Anti-PD-1 antibody-attenuated IL-2 immunoconjugates and uses thereof

Abstract

Disclosed herein are modified human interleukin-2 (hIL-2) proteins, human antibody molecules or antigen-binding fragments thereof that immunospecifically bind to human programmed cell death protein-1 (hPD-1), and immunoconjugates comprising the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 352,842 filed on June 16, 2022, U.S. Provisional Application No. 63 / 481,630 filed on January 26, 2023, and U.S. Provisional Application No. 63 / 502,746 filed on May 17, 2023, the disclosure of each of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically with this document in XML format and is hereby incorporated by reference in its entirety. The XML copy created on June 5, 2023 is named 102085.021703_SequenceListing.xml and is 696,000 bytes in size. Technical Field

[0005] Disclosed herein are modified human interleukin-2 (hIL-2) proteins, human antibody molecules or antigen-binding fragments thereof that immunospecifically bind to human programmed cell death protein-1 (hPD-1), and immunoconjugates comprising the same. Background Art

[0006] Human IL-2 (hIL-2) is a type 1 four-alpha-helical bundle, glycosylated cytokine produced by CD4+ T cells and CD8+ T cells. Autocrine and paracrine IL-2 signaling occurs through engagement of a high-affinity trimeric receptor complex containing IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132) or a moderate-affinity dimeric receptor complex containing IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-2 has dual opposing and pleiotropic effects, as it can stimulate both T-cell proliferation to generate 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, whereas high-dose IL-2 broadly stimulates cytotoxic T cells, T effectors, NK cells, and regulatory T cells. However, the use of IL-2 in the treatment of autoimmune diseases and as a cancer immunotherapy is limited by off-target effects and toxicities associated with the administration of IL-2. Summary of the invention

[0007] Disclosed herein are modified human interleukin-2 (hIL-2) proteins comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, wherein the modified hIL-2 protein exhibits reduced potency for both a high affinity hIL-2 receptor and a medium affinity hIL-2 receptor relative to unmodified hIL-2.

[0008] Also disclosed herein are modified human interleukin-2 (hIL-2) proteins comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E 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.

[0009] Also disclosed herein is a human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or the antigen-binding fragment thereof comprises:

[0010] a) a heavy chain complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423;

[0011] b) 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;

[0012] c) 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; or

[0013] d) 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.

[0014] Also disclosed herein are immunoconjugates comprising:

[0015] (a) a modified hIL-2 protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345; and

[0016] (b) a human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to hPD-1, wherein the human antibody molecule or the antigen-binding fragment thereof comprises:

[0017] (i) 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;

[0018] (ii) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 391;

[0019] (iii) 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; or

[0020] (iv) 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.

[0021] Also disclosed are pharmaceutical compositions comprising any of the modified hIL-2 proteins, human antibody molecules, or antigen-binding fragments thereof, or immunoconjugates disclosed herein.

[0022] Also disclosed herein are polynucleotides comprising a nucleic acid sequence encoding any of the modified hIL-2 proteins, human antibody molecules, or antigen-binding fragments thereof, or immunoconjugates disclosed herein, as well as vectors comprising the polynucleotides and transformed cells comprising the vectors.

[0023] Disclosed herein are methods of treating a disease or condition in a subject, the methods comprising administering to the subject a therapeutically effective amount of any of the immunoconjugates or pharmaceutical compositions disclosed herein, thereby treating the disease or condition.

[0024] Also disclosed are uses of any immunoconjugate or pharmaceutical composition disclosed herein in the preparation of a medicament for treating a disease, and uses of any immunoconjugate or pharmaceutical composition disclosed herein in treating a disease or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The Summary of the Invention and the following detailed description will be further understood when read in conjunction with the accompanying drawings. In order to illustrate the disclosed modified hIL-2 proteins, anti-hPD-1 antibodies or antigen-binding fragments thereof, and immunoconjugates, exemplary embodiments of the modified hIL-2 proteins, anti-hPD-1 antibodies or antigen-binding fragments thereof, and immunoconjugates are shown in the accompanying drawings; however, the modified hIL-2 proteins, anti-hPD-1 antibodies or antigen-binding fragments thereof, and immunoconjugates are not limited to the specific embodiments disclosed. In the accompanying drawings:

[0026] Figure 1A , Figure 1B , Figure 1C , Figure 1D , Figure 1E , Figure 1F , Figure 1G and Figure 1H Shown are exemplary antibody-hIL-2 immunoconjugates as described herein in Example 1. Unattenuated human IL-2 cytokine (grey rectangle) is fused directly (df) or via an L6 linker (L6) to the N-terminus or C-terminus of the two heavy chains or two kappa light chains of the antibody.

[0027] Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E , Figure 2F , Figure 2G and Figure 2HAn exemplary antibody-hIL-2 immunoconjugate with an extracellular domain of hCD25 (1-164) is shown, which is designed to interfere with the binding of hIL-2 of the immunoconjugate to human IL-2Rα. For the N-terminal variant, the human CD25 / IL-2Rα extracellular domain (black triangle) is fused to the unattenuated hIL-2 cytokine (gray rectangle) via an L20 linker (light gray line). The unattenuated hIL-2 cytokine is then fused directly to the antibody (df) or fused via an L6 linker. For the C-terminal variant, the hCD25 / IL-2Rα extracellular domain portion is fused directly to the antibody (df) or fused using an L6 linker, followed by an L20 linker and unattenuated hIL-2 cytokine.

[0028] Figure 3 An exemplary 1H3-hIgG1-L6-hIL-2 immunoconjugate containing a 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 hIL-2 cytokine portion (attenuated hIL-2) containing substitutions predicted to modulate binding to CD122 / IL-2Rβ as described in Example 2.

[0029] Figure 4A , Figure 4B , Figure 4C and Figure 4D Shown are the experimental results of analyzing the binding of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 to the human PD-1 receptor on Jurkat cells in the presence of saturating 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.

[0030] Figure 5 Exemplary anti-hPD-1-attenuated hIL-2 immunoconjugates with L6 linker (L6) (left) or direct fusion (df) (right) are shown. Anti-hPD-1 antibodies containing hIgG4 or hIgG1 Fc domains (with or without L235E (LE) or L235A / G237A (LAGA) modifications in the Fc domain) were fused to attenuated hIL-2 cytokines at the C-terminus of the antibody heavy chain. Various substitutions were introduced in the hIL-2 cytokine to attenuate potency.

[0031] Fig. 6A and Figure 6BResults of a competition assay are shown, demonstrating that in the presence of a saturating concentration of anti-hPD-1-attenuated hIL-2 immunoconjugate (280 nM), anti-hPD-1#1-mIgG2b-N297A ( Fig. 6A ) or anti-hPD-1#2-mIgG2b-N297A( Figure 6B ) binds to the anti-hPD-1 receptor on Jurkat cells.

[0032] Figure 7 The results of competition assays are shown, demonstrating that the anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2(D20A / R38E), C51E6-5-L6-hIgG4-hIL-2(D20A / R38E), and A2-hIgG4-df-hIL-2(D20A / R38E) do not inhibit the binding of human PD-L1 to the human PD-1 receptor using a PD-1 / PD-L1 blocking bioassay.

[0033] Figure 8 Shown are the results of an experiment analyzing the effects of administration of vehicle, alternative anti-PD-1 antibodies (anti-mPD-1RMP1-14 mIgG2b-N297A and anti-mPD-1RMP1-30 mIgG2b-N297A), and alternative anti-PD-1-attenuated hIL-2 immunoconjugates (anti-mPD-1RMP1-14mIgG2b-N297A-L6-hIL-2(F42K / Y45R / V69R) or anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2(F42K / Y45R / V69R)) on the growth of established subcutaneous MC38 syngeneic tumors in C57BL / 6 mice, as described in Example 18. Test agents were administered intraperitoneally at a dose of 5 mg / kg twice a week for 4 weeks starting on day 1. The points on the graph represent the mean tumor volume of 10 mice per group.

[0034] Fig. 9A , Fig. 9B and Fig. 9C Results of studies performed to determine the efficacy of the alternative anti-hPD-1-attenuated hIL-2 immunoconjugate, anti-mPD-1 RMP1-30 mIgG2b-N297A-hIL-2(F42K / Y45R / V69R), in the MC38 murine colon adenocarcinoma model are presented. Fig. 9A Depicted are the mean subcutaneous tumor volumes (mm) measured every 3-4 days over the 8 days after the first dose of the test agent (3 doses at 5 mg / kg on days 1, 4, and 8). 3 ). Tumor growth curves represent the average of 15 animals per group. Fig. 9BSummarizes the results of immunophenotyping of tumors by flow cytometry on day 9, which shows different CD8 + T cell subsets account for the total CD8 + Ratio of mean absolute T cell counts. Fig. 9C The results of immunophenotyping on day 9 are presented, demonstrating that CD8 + T effector memory was significantly expanded, and there was a trend toward a decrease in regulatory T cells (cells / μL) in tumors.

[0035] Fig.10 Figure 2 shows analysis of NOD-Prkdc exposed to anti-hPD-1-attenuated hIL-2 immunoconjugates. em26Cd52 IL-2rg em26Cd22 Experimental results show acceleration of graft-versus-host disease in NCG / NjuCrl (NCG) mice, as demonstrated by significant weight loss in the NCG-PBMC model.

[0036] Fig.11A and Fig. 11B Shown is the analysis of NOD-Prkdc treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) in the NCG-PBMC model. em26Cd52 IL-2rg em26Cd22 Blood CD8 + Effector memory T cells ( Fig.11A ) and CD4 + Effector memory T cells ( Fig. 11B ) cells / mL of the dose-dependent expansion experiment results.

[0037] Fig.12 NOD-Prkdc treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) in the NCG-PBMC model is shown. em26Cd52 IL-2rg em26Cd22 / NjuCrl(NCG) mice had a reduced number of blood regulatory T cells / mL.

[0038] Fig.13A and Fig. 13B showed that H7-632–hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A) (designated “H7-767”) ( Fig. 13B) continues to bind to the human PD-1 receptor on Jurkat cells.

[0039] Fig.14A and Fig. 14B is a graph showing the binding of recombinant human PD-1 captured by H7-767 immobilized to a CM5 sensor chip as assessed by surface plasmon resonance (SPR) followed by ( Fig.14A )H7-767, or or( Fig. 14B ) Diagram of the binding of PD-L1 or PD-L2.

[0040] Fig.15 It was demonstrated that H7-632-hIgG1-LAGA and H7-767 did not inhibit the binding of human PD-L1 to the human PD-1 receptor using the hPD-1 / hPD-L1 blocking bioassay.

[0041] Fig.16A , Fig. 16B , Fig. 16C and Fig.16D is a graph showing the binding of anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2(D20A / R38E), C51E6-5-hIgG4-df-hIL-2(D20A / R38E), and A2-hIgG4-df-hIL-2(D20A / R38E) to the human PD-1 receptor on Jurkat cells in the presence of saturating 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-attenuated hIL-2 immunoconjugates as assessed by flow cytometry.

[0042] Fig.17 is a graph showing the binding of 2H7-hIgG4-df-hIL-2(D20A / R38E), C51E6-5-hIgG4-df-hIL-2(D20A / R38E), A2-hIgG4-df-hIL-2(D20A / R38E), and an irrelevant antibody control 1H3-hIgG4-df-hIL-2(D20A / R38E) to HEK-293T cells recombinantly expressing cynomolgus monkey PD-1 as assessed by flow cytometry.

[0043] Fig.18A , Fig.18B , Fig. 18C and Fig.18DShown are the antagonist activities 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. Fig.18A and Fig.18B Shown are the results of titration of anti-hPD-1 #1 or anti-hPD-1 #2 in the presence of a fixed concentration of anti-hPD-1-attenuated hIL-2 immunoconjugate. Fig. 18C and Fig.18D The results show that the expression of anti-hPD-1#1 ( Fig. 18C ) or 100 nM anti-hPD-1#2 ( Fig.18D ) Results of the reverse experiment titrating the anti-hPD-1 -attenuated hIL-2 immunoconjugate.

[0044] Fig.19 Shown is the effect of administration of various test agents (including alternative anti-mouse PD-1 / attenuated IL-2 immunoconjugates) on the growth of established subcutaneous MC38 syngeneic tumors in C57BL / 6 mice. Each growth curve represents the average tumor volume of ten mice per treatment group.

[0045] Fig. 20 Shown with Fig.19 The ability of MC38 tumor cells to grow in tumor-free mice was demonstrated compared to mice in the MC38 primary tumor study that had previously been treated with anti-mPD-1-hIL-2F42K / Y45R / V69R and had demonstrated complete long-term regression of established primary tumors. Animals from two cohorts (10 mice per group) were implanted subcutaneously with 5×10 5 Mice previously exposed to the alternative agent anti-mPD-1-hIL-2F42K / Y45R / V69R showed no tumor growth because they had developed sustained immunity, whereas corresponding naive mouse controls showed typical tumor growth in their flanks. DETAILED DESCRIPTION

[0046] The disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates may be more readily understood by reference to the following detailed description in conjunction with the accompanying drawings forming a part of the present disclosure. It should be understood that the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates are not limited to the specific modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates described and / or shown herein, and that the terms used herein are only for the purpose of describing specific embodiments by way of example and are not intended to limit the claimed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates.

[0047] Unless specifically stated otherwise, any description of a possible mechanism or mode of action or reason for improvement is intended to be illustrative only, and the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates are not bound by the correctness or incorrectness of any such proposed mechanism or mode of action or reason for improvement.

[0048] Throughout this article, descriptions relate to modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates, as well as methods of using modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates. Where the present disclosure describes or claims features or embodiments related to modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates, such features or embodiments are equally applicable to methods of using modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates. Similarly, where the present disclosure describes or claims features or embodiments related to methods of using modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates, such features or embodiments are equally applicable to modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates.

[0049] In the case of enumerating or establishing a numerical range in this article, the range includes all single integers and fractions in its endpoints and range, and also includes each of the narrower ranges formed therein by all various possible combinations of the endpoints and internal integers and fractions of the subgroups forming the larger value group in the described range, and its degree is the same as the degree of clearly enumerating each narrower range. In the case where the numerical range is expressed as being greater than the value in this article, the range is still limited, and its upper limit is defined by the operable value in the context disclosed herein. In the case where the numerical range is expressed as being less than the value in this article, the lower limit of the range is still defined by a non-zero value. The scope of the modified hIL-2 protein, human antibody molecule or its antigen-binding fragment and immunoconjugate is not intended to be limited to the specific value listed when defining the range. All ranges are inclusive and combinable.

[0050] Similarly, when a value is expressed as an approximation by using the antecedent "about", it will be understood that the particular value forms another embodiment. Unless the context clearly dictates otherwise, reference to a particular numerical value includes at least the particular value. The term "about" is used to indicate that the enumerated value may differ from the listed value by as much as 10% when it comes to a numerical range, a critical value, or a particular value. Thus, the term "about" is used to cover a variation of ±10% or less, a variation of ±5% or less, a variation of ±1% or less, a variation of ±0.5% or less, or a variation of ±0.1% or less from the specified value.

[0051] It should be understood that, for the sake of clarity, certain features of the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the disclosed modified hIL-2 proteins, human antibody molecules or antigen-binding fragments thereof, and immunoconjugates described in the context of a single embodiment may also be provided separately or in any subcombination.

[0052] As used herein, the singular form "a," "an," and "the" include plural referents.

[0053] Various terms related to the various aspects of the description are used throughout the specification and claims. Unless otherwise specified, such terms have their ordinary meanings in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0054] The term "comprising" is intended to include instances encompassed by the terms "consisting essentially of and "consisting of;" similarly, the term "consisting essentially of is intended to include instances encompassed by the term "consisting of.

[0055] The term "antibody molecule" is intended to be broadly defined and includes full-length immunoglobulin molecules and antigen-binding fragments thereof.

[0056] Based on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into five major classes, namely 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 assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0057] "Antigen binding fragment" refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of a parent full-length antibody (i.e., "an antigen binding fragment thereof"). 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; heavy chain variable regions (VH); light chain variable regions (VL); and combinations thereof. Antigen binding fragments include: Fab fragments, which are monovalent fragments consisting of VL, VH, constant light chain (CL), and constant heavy chain 1 (CH1) domains; F(ab)2 fragments, which are bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; and domain antibody (dAb) fragments consisting of 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 by synthetic linkers to form various types of single-chain antibody designs, where VH and VL domains are expressed by separate single-chain antibody constructs, in which case the VH / VL domains are paired intramolecularly or intermolecularly to form a monovalent antigen binding site, such as single-chain Fv (scFv) or diabodies, which are described, for example, in International Patent Publication Nos. 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 the fragments are screened for utility in the same manner as full-length antibodies.

[0058] The phrase "immunospecific binding" refers to the ability of the disclosed antibody molecules to preferentially bind to their target (hPD-1 in the case of anti-hPD-1 antibody molecules) rather than preferentially bind to other molecules in a sample containing a mixed population of molecules. Antibody molecules that immunospecifically bind to hPD-1 are substantially free of other antibodies with different antigenic specificities (e.g., anti-hPD-1 antibodies are substantially free of antibodies that specifically bind to antigens other than hPD-1). However, antibody molecules that immunospecifically bind to hPD-1 may have 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 immunospecifically binding to both naturally produced hPD-1 and PD-1 recombinantly produced in mammals or prokaryotic cells.

[0059] The antibody variable region consists of four "framework" regions interrupted by three "antigen combining sites." Antigen binding sites are defined in various terms: (i) complementarity determining regions (CDRs), three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3), which are based on sequence variability (Wu and Kabat, J Exp Med, 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. National Institutes of Health, Department of Public Health, Bethesda, MD, 1991); and (ii) "hypervariable regions" ("HVRs" or "HVs"), three in VH (H1, H2, H3) and three in VL (L1, L2, L3), which refer to regions of antibody variable domains that are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol. Biol, 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 called because it is used by Oxford Molecular's AbM antibody modeling software (Rees, AR, Searle, SMJ, Henry, AH and Pedersen, JT (1996) Sternberg MJE (eds.), Protein Structure Prediction. Oxford University Press, 141-172). Other terms include "IMGT-CDR" (Lefranc et al., Dev Comparat Immunol, 27:55-77, 2003) and "specificity determining residue usage" (SDRU) (Almagro Mol Recognit, 17:132-43, 2004). The International Immunogenetics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definitions of 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.

[0060] "Framework" or "framework sequence" is the remaining sequence of the variable region other than the sequence defined as the antigen binding site. Because the antigen binding site can be defined in a variety of terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.

[0061] "Human antibody", "fully human antibody" and similar terms refer to antibodies with heavy and light chain variable regions, wherein the framework and antigen binding sites are derived from human sequences. If the antibody contains a constant region, the constant region is also derived from human sequences. Human antibodies comprise heavy chain variable regions and / or light chain variable regions, and if the variable regions of the antibody are obtained from a system using human germline immunoglobulins or rearranged immunoglobulin genes, the variable regions are "derived from" human sequences. Such systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals such as mice or chickens that carry 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 intentionally introduced substitutions in 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 an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, a "human antibody" may contain a consensus framework sequence derived from human framework sequence analysis, such as Knappik et al., J Mol Biol 296:57-86, 2000, or contain a synthetic HCDR3 incorporated into a human immunoglobulin gene library displayed on a phage, such as Shi et al., J Mol Biol 397:385-96, 2010, and International Patent Publication No. WO2009 / 085462. Antibodies whose antigen binding sites are derived from non-human species are not included in the definition of "human antibody".

[0062] Although derived from human immunoglobulin sequences, human antibodies can be produced using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve the antibody properties of the variable or constant regions, or both, thereby generating antibodies that do not naturally occur within the human antibody germline repertoire in vivo.

[0063] "Recombinant antibodies" include all antibodies prepared, expressed, generated or isolated by recombinant means, such as: antibodies isolated from animals transgenic or transchromosomal (e.g., mice) for human immunoglobulin genes or hybridomas prepared therefrom (described further below); antibodies isolated from host cells transformed to express antibodies; antibodies isolated from recombinant combinatorial antibody libraries; and antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences, or antibodies generated in vitro using Fab arm exchange.

[0064] "Monoclonal antibody" refers to a population of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope, or in the case of a bispecific monoclonal antibody, exhibits dual binding specificities for two different epitopes. Thus, a monoclonal antibody refers to an antibody population having a single amino acid composition in each heavy chain and each light chain, except for possible well-known changes (such as removal of the C-terminal lysine from the antibody heavy chain). Monoclonal antibodies may have heterogeneous glycosylation in the antibody population. Monoclonal antibodies may be monospecific or multispecific, or monovalent, divalent or multivalent. The term monoclonal antibody includes bispecific antibodies.

[0065] "Epitope" refers to the part of an antigen that specifically binds to an antibody. An epitope is usually composed of chemically active (such as polar, nonpolar or hydrophobic) surface groups of parts such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural properties as well as specific charge properties. An epitope may be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For a discontinuous epitope, amino acids from different parts of the linear sequence of an antigen are very close in 3-dimensional space by the folding of the protein molecule.

[0066] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications (e.g., substitutions, insertions, or deletions). The term "mutation" as used herein is intended to mean one or more intentional substitutions made to a polypeptide or polynucleotide.

[0067] "Treatment" and similar terms refer to therapeutic treatment and prophylactic or preventive measures, and include reducing the severity and / or frequency of symptoms, eliminating symptoms and / or potential causes of symptoms, reducing the frequency or likelihood of symptoms and / or their potential causes, and ameliorating or remedying damage caused directly or indirectly by the disease or condition. Treatment also includes prolonging survival compared to the expected survival of a subject not receiving treatment. Subjects to be treated include subjects already suffering from a disease or condition as well as subjects susceptible to a disease or condition or subjects for whom a disease or condition is to be prevented.

[0068] As used herein, "administering to a subject" and similar terms indicate a procedure in which the disclosed modified hIL-2 protein, immunoconjugate, or pharmaceutical composition is injected into a subject so that target cells, tissues, or fragments of the subject's body are contacted with the disclosed modified hIL-2 protein or immunoconjugate comprising the same.

[0069] The phrase "therapeutically effective amount" refers to an amount of a modified hIL-2 protein, immunoconjugate, or pharmaceutical composition as described herein that is effective to achieve a particular biological or therapeutic outcome (such as, but not limited to, a biological or therapeutic outcome disclosed, described, or exemplified herein). A therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the modified hIL-2 protein, immunoconjugate, or pharmaceutical composition to elicit a desired response in the subject. Exemplary indicators of a therapeutically effective amount include, for example, improvement in the patient's health, alleviation of disease symptoms, cessation or slowing of disease symptom progression, and / or disappearance of disease symptoms.

[0070] The term "subject" as used herein is intended to mean any animal, particularly a mammal. Thus, the methods are applicable to humans and non-human animals, but are most preferably used in humans. "Subject" and "patient" are used interchangeably herein.

[0071] Immunoconjugate and fusion protein are used interchangeably herein.

[0072] Modified human interleukin-2 (hIL-2) protein

[0073] Disclosed herein are modified hIL-2 proteins comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, wherein the modified hIL-2 protein exhibits reduced potency for both the high affinity hIL-2 receptor and the intermediate affinity hIL-2 receptor relative to the unmodified hIL-2. The disclosed modified hIL-2 proteins are also referred to herein as "attenuated" hIL-2.

[0074] Suitable substitutions at amino acid position 20 include, for example, any of a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

[0075] Suitable substitutions at amino acid position 38 include, for example, any of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D or R38K substitutions.

[0076] In some embodiments, any of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions can be combined with the R38E substitution.

[0077] The modified hIL-2 protein may comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein may comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608.In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one 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 of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

[0078] The modified hIL-2 protein may comprise a D20A substitution and a R38E substitution.

[0079] As used herein, the term "reduced potency" and related terms such as "reduced potency" or "attenuation" of IL-2 activity refers to a reduction in the potency of the modified hIL-2, as measured by EC in an IL-2-dependent assay. 50 Values ​​relative to the EC of unmodified hIL-2 50The value is increased. As described herein, the modified hIL-2 will have reduced potency for high-affinity and medium-affinity IL-2 receptors. The IL-2-dependent assay used to determine potency can be an engineered human erythroleukemia TF1 (TF1+IL-2Rβ) or human natural killer NK-92 cell proliferation assay as described herein. In one embodiment, the IL-2-dependent assay used to determine potency is an engineered human erythroleukemia TF1 (TF1+IL-2Rβ) cell proliferation assay. In another embodiment, the IL-2-dependent assay used to determine potency is a human natural killer NK-92 cell proliferation assay. Other IL-2-dependent assays used to determine potency can also be TF1+IL-2Rβ or human natural killer NK-92pSTAT5 assays as described herein. The unmodified hIL-2 can be a prokaryotically expressed hIL-2, such as (which has the native human IL-2 amino acid sequence except for the C125S substitution to remove the unbound cysteine ​​and does not have the normal human carbohydrate expression at residue T3), or the unmodified hIL-2 can be a hIL-2 having the amino acid sequence of SEQ ID NO: 345 or a hIL-2 having the amino acid sequence of SEQ ID NO: 345 and the C125S substitution, which is expressed in a mammalian cell line such as a CHO or HEK cell line.

[0080] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein can further comprise a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

[0081] Alternatively, the modified hIL-2 protein 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 comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.

[0082] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein may further comprise 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 comprises a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 219.

[0083] The modified hIL-2 protein can exhibit 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 decreased potency at the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 In some embodiments, the modified hIL-2 protein may exhibit a decrease in potency of about 10,000-fold or more for the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2. For the modified hIL-2 proteins described herein, a greater decrease in the potency of hIL-2 for the high affinity hIL-2 receptor is possible and acceptable, but due to limitations in cell proliferation assay conditions, such decreases may not be quantified using the methods described herein.

[0084] Additionally, the modified hIL-2 protein can exhibit 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 decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 In some embodiments, the modified hIL-2 protein may exhibit a potency reduction of about 10,000-fold or greater at the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0085] The modified hIL-2 protein can exhibit up to about 10,000-fold decreased potency at the high affinity IL-2 receptor (hIL-2Rαβγ) and up to about 10,000-fold decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 The modified hIL-2 protein can exhibit a potency reduction of about 10,000-fold or more for the high affinity IL-2 receptor (hIL-2Rαβγ) and a potency reduction of about 10,000-fold or more for the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0086] As demonstrated herein, the modified hIL-2 protein can be fused to an anti-PD-1 antibody or an antigen-binding fragment thereof. The hIL-2 protein can be fused to an anti-PD-1 antibody or an antigen-binding fragment thereof 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. In some embodiments, the modified hIL-2 protein is directly fused to an anti-PD-1 antibody or an antigen-binding fragment thereof via a peptide bond. The modified hIL-2 protein can be fused, for example, directly to the C-terminal amino acid residue of the anti-PD-1 antibody heavy chain via a peptide bond. In some embodiments, the modified hIL-2 protein is fused to an anti-PD-1 antibody or an antigen-binding fragment thereof via a linker.

[0087] The fusion of the modified hIL-2 protein with an antibody or an antigen-binding fragment thereof can rescue the ability of the modified hIL-2 protein to bind to and activate the human medium-affinity IL-2 receptor on PD-1 expressing cells (such as T cells, and in particular tumor-infiltrating lymphocytes). In some embodiments, the hIL-2 protein fused to an antibody or an antigen-binding fragment thereof exhibits potency comparable to that of wild-type hIL-2 for the medium-affinity IL-2 receptor on PD-1 expressing cells.

[0088] The fusion of modified hIL-2 protein with antibody or antigen binding fragment thereof can be used to selectively deliver IL-2 signaling to cells expressing the PD-1 target of the antibody or antigen binding fragment thereof. Without being bound by theory, it is believed that targeting the modified hIL-2 protein to a specific cell population can significantly enhance the therapeutic effect of IL-2 (e.g., anti-tumor immunity) without off-target systemic toxicity.

[0089] Also disclosed herein are modified human interleukin-2 (hIL-2) proteins comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E 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.

[0090] The modified hIL-2 protein may comprise the amino acid sequence of any one of SEQ ID NOs: 307, 607-611, 614, 617, or 620. 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: 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 of any one of the amino acid sequences SEQ ID NO: 307, 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 of any one of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617 or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617 or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

[0091] The modified hIL-2 protein may comprise a D20A substitution and a R38E substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149.

[0092] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein can further comprise a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

[0093] Alternatively, the modified hIL-2 protein 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 comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.

[0094] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein may further comprise 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 comprises a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 219.

[0095] The modified hIL-2 protein can exhibit 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 decreased potency at the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50Values ​​are quantified. In some embodiments, the modified hIL-2 protein may exhibit a potency reduction of about 10,000-fold or more for the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2. For the modified hIL-2 proteins described herein, a greater reduction in the potency of hIL-2 for the high affinity hIL-2 receptor is possible and acceptable, but due to limitations in cell proliferation assay conditions, such reductions may not be quantified using the methods described herein.

[0096] Additionally, the modified hIL-2 protein can exhibit 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 decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 In some embodiments, the modified hIL-2 protein may exhibit a potency reduction of about 10,000-fold or greater at the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0097] The modified hIL-2 protein can exhibit up to about 10,000-fold decreased potency at the high affinity IL-2 receptor (hIL-2Rαβγ) and up to about 10,000-fold decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 The modified hIL-2 protein can exhibit a potency reduction of about 10,000-fold or more for the high affinity IL-2 receptor (hIL-2Rαβγ) and a potency reduction of about 10,000-fold or more for the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0098] As demonstrated herein, the modified hIL-2 protein can be fused to an anti-PD-1 antibody or an antigen-binding fragment thereof. The hIL-2 protein can be fused to an anti-PD-1 antibody or an antigen-binding fragment thereof 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. In some embodiments, the modified hIL-2 protein is directly fused to an anti-PD-1 antibody or an antigen-binding fragment thereof via a peptide bond. The modified hIL-2 protein can be fused, for example, directly to the C-terminal amino acid residue of the anti-PD-1 antibody heavy chain via a peptide bond. In some embodiments, the modified hIL-2 protein is fused to an anti-PD-1 antibody or an antigen-binding fragment thereof via a linker.

[0099] The fusion of the modified hIL-2 protein with an antibody or an antigen-binding fragment thereof can rescue the ability of the modified hIL-2 protein to bind to and activate the human medium-affinity IL-2 receptor on PD-1 expressing cells (such as T cells, and in particular tumor-infiltrating lymphocytes). In some embodiments, the hIL-2 protein fused to an antibody or an antigen-binding fragment thereof exhibits potency comparable to that of wild-type hIL-2 for the medium-affinity IL-2 receptor on PD-1 expressing cells.

[0100] The fusion of modified hIL-2 protein with antibody or antigen binding fragment thereof can be used to selectively deliver IL-2 signaling to cells expressing the PD-1 target of the antibody or antigen binding fragment thereof. Without being bound by theory, it is believed that targeting the modified hIL-2 protein to a specific cell population can significantly enhance the therapeutic effect of IL-2 (e.g., anti-tumor immunity) without off-target systemic toxicity.

[0101] Human anti-human programmed cell death protein-1 (hPD-1) antibody

[0102] Disclosed herein are human antibody molecules or antigen-binding fragments thereof, which immunospecifically bind to hPD-1, wherein the human antibody molecule or antigen-binding fragment thereof comprises:

[0103] a) a heavy chain complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423;

[0104] b) 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;

[0105] c) 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; or

[0106] d) 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.

[0107] In some embodiments, the human antibody molecule or its antigen-binding fragment 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").

[0108] In some embodiments, the human antibody molecule or its antigen-binding fragment 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").

[0109] In some embodiments, the human antibody molecule or its antigen-binding fragment 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").

[0110] In some embodiments, the human antibody molecule or its antigen-binding fragment 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").

[0111] The disclosed human antibody molecules or antigen-binding fragments thereof may exhibit one or more of the following activities:

[0112] ● Binds to PD-1 without inhibiting the binding of PD-L1 to PD-1;

[0113] ● In the presence of standard-of-care anti-PD-1 antibodies used in the clinic (e.g. and ) in the case of binding to PD-1;

[0114] ●Highly selective for PD-1 and does not immunospecifically bind to other related B7 family members; and

[0115] ● Binds to PD-1 on activated human T cells (EC in flow cytometric binding assays 50 is ~0.1-0.2nM).

[0116] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417 (referred to herein as "H7-632").

[0117] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385 (referred to herein as "2H7").

[0118] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395 (referred to herein as "C51E6-5").

[0119] The human antibody molecule or antigen-binding fragment thereof may comprise: 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").

[0120] The human antibody molecule or antigen-binding fragment thereof may comprise a human IgG1 heavy chain constant region.

[0121] Human antibody molecules or their antigen-binding fragments may have substitutions or deletions in the constant region to minimize Fc-mediated immune effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by FcγRIIIA, FcγRI and FcγRIIa-dependent antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) mediated by C1q in combination. In some embodiments, human antibody molecules comprise L235A substitutions, wherein the amino acid numbering is according to EU numbering. In some embodiments, human antibody molecules comprise G237A substitutions, wherein the amino acid numbering is according to EU numbering. In some embodiments, human antibody molecules comprise L235A and G237A substitutions, wherein the amino acid numbering is according to EU numbering.

[0122] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415 (referred to herein as "H7-632-hIgG1-LAGA").

[0123] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425 (referred to herein as "2H7-hIgG4").

[0124] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427 (referred to herein as "C51E6-5-hIgG4").

[0125] The human antibody molecule or antigen-binding fragment thereof may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO: 428 and a light chain comprising the amino acid sequence of SEQ ID NO: 429 (referred to herein as "A2-hIgG4").

[0126] The human antibody molecule or antigen-binding fragment thereof can be fused to a modified hIL-2 protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345. The human antibody molecule or antigen-binding fragment thereof can be fused to any modified hIL-2 protein disclosed herein.

[0127] When not fused to an antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein may exhibit reduced potency for both the high affinity hIL-2 receptor and the intermediate affinity hIL-2 receptor relative to unmodified hIL-2.

[0128] Suitable substitutions at amino acid position 20 of the modified hIL-2 include, for example, any of a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

[0129] Suitable substitutions at amino acid position 38 of the modified hIL-2 protein include, for example, any of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D or R38K substitutions.

[0130] In some embodiments, any of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions can be combined with the R38E substitution.

[0131] The modified hIL-2 protein may comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein may comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608.In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one 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 of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

[0132] The modified hIL-2 protein may comprise a D20A substitution and a R38E substitution.

[0133] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein can further comprise a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

[0134] Alternatively, the modified hIL-2 protein 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 comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.

[0135] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein may further comprise 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 comprises a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein comprises a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 219.

[0136] When not fused to a human antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit 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 decreased potency for the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC in a hIL-2-dependent cell proliferation assay described herein. 50 Value to quantify. In some embodiments, when not fused to a human antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit a potency reduction of about 10,000-fold or more for the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2. For the modified hIL-2 proteins described herein, a greater reduction in the potency of hIL-2 for the high affinity hIL-2 receptor is possible and acceptable, but due to the limitations of the cell proliferation assay conditions, such reductions may not be quantified using the methods described herein.

[0137] Additionally, when not fused to a human antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit 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 decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC in a hIL-2-dependent cell proliferation assay described herein. 50 In some embodiments, when not fused to a human antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit a potency reduction of about 10,000-fold or more for the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0138] When not fused to a human antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit up to about 10,000-fold decreased potency at the high affinity IL-2 receptor (hIL-2Rαβγ) and up to about 10,000-fold decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC in a hIL-2-dependent cell proliferation assay described herein. 50 When not fused to a human antibody molecule or antigen-binding fragment thereof, the modified hIL-2 protein can exhibit a potency reduction of more than about 10,000-fold for the high affinity IL-2 receptor (hIL-2Rαβγ) and a potency reduction of more than about 10,000-fold for the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0139] The fusion of the modified hIL-2 protein with an antibody or an antigen-binding fragment thereof can rescue the ability of the modified hIL-2 protein to bind to and activate the human medium-affinity IL-2 receptor on PD-1 expressing cells (such as T cells, and in particular tumor-infiltrating lymphocytes). In some embodiments, the hIL-2 protein fused to an antibody or an antigen-binding fragment thereof exhibits potency comparable to that of wild-type hIL-2 for the medium-affinity IL-2 receptor on PD-1 expressing cells.

[0140] The modified hIL-2 protein can be fused to a human antibody molecule or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the hIL-2 protein is directly fused to the antibody or its antigen-binding fragment via a peptide bond. The hIL-2 can be fused, for example, directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond. In some embodiments, the hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker.

[0141] The fusion of human antibody molecules or antigen binding fragments thereof with modified hIL-2 proteins can be used to selectively deliver IL-2 signaling to cells expressing PD- 1. Without being bound by theory, it is believed that targeting modified hIL-2 proteins to specific cell populations expressing PD-1 can significantly enhance the therapeutic effect of IL-2 (e.g., anti-tumor immunity) while reducing or minimizing off-target systemic toxicity.

[0142] Immunoconjugates

[0143] Disclosed herein are immunoconjugates comprising any of the modified hIL-2 proteins disclosed herein and any of the human antibody molecules disclosed herein or antigen-binding fragments thereof. The immunoconjugates may comprise:

[0144] (a) a modified hIL-2 protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345; and

[0145] (b) a human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to hPD-1, wherein the human antibody molecule or the antigen-binding fragment thereof comprises:

[0146] (i) 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;

[0147] (ii) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 391;

[0148] (iii) 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; or

[0149] (iv) 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.

[0150] Suitable substitutions at amino acid position 20 of the modified hIL-2 portion of the immunoconjugate include, for example, any of a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

[0151] Suitable substitutions at amino acid position 38 of the modified hIL-2 portion of the immunoconjugate include, for example, any of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D or R38K substitutions.

[0152] In some embodiments, any of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions can be combined with the R38E substitution.

[0153] The modified hIL-2 protein portion of the immunoconjugate may comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein portion of the immunoconjugate may comprise the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607.In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein of any one 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 of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any one of the amino acid sequences of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

[0154] The modified hIL-2 protein portion of the immunoconjugate may comprise a D20A substitution and a R38E substitution.

[0155] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein portion of the immunoconjugate can further comprise a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO: 216.

[0156] Alternatively, the modified hIL-2 protein portion of the immunoconjugate may further comprise a deletion at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO: 218.

[0157] Relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein portion of the immunoconjugate may further comprise 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 portion of the immunoconjugate comprises a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises an amino acid sequence of SEQ ID NO: 215. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises an amino acid sequence of SEQ ID NO: 217. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO:219.

[0158] The modified hIL-2 protein portion of the immunoconjugate can exhibit 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 decreased potency for the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC in a hIL-2-dependent cell proliferation assay described herein. 50 Values ​​are quantified. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate can exhibit a potency reduction of about 10,000-fold or more for the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2. For the modified hIL-2 proteins described herein, a greater reduction in the potency of hIL-2 for the high affinity hIL-2 receptor is possible and acceptable, but due to the limitations of the cell proliferation assay conditions, such reductions may not be quantified using the methods described herein.

[0159] Additionally, the modified hIL-2 protein portion of the immunoconjugate can exhibit 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 decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 In some embodiments, the modified hIL-2 protein portion of the immunoconjugate can exhibit a greater than about 10,000-fold decrease in potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0160] The modified hIL-2 protein portion of the immunoconjugate can exhibit up to about 10,000-fold decreased potency at the high affinity IL-2 receptor (hIL-2Rαβγ) and up to about 10,000-fold decreased potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2, e.g., as measured by comparing EC 50 The modified hIL-2 protein portion of the immunoconjugate can exhibit a greater than about 10,000-fold decrease in potency for the high affinity IL-2 receptor (hIL-2Rαβγ) and a greater than about 10,000-fold decrease in potency for the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2.

[0161] The hIL-2 protein portion of the immunoconjugate can be fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the hIL-2 protein portion of the immunoconjugate is directly fused to the human antibody molecule or its antigen-binding fragment via a peptide bond. The hIL-2 protein portion of the immunoconjugate can be fused directly to the C-terminal amino acid residue of the antibody heavy chain, for example, via a peptide bond. In some embodiments, the hIL-2 protein portion of the immunoconjugate is fused to the human antibody molecule or its antigen-binding fragment via a linker.

[0162] Fusion of the modified hIL-2 protein with a human antibody molecule or antigen-binding fragment thereof can rescue the ability of the modified hIL-2 protein to activate the medium-affinity IL-2 receptor. In some embodiments, the immunoconjugate is capable of activating the medium-affinity IL-2 receptor to an extent comparable to wild-type hIL-2 activation of the medium-affinity IL-2 receptor.

[0163] In some embodiments, the human antibody molecule or antigen-binding fragment thereof 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.

[0164] In some embodiments, the human antibody molecule or antigen-binding fragment thereof 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.

[0165] In some embodiments, the human antibody molecule or antigen-binding fragment thereof 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.

[0166] In some embodiments, the human antibody molecule or antigen-binding fragment thereof 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.

[0167] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:417.

[0168] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:385.

[0169] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:395.

[0170] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: 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.

[0171] The human antibody molecule or antigen-binding fragment thereof portion of the immunoconjugate may comprise an IgG1 heavy chain constant region.

[0172] The human antibody molecule or its antigen binding fragment portion of the immunoconjugate may have a substitution or deletion in the constant region to minimize Fc-mediated immune effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by FcγRIIIA, FcγRI and FcγRIIa-dependent antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) mediated by C1q binding. In some embodiments, the human antibody molecule portion of the immunoconjugate comprises a L235A substitution, wherein the amino acid numbering is according to EU numbering. In some embodiments, the human antibody molecule portion of the immunoconjugate comprises a G237A substitution, wherein the amino acid numbering is according to EU numbering. In some embodiments, the human antibody molecule portion of the immunoconjugate comprises L235A and G237A substitutions, wherein the amino acid numbering is according to EU numbering.

[0173] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO:414 and a light chain comprising the amino acid sequence of SEQ ID NO:415.

[0174] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO:424 and a light chain comprising the amino acid sequence of SEQ ID NO:425.

[0175] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO:426 and a light chain comprising the amino acid sequence of SEQ ID NO:427.

[0176] The human antibody molecule or antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain comprising the amino acid sequence of SEQ ID NO:428 and a light chain comprising the amino acid sequence of SEQ ID NO:429.

[0177] An immunoconjugate may have one or more of the following properties:

[0178] ● Binds to PD-1 but does not inhibit the binding of PD-L1 to PD-1;

[0179] ● In the presence of standard-of-care anti-PD-1 antibodies used in the clinic (e.g. and ) in the case of binding to PD-1;

[0180] ●It is highly selective for PD-1 and does not immunospecifically bind to other related B7 family members;

[0181] ● Binds to PD-1 on activated human T cells (EC in flow cytometric binding assays 50 is ~0.1-0.2nM);

[0182] The potency at the high affinity IL-2 receptor (hIL-2Rαβγ) is reduced 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 relative to unmodified hIL-2, e.g., as determined by comparing the EC 50 Values ​​are quantified. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate can exhibit a potency reduction of about 10,000-fold or more at the high affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2. For the modified hIL-2 proteins described herein, a greater reduction in the potency of hIL-2 at the high affinity hIL-2 receptor is possible and acceptable, but due to limitations in cell proliferation assay conditions, such reductions may not be quantifiable using the methods described herein;

[0183] The potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ) is reduced 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 relative to unmodified hIL-2, e.g., as determined by comparing the EC 50 In some embodiments, the modified hIL-2 protein portion of the immunoconjugate can exhibit a potency reduction of about 10,000-fold or more for the medium affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2;

[0184] ● rescue and expansion of PD-1 expressing human memory T cell subsets in GvHD animal models; and

[0185] • Following single doses of 1 and 10 mg / kg to cynomolgus monkeys, there were minimal or no effects on body weight, blood chemistry, or hematological parameters.

[0186] In some embodiments, the immunoconjugate comprises a modified hIL-2 protein comprising a T3A substitution, an R38E substitution, a D20A substitution, and a C125A substitution, fused to the C-terminus of the antibody heavy chain of a human anti-hPD-1 antibody comprising a human IgG1 framework having a L235A substitution and a G237A substitution. In some embodiments, the immunoconjugate comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 415 and a heavy chain hIL-2 protein fusion comprising the amino acid sequence of SEQ ID NO: 532.

[0187] The disclosed immunoconjugates can selectively deliver IL-2 signaling to PD-1 expressing T cells. The human antibody molecule or antigen binding fragment portion of the immunoconjugate is only used to deliver the modified hIL-2 to PD-1 expressing cells and does not block PD-1 receptor function, as in and The primary mechanism of action of the immunoconjugates disclosed herein is 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 the modified hIL-2 protein portion of the immunoconjugate to this T cell population 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.

[0188] Pharmaceutical compositions, polynucleotides, vectors and cells

[0189] Disclosed herein are pharmaceutical compositions comprising any modified hIL-2 protein disclosed herein, any human antibody molecule or antigen binding fragment thereof disclosed herein, or any immunoconjugate disclosed herein. In some embodiments, the pharmaceutical composition comprises any modified hIL-2 protein disclosed herein. In some embodiments, the pharmaceutical composition comprises any human antibody molecule or antigen binding fragment thereof disclosed herein. In some embodiments, the pharmaceutical composition comprises any immunoconjugate disclosed herein.

[0190] Disclosed herein are polynucleotides comprising nucleic acid sequences encoding any modified hIL-2 protein disclosed herein, any human antibody molecule disclosed herein or its antigen-binding fragment, or any immunoconjugate disclosed herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any modified hIL-2 protein disclosed herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any human antibody molecule disclosed herein or its antigen-binding fragment. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any immunoconjugate disclosed herein.

[0191] Disclosed herein are vectors comprising polynucleotides comprising nucleic acid sequences encoding any modified hIL-2 protein disclosed herein, any human antibody molecule or antigen-binding fragment thereof disclosed herein, or any immunoconjugate disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding any modified hIL-2 protein disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding any human antibody molecule or antigen-binding fragment thereof disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding any immunoconjugate disclosed herein.

[0192] Also disclosed herein are transformed cells comprising any of the vectors disclosed herein.

[0193] Treatments and uses

[0194] Disclosed herein are methods of treating a disease or disorder in a subject, the methods comprising administering to the subject a therapeutically effective amount of any of the immunoconjugates or pharmaceutical compositions disclosed herein, thereby treating the disease.

[0195] Also disclosed is the use of any immunoconjugate or pharmaceutical composition disclosed herein in the preparation of a medicament for treating a disease. Also disclosed is the use of any immunoconjugate or pharmaceutical composition described herein for treating a disease or condition.

[0196] The disclosed immunoconjugates and pharmaceutical compositions can be used to treat diseases or conditions that are beneficial to the stimulation of the subject's immune system. In some embodiments, the subject's immune response is insufficient or lacking, and the disclosed immunoconjugates and pharmaceutical compositions stimulate the subject's immune response. The antibody portion of the immunoconjugate can be used to direct the modified hIL-2 protein to the subject's immune cells by, for example, binding to an antigen expressed on the surface of an immune cell. In the case of the disclosed modified hIL-2 protein-human anti-hPD-1 antibody immunoconjugate, for example, the anti-PD-1 antibody (or its antigen-binding fragment) portion of the immunoconjugate can bind to PD-1 expressed on T cells, thereby delivering the modified hIL-2 protein to T cells. Targeting the modified IL-2 protein to specific cells can significantly enhance the therapeutic effect of the IL-2 protein without off-target systemic toxicity mediated by cell populations lacking antigen expression. The disclosed methods and uses can be used to treat, for example, cancer, autoimmune diseases and inflammatory diseases, as well as chronic infections and infectious diseases. Exemplary cancers 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, gastric cancer, prostate cancer, blood cancer, skin cancer, melanoma, squamous cell carcinoma, bone cancer and kidney cancer. Exemplary autoimmune diseases and inflammatory diseases include systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriasis, Behcet's disease, granulomatosis with polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis, sclerosing cholangitis, Sjoren's syndrome, alopecia areata and inflammatory myopathy. Exemplary infectious diseases include HIV and hepatitis B.

[0197] In some embodiments, the disease is cancer. The methods and uses may comprise administering to a subject a therapeutically effective amount of any of the modified hIL-2 protein-antibody conjugates disclosed herein, thereby treating cancer. In some aspects, the cancer is melanoma. In some aspects, the cancer is non-small cell lung cancer.

[0198] Examples

[0199] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate rather than limit the disclosed embodiments.

[0200] General Methods

[0201] Protocol A. Flow cytometric screening of binding of anti-hPD-1 antibodies or anti-hPD-1 antibody-attenuated hIL-2 fusions to human PD-1

[0202] To test binding to hPD-1, antibodies and antibody-attenuated hIL-2 fusion proteins were characterized in a complete titration curve. Jurkat cell lines were transfected with a mammalian vector encoding amino acids 1-185 of human PD-1 (SEQ ID NO: 346) to stably express the extracellular domain and part of the transmembrane domain of human PD-1, and the transfected cell lines were used to determine the binding of anti-hPD-1 antibodies. Jurkat+hPD-1 cells were washed and added to FACS buffer (PBS, 0.2% heat-inactivated fetal bovine serum) in 96-well plates at 100,000 cells per well. Cells were blocked with a 1:50 dilution of human FcR blocking reagent (Miltenyi) for 10 minutes at 4°C and washed with FACS buffer.

[0203] 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 on ice in a volume of 100 μL for 1 hour. The cells were washed and resuspended in FACS buffer containing a 1:40 dilution of allophycocyanin-coupled anti-human IgG Fc monoclonal antibody. The cells were washed again, resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher) and flow cytometric analysis was performed on a BD FACS Canto II, BD Celesta or BDFortessa (BD Biosciences) flow cytometer. The geometric mean fluorescence intensity (gMFI) was calculated using FlowJo software version 10. The half-maximal effective concentration (EC) was calculated based on the gMFI of the allophycocyanin signal across the titration concentration using GraphPad Prism7 software. 50 )value.

[0204] Protocol B. Flow cytometric competition screening of binding of anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusion to human PD-1

[0205] The antibodies and antibody-attenuated hIL-2 fusion proteins were tested for their ability to bind to human PD-1 in the presence of saturating concentrations of anti-hPD-1 #1-mIgG2b-N297A (comprising the sequences of the heavy and light chain variable region sequences of nivolumab (clone 5C4), formatted on a murine IgG2b-N297A background as described in U.S. Patent Publication No. US2009 / 0217401A1) (SEQ ID NOs: 348 and 349) or anti-hPD-1 #2-mIgG2b-N297A (comprising the sequences of the heavy and light chain variable region sequences of pembrolizumab (clone 109A-H / K09A-L-11), formatted on a murine IgG2b-N297A background as described in International Publication No. WO2008 / 156712A1) (SEQ ID NOs: 350 and 351).

[0206] The antibody or antibody-attenuated hIL-2 fusion protein was serially diluted six-fold to obtain an 8-point titration curve, with or without a saturating amount of 10 μM anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A. Briefly, Jurkat cells stably expressing hPD-1 (as described in Protocol A above) were washed and resuspended in FACS buffer containing a 1:50 dilution of human FcR blocking agent. The cells were incubated at 4°C for 10 minutes and washed. The cells were then resuspended in a volume of 100 μL 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. The cells were washed and incubated for one hour at 4°C with test antibodies or antibody-attenuated hIL-2 fusion proteins serially diluted six-fold in a 100 μL volume to obtain an 8-point curve. To detect bound test anti-hPD-1 antibodies or anti-hPD-1-attenuated hIL-2 fusion proteins, the cells were washed again and incubated with a 1:40 dilution of allophycocyanin-coupled anti-human IgG Fc monoclonal antibody on ice for 45 minutes. The cells were washed and resuspended in FACS buffer containing Sytox Green (Thermo Fisher) diluted 1:1000. To generate comparisons, Jurkat cells stably expressing human PD-1 were incubated with titrated test antibodies or antibody-attenuated hIL-2 fusion proteins (without anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A) alone, and subsequently incubated with 1:40 diluted allophycocyanin-coupled anti-human IgG Fc secondary. As a control, 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 BD Canto II, BD Celesta, or BD Fortessa (BD Biosciences) flow cytometers, and gMFI was calculated using FlowJo software version 10. EC was calculated from the gMFI of the allophycocyanin signal across titrated concentrations using GraphPad Prism 7 software. 50 value.

[0207] Protocol C. Cell-based Screening for Characterization of Non-antagonist Anti-hPD-1 Antibodies or Anti-hPD-1 Antibody-Attenuated hIL-2 Fusions

[0208] Characterize the ability of human PD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins to block hPD-1 binding to the ligand hPD-L1 (SEQ ID NO: 584). Anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were characterized as antagonists or non-antagonists using an in vitro cell-based human PD-1 / PD-L1 blocking bioassay (Promega, catalog number J1255). This co-culture assay 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 express both human PD-L1 ligands and cell surface proteins to activate the cognate T cell receptor (TCR), while Jurkat effector cells express hPD-1 and luciferase reporter genes under the control of the response element of the nuclear factor of activated T cells (NFAT-RE). When these cells were co-cultured in the presence of non-antagonistic antibodies, hPD-1 / hPD-L1 interaction inhibited TCR signaling and no luminescence was detected. In the presence of antibodies that antagonize the interaction of hPD-1 with hPD-L1 (SEQ ID NO: 584), the inhibitory signal was disrupted and luminescence was detected.

[0209] Thawing and use assays were performed according to the manufacturer's instructions. In brief, aAPC / CHO-K1 cells were first thawed and plated on flat-bottomed 96-well plates at 30,000 cells per well in a 5% CO2 incubator at 37°C for 18 hours. After cell adhesion, the culture medium was removed, and 200nM or 1000nM test antibodies or antibody-attenuated hIL-2 fusion proteins were diluted in 40 μL assay buffer (RPMI 1640 culture medium + 1% FBS) and added to aAPC / CHO-K1 cells. A human IgG4 isotype control monoclonal antibody targeting keyhole limpet 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 antibodies tested was 100nM or 500nM. In some examples, a range of concentrations of anti-hPD-11 or anti-hPD-1-attenuated hIL-2 fusion proteins were tested in the co-culture assay, with the highest concentration of the five-fold titration series being 500 nM ( Figure 7 ).

[0210] The co-culture assay was incubated for an additional 18-20 hours at 37°C in a 5% CO2 incubator. To read the luminescent signal, the plate was allowed to reach room temperature and 80 μL of Bio-Glo was added to each well. TMReagents. The plates were incubated in the dark at room temperature for 15 minutes and the luminescence was read on a Victor X-ray meter (Perkin Elmer). Each three relative luminescence units (RLU) were averaged and plotted using GraphPad Prism 7 software.

[0211] Protocol D. In vitro phosphorylated STAT5 assay to test hIL-2 variant attenuation

[0212] Phosphorylated STAT5 was used to determine the attenuation level of hIL-2 receptor activation activity of the hIL-2 fusion protein characterized by antibody-attenuation. 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 high-affinity hIL-2 receptors (IL-2Rαβγ) at physiological levels, while the TF1 cell line naturally expressing IL-2Rγ (SEQ ID NO: 352) was engineered to stably express human CD122 (IL-2Rβ) (SEQ ID NO: 353) to express medium-affinity hIL-2 receptor complexes (IL-2Rβγ). This TF1+IL-2Rβ stable cell line does not express IL-2Rα (SEQ ID NO: 354). Both NK-92 cell lines 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).

[0213] For fixed concentration screening, 100,000 NK-92 cells or TF1+IL-2Rβ cells were plated into 96 wells in 50 μL of fresh growth medium lacking human IL-2 cytokine 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 test antibody-attenuated hIL-2 fusion protein for NK-92 cell assays, or 33.3 nM hIL-2 or test hIL-2 variants for TF1+IL-2Rβ cell assays. Cells were incubated at 37°C under 5% CO2 for 10 minutes. Cells were fixed with Cytofix buffer (BD Biosciences) at 37°C for 10 minutes and then permeabilized after treatment with Perm buffer III (BD Biosciences) on ice for 30 minutes. hIL-2-dependent Stat5 phosphorylation was detected after fixed and permeabilized cells were stained with Alexa Fluor-647-coupled anti-Stat5 antibody (BD Biosciences) at 0.5 μL / sample for 45 minutes in the dark at room temperature. The cells were washed and the reagents were diluted in BD Pharmingen buffer (BD Biosciences). The stained cells were acquired on a FACS-Celesta blood cell counter (BD Biosciences) and analyzed using FlowJo software version 10.7.2. The assay was performed in groups, but each plate was standardized using rhIL-2. The attenuation degree of the hIL-2 fusion protein of the selected antibody-attenuation was evaluated in an 8-point, 6-fold continuous titration curve ranging from 1200nM to 7pM on both the NK-92 cell line and the TF1+IL-2Rβ cell line. The procedure for the pStat5 curve was performed in the same manner as described above. EC was calculated using GraphPad Prism 7 software based on the geometric mean fluorescence intensity (gMFI) across the titration concentrations. 50 By adding the EC 50 The value was divided by the EC value of hIL-2 50 To calculate the fold change of rhIL-2 activity.

[0214] Protocol E. In vitro cell-based proliferation assay to test the attenuation of antibody-attenuated hIL-2 fusion proteins

[0215] The attenuated hIL-2 activity of the antibody-attenuated hIL-2 fusion protein was also tested in the hIL-2-dependent cell proliferation assay. 10,000 NK-92 cells (expressing high affinity receptor hIL-2Rαβγ) or TF1+IL-2Rβ cells (expressing medium affinity receptor hIL-2Rβγ) suspended in 50 μL of fresh growth medium without hIL-2 cytokine were plated in each well of a 96-well U-bottom cell culture plate. The antibody-attenuated hIL-2 fusion protein with an eight-point, 6-fold continuous titration at a maximum concentration of 996nM was diluted in fresh culture medium and overlaid on the cells in the wells. The cells were incubated at 37°C in a 5% CO2 incubator, TF1+IL-2Rβ cells were incubated for 3 days, or NK-92 cells were incubated for 4 days. To measure proliferation, Cell-Titer-Glo (Promega) was added to the wells, incubated for 10 min at room temperature, and the luminescence of each well was read for 0.1 sec using a VictorX multilabel plate reader (PerkinElmer). EC was calculated from relative luminescence units (RLU) across titrated concentrations using GraphPad Prism 7 software. 50 By adding the EC 50 The value was divided by the EC value of hIL-2 50 To calculate the fold change in rhIL-2 activity. 50 Values ​​were normalized for each plate.

[0216] Example 1: Optimization of antibody-attenuated hIL-2 fusion protein variants and determination of their hIL-2 activity on intermediate and high affinity hIL-2 receptor complexes

[0217] To determine the optimal structure of the antibody-attenuated hIL-2 fusion protein, unattenuated hIL-2 was fused to an anti-DNase I antibody (clone 1H3) designated 1H3-hlgG1 (SEQ ID NO:379, SEQ ID NO:374) in the antibody variable region in various ways as shown in FIG1 . Variants include hIL-2 fused to the N-terminus of the human anti-DNase I antibody (clone 1H3) immunoglobulin hIgG1 heavy chain or human kappa light chain via a direct fusion (df) denoted as hIL-2 N-terminal light chain df (SEQ ID NO:379, SEQ ID NO:356), 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) denoted as 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). Variants were also generated in which the hIL-2 portion was fused to the C-terminus of both the heavy and light chains via df or L6 and are denoted 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 NO: 374), hIL-2 C-terminal light chain df (SEQ ID NO: 379, SEQ ID NO: 362), hIL-2 C-terminal light chain L6 fusion (SEQ ID NO: 379, SEQ ID NO: 363). Additional variants were generated in which the CD25 / IL-2Rα extracellular domain (amino acids 1-164) (SEQ ID NO: 126) was fused to the N-terminus or C-terminus of the heavy chain or kappa light chain to interfere with the binding of IL-2 to the IL-2 receptor CD25 (Figure 2).In these constructs, the human CD25 extracellular domain (amino acids 1-164) (SEQ ID NO: 126) was fused to human IL-2 via a 20 amino acid linker (L20) (SEQ ID NO: 364) and then fused to the 1H3-hIgG1 heavy or light chain at the N-terminus either directly or 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: 368). Finally, a final set of variants were generated in which the CD25 / IL-2Rα extracellular domain portion (SEQ ID NO: 126) was fused to the C-terminus of the heavy and kappa light chains: 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), 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 protein A purified using standard techniques. The above 16 N-terminal or C-terminal and linker 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.

[0218] Table 1 summarizes the EC values ​​calculated from an 8-point, 6-fold continuous titration curve using the geometric mean fluorescence intensity (gMFI) calculated by FlowJo version 10 software. 50 The fold change relative to rhIL-2 was also calculated for each variant as a measure of the attenuation level compared to the activity of the rhIL-2 positive control. 50 Values ​​could not be calculated by GraphPad Prism 7 software and are marked as not calculated (NC); however, based on the dose titration curves, there was no attenuation for these variants.

[0219] Fusion of the hIL-2 portion to the N-terminus or C-terminus of the immunoglobulin heavy chain did not result in reduced IL-2 activity compared to rhIL-2 on cell lines expressing a high affinity hIL-2 receptor (NK-92) or an intermediate affinity hIL-2 receptor (TF1+IL-2Rβ). Direct fusion of hIL-2 to the antibody component of the fusion protein (df) did not result in a change 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 to the heavy or light chain of the antibody component did not result in a change 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 portion 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 showed strong attenuation of hIL-2 activity towards the high affinity IL-2 receptor (NK-92) (at least 45-fold attenuation) and 18-fold attenuation towards the intermediate affinity hIL-2 receptor (TF1+IL-2Rβ).

[0220]

[0221]

[0222] Example 2: Generation of antibody-attenuated hIL-2 fusion protein variants and determination of their binding kinetics to recombinant human CD25 and / or human CD122

[0223] Since the hIL-2 activity in various N-terminal or C-terminal immunoglobulin heavy chain fusion proteins was not reduced, the hIL-2 C-terminal heavy chain L6 fusion protein (SEQ ID NO: 361, 374) designated as "1H3-hIgG1-L6-hIL-2" was used as the basic construct of the hIL-2 fusion protein variant with substitutions in the hIL-2 portion. In order to study 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, double 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 portion were generated and evaluated for 6 rounds. These variants were first screened using a flow-based phosphorylated STAT5 (pSTAT5) assay at fixed concentrations on IL-2-dependent cell lines (NK-92 and TF1+IL-2Rβ) and in dose titration curves. Phosphorylated STAT5 is a downstream signal of IL-2 activity and is used as a snapshot measure of IL-2 potency. An IL-2-dependent cell proliferation assay was also performed to measure IL-2 activity over a 3-4 day period. Selection criteria for attenuated hIL-2 included: (1) reduced IL-2 potency in both the NK-92 cell line and the TF1+IL-2Rβ cell line, with agonist activity greater than 50% for both cell lines; and (2) moderate to high yield.

[0224] Human anti-DNase I antibody-hIL-2 fusion proteins were generated by fusing human IL-2 or human IL-2 variants (SEQ ID NOs: 1-344, 377, 378, and 575) to the C-terminus of the heavy chain of human anti-DNase I antibody (clone 1H3, with human IgG1 isotype) via an L6 linker, which was combined with a hIgG1 light chain (1H3-hkappa LC; SEQ ID NO: 374) to generate 1H3-hIgG1-L6-hIL-2 fusion protein (provided in Table 28). Mouse anti-yellow fever virus antibody-hIL-2 fusion proteins were also generated by fusing human IL-2 variants to the C-terminus of the heavy chain of mouse anti-yellow fever virus antibody (clone 2D12, with mouse IgG1 isotype) with a D265A substitution to replace reduced immune effector function via an L6 linker, which was combined with a 2D12-mIgG1 light chain (2D12-mKappa LC; SEQ ID NO: 376) to generate 2D12-mIgG1-D265A-L6-hIL-2 fusion proteins (provided in Table 28). Some of these mouse anti-yellow fever virus antibody-hIL-2 fusion proteins were formatted to human IgG1 constant regions and generated 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 were repeated for six rounds, designated Groups 1 to 6. 1H3-hlgG1-L6-hIL-2, 2D12-mIgG1-D265A-L6-hIL-2, and 2D12-hlgG1-L6-hIL-2 fusion proteins were produced, expressed, and protein A purified using standard techniques.

[0225] Group 1 contained only the initial series of 2D12-mIgG1-D265A-L6-hIL-2 or 2D12-hIgG1-L6-hIL-2 fusion proteins containing substitutions or combinations of substitutions in human IL-2 that were predicted to involve binding to only one of the IL-2 receptor subunits CD25 / IL-2Rα, CD122 / IL-2Rβ, or CD132 / IL-2Rγ. The fusion proteins in this group include the following IL-2 substitutions predicted to modulate 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: 16), (F44K / Y45R) (SEQ ID NO: 17). 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 ID NO:30).Substitutions within this group include the following substitutions predicted to modulate 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), (E15A / N88A) (SEQ ID NO:45), (E15A / N88A) (SEQ ID NO:46). NO:44) or (S87A / N88A) (SEQ ID NO:45). Group 1 also includes the following IL-2 substitutions predicted to modulate IL-2 binding 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 predicted to modulate binding to more than one IL-2 receptor subunit.

[0226] Group 2 contained a series of 1H3-hlgG1-L6-hIL-2 fusion proteins that contained one or more substitutions in human IL-2 predicted to be involved only in CD25 / IL-2Rα binding. The fusion proteins in this group include the following IL-2 substitutions predicted to modulate 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:62), E61K (SEQ ID NO:63), 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, e.g., International Publication No. WO2012 / 107417), and the substitution C125A is introduced into the IL-2 amino acid sequence to remove the unpaired cysteine ​​residue (see, e.g., International Publication No. WO2018 / 184964).The IL-2 substitutions studied in Group 2 are not expected to modulate IL-2 binding to CD132 / IL-2-Rγ, nor are these substitutions expected to modulate binding to more than one IL-2 receptor subunit.

[0227] Group 3 contained a series of 1H3-hlgG1-L6-hIL-2 fusion proteins containing one or more substitutions in human IL-2 predicted to be involved only in CD122 / IL-2Rβ binding.The fusion proteins in this group include the following IL-2 substitutions predicted to modulate 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), D84R (SEQ ID NO:99), 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).

[0228] Group 4 contained a series of fusion proteins containing 1H3-hIgG1-L6-hIL-2HC fused to a portion of the extracellular domain 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 residue substitutions predicted to be involved in binding to CD122 / IL-2Rβ. The fusion proteins in this group include the following IL-2 substitutions predicted to modulate 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:107), I92A (SEQ ID NO:108), NO: 108), E95A (SEQ ID NO: 115) or E95K (SEQ ID NO: 117). The antibody-attenuated hIL-2 fusion protein in this group is denoted as 1H3-hIgG1-L6-hCD25(1-164)-L20-hIL-2.

[0229] Group 5 contains a series of 1H3-hIgG1-L6-hIL-2, which contains substitution combinations in IL-2 that are expected to be involved in IL-2 binding to CD25 / IL-2Rα and CD122 / IL-2Rβ or CD132 / IL-2Rγ. In addition, some variants are deleted in the first three amino acids at the N-terminus of the hIL-2 portion (Δ1-3APT). The fusion proteins in Group 5 include the following IL-2 substitutions predicted to modulate IL-2 binding 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), (R38I / D20A) (SEQ ID NO: 137). 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) (SEQIDNO:156)、(L72R / D20A)(SEQ ID 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)(SEQ ID 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 IDNO: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 IDNO: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)、

[0230] (D20A / E95A / F42R)(SEQ ID NO:187)、(D20A / E95A / F42A)(SEQ ID NO:188)、(D20A / E95A / F42D)(SEQ ID NO:189)、(D20A / E95A / F42H)(SEQ ID NO:190)、(D20A / E95A / F42K)(SEQ ID NO:191)、(D20A / E95A / K43A)(SEQ ID NO:192)、(D20A / E95A / K43E)(SEQ IDNO: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 IDNO: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 IDNO: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 IDNO:216)、(T3A / D20A / R38E / C125A)(SEQ IDNO: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 predicted to modulate IL-2 binding 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). Group 5 antibody-attenuated hIL-2 fusion proteins are listed in SEQ ID NOs: 127-229 and 575. The numbering of the mutations in the hIL-2 sequence is based on the IL-2 sequence.

[0231] Group 6 contained a series of 1H3-hIgG1-L6-hIL-2 fusion proteins that included combinations of substitutions in human IL-2 predicted to be involved in 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 in IL-2 that are predicted to modulate 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), (D20A / E61F) (SEQ ID NO: 240). 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 IDNO:260)、(D20A / Y45N)(SEQ ID 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)(SEQ ID 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 IDNO: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)(SEQ ID 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 IDNO:291)、(I92D / Y45D)(SEQ ID NO:292)、(I92D / Y45Q)(SEQ ID NO:293)、(I92D / Y45E)(SEQID NO:294)、(I92D / Y45G)(SEQ ID NO:295)、(I92D / Y45H)(SEQ ID NO:296)、(I92D / Y45I)(SEQ IDNO:297)、(I92D / Y45L)(SEQ ID NO:298)、(I92D / Y45M)(SEQ ID NO:299)、(I92D / Y45F)(SEQ ID NO:300)、(I92D / Y45P)(SEQ ID 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 IDNO:308)、(F42A / N88D)(SEQ ID NO:309)、(R38E / D84A)(SEQ ID NO:310)、(R38E / D84N)(SEQID 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)(SEQ ID 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)(SEQ IDNO:325)、(R38E / I92A)(SEQ ID NO:326)、(R38E / I92R)(SEQ ID NO:327)、(R38E / I92N)(SEQID 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 IDNO: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). The numbering of the Group 6 antibody-attenuated hIL-2 fusion proteins is listed in SEQ ID NOs:230-344 according to the mutations in the hIL-2 sequence of the IL-2 sequence.

[0232] The binding kinetics of some purified 1H3-hIgG1-L6-hIL-2 variant proteins of single recombinant human CD25 and human CD122 were determined using biolayer interferometry (BLI). In brief, Octet Red96 instrument (Pall ForteBio) was used to carry out binding experiments at 25°C. Human CD25 and human CD122 extracellular domains tagged with polyhistidine at the C-terminal end were captured on anti-His2 sensors (Pall ForteBio). Starting from the highest concentration of 300nM, the sensor loaded with receptor was immersed in 7 points of continuous 3-fold dilutions of each 1H3-hIgG-L6-hIL-2 variant. 1H3-hIgG1-L6-hIL-2 fusion protein was diluted into a determination buffer consisting of phosphate buffered saline (PBMS) supplemented with 0.1% BSA, 0.02% Tween-20 (pH 7.2). The loaded sensor was regenerated using 10 mM glycine buffer (pH 1.7).The kinetic constants were calculated using a monovalent binding model.

[0233] Table 2 records the association constants (k) of 74 immunoglobulin-hIL-2 fusion protein variants binding to recombinant human CD25 or recombinant human CD122. on ), dissociation constant (k off ) and equilibrium constant (K D ).

[0234] Table 2. Binding kinetics of 1H3-hIgG-L6-hIL-2 fusion protein to recombinant human CD25 or CD122 obtained by Octet BLI

[0235]

[0236]

[0237]

[0238] Table 3 records the association constants (k) of 74 1H3-hIgG1-L6-hIL-2 fusion proteins binding to recombinant human CD122. on ), dissociation constant (k off ) and equilibrium constant (K D ).

[0239]

[0240]

[0241]

[0242]

[0243] Example 3: Testing the Attenuation of High and Intermediate Affinity hIL-2 Receptors Using a Fixed Concentration Cell-Based Potency pSTAT5 Screen

[0244] As described in Protocol D, the attenuation of the antibody-attenuated hIL-2 fusion proteins described in Example 2 was tested in a fixed concentration pSTAT5 screen using the NK-92 (expressing a high affinity hIL-2 receptor) and TF1+IL-2Rβ (expressing a 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 proteins from free cytokine wild-type rhIL-2, a measure of the reduction in IL-2 activity. For fixed concentration screens, the fold change was calculated by dividing the gMFI of rhIL-2 by the gMFI of the variant. For experiments with a complete titration curve, the EC values ​​of rhIL-2 were calculated by dividing the EC values ​​of the variant by the EC values ​​of the variant. 50 The value is divided by the EC of the variant 50 The fold change relative to rhIL-2 was calculated. The fold change was rounded to the nearest integer. The gMFI reduction in both the NK-92 cell line and the TF1+IL-2Rβ cell line compared to the gMFI produced by rhIL-2 indicated attenuation of IL-2 activity of high-affinity and medium-affinity receptors. The first set of variants described in Example 2 was not tested in the potency pSTAT5 screen based on fixed concentration cells.

[0245] The IL-2 agonistic activity of each variant tested was also evaluated and characterized as a full or partial IL-2 agonist, or as having no IL-2 activity (inactive). 1H3-hIgG1-L6-hIL-2 fusion protein dose titration curves that reached the maximum gMFI level exhibited by the rhIL-2 positive control were considered to be antibody-attenuated hIL-2 fusion proteins with full agonist activity. The percentage of partial agonist activity to full activity was calculated using the rhIL-2 maximum gMFI as 100%. Antibody-attenuated hIL-2 fusion proteins with less than 10% of the rhIL-2 maximum gMFI at the highest concentration of 1200nM were considered to have no agonist activity (inactive). Some EC values ​​could not be accurately calculated using GraphPad Prism 7 software. 50 values ​​and decay levels because the activity did not reach its maximum value and therefore these values ​​are estimates.

[0246] The pSTAT5 fixed concentration results demonstrated that while some single residue substitutions attenuated IL-2 activity on a high affinity cell line (NK-92), combinations of substitutions that modulated the binding of both the α and β chains or both the α and γ chains were required to significantly attenuate IL-2 activity on the high affinity IL-2 receptor (more than 20-fold attenuation for recombinant hIL-2).

[0247] Table 4. Fold changes relative to rhIL-2 in fixed concentration pSTAT5 screen on 1H3-hlgG1-L6-hIL-2 fusion protein from Group 2

[0248]

[0249]

[0250] NT-1 = First tested in a full titration of pSTAT5, no data for fixed concentration assays.

[0251] Table 5. Fold changes relative to rhIL-2 in a fixed concentration pSTAT5 screen on 1H3-hlgG1-L6-hIL-2 fusion protein from Group 3

[0252]

[0253]

[0254]

[0255] NT-1 = First tested in a full titration of pSTAT5, no data for fixed concentration assays.

[0256] Table 6. Fold changes relative to rhIL-2 in fixed concentration pSTAT5 screen on 1H3-hlgG1-L6-hIL-2 fusion protein from Group 4

[0257]

[0258]

[0259] Table 7. Fold changes relative to rhIL-2 in fixed concentration pSTAT5 screen on 1H3-hlgG1-L6-hIL-2 fusion protein from Group 5

[0260]

[0261]

[0262]

[0263]

[0264] Table 8. Fold changes relative to rhIL-2 in fixed concentration pSTAT5 screen on 1H3-hlgG1-L6-hIL-2 fusion protein from Group 6

[0265]

[0266]

[0267]

[0268]

[0269] Example 4: Testing the Attenuation of IL-2 Fusion Proteins for Each of the High and Intermediate Affinity hIL-2 Receptors Using a Cell-Based Potency pSTAT5 Dose Titration Screen

[0270] Selected antibody-attenuated hIL-2 fusion proteins described in Example 2 (1H3-hIgG1-L6-hIL-2 fusion proteins from Groups 2-6) were tested for attenuation in a pSTAT5 titration curve using the NK-92 and TF1+IL-2Rβ cell lines as described in Protocol D.

[0271] A four-parameter logistic curve was generated using the gMFI of Alexa Fluor 647pSTAT5-positive signals, and the EC was then calculated using GraphPad Prism 7 software. 50These values ​​were compared to a recombinant hIL-2 (rhIL-2) control as a measure of attenuation. Tables 9-13 summarize the fold change in rhIL-2 activity calculated using the gMFI of the Alexa Fluor 647 signal.

[0272] The increase of the change multiple of rhIL-2 indicates the attenuation degree of hIL-2 activity. The agonistic activity of the hIL-2 fusion protein of each antibody-attenuation tested in the pSTAT5 titration curve was also assessed, and was characterized as complete, partial or inactive (inactive). The hIL-2 fusion protein dose titration curve of the antibody-attenuation that reaches the maximum gMFI level like rhIL-2 is considered to be a variant with complete agonist activity. Calculate partial agonist activity as described in Example 3. Compared with rhIL-2, the hIL-2 fusion protein of the inactive antibody-attenuation is classified as active less than 10%. Using GraphPad Prism 7 software, it is impossible to accurately calculate some change multiples (expressed as not calculated or " NC ") relative to rhIL-2, because a complete four-parameter logistic curve is not produced, and therefore these values ​​are estimated values ​​(marked as in Table 9-13). a ). However, on the graph, these variants were attenuated by more than 10,000-fold relative to rhIL-2 (data not shown). This is indicated in Tables 9-13 as "on graph>10,000; NC".

[0273] The fully titrated pSTAT5 curves demonstrated results similar to those presented in Example 3, where substitutions that modulated binding to both the α and β chains significantly attenuated IL-2 activity on the high affinity IL-2 receptor compared to single substitutions that bound only the α or β chains. In addition, the fully titrated pSTAT5 assay was also able to distinguish variants with substitutions that resulted in inactivation from highly attenuated variants. Finally, comparison of the dose titration curves demonstrated more precise attenuation levels than fixed concentration assays.

[0274] Table 9. Fold change relative to rhIL-2 and agonistic activity on 1H3-hIgG1-L6-hIL-2 fusion protein from Group 2

[0275]

[0276]

[0277] NT = Not Tested

[0278] a = Fold changes are only estimates, as a full four-parameter logistic curve was not achieved

[0279] Table 10. Fold change relative to rhIL-2 and agonistic activity on 1H3-hIgG1-L6-hIL-2 fusion protein variants from Group 3

[0280]

[0281]

[0282]

[0283] NT = not tested; NC = not calculated by GraphPad Prism 7

[0284] a = Fold changes are only estimates, as a full four-parameter logistic curve was not achieved

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] Example 5: Testing the Attenuation of High and Medium Affinity hIL-2 Receptors Using Cell-Based Proliferation Assays

[0292] Antibody-attenuated hIL-2 fusion proteins from Groups 1-6 in Example 2 (2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, and 1H3-hIgG1-L6-hIL-2 fusion proteins) were tested for attenuation of IL-2 activity in proliferation assays of both the NK-92 cell line and the TF1+IL-2Rβ cell line as described in Protocol E. The assay results are provided in Tables 14-19.

[0293] The selected 1H3-hIgG1-L6-hIL-2 fusion proteins from Example 4 with significant attenuation in the pSTAT5 titration curve were tested in this cell-based proliferation assay. pSTAT5 is a downstream readout of IL-2 activity, and the assay only requires 10 minutes of stimulation, which may be a small snapshot of IL-2 dependent activity. For proliferation assays, 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 recombinant hIL-2 controls, thereby providing a more physiologically relevant reading of IL-2 dependent activity in vivo. The IL-2 dependent activity of other 2D12-mIgG1-D265A-L6-hIL-2 and 2D12-hIgG1-L6-hIL-2 fusion proteins produced but not tested in the pSTAT5 assay was determined using this proliferation assay.

[0294] Similar to the cell-based pSTAT5 dose titration experiments, the calculated EC was determined based on relative luminescence units (RLU) rather than gMFI. 50 , and once the EC is calculated 50 , the results were analyzed as in Example 4. Similar to the results identified in Example 4, the proliferation curves demonstrated that some substitutions that modulated binding to both the α chain and the β chain significantly attenuated IL-2 activity on high affinity receptors compared to single substitutions that bound only the α chain or the β chain. These selected 1H3-hIgG1-L6-hIL-2 fusion proteins were also tested for proliferation on the TF1+IL-2Rβ cell line, and demonstrated that some of these fusion proteins significantly attenuated IL-2 activity on medium affinity receptors.

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] Example 6: Generation of anti-hPD-1 antibodies

[0310] Several approaches were used to generate a variety of anti-hPD-1 antibodies with desired properties.

[0311] In one approach, transgenic chickens (OmniChicken) expressing human antibody genes (human light chain (VLCL or VKCK) and human VH) and chicken heavy chain constant regions are used. TM ) to generate anti-hPD-1 human monoclonal antibodies (Ching et al., mAbs2018). Transgenic chickens were immunized with 100 μg of Fc-tagged human PD-1 protein (huPD-1-Fc) (SEQ ID NO: 380) every 14 days for 14 weeks. In another approach, transgenic chickens were genetically immunized six times with DNA encoding human PD-1 (SEQ ID NO: 347), followed by a final boost with 100 μg of huPD-1-Fc (SEQ ID NO: 380). The serum immune response of each animal was monitored by ELISA against biotinylated human PD-1 on streptavidin-coated plates.

[0312] Splenocytes were isolated from each immunized animal and positive antibody clones were tested using a gel-encapsulated microenvironment (GEM) assay (e.g. Mettler Izquierdo, S., Varela, S., Park, M., Collarini, E. J., Lu, D., Pramanick, S., Rucker, J., Lopalco, L., Etches, R., & Harriman, W. (2016). High-efficiency antibody discovery achieved with multiplexed microscopy Microscopy (Oxford, UK), 65(4), 341–352) and screened against human PD-1 labeled 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 to the hinge and Fc regions of immunoglobulins (ScFv-Fc). These unique scFv-Fc fusion proteins were transiently expressed in Expi293 cells and the supernatants were tested for binding activity by ELISA on plates coated with huPD-1-Fc (SEQ ID NO: 380) or cynomolgus monkey PD-1-Fc (SEQ ID NO: 381). A total of 102 unique anti-human PD-1 variable heavy and variable light pairs 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 in this method.

[0313] Additional methods led to the identification of an anti-hPD-1 antibody denoted C51E6-hIgG4, which was germline optimized to become an antibody designated C51E6-5-hIgG4 (SEQ ID NOs: 392-401, 426, 427), and humanized and further sequence optimized to become an antibody designated Abz1mod-hIgG4 (SEQ ID NOs: 449, 450).

[0314] As described in General Methods Protocol A, anti-PD-1 variable region sequences were expressed as human IgG4κ antibodies and their ability to bind to PD-1 expressing cells was assessed using flow cytometry. The antibodies to be tested were first 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 from a maximum concentration of 280 nM, and then an allophycocyanin-coupled anti-human IgG secondary antibody was added to the cells for detection. Of the 92 hits, 79 tested anti-PD-1 antibodies had an EC of 1.0 for 1.0 and 1.0 for 1.0. 50Binding (by flow cytometry) <30 nM. 2H7-hIgG4 (SEQ ID NOs: 382-391, 424 and 425), C51E6-5-hIgG4 (SEQ ID NOs: 392-401, 426 and 427), A2-hIgG4 (SEQ ID NOs: 402-411, 428 and 429), OMC.1.B6-hIgG4 (SEQ ID NOs: 438 and 439), OMC.1.D6-hIgG4 (SEQ ID NOs: 442 and 443), OMC.2.C6-hIgG4 (SEQ ID NOs: 440 and 441), 1H9-hIgG4 (SEQ ID NOs: 576 and 525), 1D5-hIgG4 (SEQ ID NOs: 577 and 527) and 2A3.H7-hIgG4 (SEQ ID NOs: 424 and 523) are among a panel of antibodies produced using a Jurkat cell line expressing human PD-1 (SEQ ID NOs: NO:346) as antibodies with medium to high affinity binding to hPD-1. For 2H7-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4, the EC binding to Jurkat cells recombinantly expressing hPD-1 was calculated by flow cytometry in multiple experiments. 50 EC binding to hPD-1 expressing Jurkat cells calculated by flow cytometry 50 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 for hPD-1, as 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 antibody titrations did not bind to the parental Jurkat cell line that does not express hPD-1 (data not shown).

[0315] 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

[0316] As described in General Methods Protocol B, 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 were evaluated for binding competition with hPD-1 in the presence of anti-hPD-1#1-mIgG2b-N297A and anti-hPD-1#2-mIgG2b-N297A.

[0317] As a control, titration was performed in the presence of a saturating concentration of 10 μM anti-hPD-1#1-mIgG2b-N297A (Nivolumab)( Figure 4A ). Compared with the one without anti-hPD-1#1-mIgG2b-N297A competitor Compared to the dose titration curves of 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4, the dose titration curves in the presence of anti-hPD-1#1-mIgG2b-N297A competitor were greatly reduced (the dose titration curves were shifted 100-fold to the right of the graph) . Addition of saturating concentrations (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 shown in Figure 2 . Figures 4B-4D This is demonstrated by a less than 10-fold shift in the PD-1 matrix, indicating that 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 did not compete for binding to PD-1 in the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A.

[0318] Example 8: Characterization of non-antagonist hPD-1 antibodies

[0319] 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 as described in General Methods Protocol C. All antibodies except A2-hIgG4 were tested at 200 nM final concentration. A2-hIgG4 was tested at 500 nM final concentration.

[0320] 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 an average luminescence level of 3000 relative luminescence units (RLU) and showed RLUs similar to 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, showed a luminescence of more than 14,000 RLU (data not shown).

[0321] Example 9: Anti-hPD-1-attenuated hIL-2 fusion protein binds to Jurkat cells expressing human PD-1

[0322] To construct various antibodies and antibody-attenuated hIL-2 fusion protein expression vectors, corresponding polynucleotides encoding antibodies, cytokines, cytokine receptors and linker sequences were generated and cloned into expression vectors. The antibodies or antibody fusion proteins were transiently expressed in human embryonic kidney (HEK) 293 cells and then purified by affinity chromatography using protein A or protein G agarose. The purified protein was concentrated and buffer-exchanged into phosphate-buffered saline or phosphate-buffered saline containing 100 mM L-arginine and 10 mM L-histidine using ultracentrifugal filtration, after which the protein concentration was determined.

[0323] In some methods, 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 carrying the S228P hinge stabilizing mutation were fused to hIL-2 directly (df) or at the C-terminus of the immunoglobulin heavy chain using an L6 linker. Figure 5The description of these anti-PD-1-attenuated hIL-2 fusion proteins is summarized. As described in Example 2, various constructs were generated with substitutions in hIL-2 that attenuated hIL-2 activity. As described in General Methods Protocol A, the hPD-1-expressing Jurkat cell line was used to test the binding of the anti-hPD-1-attenuated hIL-2 fusion proteins listed in Table 20 to 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 effector function 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) to a hIL-2 variant with attenuated hIL-2 activity (hIL-2T3A / D20A / R38E / C125A; SEQ ID NO: 217) to become H7-767 (SEQ ID NO: 412-413, 415-423, 532), and both H7-632-hIgG1-LAGA and H7-767 were tested for binding to hPD-1 (Table 20). EC was calculated using GraphPad Prism 7 software based on the geometric mean fluorescence intensity (gMFI) across titration concentrations. 50 value.

[0324] The production of anti-hPD-1-attenuated hIL-2 fusion proteins did not reduce binding to hPD-1, and the anti-hPD-1-attenuated hIL-2 fusion proteins were still able to bind to Jurkat cells expressing human PD-1. The calculated EC values ​​of the tested anti-hPD-1-attenuated hIL-2 fusion proteins compared to the corresponding anti-hPD-1 antibodies without the attenuated hIL-2 portion are summarized in Table 20. 50 .

[0325]

[0326] Compared with the anti-hPD-1 antibody without attenuated hIL-2 moiety, the anti-hPD-1-hIL-2 fusion protein has a stronger affinity for the EC of Jurkat+hPD-1 cells. 50 The binding increase was less than 2-fold, demonstrating that the addition of the attenuated hIL-2 moiety to the anti-hPD-1 antibody did not abrogate binding to human PD-1.

[0327] Example 10: Anti-hPD-1-attenuated hIL-2 fusion protein binds hPD-1 in the presence of anti-hPD-1 #1 and anti-hPD-1 #2 antibodies

[0328] As described in General Methods Protocol B and Example 7, the binding of anti-hPD-1-attenuated hIL-2 fusion proteins to the hPD-1 receptor was tested in the presence of anti-hPD-1#1 and anti-hPD-1#2. The opposite 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 saturating concentrations of the test antibody-attenuated hIL-2 fusion protein. In this format, Jurkat cells expressing hPD-1 were plated at 100,000 cells per well in FACS buffer, blocked with anti-human FcγR blocking reagent (Miltenyi Biotec) at 4°C for 10 minutes 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 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 with Jurkat cells expressing hPD-1 cells on ice for 1 hour. The cells were washed and resuspended on ice in FACS buffer containing six-fold serial titrations of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A starting at a maximum concentration of 50 nM for 1 hour. The cells were washed and resuspended on ice in a 1:100 dilution of phycoerythrin-conjugated anti-mouse IgG light chain κ monoclonal antibody for 45 minutes. The cells were washed again and resuspended in FACS buffer with a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific). Flow cytometric analysis was performed using a 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 the gMFI of the phycoerythrin signal across the titration concentrations. 50 value.

[0329] In the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A, adding attenuated hIL-2 to the anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 did not impair the ability of the anti-hPD-1 proteins to bind to human PD-1, similar to the results described in Example 7 ( Figures 16B-16D ). H7-767 was also tested in this competition assay, and Fig. 13B H7-767 continued to bind to the hPD-1 receptor in the presence of anti-hPD-1-#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A. In contrast, binding of the positive control anti-hPD-1#1 was significantly reduced in the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A ( Fig.16A , 13A).

[0330] Fig. 6A and Figure 6B The results show that for the reverse competition assay, anti-hPD-1#1-mIgG2b-N297A ( Fig. 6A ) and anti-hPD-1#2-mIgG2b-N297A( Figure 6B ) were still able to bind to hPD-1 on Jurkat cells. Prior to exposure to anti-hPD-1 fusion proteins, the binding curves of these hIL-2 fusion proteins with saturated anti-hPD-1-attenuated proteins overlapped with the binding curves of anti-hPD-1#1-mIgG2b-N297A (no competition) or anti-hPD-1#2-mIgG2b-N297A (no competition). The binding curves also overlapped with the saturated negative control fusion protein 1H3-hIgG4-df-hIL-2 (D20A / R38E) that did not bind to hPD-1.

[0331] Example 11: Anti-hPD-1-attenuated hIL-2 fusion protein binds to recombinantly expressed cynomolgus monkey PD-1

[0332] The binding of hIL-2 fusion protein of anti-hPD-1-attenuation to cynomolgus monkey PD-1 was tested in flow cytometry using a human embryonic kidney 293 cell line expressing SV40 large T cell antigen (HEK-293T), which was transiently transfected to recombinantly express cynomolgus monkey PD-1. For each transfection reaction, 2 million HEK-293T cells were transfected with 2 μg of pCMV6-hygro-HA-cyno-PD-1 (1-185) (SEQ ID NO: 448) (which is a mammalian vector containing a sequence encoding hygromycin resistance and a cynomolgus monkey PD-1 extracellular domain labeled with human influenza hemagglutinin). Transfection was performed by electroporation. Transfected cells were blocked with human FcγR blocking agents and stained with titrated anti-hPD-1-attenuated hIL-2 fusion proteins. In addition, phycoerythrin-coupled anti-hemagglutinin clone 15B12 was added to the cells to stain the transfected cells, and allophycocyanin-coupled anti-human IgG Fc secondary clone HP6017 (BioLegend, catalog number 409306) was added to the cells to stain the bound antibodies. Cells were analyzed on a BD Canto II, and FlowJo software version 10 was used to gate live, transfected (hemagglutinin-positive) cells and calculate the gMFI of the allophycocyanin signal. EC was calculated based on the gMFI across titration concentrations using GraphPad Prism 7 software. 50 value.

[0333] The anti-hPD-1-attenuated hIL-2 fusion protein bound to HEK-293T cells expressing cynomolgus monkey PD-1 in a pattern similar to that seen on Jurkat T cells expressing human PD-1 ( Fig.17 ). EC binding to HEK-293T cells expressing cynomolgus monkey PD-1 50 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 comparator anti-hPD-1-attenuated hIL-2 fusion protein) were also tested with EC values ​​of 9 nM and 2 nM, respectively. 50 values ​​for binding to cynomolgus monkey PD-1, indicating that the addition of the attenuated hIL-2 moiety to the anti-hPD-1 antibody did not abolish the binding to cynomolgus monkey PD-1.

[0334] Example 12: Anti-hPD-1-attenuated hIL-2 fusion protein binds activated primary human and cynomolgus monkey PD-1

[0335] The binding of anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins on activated primary T cells expressing hPD-1 was examined by flow cytometry. To test whether 2H7-hIgG4, C51E6-5-hIgG4 or A2-hIgG4 bind 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 ionomycin to upregulate the hPD-1 receptor. Activated PBMCs were collected, blocked with a 1:50 dilution of human FcγR blocking reagent (Miltenyi Biotec) at 4°C for 10 minutes, and stained with titrated concentrations of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, anti-hPD-1#1, and isotype controls. The cells were then stained with allophycocyanin-conjugated anti-human IgG Fc at a dilution of 1:20 to detect bound antibodies. To delineate immune subsets, a surface marker cocktail including anti-human CD3, anti-CD4, and anti-CD8 antibodies was used. In addition, sample portions were examined for cellular expression of hPD-1, hCD25, hCD122, and hCD132. Cells were analyzed on a BD Fortessa (BD Biosciences) using FlowJo software version 10 to gate T cell subsets and then calculate the gMFI of the allophycocyanin signal. EC was calculated based on the gMFI across titration concentrations using GraphPadPrism 7 software. 50 To test the binding of anti-hPD-1-hIL-2 fusion protein, cryopreserved CD3+ T cells were activated with PMA / ionomycin and flow cytometry binding was performed identically as above.

[0336] Flow cytometry was also used to test the binding of human PD-1 antibody-attenuated hIL-2 fusion protein to activated cynomolgus monkey T cells. Cynomolgus monkey PBMCs were activated with a mixture of 0.081 μM PMA and 1.34 μM ionomycin. After 24 hours, cells were stained using the same procedure as described above for binding human PD-1 primary cells, except that a cynomolgus monkey cross-reactive marker was used. FlowJo software version 10 was used to gate live CD3 + CD4 + or CD3 + CD8 + EC was calculated from the gMFI of anti-hPD-1 antibody or hPD-1 antibody-attenuated hIL-2 fusion protein across titrated concentrations using GraphPad Prism 7 software. 50 value.

[0337] In some of the variants tested, the attenuated hIL-2 also included the substitutions T3A and C125A, which remove the site of O-linked glycosylation and replace a free cysteine ​​residue, respectively.

[0338] After activation with PMA and ionomycin, 40-50% of CD4 + T cells are PD-1 + , while 30-40% of CD8 + T cells are PD-1 + (Data not shown). Calculated by flow cytometry with activated human CD3 + CD4 + T cell-bound EC 50 0.1-0.7 nM for 2H7-hIgG4, 12 nM for C51E6-5-hIgG4, 30 nM for A2-hIgG4, and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A / D20A / R38E / C125A). + CD8+ T cell-bound EC 50 For 2H7-hIgG4, it was 0.1-0.8 nM, for C51E6-5-hIgG4, it was 16 nM, for A2-hIgG4, it was 22 nM, and for 2H7-hIgG4-df-hIL-2, it was 0.03 nM (T3A / D20A / R38E / C125A). For H7-767, it was inversely proportional to activated human CD3 + CD4 + T cell-bound EC 50 0.19 nM, and activated human CD3 + CD8 + T cells were 0.12 nM. + CD4+ T cell-bound EC 50 0.09 nM for 2H7-hIgG4 and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A / D20A / R38E / C125A). + CD8+ T cell-bound 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, with activated cynomolgus monkey CD3 + CD4 + T cell-bound EC 50 0.26nM, and activated cynomolgus monkey CD3 +CD8 + The data demonstrate that when hPD-1 antibodies are switched to anti-hPD-1-attenuated hIL-2 fusion proteins, the calculated EC binding to activated hPD-1 is 50 The value is still consistent with the calculated EC of hPD-1 naked antibody binding to hPD-1 50 The values ​​are similar.

[0339] Testing of H7-767 and H7-632-hIgG1-LAGA naked anti-PD-1 antibodies against primary non-activated human CD4 + and CD8 + Combination of frozen human CD3 + T cells were thawed and flow cytometry was performed as described above. Neither H7-767 nor H7-632-hIgG1-LAGA naked anti-PD-1 antibodies bind to unactivated human CD4 + and CD8 + T cells (data not shown).

[0340] Example 13: Quantification of Binding of Anti-hPD-1 Antibodies and Anti-hPD-1-Attenuated hIL-2 Fusion Proteins to Recombinant Human or Cynomolgus Monkey PD-1 by Surface Plasmon Resonance (SPR)

[0341] Using high-throughput SPR LSA TM Surface plasmon resonance binding analysis was performed to determine the binding affinity of anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins. 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 HC30M (Carterra Bio) chips using sulfo-N-hydroxysuccinimide / 1-ethyl-3-(3-dimethylamino)propylcarbodiimide (sulfo-NHS / EDC) coupling chemistry and blocked with ethanolamine. The non-regenerative kinetic coupling process was used to determine the binding kinetics with commercially derived recombinant His-tagged human PD-1 and His-tagged cynomolgus monkey PD-1 (Acro Biosystems).

[0342] Anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins were expressed with modified human IgG1 or modified IgG4 isotypes with a kappa light chain framework. Other substitutions L235E or L235A / G237A (LAGA, as described in International Publication WO1998 / 006248) (numbering based on the EU numbering system) were introduced into the Fc region to eliminate the effector function of the immunoglobulin component.

[0343] Binding constants (k) of various anti-hPD-1 antibodies and anti-hPD-1 antibody-attenuated hIL-2 fusion proteins binding to recombinant human or cynomolgus monkey PD-1 protein a ), dissociation constant (k d ) and equilibrium constant (K D ) was determined from the titration curve and Carterra kinetics software. The maximum feasible SPR signal generated (R max ) and residual standard deviation (Res SD). Table 21 summarizes the results of the kinetic screening and demonstrates that the addition of the attenuated hIL-2 moiety to the anti-hPD-1 antibody does not modulate the binding of the PD-1 antibody to either human PD-1 or cynomolgus monkey PD-1 antigen. In a separate experiment, H7-632-hIgG1-LAGA (SEQ ID NOs: 414 and 415) was measured by SPR and its steady-state equilibrium dissociation constant (K D ) is 1.23×10 -9 M, while H7-767's K D =1.93×10 -9 M.

[0344]

[0345]

[0346] Example 14: Determination by surface plasmon resonance (SPR) whether anti-hPD-1 antibodies and anti-hPD-1-attenuated hIL-2 fusion proteins compete with anti-hPD-1#1 and anti-hPD-1#2 for binding to PD-1

[0347] The competition between anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins was determined using a sandwich method. The antibodies and corresponding antibody-IL-2 cytokine fusion proteins were fixed to the HC30M chip using the amine coupling chemistry described in Example 13. After the kinetic analysis described in Example 13, 80nM human PD-1 (Bipsix, catalog number PD-1-H5221-100 ug) 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 then injected into the array, and the binding parameters were evaluated using SPR. The evaluation of all anti-hPD-1 and anti-hPD-1-hIL-2 fusion proteins was repeated. Some variants tested have modified human IgG1 or IgG4 light chain frameworks with additional L235E or L235A / G237A (LAGA) substitutions to eliminate the effector function of immunoglobulins.

[0348] Screening of pairs of anti-hPD-1 or anti-hPD-1-attenuated hIL-2 fusion proteins allowed the identification of two bins, as shown in Table 22. The antibodies and fusion proteins of Group 1 were able to bind hPD-1 in the presence of all antibodies and fusion proteins of Group 2, but competed with all members of the same group. The antibodies and fusion proteins of Group 2 were able to bind hPD-1 in the presence of all antibodies and fusion proteins of Group 1, but competed with all members of the same group. None of the anti-hPD-1 listed in Group 1 of Table 22 was able to bind hPD-1 in the presence of all antibodies and fusion proteins of Group 1, but competed with all members of the same group. and compete.

[0349] Table 22. Binning screen of groups 1 and 2 from anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins by SPR

[0350]

[0351]

[0352] *Commercial origin, no sequence available

[0353] Example 15: Antagonism of hPD-1 by anti-hPD-1-attenuated hIL-2 fusion protein in the presence of anti-hPD-1#1 and anti-hPD-1#2

[0354] The anti-hPD-1-attenuated hIL-2 fusion proteins were tested for their antagonistic effect on hPD-1. The anti-hPD-1-attenuated hIL-2 fusion proteins were characterized according to General Methods Protocol C. Figure 7 These results were shown when combined with PD-1 antagonists or When compared, 2H7-hIgG4-df-hIL-2 (D20A / R38E), C51E6-5-hIgG4-L6-hIL-2 (D20A / R38E) and A2-hIgG4-df-hIL-2 (D20A / R38E) were non-antagonists to human PD-1, as evidenced by low levels of detectable luminescence. H7-632-hIgG1-LAGA and H7-767 were also tested for antagonist activity as described in General Protocol C. Fig.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.

[0355] For the competition 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 the CHO cells. Forty (40) μL of Jurkat PD-1 effector cells were overlaid on the mixture of CHO cells and anti-hPD-1-attenuated hIL-2 fusion protein. In this competition assay, a saturating final concentration of 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. Fig.18A and Fig.18B It was demonstrated that the addition of 100 nM anti-hPD-1-attenuated hIL-2 fusion protein did not compete with the blocking titration of anti-hPD-1#1 binding to hPD-L1 (SEQ ID NO: 584). The dose titration curve of anti-hPD-1#1 remained unchanged compared to the curve without competing antibody, indicating that the presence of anti-hPD-1-attenuated hIL-2 fusion protein did not compete with the function of anti-hPD-1#1 even at high concentrations. In the presence of 100 nM 2H7-hIgG4-df-hIL-2 (D20A / R38E) and 100 nM C51E6-5-hIgG4-L6-hIL-2 (D20A / R38E), at higher concentrations of anti-hPD-1#2, anti-hPD-1#2 exhibited a 35% decrease in luminescence (RLU) ( Fig.18B ), but due to the magnitude of the standard deviation it is unclear whether this reduction is significant.

[0356] 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. Anti-hPD-1-attenuated hIL-2 fusion protein was titrated continuously and 40 μL of the mixture was added to CHO cells and then overlaid with 40 μL of jur kat PD-1 effector cells. The rest of the assay was performed as described in General Protocol C. Fig. 18C and Fig.18D It was shown that adding 100nM anti-hPD-1#1 ( Fig. 18C ) or 100 nM anti-hPD-1#2 ( Fig.18D) does not impair the ability of the anti-hPD-1-attenuated hIL-2 fusion protein to act as an antagonist. The flat curve observed above 18,000 relative luminescence units (RLU) indicates that there is no competition for antagonistic activity, and the tested anti-hPD-1-attenuated hIL-2 fusion protein can still show antagonistic function even in the presence of anti-hPD-1#1 or anti-hPD-1#2.

[0357] Example 16: Testing the attenuation of anti-hPD-1-attenuated hIL-2 fusion proteins at high and intermediate affinity hIL-2 receptors using a cell-based proliferation assay

[0358] As described in General Protocol E, the level of attenuation of hIL-2 activity of anti-hPD-1-attenuated hIL-2 fusion proteins was assessed using cell proliferation assays on NK-92 and TF1+IL-2Rβ cell lines. Control fusion proteins included fusion proteins incorporating an anti-DNase I antibody (designated 1H3) having a human IgG4 or human IgG1 backbone fused directly to hIL-2 or having a linker (SEQ ID NO: 355) to demonstrate the effect of non-targeted attenuated hIL-2 fusion proteins. As described in Example 2, the hIL-2 sequences of these constructs contained substitutions for attenuated hIL-2 activity. Similar to Example 3, complete, partial or no agonistic IL-2 activity (inactivity) was also assessed. Some of the tested variants were expressed on modified human IgG1 or IgG4 isotypes with a kappa light chain, with additional L235E or L235A / G237A (LAGA) substitutions in the Fc region to eliminate immunoglobulin effector functions. In some antibody-cytokine fusion proteins, the hIL-2 cytokine is fused to the C-terminus of the light chain (LC fusion).

[0359] Calculated EC for each antibody-cytokine fusion protein 50 The relative luminescence units (RLU) were used to determine the EC value when compared to recombinant human IL-2 (rhIL-2). 50 Table 23 summarizes the fold change and agonist activity relative to rhIL-2. The maximum luminescence of the antibody-attenuated hIL-2 fusion protein compared to the maximum luminescence of rhIL-2 was used to determine whether the agonist activity was complete, partial, or inactive. Antibody-attenuated hIL-2 fusion protein dose titration curves that reached maximum luminescence like rhIL-2 were considered to be fully active variants. The percentage of partial activity to full activity was calculated using the rhIL-2 maximum luminescence as 100%. Antibody-attenuated hIL-2 fusion proteins with a maximum RLU of less than 10% of the maximum RLU of rhIL-2 at the highest concentration of 1200 nM were considered to have no agonist activity or to be inactive. As shown in Table 23a As shown, the EC of some variants was estimated only because the maximum luminescence was not reached. 50 value.

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Example 17: Rescue of IL-2 activity of hPD-1-attenuated hIL-2 fusion protein on a cell line expressing the intermediate affinity hIL-2 receptor and hPD-1

[0367] The rescue of hIL-2 activity by anti-hPD-1-attenuated hIL-2 fusion proteins was evaluated using a targeted cell line expressing hPD-1. In short, the TF1+IL-2Rβ cell line described in General Methods Scheme D was modified to express the hPD-1 receptor (SEQ ID NO: 580) by lentiviral transduction. Flow cytometry using hPD-1 antibodies (BD Biosciences, catalog number 565936) combined with Brilliant Blue 515 was used to detect TF1+IL-2Rβ cells expressing hPD-1. Low hPD-1 expression of sorted cells (intensity below 103 on Brilliant Blue 515 fluorophores). The collection was sorted twice more to collect cells close to the expression level of hPD-1 on activated primary cells. The cell line (TF1+IL-2Rβ+hPD-1) was amplified and frozen in equal parts for cell-based proliferation assays. Proliferation assays were performed as described in General Methods Scheme E with an incubation period of 3 days. Some of the variants tested had modified human IgG1 or IgG4 light chain frameworks with additional L235E or L235A / G237A (LAGA) substitutions to eliminate the effector functions of the immunoglobulin.

[0368] Table 24 summarizes the results of the proliferation assay targeting the TF1+IL-2Rβ+hPD-1 cell line. Whether the agonist activity is full, partial, or inactive is determined by the maximum luminescence of the antibody-attenuated hIL-2 fusion protein compared to the maximum luminescence of rhIL-2. When rhIL-2 reaches the maximum luminescence, the antibody-attenuated hIL-2 fusion protein dose titration curve is considered to be a variant with full activity. The percentage of partial activity to full activity is calculated using the rhIL-2 maximum luminescence as 100%. Antibody-attenuated hIL-2 fusion proteins with a maximum RLU of less than 10% of the maximum RLU of rhIL-2 at the highest concentration of 1200nM are considered to have no agonist activity or be inactive. For some variants, EC 50 The values ​​are only estimates, as the full curve was not achieved. Many examples of anti-hPD-1-hIL-2 fusion proteins with attenuated hIL-2 showed rescued hIL-2 activity on the targeted cell lines, while the non-targeting antibody control (denoted by 1H3) did not demonstrate rescued hIL-2 activity. Complete rescue was indicated by a reduction in the fold change relative to rhIL-2 to a value of 0 or 1.

[0369] Table 24. Fold change relative to rhIL-2 and agonistic activity of antibody-hIL-2 fusion proteins on TF1+IL-2Rβ+hPD-1 cell line (human PD-1 expressing cell line with medium affinity IL-2R).

[0370]

[0371]

[0372]

[0373]

[0374]

[0375] NC = Not calculated by GraphPad Prism 7

[0376] a = Fold changes are only estimates, as a full four-parameter logistic curve was not achieved

[0377] Example 18: Evaluation of alternative anti-hPD-1-attenuated hIL-2 fusion proteins with or without blocking mouse PD-L1 in an in vivo murine colon adenocarcinoma (MC38) model

[0378] Since there are no recognized models to explore the in vivo efficacy of tumor treatments in primates, alternative anti-mPD-1-attenuated hIL-2 fusion proteins were generated and tested in a syngeneic murine tumor model. This MC38 colon adenocarcinoma model is often used to test the efficacy of immuno-oncology therapies. To investigate the in vivo effects of the anti-PD-1-attenuated hIL-2 fusion proteins, an alternative anti-mouse PD-1 antibody, RMP1-14 (known to block mouse PD-L1 binding) and RMP1-30 (described as a mouse PD-L1 non-blocker) were fused to the attenuated hIL-2 at the C-terminus of the mouse IgG2b-N297A heavy chain and tested in the MC38 colon adenocarcinoma model. The hIL-2 portion included substitutions F42K, Y45R, and V69R, which were tested in an IL-2-dependent mouse T lymphoblastoid cell line (CTLL-2) and demonstrated to be attenuated for mouse IL-2 activity. Human IL-2 can stimulate the proliferation of mouse T cells at similar concentrations, however, the same substitution that weakens the activity of human IL-2-dependent cell lines does not weaken the activity of CTLL-2 cell lines (data not shown). Therefore, F42K / Y45R / V69R substitutions are used as substitutes for hIL-2 because they show attenuated IL-2 activity on mouse cell lines. Sequences containing heavy and light chain variable region sequences of anti-mouse PD-1 antibodies RMP1-14 and RMP1-30 (such as Matsumoto K et al., J Immunol. February 15, 2004; 172 (4): 2530-41) are also formed on the background of mouse IgG2b-N297A to produce anti-mPD-1RMP1-14 mIgG2b-N297A (SEQ ID NO: 564 and 566) and anti-mPD-1RMP1-30 mIgG2b-N297A (SEQ ID NO: 567 and 568). The mouse IgG2b isotype with the N297A substitution is the mouse equivalent of the Fc isotype with Fc immune effector functions abolished. Surrogate antibodies and antibody-attenuated hIL-2 fusion proteins were generated, expressed, and protein A purified using standard techniques.

[0379] In this mouse tumor model, 5×10 5 MC38 colorectal cancer cells were injected into the right flank of ten-week-old female C57BL / 6NCrl (Charles River) mice. When the tumor reached 80-120 mm 3At 1:10, mice were sorted into groups (10 mice / group) and treatment was started on day 1 of the study. Anti-mPD-1RMP1-14mIgG2b-N297A, anti-mPD-1RMP1-30 mIgG2b-N297A, anti-mPD-1RMP1-14mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) (SEQ ID NO: 565 and 566) and anti-mPD-1RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) (SEQ ID NO: 568 and 569) were intraperitoneally administered at a dose of 5 mg / kg along with vehicle control (phosphate buffered saline) twice a week for 4 weeks. During the study, the formula (w 2 × L) / 2 Tumor size was measured twice weekly with a caliper, where w = width and L = length. The study endpoint was 1000 mm 3 tumor volume or survival on day 50, whichever comes first.

[0380] Figure 8 It was shown that although the administration of anti-mPD-1RMP1-14-mIgG2b-N297A or anti-mPD-1RMP1-30-mIgG2b-N297A antibodies alone had no significant therapeutic effect compared to vehicle control, the administration of anti-mPD-1RMP1-14 mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) or anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) anti-PD-1-attenuated hIL-2 fusion proteins was associated with 90% and 100% complete tumor regression, respectively. These data demonstrate that the anti-tumor efficacy mediated by anti-mPD-1-hIL-2 (F42K / Y45R / V69R) fusion proteins does not require PD-1 checkpoint blockade and that the efficacy is dependent on hIL-2 activity. The data further demonstrated that antibody-mediated targeting of PD-1-expressing T cells was sufficient to promote potent antitumor efficacy in the MC38 tumor model.

[0381] Example 19: Alternative anti-hPD-1-attenuated hIL-2 fusion protein expands effector memory CD8+ T cells in an in vivo murine colon adenocarcinoma model

[0382] To understand the mechanism of action of the alternative anti-hPD-1-attenuated hIL-2 fusion protein in vivo, an in vivo experiment similar to Example 18 was performed, followed by immunophenotyping of the resulting T cell populations in tumors, blood, spleen, and lymph nodes after three doses. 5Murine MC38 colon adenocarcinoma tumor cells were implanted subcutaneously into the right flank of ten-week-old female C57BL / 6NCrl (Chasliva) mice, and tumor growth was monitored. 3 Animals between 1 and 2 were divided into four groups, with 10 mice in each group for study. After 21 days after implantation, 0.2 mL / dose of phosphate buffered saline (PBS) was intraperitoneally administered to animals for vehicle control, 5 mg / kg anti-KLH-C3-mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R), 5 mg / kg anti-mPD-1RMP1-30mIgG2b-N297A or 5 mg / kg anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2 (F42K / Y45R / V69R) on the 1st, 4th and 8th days. On the 9th day, tumors, spleens and inguinal lymph nodes were collected from all mice and processed into single cell suspensions for subsequent flow cytometry analysis.

[0383] Fig. 9A Tumor volume growth (mm) was plotted over 9 days from the first dose on day 1. 3 ), where each point represents the mean of 10 mice. By day 8, tumor volume was reduced in anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2(F42K / Y45R / V69R) compared with other treatment groups. Fig. 9B The various CD8 + The contribution of T cell subsets in tumors, among which the central memory phenotype of T cells is CD45 + CD3 + CD4 - CD8 + CD44 + CD127 + CD69 - CD103, CD45 for T effector memory + 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 the other treatment groups, CD8 + T effector memory subsets have expanded, such as Fig. 9B This is also shown in the increase of light grey slices. Fig. 9C The absolute counts (cells / μL) in MC38-cleaved tumors are used to illustrate. In addition, the absolute counts (cells / μL) of regulatory T cells defined as expressing the CD45+CD3+CD4+CD8-CD25+FoxP3+ marker were reduced in the tumor.

[0384] CD8 + Expansion of T effector cell memory and reduction of regulatory T cells are associated with effective immunotherapy in mice and humans.

[0385] Example 20: Anti-hPD-1-attenuated hIL-2 fusion protein is active in vivo in the NCG-PBMC model

[0386] Transplantation of human immune cells into NOD-Prkdc cells lacking functional T, B, and NK cells em26Cd52 IL-2rg em26Cd22 The NjuCrl / NCG mouse has become a valuable tool for evaluating the efficacy of therapeutics that are hypothesized to stimulate human T cells. In this model, if therapeutic agents activate human T cells, this will lead to T cell expansion and acceleration of graft-versus-host disease (GvHD).

[0387] Over a 4-week period, three independent donors for human peripheral mononuclear cell (hPBMC) transplantation were evaluated for engraftment kinetics and expression of human PD-1 and human IL-2 receptors on T cells. Of the three donors tested, the donor that induced the most T cells and had an intermediate window for GvHD was selected. 1.5x107 hPBMCs were injected intravenously into NCG mice and divided into 8 groups of 8-16 mice each. On days 7, 10, and 14, mice were injected intraperitoneally 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

[0388] (T3A / R38E / I92K / C125A) (SEQ ID NO: 474, 425) (5 mg / kg). Anti-DNase fusion proteins as wild-type hIL-2 (1H3-hIgG1-LAGA-df-hIL-2(T3A / C125A)) and hIL-2 with attenuated moiety (1H3-hIgG1-LAGA-df-hIL-2(T3A / D20A / R38E / C125A)) were used as non-targeting antibody controls. Although the 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A) fusion protein has no changes in the hIL-2 portion that reduces hIL-2 activity, it does contain T3A and C125A substitutions to remove predicted O-linked glycosylation sites on human IL-2 (see, e.g., International Publication No. WO2012 / 107417) and unpaired cysteine ​​residues (see, e.g., International Publication No. WO2018 / 184964), respectively. These substitutes have not shown reduced hIL-2 efficacy clinically. On day 21, blood, spleen, and lungs were collected, where blood and spleen were processed for flow cytometry immunophenotyping, and lungs were weighed.

[0389] After 21 days, blood and spleen of the animals were subjected to flow cytometry immunophenotyping. Table 25 summarizes the markers used to characterize human T cell populations for subsequent analysis.

[0390] Table 25. Phenotypic markers defining human T cell subsets in NCG-PBMC mice

[0391] Cell population Phenotypic markers Pan T cells CD3+ CD8+ naive CD3+CD4-CD8+CD45RO-CCR7+ CD8+ effector CD3+CD4-CD8+CD45RO-CCR7- CD8+ effector memory CD3+CD4-CD8+CD45RO+CCR7- CD8+ central memory CD3+CD4-CD8+CD45RO+CCR7+ CD4+primary CD3+CD4+CD8-CD45RO-CCR7+ CD4+ effector CD3+CD4+CD8-CD45RO-CCR7- CD4+ effector memory CD3+CD4+CD8-CD45RO+CCR7- CD4+ Central Memory CD3+CD4+CD8-CD45RO+CCR7+ Regulatory T cells CD3+CD4+CD8-CD25+Foxp3+ NK cells CD3-CD56+

[0392] like Fig.10 As shown, the body weight of each individual animal was measured for 21 days and normalized to day 1 as an assessment of graft-versus-host disease (GvHD). Accelerated GvHD was observed in mice treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) at a dose of 10 mg / kg. A slight decrease in body weight was also observed in mice treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) at 2.5 mg / kg and 5 mg / kg, and in mice treated with 2H7-hIgG1-LAGA-df-hIL-2 (T3A / R38E / I92K / C125A) at 5 mg / kg. Although weight loss was observed in 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A), this did not last.

[0393] Flow cytometry analysis correlated with the observed accelerated graft-versus-host disease (GvHD). Using the phenotypic markers depicted by the human T cell subsets 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 mice treated with 10 mg / kg of 1H3-hIgG1-LAGA-df-hIL-2 (T3A / C125A), CD3+, CD4+, and CD8+ T cell subsets were only slightly expanded (quantified by the change fold of CD3+ T cells between 10 and 50 times relative to the vehicle control). In addition, in mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2

[0394] In the peripheral blood of mice treated with (T3A / D20A / R38E / C125A), CD3 + 、CD4 + and CD8 + T cell subsets are greatly expanded (for CD3 + T cells, fold change greater than 50-fold relative to vehicle control). Table 26 summarizes the expanded human T cell subsets.

[0395]

[0396] In addition to evaluating CD3 + 、CD4 + and CD8 +In addition to T cells, CD4 + and CD8 + Memory and naive subsets of T cell subsets. Table 25 summarizes the phenotypic markers used to describe the naive, effector, effector memory, and central memory of CD4+ and CD8+ T cells. There was no change in naive, effector, or central memory T cells between treatment groups (data not shown). However, mice treated with 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) greatly expanded CD4+ and CD8+ effector memory (EM) T cells in peripheral blood, with an average cell count of more than 5 million per ml for CD8+ T cells and more than 50 million (95% CI, 2.0-3.0) for CD4+ T cells. Fig.11A and 11B ). Box and whisker plots are drawn with boxes around the first and third quartiles, the horizontal line is the median, and the lines indicate 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 + There was a moderate expansion of effector memory (EM) T cells, defined as a mean cell count between 1 and 5 million cells per milliliter. In mice treated with 2.5 and 5 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A), CD4 + T cells, CD4 + Effector memory (EM) T cells were present with a moderate expansion between 6 and 13 million per ml.

[0397] In addition to stimulating effector T cells, IL-2 has also been described to stimulate NK cells and regulatory T cells (Tregs), and since Tregs express high levels of CD25 and NK cells express CD122, these immune cell types were also evaluated. Fig.12It is illustrated 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 had the lowest percentage of regulatory T cells (as phenotypically defined in Table 25) in the peripheral blood of the animals. There was a dose-dependent reduction in human regulatory T cells, and compared to the vehicle control, which had an average of 1.6% of human CD3+ T cells as tregs, 10 mg / kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) had an average of 0.16% of human CD3+ T cells as tregs. + The T cells were tregs. The percentage of human NK cells (phenotype defined in Table 25) in peripheral blood did not change in all treatment groups compared to vehicle control (data not shown).

[0398] Example 21: Nonclinical safety of anti-hPD-1-attenuated hIL-2 fusion protein

[0399] Cynomolgus monkeys have previously been used to evaluate the toxicity of unmodified IL-2. Lethality was observed in cynomolgus monkeys at doses as low as 50 μg / kg / day of exogenous recombinant IL-2. Since the binding of H7-767 to cynomolgus monkey hPD-1 on primary activated PBMCs was confirmed by flow cytometry (Example 12), a single-dose study of H7-767 variants (H7-02-hIgG1-LAGA-df-hIL-2) (T3A / D20A / R38E / C125A) (SEQ ID NO: 582 and 583) and H7-767 was conducted for preliminary safety evaluation. H7-02-hIgG1-LAGA-df-hIL-2 (T3A / D20A / R38E / C125A) was delivered to 8 monkeys by 15-minute intravenous infusion at a dose of 1 mg / kg (4 animals) or 10 mg / kg (4 animals). Sampling was performed at the time point of 360 hours after infusion. No adverse reactions, total toxicity, weight loss or lethality (data not shown) were observed. The subsequent single-dose study using H7-767 was performed with a higher dosage of 5mg / kg and 50mg / kg similar to the first study, and the sampling time point was 360 hours after infusion. Equally, no adverse reactions, total toxicity, weight loss or lethality (data not shown) were observed.

[0400] Example 22: Attenuation of IL-2 activity of modified hIL-2 proteins

[0401] As described in Example 5 above, the attenuation of IL-2 activity of modified hIL-2 proteins comprising a substitution at amino acid position 20 (D20) and a substitution at amino acid position 38 (R38) was tested in a proliferation assay of NK-92 and TF1+IL-2Rβ cell lines. Based on the maximum agonist activity of the modified hIL-2 proteins and the level of potency attenuation relative to unmodified recombinant hIL-2 for medium and high affinity receptors (Table 27), the modified hIL-2 proteins were divided into 7 groups (1 to 7). The criteria for grouping the modified hIL-2 proteins were:

[0402] • Group 1: variants with the highest attenuation (ie, >10,000-fold) and at least about 80% activity at the intermediate affinity receptor, but also with high attenuation and at least about 70% activity at the high affinity receptor.

[0403] • Group 2: Variants with at least about 70% activity and >1,000-fold attenuation at the medium affinity receptor and about 20% to about 30% activity at the high affinity receptor.

[0404] • Group 3: Variants with about 50% to about 70% activity and >1,000-fold attenuation at the medium affinity receptor and about 20% activity at the high affinity receptor.

[0405] • Group 4: Variants with at least about 70% activity but only >500-fold attenuation at the medium affinity receptor and about 50% activity at the high affinity receptor.

[0406] • Group 5: Variants that have at least about 70% activity at both receptors but have a >10-fold to >300-fold rolloff at the intermediate affinity receptor and also a 70-fold to 1500-fold rolloff at the high affinity receptor.

[0407] • Group 6: Variants with only about 30% activity and >2,500-fold attenuation at the medium affinity receptor and no activity at the high affinity receptor.

[0408] • Group 7: Variants that are inactive against both the medium and high affinity receptors.

[0409] Table 27. Fold change relative to rhIL-2 and agonist activity of modified hIL-2 proteins comprising a substitution at amino acid position 20 (D20) and a substitution at amino acid position 38 (R38) in a cell-based proliferation assay

[0410]

[0411] Example 23: Activity of alternative fusion proteins in the mouse MC38 colorectal tumor model

[0412] 5 × 10 5 Syngeneic MC38 colorectal cancer cells. When the tumor reaches 80-120mm 3 Mice were sorted into groups (10 mice / group) at 4 pm and treatment was initiated on day 1 of the study. All drugs except hIL-2 were administered intraperitoneally at a dose of 5 mg / kg twice a week for 4 weeks starting on day 1. hIL-2 was administered intraperitoneally at 36,000 IU once a day starting on days 1-5. Tumor size was measured twice a week with calipers during the study. The study endpoint was 1000 mm 3 Patients were considered to have either a tumor volume of ≥50% or survival on day 50 or progression-free survival on day 70, whichever came first.

[0413] All tested agents, including antibody molecules and antibody-hIL-2 fusion proteins, were generated using a mouse IgG2b Fc region with a single N297A amino acid substitution at position 297, which prevents glycosylation of the Fc region and significantly reduces any Fc region-mediated immune effector function, thereby preventing in vivo cell exhaustion. Anti-mPD-1 RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (Matsumoto, J Immunol 172:2530-2541, 2004). Anti-mPD-1RMP1-14-hIL-2F42K / Y45R / V69R is a bifunctional fusion protein consisting of a monoclonal RMP1-14 antibody antagonist of the mouse PD-1 receptor, fused to hIL-2F42K / Y45R / V69R (SEQ ID NO: 621) at its C-terminus via a flexible six-amino acid glycine / serine linker, which is a reduced potency IL-2 variant. This molecule is designed to directly target hIL-2 variants with reduced potency to T cells expressing PD-1 in mice. Anti-KLH-hIL-2F42K, Y45R, V69R is a control fusion protein consisting of an isotype control monoclonal antibody that recognizes a non-mammalian antigen (keyhole limpet hemocyanin, KLH), which is fused to a reduced potency IL-2 variant hIL-2F42K, Y45R, V69R at its C-terminus via a flexible six-amino acid glycine / serine linker.

[0414] The results are Fig.19The MC38 colorectal tumor model was particularly sensitive to antibody-mediated inhibition of the PD-1 receptor. Although tumors grown in vehicle-treated mice rapidly reached study endpoints, 50% of mice treated with anti-mPD-1RMP1-14 experienced complete tumor regression. In contrast, 100% of mice treated with anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R fusion protein experienced durable, long-term tumor regression. Mice treated with various combinations of the individual components of the anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R fusion protein, including the combination of anti-mPD-1RMP1-14 with hIL-2 free cytokine (administered at doses and schedules comparable to human therapeutic doses) or the combination of anti-mPD-1RMP1-14 with a non-targeted anti-KLH-hIL-2F42K, Y45R, V69R fusion protein, did not recapitulate the efficacy seen with anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R. These data suggest that targeting the reduced potency hIL-2 to PD-1 expressing cells significantly improves anti-tumor efficacy relative to anti-PD-1 receptor antagonists and that the activity of the fusion protein is not due to additive effects of the individual components of the molecule.

[0415] Example 24: Evaluation of protective anti-tumor immunity induced by alternative anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R in the MC38 colorectal tumor model

[0416] Mice that experienced complete tumor regression and survived to day 50 in the primary tumor study described in Example 23 were subjected to secondary tumor challenge without any additional drug treatment. For tumor rechallenge, mice were transplanted with 5×10 5 MC38 tumor cells were implanted in the left flank contralateral to the primary tumor location. As a control group, 10 age-matched tumor-naive mice were also implanted with MC38 tumor cells.

[0417] Fig. 20 It was shown that all mice that had previously experienced complete tumor regression in previous primary tumor studies and survived to day 50 after treatment with anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R were completely protected from secondary tumor development. In contrast, all tumor-free mice transplanted with MC38 tumor cells continued to develop tumors, rapidly reaching a tumor volume of 100 mm 3 The development of protective antitumor immunity in the absence of continued drug treatment indicated that anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R induced antitumor memory T cell responses.

[0418] Those skilled in the art will appreciate that many changes and modifications may be made to the preferred embodiments disclosed herein, and that these changes and modifications may 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.

[0419] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated by reference in their entirety.

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[0516] Example

[0517] The following list of examples is intended to supplement, not replace or substitute for, what has been previously described.

[0518] Example 1. A modified human interleukin-2 (hIL-2) protein, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, wherein the modified hIL-2 protein exhibits reduced potency for both a high affinity hIL-2 receptor and a medium affinity hIL-2 receptor relative to the unmodified hIL-2.

[0519] Embodiment 2. The modified hIL-2 protein according to embodiment 1, wherein the substitution at amino acid position 20 is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

[0520] Embodiment 3. A modified hIL-2 protein according to embodiment 1 or 2, wherein the substitution at amino acid position 38 is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D and R38K substitutions.

[0521] Embodiment 4. The modified hIL-2 protein according to any one of the preceding embodiments, further comprising a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0522] Embodiment 5. The modified hIL-2 protein according to embodiment 4, wherein the substitution at amino acid position 3 is T3A.

[0523] Embodiment 6. The modified hIL-2 protein according to any one of the preceding embodiments, further comprising a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0524] Embodiment 7. The modified hIL-2 protein according to embodiment 6, wherein the substitution at amino acid position 125 is C125A.

[0525] Embodiment 8. The modified hIL-2 protein according to any one of the preceding embodiments, wherein the modified hIL-2 protein exhibits approximately 1,000-fold reduced potency for the high affinity IL-2 receptor (hIL-2Rαβγ).

[0526] Embodiment 9. The modified hIL-2 protein according to any one of the preceding embodiments, wherein the modified hIL-2 protein exhibits approximately 10,000-fold reduced potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ).

[0527] Embodiment 10. The modified hIL-2 protein according to any one of embodiments 1 to 9, wherein the modified hIL-2 protein is fused to an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0528] Embodiment 11. The modified hIL-2 protein according to Embodiment 10, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

[0529] Embodiment 12. The modified hIL-2 protein according to embodiment 10 or 11, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof through a peptide bond.

[0530] Embodiment 13. The modified hIL-2 protein according to embodiment 12, wherein the modified hIL-2 is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

[0531] Embodiment 14. The modified hIL-2 protein according to embodiment 10 or 11, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof via a linker.

[0532] Embodiment 15. A modified human interleukin-2 (hIL-2) protein, comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E 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.

[0533] Embodiment 16. The modified hIL-2 protein according to embodiment 15, comprising the amino acid sequence of any one of SEQ ID NO: 307, 607-611, 614, 617 or 620.

[0534] Embodiment 17. The modified hIL-2 protein according to embodiment 15 or 16, comprising a D20A substitution and a R38E substitution.

[0535] Embodiment 18. The modified hIL-2 protein according to Embodiment 17, comprising the amino acid sequence of SEQ ID NO:149.

[0536] Embodiment 19. The modified hIL-2 protein according to any one of embodiments 15 to 18, further comprising a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0537] Embodiment 20. The modified hIL-2 protein according to embodiment 19, wherein the substitution at amino acid position 3 is T3A.

[0538] Embodiment 21. The modified hIL-2 protein according to Embodiment 20, comprising the amino acid sequence of SEQ ID NO: 216.

[0539] Embodiment 22. The modified hIL-2 protein according to Embodiment 19, comprising the amino acid sequence of SEQ ID NO: 218.

[0540] Embodiment 23. The modified hIL-2 protein according to any one of embodiments 15 to 22, further comprising a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0541] Embodiment 24. The modified hIL-2 protein according to embodiment 23, wherein the substitution at amino acid position 125 is C125A.

[0542] Embodiment 25. The modified hIL-2 protein according to Embodiment 24, comprising the amino acid sequence of SEQ ID NO: 215, 217 or 219.

[0543] Embodiment 26. The modified hIL-2 protein according to Embodiment 25, comprising the amino acid sequence of SEQ ID NO: 217.

[0544] Embodiment 27. The modified hIL-2 protein according to any one of embodiments 15 to 26, wherein the modified hIL-2 protein is fused to an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0545] Embodiment 28. A modified hIL-2 protein according to Embodiment 27, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

[0546] Embodiment 29. The modified hIL-2 protein according to embodiment 27 or 28, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

[0547] Embodiment 30. The modified hIL-2 protein according to embodiment 29, wherein the modified hIL-2 is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

[0548] Embodiment 31. The modified hIL-2 protein according to embodiment 27 or 28, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof via a linker.

[0549] Embodiment 32. A human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or the antigen-binding fragment thereof comprises:

[0550] a) a heavy chain complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423;

[0551] b) 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;

[0552] c) 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; or

[0553] d) 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.

[0554] Embodiment 33. The human antibody molecule or antigen-binding fragment thereof according to embodiment 32, comprising:

[0555] a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417;

[0556] b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385;

[0557] c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395; or

[0558] d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 405.

[0559] Embodiment 34. The human antibody molecule or antigen-binding fragment thereof according to embodiment 32 or 33, which comprises a human IgG1 heavy chain constant region.

[0560] Embodiment 35. The human antibody molecule or antigen-binding fragment thereof according to embodiment 34, comprising a L235A substitution and a G237A substitution according to EU numbering.

[0561] Embodiment 36. The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 32 to 35, comprising:

[0562] a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415;

[0563] b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425;

[0564] c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427; or

[0565] d) a heavy chain comprising the amino acid sequence of SEQ ID NO:428 and a light chain comprising the amino acid sequence of SEQ ID NO:429.

[0566] Embodiment 37. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 36 comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415.

[0567] Embodiment 38. A human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 32 to 37, which is fused to a modified human interleukin-2 (hIL-2) protein, wherein the modified hIL-2 protein comprises a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

[0568] Embodiment 39. The human antibody molecule or antigen-binding fragment thereof according to embodiment 38, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620.

[0569] Embodiment 40. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 39, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149.

[0570] Embodiment 41. The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 38 to 40, 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.

[0571] Embodiment 42. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 41, wherein the substitution at amino acid position 3 is T3A.

[0572] Embodiment 43. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 42, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 216.

[0573] Embodiment 44. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 41, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 218.

[0574] Embodiment 45. The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 38 to 44, 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.

[0575] Embodiment 46. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 45, wherein the substitution at amino acid position 125 is C125A.

[0576] Embodiment 47. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 46, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

[0577] Embodiment 48. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 47, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217.

[0578] Embodiment 49. A human antibody molecule or antigen-binding fragment thereof according to any one of Embodiments 38 to 48, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof 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.

[0579] Embodiment 50. The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 38 to 49, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

[0580] Embodiment 51. The human antibody molecule or antigen-binding fragment thereof according to Embodiment 50, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

[0581] Embodiment 52. The human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 38 to 49, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment via a linker.

[0582] Embodiment 53. An immunoconjugate comprising:

[0583] (a) a modified human interleukin-2 (hIL-2) protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345; and

[0584] (b) a human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or the antigen-binding fragment thereof comprises:

[0585] (i) a heavy chain complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423;

[0586] (ii) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 391;

[0587] (iii) 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; or

[0588] (iv) 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.

[0589] Embodiment 54. The immunoconjugate of embodiment 53, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

[0590] Embodiment 55. The immunoconjugate of embodiment 53 or 54, wherein the substitution at amino acid position 38 of the modified hIL-2 protein is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D and R38K substitutions.

[0591] Embodiment 56. The immunoconjugate of any one of embodiments 53 to 55, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution, and the amino acid substitution at amino acid position 38 of the modified hIL-2 protein is R38E.

[0592] Embodiment 57. The immunoconjugate of any one of embodiments 53 to 56, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620.

[0593] Embodiment 58. The immunoconjugate of any one of embodiments 53 to 56, wherein the modified hIL-2 protein comprises D20A and R38E substitutions.

[0594] Embodiment 59. The immunoconjugate of Embodiment 58, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149.

[0595] Embodiment 60. An immunoconjugate according to any one of embodiments 53 to 57, comprising the amino acid sequence of any one of SEQ ID NO: 608, 614, 611, 620, 607, 610, 617, 609 or 307.

[0596] Embodiment 61. The immunoconjugate of any one of embodiments 53 to 60, 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.

[0597] Embodiment 62. The immunoconjugate of embodiment 61, wherein the substitution at amino acid position 3 of the modified hIL-2 protein is T3A.

[0598] Embodiment 63. The immunoconjugate of Embodiment 62, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

[0599] Embodiment 64. The immunoconjugate of Embodiment 61, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218.

[0600] Embodiment 65. The immunoconjugate of any one of embodiments 53 to 64, 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.

[0601] Embodiment 66. The immunoconjugate of Embodiment 65, wherein the substitution at amino acid position 125 is C125A.

[0602] Embodiment 67. The immunoconjugate of Embodiment 66, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

[0603] Embodiment 68. The immunoconjugate of Embodiment 67, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 217.

[0604] Embodiment 69. An immunoconjugate according to any one of Embodiments 53 to 68, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

[0605] Embodiment 70. The immunoconjugate of any one of embodiments 53 to 69, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

[0606] Embodiment 71. The immunoconjugate of embodiment 70, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

[0607] Embodiment 72. The immunoconjugate of any one of embodiments 53 to 69, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof via a linker.

[0608] Embodiment 73. The immunoconjugate of any one of embodiments 53 to 72, wherein the human antibody molecule or antigen-binding fragment thereof comprises:

[0609] a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417;

[0610] b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385;

[0611] c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395; or

[0612] d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 405.

[0613] Embodiment 74. An immunoconjugate according to any one of Embodiments 53 to 73, wherein the human antibody molecule or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region.

[0614] Embodiment 75. The immunoconjugate of Embodiment 74, wherein the human antibody molecule or antigen-binding fragment thereof comprises a L235A substitution and a G237A substitution according to EU numbering.

[0615] Embodiment 76. The immunoconjugate of any one of Embodiments 53 to 75, wherein the human antibody molecule or antigen-binding fragment thereof comprises:

[0616] a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415;

[0617] b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425;

[0618] c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427; or

[0619] d) a heavy chain comprising the amino acid sequence of SEQ ID NO:428 and a light chain comprising the amino acid sequence of SEQ ID NO:429.

[0620] Embodiment 77. The immunoconjugate of Embodiment 76, wherein the human antibody molecule or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415.

[0621] Embodiment 78. The immunoconjugate according to any one of embodiments 53 to 77, comprising

[0622] A light chain comprising the amino acid sequence of SEQ ID NO: 415; and

[0623] A heavy chain modified hIL-2 protein fusion comprising the amino acid sequence of SEQ ID NO:532.

[0624] Embodiment 79. A pharmaceutical composition comprising a modified hIL-2 protein according to any one of embodiments 1 to 31, a human antibody molecule or an antigen-binding fragment thereof according to any one of embodiments 32 to 52, or an immunoconjugate according to any one of embodiments 53 to 78.

[0625] Embodiment 80. A polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of embodiments 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of embodiments 32 to 52, or an immunoconjugate according to any one of embodiments 53 to 78.

[0626] Embodiment 81. A vector comprising a polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of embodiments 1 to 31, a human antibody molecule or an antigen-binding fragment thereof according to any one of embodiments 32 to 52, or an immunoconjugate according to any one of embodiments 53 to 78.

[0627] Embodiment 82. A transformed cell comprising the vector according to embodiment 81.

[0628] Embodiment 83. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a modified hIL-2 protein according to any one of Embodiments 10 to 14 and 27 to 31, an immunoconjugate according to any one of Embodiments 53 to 78, or a pharmaceutical composition according to Embodiment 79, thereby treating the disease or condition.

[0629] Embodiment 84. The method of Embodiment 83, wherein the disease or condition is cancer.

[0630] Embodiment 85. The method of Embodiment 84, wherein the cancer is melanoma.

[0631] Embodiment 86. A method according to Embodiment 84, wherein the cancer is non-small cell lung cancer.

[0632] Embodiment 87. Use of a modified hIL-2 protein according to any one of embodiments 10 to 14 and 27 to 31, an immunoconjugate according to any one of embodiments 53 to 78, or a pharmaceutical composition according to embodiment 79 for the preparation of a medicament for treating a disease or condition.

[0633] Embodiment 88. The use according to Embodiment 87, wherein the disease or condition is cancer.

[0634] Embodiment 89. The use according to Embodiment 88, wherein the cancer is melanoma.

[0635] Embodiment 90. The use according to Embodiment 88, wherein the cancer is non-small cell lung cancer.

[0636] Embodiment 91. A use of a modified hIL-2 protein according to any one of embodiments 10 to 14 and 27 to 31, an immunoconjugate according to any one of embodiments 53 to 78, or a pharmaceutical composition according to embodiment 79 for treating a disease or condition.

[0637] Embodiment 92. The use according to Embodiment 91, wherein the disease or condition is cancer.

[0638] Embodiment 93. The use according to Embodiment 92, wherein the cancer is melanoma.

[0639] Embodiment 94. The use according to Embodiment 92, wherein the cancer is non-small cell lung cancer.

Claims

1. A modified human interleukin-2 (hIL-2) protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, wherein the modified hIL-2 protein exhibits reduced potency for both the high affinity hIL-2 receptor and the intermediate affinity hIL-2 receptor relative to the unmodified hIL-2.

2. The modified hIL-2 protein according to claim 1, wherein the substitution at amino acid position 20 is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

3. The modified hIL-2 protein according to claim 1 or 2, wherein the substitution at amino acid position 38 is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D and R38K substitutions.

4. The modified hIL-2 protein according to any one of the preceding claims, further comprising a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345. The modified hIL-2 protein according to claim 4 , wherein the substitution at amino acid position 3 is T3A.

6. The modified hIL-2 protein according to any one of the preceding claims, further comprising a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345.

7. The modified hIL-2 protein according to claim 6, wherein the substitution at amino acid position 125 is C125A.

8. The modified hIL-2 protein according to any one of the preceding claims, wherein the modified hIL-2 protein exhibits approximately 1,000-fold reduced potency for the high affinity IL-2 receptor (hIL-2Rαβγ).

9. The modified hIL-2 protein according to any one of the preceding claims, wherein the modified hIL-2 protein exhibits approximately 10,000-fold reduced potency at the intermediate affinity IL-2 receptor (hIL-2Rβγ).

10. The modified hIL-2 protein according to any one of claims 1 to 9, wherein the modified hIL-2 protein is fused to an anti-PD-1 antibody or an antigen-binding fragment thereof.

11. The modified hIL-2 protein according to claim 10, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

12. The modified hIL-2 protein according to claim 10 or 11, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

13. The modified hIL-2 protein according to claim 12, wherein the modified hIL-2 is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

14. The modified hIL-2 protein according to claim 10 or 11, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof via a linker.

15. A modified human interleukin-2 (hIL-2) protein comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E 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.

16. The modified hIL-2 protein according to claim 15, comprising the amino acid sequence of any one of SEQ ID NO: 307, 607-611, 614, 617 or 620.

17. The modified hIL-2 protein according to claim 15 or 16, comprising a D20A substitution and a R38E substitution. The modified hIL-2 protein according to claim 17 , comprising the amino acid sequence of SEQ ID NO:

149.

19. The modified hIL-2 protein according to any one of claims 15 to 18, further comprising a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345.

20. The modified hIL-2 protein according to claim 19, wherein the substitution at amino acid position 3 is T3A.

21. The modified hIL-2 protein according to claim 20, comprising the amino acid sequence of SEQ ID NO:

216.

22. The modified hIL-2 protein according to claim 19, comprising the amino acid sequence of SEQ ID NO:

218.

23. The modified hIL-2 protein according to any one of claims 15 to 22, further comprising a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345.

24. The modified hIL-2 protein of claim 23, wherein the substitution at amino acid position 125 is C125A.

25. The modified hIL-2 protein according to claim 24, comprising the amino acid sequence of SEQ ID NO: 215, 217 or 219.

26. The modified hIL-2 protein according to claim 25, comprising the amino acid sequence of SEQ ID NO:

217.

27. The modified hIL-2 protein according to any one of claims 15 to 26, wherein the modified hIL-2 protein is fused to an anti-PD-1 antibody or an antigen-binding fragment thereof.

28. The modified hIL-2 protein of claim 27, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

29. The modified hIL-2 protein according to claim 27 or 28, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

30. The modified hIL-2 protein of claim 29, wherein the modified hIL-2 is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

31. The modified hIL-2 protein according to claim 27 or 28, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof via a linker.

32. A human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or the antigen-binding fragment thereof comprises: a) a heavy chain complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423; b) 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; c) 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; or d) 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.

33. The human antibody molecule or antigen-binding fragment thereof according to claim 32, comprising: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385; c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395; or d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

405.

34. The human antibody molecule or antigen-binding fragment thereof according to claim 32 or 33, comprising a human IgG1 heavy chain constant region.

35. The human antibody molecule or antigen-binding fragment thereof according to claim 34, comprising a L235A substitution and a G237A substitution according to EU numbering.

36. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 35, comprising: a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415; b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425; c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427; or d) a heavy chain comprising the amino acid sequence of SEQ ID NO:428 and a light chain comprising the amino acid sequence of SEQ ID NO:

429.

37. The human antibody molecule or antigen-binding fragment thereof according to claim 36, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO:

415.

38. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 37, which is fused to a modified human interleukin-2 (hIL-2) protein, wherein the modified hIL-2 protein comprises a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:

345.

39. The human antibody molecule or antigen-binding fragment thereof according to claim 38, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620.

40. The human antibody molecule or antigen-binding fragment thereof according to claim 39, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

149.

41. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 40, 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.

42. The human antibody molecule or antigen-binding fragment thereof according to claim 41, wherein the substitution at amino acid position 3 is T3A.

43. The human antibody molecule or antigen-binding fragment thereof according to claim 42, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

216.

44. The human antibody molecule or antigen-binding fragment thereof according to claim 41, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

218.

45. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 44, 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.

46. ​​The human antibody molecule or antigen-binding fragment thereof according to claim 45, wherein the substitution at amino acid position 125 is C125A.

47. The human antibody molecule or antigen-binding fragment thereof according to claim 46, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

48. The human antibody molecule or antigen-binding fragment thereof according to claim 47, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

217.

49. The human antibody molecule or antigen binding fragment thereof according to any one of claims 38 to 48, wherein the modified hIL-2 protein is fused to the antibody or antigen binding fragment thereof 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.

50. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 49, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

51. The human antibody molecule or antigen-binding fragment thereof according to claim 50, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

52. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 49, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment via a linker.

53. An immunoconjugate comprising: (a) a modified human interleukin-2 (hIL-2) protein comprising a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345; and (b) a human antibody molecule or an antigen-binding fragment thereof, which immunospecifically binds to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or the antigen-binding fragment thereof comprises: (i) a heavy chain complementary determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 423; (ii) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 391; (iii) 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; or (iv) 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.

54. The immunoconjugate of claim 53, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

55. The immunoconjugate of claim 53 or 54, wherein the substitution at amino acid position 38 of the modified hIL-2 protein is selected from the group consisting of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D and R38K substitutions.

56. The immunoconjugate of any one of claims 53 to 55, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution, and the amino acid substitution at amino acid position 38 of the modified hIL-2 protein is R38E.

57. The immunoconjugate of any one of claims 53 to 56, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NOs: 134-150, 307, 344, 607-611, 614, 617 or 620.

58. The immunoconjugate of any one of claims 53 to 56, wherein the modified hIL-2 protein comprises D20A and R38E substitutions.

59. The immunoconjugate of claim 58, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

149.

60. The immunoconjugate of any one of claims 53 to 57, comprising the amino acid sequence of any one of SEQ ID NO: 608, 614, 611, 620, 607, 610, 617, 609 or 307.

61. The immunoconjugate of any one of claims 53 to 60, 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.

62. The immunoconjugate of claim 61, wherein the substitution at amino acid position 3 of the modified hIL-2 protein is T3A.

63. The immunoconjugate of claim 62, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

216.

64. The immunoconjugate of claim 61, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

218.

65. The immunoconjugate of any one of claims 53 to 64, 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.

66. The immunoconjugate of claim 65, wherein the substitution at amino acid position 125 is C125A.

67. The immunoconjugate of claim 66, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

68. The immunoconjugate of claim 67, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:

217.

69. The immunoconjugate of any one of claims 53 to 68, wherein the modified hIL-2 protein is fused to the antibody or its antigen binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the Fab, or the C-terminus of the Fab.

70. The immunoconjugate of any one of claims 53 to 69, wherein the modified hIL-2 protein is fused directly to the antibody or antigen-binding fragment thereof via a peptide bond.

71. The immunoconjugate of claim 70, wherein the modified hIL-2 protein is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

72. The immunoconjugate of any one of claims 53 to 69, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof via a linker.

73. The immunoconjugate of any one of claims 53 to 72, wherein the human antibody molecule or antigen-binding fragment thereof comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 416 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 417; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 384 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 385; c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 394 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 395; or d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

405.

74. The immunoconjugate of any one of claims 53 to 73, wherein the human antibody molecule or antigen-binding fragment thereof comprises an IgG1 heavy chain constant region.

75. The immunoconjugate of claim 74, wherein the human antibody molecule or antigen-binding fragment thereof comprises a L235A substitution and a G237A substitution according to EU numbering.

76. The immunoconjugate of any one of claims 53 to 75, wherein the human antibody molecule or antigen-binding fragment thereof comprises: a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 414 and a light chain comprising the amino acid sequence of SEQ ID NO: 415; b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 424 and a light chain comprising the amino acid sequence of SEQ ID NO: 425; c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 426 and a light chain comprising the amino acid sequence of SEQ ID NO: 427; or d) a heavy chain comprising the amino acid sequence of SEQ ID NO:428 and a light chain comprising the amino acid sequence of SEQ ID NO:

429.

77. The immunoconjugate of claim 76, wherein the human antibody molecule or antigen-binding fragment thereof comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:414 and a light chain comprising the amino acid sequence of SEQ ID NO:

415.

78. The immunoconjugate according to any one of claims 53 to 77, comprising A light chain comprising the amino acid sequence of SEQ ID NO: 415; and A heavy chain modified hIL-2 protein fusion comprising the amino acid sequence of SEQ ID NO:

532.

79. A pharmaceutical composition comprising a modified hIL-2 protein according to any one of claims 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 52, or an immunoconjugate according to any one of claims 53 to 78.

80. A polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of claims 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 52, or an immunoconjugate according to any one of claims 53 to 78.

81. A vector comprising a polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of claims 1 to 31, a human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 52, or an immunoconjugate according to any one of claims 53 to 78.

82. A transformed cell comprising the vector according to claim 81.

83. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a modified hIL-2 protein according to any one of claims 10 to 14 and 27 to 31, an immunoconjugate according to any one of claims 53 to 78, or a pharmaceutical composition according to claim 79, thereby treating the disease or condition.

84. The method of claim 83, wherein the disease or condition is cancer.

85. The method of claim 84, wherein the cancer is melanoma.

86. The method of claim 84, wherein the cancer is non-small cell lung cancer.

87. Use of a modified hIL-2 protein according to any one of claims 10 to 14 and 27 to 31, an immunoconjugate according to any one of claims 53 to 78, or a pharmaceutical composition according to claim 79 for the preparation of a medicament for treating a disease or condition.

88. The use of claim 87, wherein the disease or disorder is cancer.

89. The use according to claim 88, wherein the cancer is melanoma.

90. The use of claim 88, wherein the cancer is non-small cell lung cancer.

91. Use of a modified hIL-2 protein according to any one of claims 10 to 14 and 27 to 31, an immunoconjugate according to any one of claims 53 to 78, or a pharmaceutical composition according to claim 79 for treating a disease or disorder.

92. The use according to claim 91, wherein the disease or disorder is cancer.

93. The use according to claim 92, wherein the cancer is melanoma.

94. The use of claim 92, wherein the cancer is non-small cell lung cancer.

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