wherein the substitution at amino acid position 3 of the modified hIL-2 protein is T3A.

8. The method of claim 7, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

216.

9. The method of claim 6, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

218.

10. The method according to any one of the preceding claims, 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.

11. The method according to claim 10, wherein the substitution at position 125 of the amino acid is C125A.

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.

13. The method of claim 12, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

217.

14. The method according to any one of the preceding claims, wherein the modified hIL-2 protein is fused with the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate 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.

15. The method according to any one of the preceding claims, wherein the modified hIL-2 protein is directly fused to the anti-hPD-1 antibody or its antigen-binding fragment of the immunoconjugate via a peptide bond.

16. The method of claim 15, wherein 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 immunoconjugate via a peptide bond.

17. The method according to any one of claims 1 to 14, wherein the modified hIL-2 protein is fused via a linker to the anti-hPD-1 antibody or its antigen-binding fragment thereof of the immunoconjugate.

18. The method according to any one of the preceding claims, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 416 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 417; b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 384 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 385; c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 394 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 395; or d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 404 and the light chain variable region containing the amino acid sequence of SEQ ID NO:

405.

19. The method according to any one of the preceding claims, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises the IgG1 heavy chain constant region.

20. The method of claim 19, wherein, according to EU designation, the anti-hPD-1 antibody or antigen-binding fragment portion thereof of the immunoconjugate comprises an L235A substitution and a G237A substitution.

21. The method according to any one of the preceding claims, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a) The heavy chain containing the amino acid sequence of SEQ ID NO: 414 and the light chain containing the amino acid sequence of SEQ ID NO: 415; b) The heavy chain containing the amino acid sequence of SEQ ID NO: 424 and the light chain containing the amino acid sequence of SEQ ID NO: 425; c) The heavy chain containing the amino acid sequence of SEQ ID NO: 426 and the light chain containing the amino acid sequence of SEQ ID NO: 427; or d) The heavy chain containing the amino acid sequence of SEQ ID NO: 428 and the light chain containing the amino acid sequence of SEQ ID NO:

429.

22. The method of claim 21, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 414, and the light chain comprising the amino acid sequence of SEQ ID NO:

415.

23. The method according to any one of the preceding claims, wherein the immunoconjugate comprises: A light chain comprising the amino acid sequence of SEQ ID NO: 415; and A heavy chain modified hIL-2 protein fusion containing the amino acid sequence of SEQ ID NO:

532.

24. The method according to any one of the preceding claims, wherein the antagonistic anti-PD-1 antibody and the immunoconjugate are administered to the subject together in a mixture, as a single agent simultaneously or as a single agent in any order.

25. The method of claim 24, comprising: The immunoconjugate was administered prior to the administration of the antagonistic anti-PD-1 antibody; Administer the antagonistic anti-PD-1 antibody prior to the administration of the immunoconjugate; or Essentially, the immunoconjugate is administered simultaneously with the administration of the antagonistic anti-PD-1 antibody.

26. The method according to any one of the preceding claims, wherein the cancer is melanoma, Merkel cell carcinoma, non-small cell lung cancer, renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, hepatocellular carcinoma, or a tumor with high microsatellite instability or a tumor with DNA mismatch repair defects.

27. A method of treating a subject with renal cell carcinoma, triple-negative breast cancer, head / neck squamous cell carcinoma, Merkel cell carcinoma, hepatocellular carcinoma, or a tumor with high microsatellite instability or a tumor with DNA mismatch repair deficiency, the method comprising administering to the subject the following: A human interleukin-2 (hIL-2) immunoconjugate modified with an anti-human PD-1 (hPD-1) antibody, said immunoconjugate comprising: A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises: (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO: 421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO: 422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO: 423; (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391; (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or (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.

28. The method of claim 27, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 149, 307, 607-611, 614, 617 or 620.

29. The method according to claim 27 or 28, wherein the modified hIL-2 protein contains a D20A substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, and contains an R38E substitution relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345.

30. The method of claim 29, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

149.

31. The 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.

32. The method of claim 31, wherein the substitution at amino acid position 3 of the modified hIL-2 protein is T3A.

33. The method of claim 32, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

216.

34. The method of claim 31, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

218.

35. The 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.

36. The method of claim 35, wherein the substitution at position 125 of the amino acid is C125A.

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

38. The method of claim 37, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

217.

39. The method according to any one of claims 27 to 38, wherein the modified hIL-2 protein is fused with the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate 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.

40. The method according to any one of claims 27 to 39, wherein the modified hIL-2 protein is fused directly to the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate via a peptide bond.

41. The method of claim 40, wherein 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 immunoconjugate via a peptide bond.

42. The method according to any one of claims 27 to 39, wherein the modified hIL-2 protein is fused via a linker to the anti-hPD-1 antibody or its antigen-binding fragment thereof of the immunoconjugate.

43. The method according to any one of claims 27 to 42, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 416 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 417; b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 384 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 385; c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 394 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 395; or d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO: 404 and the light chain variable region containing the amino acid sequence of SEQ ID NO:

405.

44. The method according to any one of claims 27 to 43, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises a constant region of the IgG1 heavy chain.

45. The method of claim 44, wherein, according to EU designation, the anti-hPD-1 antibody or antigen-binding fragment portion thereof of the immunoconjugate comprises an L235A substitution and a G237A substitution.

46. ​​The method according to any one of claims 27 to 45, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises: a) The heavy chain containing the amino acid sequence of SEQ ID NO: 414 and the light chain containing the amino acid sequence of SEQ ID NO: 415; b) The heavy chain containing the amino acid sequence of SEQ ID NO: 424 and the light chain containing the amino acid sequence of SEQ ID NO: 425; c) The heavy chain containing the amino acid sequence of SEQ ID NO: 426 and the light chain containing the amino acid sequence of SEQ ID NO: 427; or d) The heavy chain containing the amino acid sequence of SEQ ID NO: 428 and the light chain containing the amino acid sequence of SEQ ID NO:

429.

47. The method of claim 46, wherein the anti-hPD-1 antibody or its antigen-binding fragment portion of the immunoconjugate comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 414, and the light chain comprising the amino acid sequence of SEQ ID NO:

415.

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

532.

49. The uses of the following items: (A) An anti-hPD-1 antibody-modified human interleukin-2 (hIL-2) immunoconjugate, said immunoconjugate comprising: A modified hIL-2 protein, wherein the modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitution at amino acid position 20, and, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, comprises an R38E substitution at amino acid position 38; and An anti-hPD-1 antibody or its antigen-binding fragment thereof, wherein the anti-hPD-1 antibody or its antigen-binding fragment binds specifically to hPD-1 immune response, wherein the antibody or its antigen-binding fragment comprises: (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO: 421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO: 422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO: 423; (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 388, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 389, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 391; (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 398, light chain CDR1 containing the amino acid sequence of SEQ ID NO: 399, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 401; or (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; and (B) Anti-PD-1 antibodies that antagonize PD-1 in the presence of the aforementioned immunoconjugate It is used to treat cancer.

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