Immunoconjugates and uses thereof

Abstract

The present disclosure relates to immunoconjugates and uses thereof. In particular, the present disclosure relates to immunoconjugates comprising an antibody to PD-1 or an antigen-binding fragment thereof and IL-2, and uses thereof for treating cancer.

Description

[Technical field]

[0001] This disclosure claims priority to a Chinese patent application filed on February 11, 2022, bearing application number CN202210127604.7 and entitled "Immunoconjugate and Use Thereof," the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the field of biomedicine, and in particular to immunoconjugates comprising PD-1 antibodies and IL-2, and methods and related pharmaceutical uses thereof for treating cancer. [Background technology]

[0003] Human interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is located on chromosome 4 (4q27), contains a total sequence of 7 kb, consists of 133 amino acids, and has a molecular weight of about 15 kD. Initial in vitro cell experiments showed that T cells can secrete IL-2 and express IL-2 receptors (IL-2R) on the cell surface after activation by TCR and CD28. The binding of IL-2 to its receptor can cause T cell proliferation and produce effects on T cells. This model makes IL-2 a molecule that plays a central role in T cell immune responses. However, subsequent in vivo experiments found that when IL-2 or its receptor was knocked out, animals developed autoimmunity. Subsequent experiments showed that IL-2 can not only activate effector cells such as T cells and NK cells, but also activate regulatory T cells (Treg), suppressing excessive autoimmunity.

[0004] IL-2 functions through IL-2R on the cell surface. IL-2R contains three subunits: IL-2Rα (i.e., CD25), IL-2Rβ (i.e., CD122), and IL-2Rγ (i.e., CD132). The three subunits can form three receptor forms: high avidity receptors, which contain all three subunits IL-2Rα / β / γ; intermediate avidity receptors, which contain IL-2Rβ / γ; and low avidity receptors, which are IL-2Rα. Among them, IL-2Rβ and IL-2Rγ are necessary for the activation of downstream signaling pathways by IL-2. When IL-2 binds to IL-2Rβ and IL-2Rγ simultaneously, the two receptor subunits form heterodimers, phosphorylate STAT5 in cells, and enter the cell nucleus to cause the transcription and expression of corresponding genes. IL-2Rα is not necessary for signal transmission, but can enhance the binding of IL-2 to IL-2Rβ and IL-2Rγ. Tregs continuously express the high avidity receptors IL-2Rα / β / γ but are not activated CD8 + T cells and NK cells express only the intermediate avidity receptor IL-2Rβ / γ. Thus, low doses of IL-2 preferentially bind to and activate Tregs, whereas high doses of IL-2 preferentially bind to and activate CD8 + It can also activate immune effector cells such as T cells and NK cells. Due to its effect of activating immune effector cells, high doses of IL-2 are applicable to tumor immunotherapy. However, treatment with high doses of IL-2 is accompanied by severe toxicity and side effects, including fever, diarrhea, capillary leak syndrome, etc. These side effects are associated with the excessive activation of peripheral T cells and NK cells. By reducing or eliminating the activity of IL-2 on peripheral T cells and NK cells and specifically targeting the tumor microenvironment or tumor-associated immune cells, the systemic toxicity of IL-2 can be avoided while achieving its antitumor effect.

[0005] PD-1 (Programmed Cell Death-1) belongs to the CD28 receptor family and is an immunosuppressive receptor (Riley et al., 2009, Immunol. Rev. 29:114-25). The family also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. PD-1 is expressed primarily on activated B cells, T cells, and myeloid cells (Chen et al., 2013, Nat. Rev. Immunol. 13:227-42) and has two cell surface glycoprotein ligands, PD-L1 and PD-L2, respectively. PD-L1 is expressed on lymphocytes (e.g., CD4 + T cells and CD8 + PD-1 is widely expressed in various types of cells (e.g., T cells, macrophages, etc.) and in peripheral tissues, various tumor cells, and virus-infected cells, etc. PD-L2 is mainly expressed in activated dendritic cells and macrophages (Dong et al., 1999, Nat. Med. 5:1365-9). Upon binding to its ligand PD-L1 or PD-L2, PD-1 upregulates T cell functions, including T cell activation, differentiation, proliferation, and reduced cytokine secretion.

[0006] PD-1 is characterized by low or no expression on the surface of peripheral T cells, but high expression on the surface of tumor-associated T cells. Currently, several PD-1 monoclonal antibodies, such as pembrolizumab, nivolumab, and camrelizumab, have been developed for tumor immunotherapy. These monoclonal antibodies exert their pharmacological effects by blocking the interaction between PD-1 and its ligand in the tumor microenvironment, and have achieved good therapeutic effects in multiple tumor types. However, there is still a significant proportion of patients who are poorly responsive to PD-1 monoclonal antibodies. For example, the response rate of nivolumab in advanced non-squamous non-small cell lung cancer is 19% (Borghaei et al., 2015, N Engl J Med. 373: 1627-39). Part of the reason for the low response may be the low level of tumor-infiltrating lymphocytes (Daud et al., 2016, J. Clin. Invest. 126:3447-52) and the lack of a T cell-activating tumor microenvironment (Ayers et al., 2017, J. Clin. Invest. 127:2930-2940). Clinical studies have revealed that the combination of PD-1 and IL-2 can be very effective. However, such IL-2 has certain systemic toxicities, such as flu-like symptoms (86.8%), rash (78.9%), and fatigue (73.7%) (Diab et al., 2020, Cancer Discov. 10:1158-1173), limiting its efficacy.

[0007] The prior art lacks a method for simultaneously improving the anti-tumor effect of PD-1 and avoiding IL-2-related toxicity by using IL-2. The present disclosure provides an immune complex comprising a PD-1 antibody and IL-2 or a variant thereof, in which the IL-2 variant has reduced affinity to high affinity receptors (IL-2Rα / β / γ) and medium affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, has eliminated affinity to low affinity receptors (IL-2Rα) compared to wild-type IL-2, and has reduced affinity to high affinity receptors more than affinity to medium affinity receptors. Because of reduced affinity to IL-2Rα / β / γ and IL-2Rβ / γ compared to wild-type IL-2, the immune complex has no activity on immune cells with low or no expression of PD-1, but can activate immune cells with high expression of PD-1 from two aspects. The immune complex uses the PD-1 antibody to block the interaction between PD-1 / PD-L1 and PD-1 / PD-L2, releasing the immune inhibition of tumor T cells, while binding to PD-1 on the surface of T cells, and only after binding to PD-1 can the IL-2 mutant bind to the IL-2 receptor on the cell surface through in-cis action, thereby exerting the activation effect of the IL-2 mutant on PD-1 positive T cells. This design makes it possible to use the IL-2 mutant to improve the activation, proliferation and anti-tumor effect of the PD-1 antibody on T cells, while increasing the specificity of IL-2 to PD-1 positive immune cells and avoiding the systemic toxicity commonly seen in wild-type IL-2, thereby providing a better solution for the clinical treatment of tumors. Summary of the Invention

[0008] The present disclosure provides PD-1 antibodies or antigen-binding fragments thereof. Also provided are IL-2 variants. Also provided are immunoconjugates comprising an antigen-binding domain (e.g., a PD-1 binding domain) and a cytokine (e.g., IL-2 or a variant thereof). Also provided are nucleic acids encoding the PD-1 antibodies or antigen-binding fragments thereof, the IL-2 variants, and the immunoconjugates, vectors, host cells, pharmaceutical compositions, methods for treating or preventing a disease or condition (e.g., cancer) thereof, and related pharmaceutical uses. PD-1 antibody or antigen-binding fragment thereof

[0009] The disclosure provides a PD-1 antibody, or antigen-binding fragment thereof, comprising a PD-1 binding domain comprising at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprising: CDR1, CDR2 and CDR3 in an amino acid sequence represented by any one of SEQ ID NOs: 3, 13 to 15, 17 to 35, and 88 to 92, or It contains CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2 and 16, The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems. The immunoglobulin single variable domain specifically binds to the PD-1 antigen or a fragment thereof.

[0010] The present disclosure provides a PD-1 antibody, or antigen-binding fragment thereof, that comprises a PD-1-binding domain that includes any one or any combination of CDR1, CDR2, and CDR3 described above.

[0011] In some embodiments, the PD-1 binding domain has the amino acid sequences of CDR1, CDR2, and CDR3, respectively, of the immunoglobulin single variable domain, according to the Kabat numbering system: Represented by SEQ ID NOs: 97, 100, and 101; Represented by SEQ ID NOs: 7, 36, and 37; As shown in SEQ ID NO: 97, 98, 99, or Represented by sequence numbers 4, 5 and 6.

[0012] In some embodiments, the immunoglobulin single variable domain in the PD-1-binding domain has a CDR1 amino acid sequence represented by SEQ ID NO: 7 or 93, a CDR2 amino acid sequence represented by any one of SEQ ID NOs: 8, 38 to 40, and 94 to 95, and a CDR3 amino acid sequence represented by any one of SEQ ID NOs: 9, 41, 42, and 96; In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 38, and 41, respectively.

[0013] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 38, and 96, respectively.

[0014] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 94, and 41, respectively.

[0015] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 93, 94, and 41, respectively.

[0016] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 93, 94, and 96, respectively.

[0017] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 93, 95, and 96, respectively.

[0018] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.

[0019] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 42, respectively.

[0020] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 38, and 6, respectively.

[0021] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 39, and 9, respectively.

[0022] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 40, and 6, respectively.

[0023] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 41, respectively.

[0024] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 42, respectively.

[0025] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 38, and 41, respectively.

[0026] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 38, and 42, respectively.

[0027] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 39, and 41, respectively.

[0028] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 39, and 42, respectively.

[0029] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 40, and 41, respectively.

[0030] In some embodiments, the immunoglobulin single variable domain in the PD-1 binding domain has CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 40, and 42, respectively.

[0031] In some embodiments, the immunoglobulin single variable domain in the PD-1 antibody or antigen-binding fragment thereof has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduced antibody deamidation, and / or reduced antibody isomerization. In some embodiments, the immunoglobulin single variable domain is obtained by affinity maturation and has one or more changes in one or more CDRs that result in increased affinity for PD-1 over the parent.

[0032] In some specific embodiments, the heavy chain framework regions of the human germline templates IGHV3-23*01 or IGHV3-23*04 are used in the humanization engineering process.

[0033] In some embodiments, the immunoglobulin single variable domain in the PD-1-binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3, 13-15, 17-35, 88-92, or an amino acid sequence set forth in any one of SEQ ID NOs: 2 and 16, or a sequence having at least 80%, at least 85%, or at least 90% sequence identity to any one of the above sequences.

[0034] In some embodiments, the PD-1 antibody or antigen-binding fragment thereof comprises or is an antibody or antigen-binding fragment thereof that specifically binds to PD-1 or a fragment thereof, the antibody or antigen-binding fragment thereof being, for example, a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof being, for example, a recombinant antibody or fragment thereof. In some specific embodiments, the antigen-binding fragment is a linear antibody, a single chain antibody, a nanobody, a peptide antibody, a domain antibody, and a multispecific antibody (such as a bispecific antibody, a diabody, a triabody, and a tetrabody, a tandem di-scFv, a tandem tri-scFv).

[0035] In some embodiments, at least one immunoglobulin single variable domain in the PD-1 antibody, or antigen-binding fragment thereof, is a VHH.

[0036] In some embodiments, the PD-1 antibody, or antigen-binding fragment thereof, comprises one or more (e.g., 2, 3, 4, 5, 6) of the immunoglobulin single variable domains, which may be the same or different, and which may form a dimeric or multimeric molecule.

[0037] In some embodiments, the PD-1 binding domain of the PD-1 antibody or antigen-binding fragment thereof comprises a PD-1 binding domain of a heavy chain variable region (VH) and a PD-1 binding domain of a light chain variable region (VL), e.g., the PD-1 binding domain comprises or is a Fab, Fab', (Fab')2, ScFv, Fv, etc.

[0038] In some specific embodiments, the PD-1 binding domain comprising a heavy chain variable region and a light chain variable region is a Fab.

[0039] In some specific embodiments, the PD-1 antibody or antigen-binding fragment thereof comprises a PD-1-binding domain 1 and a PD-1-binding domain 2, or comprises a PD-1-binding domain 1, or comprises a PD-1-binding domain 2, wherein the PD-1-binding domain 1 comprises at least one of any one of the above immunoglobulin single variable domains, and the PD-1-binding domain 2 comprises a heavy chain variable region (VH) and a light chain variable region (VL) that bind to PD-1.

[0040] Exemplarily, the PD-1 binding domain 2 is selected from the group consisting of pembrolizumab, nivolumab, sintilimab, tislelizumab, cemiplimab, toripalimab, camrelizumab, penprimab, zimblerimab, sasanlimab, spartalizumab, and the like. The PD-1 binding domain is selected from the PD-1 antibodies: umab, retifanlimab, balstilimab, cetrelimab, dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, and AK-103.

[0041] In some further embodiments, the PD-1 binding domain 2 in the immune conjugate is selected from the antigen-binding domain of an antibody: a PD-L1 antibody (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072), a CTLA-4 antibody (e.g., Tremelimumab, Ipilimumab), or a 4-1BB antibody (e.g., Urelumab, Utomilumab).

[0042] The antigen-binding domain (e.g., PD-1-binding domain 2) is a portion of the antibody (e.g., PD-1 antibody) that binds to an antigen (e.g., PD-1), such as its Fab', F(ab')2, or its heavy chain variable region and light chain variable region, or a domain comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region.

[0043] In some embodiments, the PD-1-binding domain 2 comprises pembrolizumab or an antigen-binding fragment thereof (e.g., Fab), e.g., comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54, 55, and 56, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 57, 58, and 59. In some specific embodiments, the heavy chain variable region is a heavy chain variable region in a Fab heavy chain having the amino acid sequence set forth in SEQ ID NO: 52, and the light chain variable region comprises a light chain variable region in a Fab light chain having the amino acid sequence set forth in SEQ ID NO: 53.

[0044] In some embodiments, the PD-1 antibody or antigen-binding fragment thereof further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2, or IgG4 Fc region. The Fc region may have mutations, exemplary mutations being L234A / L235A in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in IgG1, IgG2, IgG3, or IgG4, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G237A in IgG1 ... IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S in IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG4. A hybrid IgG2 / 4 Fc domain may also be used, for example an Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4. In some specific embodiments, the human IgG4 Fc region has S228P, F234A, L235A and / or K447A mutations. In some specific embodiments, the human IgG1 Fc region has L234A / L235A or L234A / L235A / P329G mutations. In some specific embodiments, the human IgG1 Fc region has or does not have a C220S mutation.

[0045] The mutations in the Fc region are numbered according to the EU numbering rules.

[0046] In some embodiments, the Fc region contained in the PD-1 antibody, or antigen-binding fragment thereof, can enable the binding protein to form a dimeric molecule and increase the in vivo half-life of the binding protein.

[0047] In some embodiments, the immunoglobulin single variable domain in the PD-1 antibody or antigen-binding fragment thereof is linked to the Fc region via a linker. The linker may be a non-functional amino acid sequence having a length of 1 to 20 or more amino acids and no secondary or higher structure. For example, the linker is a flexible linker, such as G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, etc.

[0048] In some embodiments, the PD-1 antibody, or antigen-binding fragment thereof, has at least one activity selected from the following: (a)≦10 -7 K D binding to human PD-1 or an epitope thereof at a value (b) inhibiting the binding of PD-1 to PD-L1; (c) inhibiting the binding of PD-1 to PD-L2; (d) CD4 + Inducing the secretion of IFN-γ by T cells; (e) enhancing activation of PBMCs; (f) enhancing T cell activation; and (g) inhibiting tumor growth.

[0049] In some embodiments, the PD-1 antibody or antigen-binding fragment thereof of the disclosure has a K that binds to PD-1. D Value is ≦1×10 -7 M, for example, ≦1×10 -8 M, or ≦1×10 -9 M, or ≦1×10 -10 It's M.

[0050] In some embodiments, the PD-1 antibodies, or antigen-binding fragments thereof, of the disclosure can specifically bind to human PD-1 and block the interaction of PD-1 with PD-L1 and / or PD-1 with PD-L2.

[0051] In some embodiments, the PD-1 antibodies or antigen-binding fragments thereof of the present disclosure can inhibit tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.

[0052] In some embodiments, the PD-1 antibodies or antigen-binding fragments thereof of the present disclosure include variants, such as those that include one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs: 2-3, 13-35, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, which may occur in the CDR and / or FR regions.

[0053] In some embodiments, the PD-1 antibodies or antigen-binding fragments thereof disclosed herein are resistant to or highly stable against heat treatment, e.g., they are stable at least at 60°C, with no obvious aggregation or degradation observed after 30 days at 40°C.

[0054] In some embodiments, a PD-1 antibody or antigen-binding fragment thereof is provided that binds to or competitively binds to the same epitope as an immunoglobulin single variable domain in the PD-1 antibody or antigen-binding fragment thereof of the present disclosure.

[0055] In some embodiments, a PD-1 antibody or antigen-binding fragment thereof is provided that blocks binding of an immunoglobulin single variable domain in the PD-1 antibody or antigen-binding fragment thereof disclosed herein to PD-1 (e.g., human PD-1).

[0056] In some embodiments, a PD-1 antibody or antigen-binding fragment thereof is provided whose binding to PD-1 (e.g., human PD-1) is blocked by an immunoglobulin single variable domain in the PD-1 antibody or antigen-binding fragment thereof disclosed herein.

[0057] In some embodiments, the PD-1 antibodies, or antigen-binding fragments thereof, of the present disclosure reduce binding of PD-1 to PD-L1 and / or PD-L2 by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%.

[0058] In some embodiments, the disclosure provides a PD-1 binding protein that comprises at least one binding PD-1 immunoglobulin single variable domain (e.g., VHH) as described above.

[0059] As used above and in this disclosure, "at least 90% identity" includes at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity.

[0060] IL-2 and its variants The present disclosure provides IL-2 and variants thereof. The IL-2 is preferably human IL-2, and an example of the amino acid sequence of wild-type human IL-2 is, for example, SEQ ID NO: 87. The positions of the IL-2 variants of the present disclosure are numbered according to the wild-type IL-2 amino acid sequence shown in SEQ ID NO: 87.

[0061] In some embodiments, the present disclosure provides IL-2 variants that have reduced affinity for the high affinity receptors (IL-2Rα / β / γ) and the medium affinity receptors (IL-2Rβ / γ) compared to wild-type IL-2, and / or have ablated affinity for the low affinity receptor (IL-2Rα) compared to wild-type IL-2. In some specific embodiments, the IL-2 variants have reduced affinity for IL-2Rα / β / γ more than for IL-2Rβ / γ. The affinity can be detected by conventional methods, e.g., ELISA, ForteBio, SPR, e.g., the SPR detection method provided in Example 13 of the present disclosure.

[0062] In some embodiments, the disclosure provides an IL-2 variant comprising, or consisting of, the following mutation (i), mutation (ii), mutation (iii), mutation (iv), or any combination thereof:

[0063] Regarding mutation (i): The present disclosure provides mutations that confer on IL-2 variants altered (eg, improved or decreased) affinity for IL-2Rβ / γ compared to wild-type IL-2.

[0064] In some embodiments, the IL-2 variant has a mutation (i) that reduces affinity for IL-2Rβ / γ compared to wild-type IL-2. In some specific embodiments, the affinity of the IL-2 variant to IL-2Rβ / γ is reduced by 1 to 1000 times, 2 to 800 times, 5 to 500 times, 5 to 200 times, 10 to 200 times, 10 to 100 times, 10 to 50 times, 20 to 200 times, 20 to 100 times, 5 to 20 times, 2 to 20 times, 50 to 100 times, or 50 to 70 times, for example, about 5, about 10, about 13, about 15, about 20, about 30, about 40, about 50, about 60, about 63, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 180, about 200, about 250, about 300, about 400, or about 500 times, compared to that of wild-type IL-2. In some specific embodiments, the IL-2 mutant has a 10- to 100-fold reduced affinity for IL-2Rβ / γ compared to wild-type IL-2. The affinity can be detected by a conventional method, for example, the SPR detection method provided in Example 13 of the present disclosure.

[0065] In some embodiments, the mutations (i) are at positions 12-20, 84-95, and 126-130 of IL-2.

[0066] In some embodiments, the mutation (i) is selected from positions 12, 15, 16, 18, 19, 20, 23, 43, 69, 84, 87, 88, 91, 92, 95, 123, 126, 127, 129, 130, or any combination thereof; in some embodiments, the mutation is selected from positions L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129, and S130, or any combination thereof, e.g., N88 / D20; In some specific embodiments, the mutations are 12R, 12K, 12E, 12Q, 15Q, 15R, 15A, 15S, 16N, 16A, 16E, 16D, 16G, 16S, 16T, 16V, 16P, 20A, 20H, 20Y, 20N, 20Q, 20E, 20R, 20S, 23A, 23R, 23K, 23G, 23S, 23T, 23V, 23P, 87K, 87A, 84S, 84L, 84N, 84V, 84H, 84Y, 84R, 84K, 84G, 84A, 84T, 84P, 88D, 88A, 88S, 88E, 88T, 88R, 88I, 91A, 91T, 91E , 91I, 91L, 91D, 91N, 91Q, 91S, 91H, 92E, 92T, 92K, 92R, 92L, 95S, 95A, 95R, 9 5Q, 95G, 95T, 95V, 95P, 95H, 95N, 123A, 123E, 123K, 123Q, 126D, 126L, 126A, Selected from 126S, 126T, 126E, 127A, 127E, 127K, 127Q, 84N / 95Q, 84E / 95Q, 84T / 95Q, 84Q / 95Q, 84T / 16T, 84N / 91I, 84T / 126E, 84N / 126E, 16T / 84Q, 16T / 91I, and some In a specific embodiment, the mutations are L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, D20Q, D20E, D20R, D20S, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G, D84A, D84T,D84P, N88D, N88A, N88S, N88E, N88T, N88R, N88I, V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S, V91H, I92E, I92T, I92K, I92R, I92L, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S, Q126T, Q126E, S127A , S127E, S127K, S127Q, D84N / E95Q, D84E / E95Q, D84T / E95Q, D84Q / E95Q, D84T / H16T, D84N / V91I, D84T / Q126E, D84N / Q126E, H16T / D84Q, H16T / V91I, in some embodiments, said mutation (i) is selected from N88D, N88R, D20A, D20N, V91T, Q126D or H16A / D84S, in some embodiments, said mutation (i) is N88D or D20A.

[0067] Regarding mutation (ii): The present disclosure provides mutations that confer on IL-2 mutants altered (eg, reduced) affinity for IL-2Rα compared to wild-type IL-2, or that do not bind IL-2Rα.

[0068] In some embodiments, the IL-2 mutant has a mutation (ii) that reduces affinity for IL-2Rα compared to wild-type IL-2 or does not bind to IL-2Rα, which can be detected by conventional methods, such as the SPR detection method provided in Example 13 of the present disclosure.

[0069] In some specific embodiments, the mutation (ii) is selected from positions 35, 37, 38, 41, 42, 43, 45, 61, 62, 65, 68, 72, or any combination thereof, and in some embodiments, the mutation is selected from positions 38A, 38D, 38E, 62Q, 42A, 42G, 42S, 42T, 42Q, 42E, 42N, 42D, 42R, 42K, 45A, 45G, 45S, 45T, 45Q, 45E , 45N, 45D, 45R, 45K, 65R, 65E, 65K, 65H, 65Y, 65Q, 65D, 65N, 68A, 68Q, 68K, 68R, 72G, 72A, 72S, 72T, 72Q, 72E, 72N, 72D, 72R, 72K, and in some embodiments, the mutation (ii) is selected from the positions K35, T37, R38, E62, P65, E68, F42, Y45, L72, or any combination thereof. In some embodiments, the mutation is selected from the group consisting of R38A, R38D, R38E, E62Q, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, P65R, P65E, P65K, P65H, P65Y, P65Q, P65D , P65N, E68A, E68Q, E68K, E68R, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K, and in some embodiments, the mutation (ii) is selected from F42A, Y45A, L72G, R38E / F42A, F42A / L72G, F42A / Y45A, Y45A / L72G or F42A / Y45A / L72G.

[0070] Regarding mutation (iii): The present disclosure provides mutation (iii) that confers improved stability, expression level, purity and druggability to the IL-2 variant.

[0071] This disclosure incorporates all of the IL-2 variants in WO2020125743A.

[0072] In some embodiments, the IL-2 variant has a mutation (iii), wherein said mutation (iii) is selected from positions 11, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 70, 71, 72, 78, 82, 88, 132, or any combination thereof; in some embodiments, said mutation (iii) is selected from 26Q, 29S, 30S, 71Q, 11C, 132C, 70C, 82C, 27C, 78C, or any combination thereof; in some embodiments, said mutation (iii) is selected from positions N26, N29, N30, N71, Q11, L In some embodiments, said mutation (iii) is selected from N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, G27C / F78C, or any combination thereof; in some embodiments, said mutation (iii) is selected from N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, N26Q / N30S / G27C / F78C.

[0073] Regarding mutation (iv): In some embodiments the IL-2 variant comprises a mutation (iv), said mutation (iv) being selected from positions 3 and / or 125, in some embodiments said mutation (iv) being selected from positions T3 and / or C125, in some embodiments said mutation (iv) being selected from 3A, 3G, 3Q, 3E, 3N, 3D, 3R, 3K, 3P and / or 125A, 125S, 125T, 125V mutations. In some embodiments said mutation (iv) is selected from T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, T3P and / or C125A, C125S, C125T, C125V mutations.

[0074] For example, "mutation 1 / mutation 2" indicates that mutation 1 and mutation 2 are included simultaneously.

[0075] Exemplary IL-2 Mutants The present disclosure provides IL-2 that includes, or includes only, the following mutations: (a) F42A and N88D, N88A, N88S, N88E, N88T, N88R or N88I, (b) F42A and D20A, D20H, D20Y, D20N, D20Q, D20E, D20R or D20S, (c) F42A and Q126D, Q126L, Q126A, Q126S, Q126T or Q126E, (d) F42A and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S or V91H, (e) F42A / L72G and N88D, N88A, N88S, N88E, N88T, N88R or N88I, (f) F42A / L72G and D20A, D20H, D20Y, D20N, D20Q, D20E, D20R or D20S, (g) F42A / L72G and Q126D, Q126L, Q126A, Q126S, Q126T or Q126E, (h) F42A / L72G and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S or V91H, (s) F42A / R38E and V91A, V91T, V91E, V91I, V91L, V91D, V91N, V91Q, V91S or V91H, (i) Any one of the above (a) to (h) and (s) and N26Q, (j) any one of (a) to (h) and (s) above and N29S, (k) Any one of the above (a) to (h) and (s) and N71Q, (l) Any one of the above (a) to (h) and (s) and N26Q / N29S, (m) Any one of the above (a) to (h) and (s) and N26Q / N29S / N71Q, (n) Any one of the above (a) to (h) and (s) and Q11C / L132C, (o) Any one of the above (a) to (h) and (s) and L70C / P82C, (p) Any one of the above (a) to (h) and (s) and G27C / F78C, (q) Any one of the above (a) to (h) and (s) and T3A, (r) Any one of (a) to (h) and (s) above and T3A / C125A.

[0076] In some embodiments, the present disclosure provides an IL-2 variant that comprises or has an amino acid sequence set forth in any one of SEQ ID NOs: 60-68, or has at least 80%, at least 85%, or at least 90% sequence identity thereto.

[0077] In some embodiments, any IL-2 variant comprising the above mutation (i) has any one or more of 1) to 5) relative to wild-type IL-2: 1) having reduced affinity for the high affinity IL-2R receptor (IL-2Rαβγ); 2) having reduced affinity for the intermediate affinity IL-2R receptor (IL-2Rβγ); 3) Reduced T cell (e.g., CD4 + and CD8 + T cell) activation, 4) having reduced activation of NK cells; and 5) The release of inflammatory factors from T cells and / or NK cells stimulated with IL-2 is reduced.

[0078] In some embodiments, any of the IL-2 mutants including the above mutation (i) and mutation (ii) has any one or more of the above 1) to 5) and the following 6) to 8) compared to wild-type IL-2: 6) having reduced affinity for IL-2Rα or not binding to IL-2Rα; 7) The affinity for IL-2Rαβγ is reduced more than the affinity for IL-2Rβγ; and 8) CD25 + Cells (e.g., CD25 + CD8 + Reduces activation of T cells and Treg cells.

[0079] In some embodiments, any IL-2 mutant comprising the above mutation (i), mutation (ii), mutation (iii) and / or mutation (iv) has any one or more of the above 1) to 8) and the following 9) relative to wild-type IL-2: 9) Improved stability, druggability, expression level, purity and / or extended in vivo half-life.

[0080] immune complex The present disclosure provides an immunoconjugate comprising an antigen-binding domain and IL-2 or a variant thereof.

[0081] In some embodiments, the antigen-binding domain in the immune complex targets an immune effector cell or a tumor cell, and in some specific embodiments, the immune effector cell is capable of responding to stimulation or activation by IL-2 in the immune complex, and in some specific embodiments, the immune effector cell has IL-2Rβγ, and in some specific embodiments, the immune effector cell is a cell selected from the group consisting of, for example, NK cells, CD4 + CD25 - T cells, CD8 + CD25 - T cells.

[0082] In some embodiments, the antigen-binding domain in the immune complex targets PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP, or CD16a, and in some specific embodiments, the antigen-binding domain targets PD-1 (i.e., a PD-1 binding domain).

[0083] In some embodiments, the PD-1 binding domain is any PD-1 binding domain provided herein.

[0084] Illustratively, in some specific embodiments, the PD-1-binding domain comprises at least one immunoglobulin single variable domain, which comprises CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 3, 13-15, 17-35, and 88-92, or CDR1, CDR2, and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 2 and 16, wherein the CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

[0085] Exemplarily, in some specific embodiments, the PD-1 binding domain has the amino acid sequences of CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain set forth in SEQ ID NOs: 7, 36 and 37, respectively, or SEQ ID NOs: 4, 5 and 6, according to the Kabat numbering system.

[0086] For example, in some specific embodiments, the immunoglobulin single variable domain in the PD-1-binding domain has a CDR1 amino acid sequence represented by SEQ ID NO:7, a CDR2 amino acid sequence represented by any one of SEQ ID NOs:8, 38 to 40, and a CDR3 amino acid sequence represented by any one of SEQ ID NOs:9, 41, and 42.

[0087] Exemplarily, in some specific embodiments, the immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 36 and 37, or shown in SEQ ID NOs: 4, 5 and 6, or shown in SEQ ID NOs: 7, 38 and 41.

[0088] Exemplarily, in some specific embodiments, the amino acid sequence of the immunoglobulin single variable domain comprises any one of the amino acid sequences set forth in SEQ ID NOs: 3, 13-15, 17-35, 88-92, or any one of the amino acid sequences set forth in SEQ ID NOs: 2, 16, or an amino acid sequence having at least 90% sequence identity to any one of the above sequences.

[0089] In some embodiments, the PD-1 binding domain may comprise an antigen-binding fragment that targets PD-1 other than the immunoglobulin single variable domain, which comprises a heavy chain variable region (VH) and a light chain variable region (VL). Exemplary antigen-binding fragments that target PD-1 include pembrolizumab, nivolumab, sintilimab, tislelizumab, cemiplimab, toripalimab, camrelizumab, penprimab, zimbelelimab, sasanlimab, spartalizumab, spartan-1ib, spartan-2ib, spartan-3ib, spartan-4ib, spartan-5ib, spartan-6ib, spartan-7ib, spartan-8ib, spartan-9ib, spartan-1b ... The PD-1 binding domain is selected from the PD-1 antibodies: lizumab, retifanlimab, balstilimab, cetrelimab, dostarlimab (TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680 (AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, and AK-103.

[0090] In some further embodiments, the antigen-binding domain in the immune complex is selected from the antigen-binding domain of an antibody: a PD-L1 antibody (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072), a CTLA-4 antibody (e.g., Tremelimumab, Ipilimumab), or a 4-1BB antibody (e.g., Urelumab, Utomilumab).

[0091] The antigen-binding domain (e.g., a PD-1-binding domain) is a portion of the antibody (e.g., a PD-1 antibody) that binds to an antigen (e.g., PD-1), such as its Fab', F(ab')2, or its heavy chain variable region and light chain variable region, or a domain comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region and LCDR1, LCDR2, and LCDR3 of the light chain variable region.

[0092] In some embodiments, the PD-1-binding domain comprises pembrolizumab or an antigen-binding fragment thereof (e.g., Fab), e.g., comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54, 55, and 56, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 57, 58, and 59. In some specific embodiments, the heavy chain variable region is a heavy chain variable region in a Fab heavy chain having the amino acid sequence set forth in SEQ ID NO: 52, and the light chain variable region comprises a light chain variable region in a Fab light chain having the amino acid sequence set forth in SEQ ID NO: 53.

[0093] In some embodiments, the immunoconjugate comprises any one of the following: 1) a PD-1-binding domain 1 and IL-2 or a variant thereof, wherein said PD-1-binding domain 1 comprises at least one of any of the immunoglobulin single variable domains provided herein; 2) PD-1-binding domain 1, PD-1-binding domain 2, and IL-2 or a variant thereof, wherein the PD-1-binding domain 1 comprises at least one of any of the immunoglobulin single variable domains provided in the present disclosure, and the PD-1-binding domain 2 is any of the PD-1-binding domains comprising a VH and a VL provided in the present disclosure.

[0094] In some embodiments, the IL-2 or variant thereof in the immune complex is any IL-2 or variant thereof provided herein.

[0095] Illustratively, in some specific embodiments, the IL-2 is wild-type human IL-2.

[0096] Exemplarily, in some specific embodiments, the IL-2 variant comprises mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof provided in the present disclosure, or consists of mutation (i), mutation (ii), mutation (iii), mutation (iv) or any combination thereof.

[0097] Illustratively, in some specific embodiments, the IL-2 mutant comprises any one of the mutations (a)-(r), (s) provided in the present disclosure, or comprises only one of them.

[0098] Exemplarily, in some specific embodiments, the IL-2 mutant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 60 to 68, or a sequence having at least 80%, at least 85%, or at least 90% identity thereto, or consists of the above sequence.

[0099] In some embodiments, the immune complex further comprises a human immunoglobulin Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region.

[0100] In some embodiments, the antigen-binding domain (e.g., the PD-1-binding domain) in the immune complex is at the N-terminus of the Fc region and the IL-2 or variant thereof is at the N-terminus or C-terminus of the Fc region. In some specific embodiments, the antigen-binding domain is at the N-terminus of the Fc and the IL-2 or variant thereof is at the C-terminus of the Fc region.

[0101] In some embodiments, the valency ratio of the antigen-binding domain (e.g., the PD-1-binding domain) to IL-2 or a variant thereof in the immune complex is between 6:1 and 1:3 (e.g., 4:1 and 1:2), specifically, for example, 4:1, 2:1, 3:1, or 1:1.

[0102] As used herein, the valency ratio of PD-1 binding domains to IL-2 or variants thereof refers to the ratio of the number of PD-1 binding domains to IL-2 or variants thereof contained in one immunoconjugate molecule. In some embodiments, the valency ratio comprises the ratio of the number of PD-1 binding domains 1 of at least one immunoglobulin single variable domain to IL-2 or variants thereof. In some embodiments, the valency ratio is the ratio of the sum of the number of PD-1 binding domains 1 and PD-1 binding domains 2 to the number of IL-2 or variants thereof.

[0103] In some embodiments, the immune complex comprises one or two IL-2 or variants thereof and one, two, three, four, five, or six antigen-binding domains (e.g., PD-1 binding domains) per immune complex.

[0104] In some embodiments, the immune complex further comprises an Fc region comprising a knob-into-hole modification of a first subunit, Fc1, and a second subunit, Fc2, that are capable of associating with each other.

[0105] In some embodiments, Fc1 and Fc2 contain amino acid mutations such that Fc1 preferentially pairs with Fc2 or forms heterodimers over Fc1. In some embodiments, the mutations are in CH3 of Fc1 and Fc2. In some embodiments, the amino acid mutations in Fc1 and Fc2 generate greater electrostatic complementarity than wild-type variants that do not contain the mutations. In some embodiments, the amino acid mutations in Fc1 and Fc2 generate greater spatial complementarity than wild-type variants that do not contain the mutations.

[0106] In some embodiments, for Fc1 and Fc2, for example, one or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume in the CH3 / CH3 interface to generate a protrusion (or knob) on the surface of the CH3 domain of Fc1, and one or more, preferably two or three, amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume to generate a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some embodiments, the input residue with the larger side chain volume is phenylalanine (F), tyrosine (Y), arginine (R), or tryptophan (W). In some embodiments, the input residue with the smaller side chain volume is serine (S), alanine (A), valine (V), or threonine (T).

[0107] In some embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366, L368, and Y407 (hole mutation modification, also referred to as a hole modification), and the Fc2 comprises T366 (knob mutation modification, also referred to as a Knok modification), or the Fc1 comprises T366 (knob mutation modification) and the Fc2 comprises at least one or at least two amino acid mutations selected from T366, L368, and Y407 (hole mutation modification).

[0108] In some specific embodiments, the Fc1 comprises at least one or at least two amino acid mutations selected from T366S, L368A, and Y407V (hole mutation modifications) and the Fc2 comprises T366W (knob mutation modification), or the Fc1 comprises T366W (knob mutation modification) and the Fc2 comprises at least one or at least two amino acid mutations selected from T366S, L368A, and Y407V (hole mutation modifications).

[0109] In some specific embodiments, the IL-2 or variant thereof is in a polypeptide chain in the presence of Fc1.

[0110] In some specific embodiments, in Fc1 and Fc2, for example, CH3 may comprise a mutation of a native non-cysteine ​​to a cysteine, e.g., Fc1 comprises S354C and Fc2 comprises Y349C, or Fc1 comprises Y349C and Fc2 comprises S354C.

[0111] In some specific embodiments, Fc1 and Fc2, for example within the Fc1 CH3 / Fc2 CH3 interface, comprise the following amino acid mutations or combinations thereof: T366Y / Y407T, T366W / Y407A, T366Y / Y407T, T394W / F405A, T366Y / F405A, T394W / Y407T, T366W / F405W / T394S / Y407A, F405W / T394S, D399C / K392C, T366W / T366S / L368A / Y407V, T366W / D399C / T366S / L368A / K392C / Y407V, T366W / K392C / T366S / D399C / L368A / Y407V, S354C / T366W / Y349C / T366S / L368A / Y407V, Y349C / T366W / S354C / T366S / L368A / Y407V, E356C / T366W / Y349C / T366S / L368A / Y407V, Y349C / T366W / E356C / T366S / L368A / Y407V, E357C / T366W / Y349C / T366S / L368A / Y407V, and Y349C / T366W / E357C / T366S / L368A / Y407V.

[0112] In some specific embodiments, Fc1 and Fc2 further comprise amino acid mutations that form an electrostatic interaction interface between Fc1 and Fc2 (e.g., CH3 and CH3). The amino acid mutations that form the electrostatic interaction interface are, for example, selected from the following: K370E / D399K / K439D / D356K / E357K / K409D, K409D / D399K, K409E / D399K, K409E / D399R, K409D / D399R, D339K / E356K, D399K / E356K / K409D / K392D, D399K / E356K / K409D / K439D, D399K / E357K / K409D / K370D, D399K / E356K / E357K / K409D / K392D / K370D, D399K / E357K / K409D / K392D, K392D / K409D / D399K, and K409D / K360D / D399K.

[0113] In some embodiments, Fc1 and / or Fc2 comprise domains from different antibody subtypes, eg, from different antibody subtype CH3s.

[0114] Furthermore, the present disclosure incorporates approaches in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, and WO2013096291 for enhancing heterodimerization by modifying the CH3 region of the Fc region.

[0115] In some embodiments, Fc1 and / or Fc2, e.g., CH3, include amino acid mutations to modify effector functions. The effector functions of the antibody include, but are not limited to, C1q binding and complement-dependent cytotoxicity, Fc receptor (e.g., FcγR) binding, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Amino acid mutations that modify effector functions are, for example, selected from the following: S298A / E333A / K334A, S239D / I332E / A330L, S239D / I332E / G236A, G236A / S239D / A330L / I332E, F243L / R292P / Y300L / V305I / P396L, K326A / E333A, K326W / E333S, K326M / E333S, C221D / D222C, S267E / H268F / S324T, E345R, S298A / E333A / K334A / N434A, E294 deletion / T307P / N434Y, T256N / A378V / S383N / N434Y, T252L / T253S / T254F, M252Y / S254T / T256E, M428L / N434S, L234A / L235A, S228P / L235E, L234A / L235A / P331S, L234A / L235A / P329G, D265A / E233P, H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A300S / P331S, L234A / L235A / G237A / P238S / H268A / V309L / A300S / P331S, S228P / F234A / L235A, D270A / P329A, L234F / L235E, L234F / L235E / P331S, F241A / V264A / D265A, N297G / D265A, and L234Y / G236W / S298A.

[0116] In some specific embodiments, the Fc1 and / or the Fc2 comprise a mutation that reduces effector function (eg, ADCC), e.g., L234A / L235A, L234A / L235A / P329G.

[0117] In some embodiments, Fc1 and Fc2, e.g., CH3 thereof, comprise amino acid mutations to modify (e.g., extend) half-life. In some embodiments, the half-life is determined by FcRn binding affinity. Extending the half-life allows for lower drug dosages and / or less frequent administration to patients. In some specific embodiments, the Fc region comprises M252Y, S254T and / or T256E mutations. In some embodiments, the immune complex comprises a PEGylation modification or is conjugated / fused to (human serum) albumin, an albumin binding protein.

[0118] In some embodiments, Fc1 and / or Fc2, e.g., CH3, include one or more allotypic mutations, hi some embodiments, the allotypic mutations are D356E and L358M.

[0119] In some embodiments, Fc1 and / or Fc2 comprise a C220 mutation, e.g., a C220S mutation.

[0120] In some embodiments, the immunoconjugate comprises a combination of polypeptide chains of the amino acid sequences SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47, 44 and 48, SEQ ID NOs: 47, 46 and 48, SEQ ID NOs: 49, 50 and 48, SEQ ID NOs: 51 and 46, and SEQ ID NOs: 102 and 103, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of the above polypeptide chain combinations.

[0121] Exemplary Immunoconjugates For immunoconjugates, the present disclosure provides embodiments such as: Embodiment 1. 1) comprising a PD-1 binding domain and an IL-2 mutant, the IL-2 mutant comprising a mutation selected from N88D or D20A; or 2) comprising a PD-1 binding domain and an IL-2 variant, wherein the IL-2 variant has reduced or eliminated affinity for the high affinity receptor (IL-2Rα / β / γ) compared to wild-type IL-2 and has reduced affinity for the intermediate affinity receptor (IL-2Rβ / γ) compared to wild-type IL-2, and wherein the affinity for the high affinity receptor is reduced more than the affinity for the intermediate affinity receptor, e.g., the IL-2 variant comprises a mutation selected from N88D or D20A; immune complex.

[0122] Embodiment 2. The IL-2 variants include mutations selected from F42, Y45, L72, or any combination thereof; For example, the mutations include those selected from F42A, Y45A, L72G, or any combination thereof; Further, the present invention further includes, for example, the following mutations: F42A / L72G, F42A / Y45A, Y45A / L72G, F42A / Y45A / L72G. 2. The immunoconjugate of embodiment 1.

[0123] Embodiment 3. the IL-2 variant comprises a mutation selected from N26Q, N29S, N30S, N71Q, Q11C and L132C, L70C and P82C, G27C and F78C, or any combination thereof; for example, including mutations or combinations selected from N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, and N26Q / N30S / G27C / F78C; 3. The immune complex of embodiment 1 or 2.

[0124] Embodiment 4. an antigen-binding domain; and IL-2 or a variant thereof, the IL-2 variant comprising a first type of mutation that confers on the IL-2 variant an affinity for the intermediate affinity receptor (IL-2Rβ / γ) that is reduced relative to wild-type IL-2; For example, the first type mutation is selected from L12, E15, H16, L18, L19, D20, M23, Q22, K43, V69, D84, S87, N88, V91, I92, E95, T123, Q126, S127, I129, and S130; Furthermore, for example, the first type mutations include L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D selected from 84G, D84A, D84T, D84P, N88D, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, and S127Q; Also, for example, the first type mutation is selected from N88D, N88R, D20A, D20N, V91T, Q126D, and H16A / D84S; Further, for example, the first mutation is selected from N88D or D20A; Immune complex.

[0125] Embodiment 5. the IL-2 variant comprises a second type of mutation that confers on the IL-2 variant an affinity for the high affinity receptor (IL-2Rα / β / γ) that is reduced or eliminated compared to wild-type IL-2; For example, the second type of mutation is selected from R38, E62, P65, E68, F42, Y45, L72, or any combination thereof; Furthermore, for example, the second type of mutation is selected from R38A, R38D, R38E, E62Q, P65R, P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K; Further, for example, the second type of mutation is selected from F42A, Y45A, L72G, F42A / L72G, F42A / Y45A, Y45A / L72G, or F42A / Y45A / L72G; 5. The immunoconjugate of embodiment 4.

[0126] Embodiment 6. the IL-2 variant comprises a third mutation that confers increased stability to the IL-2 variant relative to wild-type IL-2; For example, the third type mutation is selected from N26, N29, N30, N71, Q11, L132, L70, P82, G27, F78, or any combination thereof; Furthermore, for example, the third type of mutation is selected from N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, G27C / F78C, or any combination thereof; Further, for example, the third type mutation is selected from N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, and N26Q / N30S / G27C / F78C; 6. The immune complex of embodiment 4 or 5.

[0127] Embodiment 7. a PD-1 binding domain and IL-2 or a variant thereof; The PD-1 binding domain comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprising: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 13 to 15, and 17 to 35, or It contains CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2 and 16, wherein CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; For example, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the above immunoglobulin single variable domains are respectively: As shown in SEQ ID NO: 7, 36, 37, or Represented by SEQ ID NOs: 4, 5, and 6, For example, the immunoglobulin single variable domain has an amino acid sequence of CDR1 represented by SEQ ID NO: 7, an amino acid sequence of CDR2 represented by any one of SEQ ID NOs: 8, 38 to 40, and an amino acid sequence of CDR3 represented by any one of SEQ ID NOs: 9, 41, and 42, Further, for example, the above immunoglobulin single variable domains have amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 7, 38, and 41, respectively. immune complex.

[0128] Embodiment 8. The IL-2 variants include a combination of mutations selected from group 1) and group 2): the first) group of mutations is selected from F42A / L72G, F42A / Y45A, Y45A / L72G and F42A / Y45A / L72G; Group 2) mutations are selected from N88R, N88G, N88I, N88D, D20H, D20Y, V91T, Q126L, Q126D, and H16A / D84S; For example, the IL-2 mutants include the following combinations of mutations: F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / L72G / Q126D, F42A / L72G / H16A / D84S, The immune complex according to any one of embodiments 1 to 7.

[0129] EMBODIMENT 9. the IL-2 variant further comprises a mutation of group 3), the mutations of group 3 being selected from N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C and N26Q / N30S / G27C / F78C; For example, the IL-2 mutant may be F42A / L72G / N88D / N26Q / N29S / N71Q, F42A / L72G / N88R / N26Q / N29S / N71Q, F42A / L72G / D20A / N26Q / N29S / N71Q, F42A / L72G / D20N / N26Q / N29S / N71Q, F42A / L72G / V91T / N26Q / N29S / N71Q, F42A / L72G / Q126D / N26Q / N29S / N71Q, Contains the combination of mutations F42A / L72G / H16A / D84S / N26Q / N29S / N71Q The immunoconjugate of embodiment 8.

[0130] Embodiment 10. The IL-2 variant is selected from T3 and / or C125 or a combination thereof; For example, including T3A and / or C125A, C125S, C125T, C125V mutations. The immune complex according to any one of embodiments 1 to 9.

[0131] Embodiment 11. The immune complex according to any one of embodiments 1 to 10, wherein the IL-2 mutant has an amino acid sequence shown in any one of SEQ ID NOs: 60 to 68.

[0132] Embodiment 12. 1) The above antigen is 1-1) Antigens on tumor cells or immune cells in the tumor microenvironment, 1-2) PD-1, CTLA-4, LAG3, TIM3, 4-1BB, OX40, GITR, CD8a, CD8b, CD4, NKp30, NKG2A, TIGIT, TGFβR1, TGFβR2, Fas, NKG2D, NKp46, PD-L1, CD107a, ICOS, TNFR2, FAP, TNC A1, TNC A2, CEA, EDB, MCSP or CD16a, 1-3) Immune checkpoint antigens, such as PD-1, PD-L1, CTLA-4, Selected from 2) The antigen-binding domain is PD-1 antibodies (e.g., Pembrolizumab, Nivolumab, Sintilimab, Tislelizumab, Cemiplimab, Toripalimab, Camrelizumab, Penprimimab, Zimberelimab, Sasanlimab, Spartalizumab) (Spartalizumab), Retifanlimab, Balstilimab, Cetrelimab, Dostarlimab , TSR-042), CS-1003, SCT-I10A, SSI-361, MEDI-0680(AMP-514), BAT-1306, JTX-4014, JTX-4014, BI-754091, AK-103), CTLA-4 antibodies (e.g., Tremelimumab, Ipilimumab), 4-1BB antibodies (e.g., Urelumab, Utomilumab), PD-L1 antibodies (e.g., Durvalumab, Atezolizumab, Durvalumab, Avelumab, Adebrelimab, Envafolimab, STI-A1110, CS-1001, TQB-2450, KL-A167, IMC-001, CX-072), The immune complex according to any one of embodiments 4 to 6 and 8 to 10, comprising a VH domain and a VL domain, or HCDR1, HCDR2, and HCDR3 of VH and LCDR1, LCDR2, and LCDR3 of VL, of an antibody selected from the group consisting of:

[0133] Embodiment 13. The PD-1 binding domain comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprising: CDR1, CDR2 and CDR3 in an amino acid sequence represented by any one of SEQ ID NOs: 3, 13 to 15, 17 to 35, and 88 to 92, or It contains CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 2 and 16, wherein CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; For example, according to the Kabat numbering system, the amino acid sequences of CDR1, CDR2 and CDR3 of the above immunoglobulin single variable domains are respectively: Represented by SEQ ID NOs: 97, 100, and 101; Represented by SEQ ID NOs: 7, 36, and 37; As shown in SEQ ID NO: 97, 98, 99, or Represented by SEQ ID NOs: 4, 5, and 6, Also, for example, the immunoglobulin single variable domain has an amino acid sequence of CDR1 represented by SEQ ID NO: 7 or 93, an amino acid sequence of CDR2 represented by any one of SEQ ID NOs: 8, 38 to 40, and 94 to 95, and an amino acid sequence of CDR3 represented by any one of SEQ ID NOs: 9, 41, 42, and 96, Also, for example, the immunoglobulin single variable domain may be 1) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 41; 2) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 96; 3) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 94 and 41; 4) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 94 and 41; 5) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 94 and 96; 6) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 95 and 96; 7) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 9; 8) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 42; 9) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 6; 10) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 9; 11) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 6; 12) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 41; 13) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 42; 14) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 41; 15) comprising CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 42; 16) comprising CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 41; 17) comprising CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 42; 18) containing CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 41; and 19) comprising CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 42; Any one selected from Further, for example, the above immunoglobulin single variable domains have amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NOs: 7, 38, and 41, respectively. The immune complex according to any one of embodiments 1 to 3 and 12.

[0134] Embodiment 14. The immunoglobulin single variable domain comprises: 1) modified by humanization, affinity maturation, removal of T cell epitopes, reducing antibody deamidation, and / or reducing antibody isomerization, for example, the heavy chain framework region of the human germline template used in the humanization modification process is IGHV3-23*01 or IGHV3-23*04; 2) A camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof; 14. The immunoconjugate of embodiment 7 or 13.

[0135] EMBODIMENT 15. The immunoglobulin single variable domain comprises: An amino acid sequence represented by any one of SEQ ID NOs: 3, 13 to 15, 17 to 35, and 88 to 92, or An amino acid sequence shown in any one of SEQ ID NOs: 2 and 16, or Contains an amino acid sequence having at least 90% sequence identity to any one of the above sequences, 15. The immunoconjugate of embodiment 13 or 14.

[0136] Embodiment 16. the PD-1-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences set forth in SEQ ID NOs: 54, 55, and 56, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences set forth in SEQ ID NOs: 57, 58, and 59, For example, the heavy chain variable region is a heavy chain variable region in a Fab heavy chain having an amino acid sequence as shown in SEQ ID NO: 52, and the light chain variable region comprises a light chain variable region in a Fab light chain having an amino acid sequence as shown in SEQ ID NO: 53. The immune complex according to any one of embodiments 1 to 3 and 12.

[0137] EMBODIMENT 17. The immune complex according to any one of the preceding embodiments, further comprising a human immunoglobulin Fc region, e.g., said Fc region is a human IgG1, IgG2, IgG3 or IgG4 Fc region.

[0138] Embodiment 18. the Fc region of said human IgG1 comprises one or more amino acid mutations that reduce binding to an Fc receptor (FcR, e.g., FcγR) and / or reduce effector function; For example, the Fc region of the human IgG1 comprises amino acid mutations that reduce binding to one or more Fcγ receptors (FcγR) and / or reduce ADCC effect, Furthermore, for example, the Fc region of human IgG1 contains mutations at L234, L235, P329, or any combination thereof, Further, for example, the Fc region of the human IgG1 comprises L234A / L235A or L234A / L235A / P329G mutations. 18. The immunoconjugate of embodiment 17.

[0139] EMBODIMENT 19. the Fc region comprises a knob-into-hole modification that promotes binding (or heterodimerization) of the first and second subunits of the Fc region; As described herein, the knob-into-hole modification refers to, for example, replacing an amino acid residue in the CH3 region of a first subunit of the Fc region with an amino acid residue having a larger side chain volume, such that a protrusion is generated in the CH3 region of the first subunit that can fit into a cavity in the CH3 region of the second subunit, and replacing an amino acid residue in the CH3 region of a second subunit of the Fc region with an amino acid residue having a smaller side chain volume, such that a cavity is generated in the CH3 region of the second subunit to fit the protuberance in the CH3 region of the first subunit, and, for example, the amino acid residue having the larger side chain volume is selected from R, F, Y, W, and the amino acid residue having the smaller side chain volume is selected from A, S, T, V. 19. The immunoconjugate of embodiment 17 or 18.

[0140] EMBODIMENT 20. the first subunit of the Fc region comprises a T366 mutation, and the second subunit comprises a mutation selected from T366, L368, Y407, or any combination thereof; a first subunit of the Fc region comprises a mutation selected from S354 or E356 and a second subunit of the Fc region comprises a mutation selected from Y349; or a first subunit of the Fc region comprises S354 and T366 mutations, and a second subunit comprises Y349, T366, L368 and Y407 mutations; for example, the first subunit of the Fc region comprises a T366W mutation, and the second subunit comprises a mutation selected from T366S, L368A, Y407V, or any combination thereof; a first subunit of the Fc region comprises a mutation selected from S354C or E356C and a second subunit of the Fc region comprises a mutation selected from Y349C; or The first subunit of the Fc region comprises an S354C / T366W mutation and the second subunit comprises a Y349C / T366S / L368A / Y407V mutation; 20. The immunoconjugate of embodiment 19.

[0141] EMBODIMENT 21. The immune complex according to embodiment 19 or 20, wherein the second subunit of the Fc region further comprises a H435 and / or R436 mutation, preferably a H435R / Y436F, H435R, H435K mutation.

[0142] EMBODIMENT 22. the Fc region of said human IgG1 comprises a mutation that enhances FcRn-mediated recycling and / or extends half-life; For example, the human IgG1 Fc region comprises the M252 and / or M428 mutations, For example, the Fc region of the human IgG1 includes the M252Y / M428V and M252Y / M428L mutations. The immune complex according to any one of embodiments 17 to 21.

[0143] EMBODIMENT 23. The immune complex according to any one of embodiments 17 to 22, wherein the first subunit and / or the second subunit of the Fc region of human IgG1 comprises a C220 mutation, for example, a C220S mutation.

[0144] EMBODIMENT 24. the PD-1-binding domain or antigen-binding domain is at the N-terminus of the Fc region, and the IL-2 variant is at the N-terminus or C-terminus of the Fc region; For example, the PD-1 binding domain or antigen binding domain is at the N-terminus of the Fc region and the IL-2 variant is at the C-terminus of the Fc region. 10. The immune complex according to any one of the preceding embodiments.

[0145] EMBODIMENT 25. the valency ratio of the PD-1-binding domain or antigen-binding domain to IL-2 or its variant is between 6:1 and 1:3 (e.g., 4:1 and 1:2), preferably 4:1, 2:1, 3:1 or 1:1; or an immune complex contains one or two IL-2 or its variants and one, two, three, four, five or six PD-1-binding domains or antigen-binding domains. 10. The immune complex according to any one of the preceding embodiments.

[0146] 26. The following I) to VI), namely: I) First polypeptide chain: [PD-1 binding domain 1]-[First subunit of Fc region]-[Linker 1]-[IL-2 or a variant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[second subunit of Fc region], II) First polypeptide chain: [PD-1 binding domain 1]-[Linker 2]-[PD-1 binding domain 1]-[First subunit of Fc domain]-[Linker 1]-[IL-2 or a variant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[second subunit of Fc domain]; The present invention relates to a combination of polypeptide chains represented by any one of the following: III) First polypeptide chain: [Fab heavy chain of PD-1 binding domain 2]-[First subunit of Fc region]-[Linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[second subunit of Fc region], Third polypeptide chain: [Fab light chain of PD-1 binding domain 2], IV) first polypeptide chain: [Fab heavy chain of PD-1 binding domain 2]-[first subunit of Fc region]-[linker 1]-[IL-2 or a variant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[second subunit of Fc domain]; Third polypeptide chain: [Fab light chain of PD-1 binding domain 2], V) First polypeptide chain: [PD-1 binding domain 1]-[Linker 2]-[Fab heavy chain of PD-1 binding domain 2]-[First subunit of Fc region]-[Linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[Fab light chain of PD-1 binding domain 2]-[second subunit of Fc region]; Third polypeptide chain: [Fab light chain of PD-1 binding domain 2], VI) first polypeptide chain: [IL-2 or a variant thereof]-[linker 3]-[first subunit of the Fc region]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[second subunit of Fc domain]; The present invention relates to a combination of polypeptide chains represented by any one of the following: wherein - represents a peptide bond, linker is a polypeptide capable of fulfilling a linking function, linker 1, linker 2, and linker 3 may be the same or different, [PD-1-binding domain 1] is any PD-1-binding domain selected from embodiments 11 to 13, and [PD-1-binding domain 2] is any PD-1-binding domain selected from embodiment 14; For example, the linker is (G x S) y linkers, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6, and for example, linker 1 is (G4S)3 (SEQ ID NO: 104), linker 2 is (G4S)2 (SEQ ID NO: 105), and linker 3 is G4S (SEQ ID NO: 106); The immune complex according to any one of embodiments 1 to 3, 7, and 11 to 25.

[0147] Some other linkers include (G m S n ) h or (GGNGT) h (SEQ ID NO: 107) or (YGNGT) h (SEQ ID NO: 108) or (EPKSS) h(SEQ ID NO: 109), but is not limited to, wherein m and n are each independently selected from integers of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers of 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0148] The order of the polypeptide chain is from N-terminus to C-terminus.

[0149] EMBODIMENT 27. SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46, SEQ ID NOs: 47, 44 and 48, SEQ ID NOs: 47, 46 and 48, SEQ ID NOs: 49, 50 and 48, SEQ ID NOs: 51 and 46, and SEQ ID NOs: 102 and 103, A combination of polypeptide chains having the amino acid sequence 10. The immune complex according to any one of the preceding embodiments.

[0150] Or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the above polypeptide chain combinations.

[0151] Unless otherwise stated, the numbering convention for all of the above sites is the Kabat EU index.

[0152] The present disclosure provides a fusion protein that comprises or is any of the above immunoconjugates.

[0153] Polynucleotides and Vectors The present disclosure provides polynucleotides encoding the PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, and immunoconjugates of the present disclosure. The nucleic acids of the present disclosure may be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the nucleic acids of the present disclosure are essentially isolated nucleic acids.

[0154] The nucleic acids of the present disclosure may be in the form of, present in, and / or part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, i.e., a vector that allows for the expression of the PD-1 antibody or antigen-binding fragment thereof in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors generally contain at least one nucleic acid of the present disclosure, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory elements and other elements useful or necessary for expression of the PD-1 antibody or antigen-binding fragment thereof of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0155] The nucleic acids according to the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptide according to the present disclosure, and / or may be isolated from a suitable natural source.

[0156] host cell The present disclosure provides recombinant host cells that express or are capable of expressing one or more of the PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, immunoconjugates of this disclosure, and / or that comprise a nucleic acid or vector of this disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0157] Bacterial cells include, for example, cells of gram-negative strains (e.g., Escherichia coli, Proteus and Pseudomonas strains) and gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus and Lactococcus strains).

[0158] Fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0159] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0160] However, the present disclosure may be used with amphibian cells, insect cells, plant cells and any other cells known in the art for expressing heterologous proteins.

[0161] Preparation method The present disclosure provides a method for preparing a PD-1 antibody or antigen-binding fragment thereof, an IL-2 variant, or an immunoconjugate, comprising expressing the target protein in a host cell as described above and isolating the target protein from the host cell. Optionally, a purification step may be included, for example, purification on an A or G Sepharose FF column with a conditioned buffer, washing out non-specifically bound components, and then eluting the bound antibody by a pH gradient method, detecting it by SDS-PAGE, and collecting it. Optionally, filtering and concentrating it by conventional methods may be used. Soluble compounds and polymers may be removed by conventional methods such as molecular sieving, ion exchange, etc. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0162] Methods for producing and purifying antibodies are well known in the art and can be found, for example, in the Cold Spring Harbor Antibody Laboratory Techniques Manual (Chapters 5-8 and 15).

[0163] The engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in a bioreactor to produce the antibody. The culture medium into which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can be removed by conventional methods such as molecular sieving, ion exchange, etc.

[0164] composition The present disclosure provides compositions comprising the above-described PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants and / or immunoconjugates, for example pharmaceutical compositions comprising an effective amount of the above-described PD-1 antibodies or antigen-binding fragments thereof, immunoconjugates, and at least one medicamentable excipient, diluent, or vector for treating, ameliorating, or preventing cancer.

[0165] In some specific embodiments, the pharmaceutical composition may contain 0.01-99% by weight of the PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, and / or immune complex per unit dose, or the content of the PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, and / or immune complex per unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0166] In some embodiments, an article of manufacture or product is provided that includes the PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, and / or immunoconjugate. Optionally, the article of manufacture includes a container and a label. The container is, for example, a vial, syringe, and test tube. The container holds a composition that is effective for treating a medical condition. A label on or associated with the container indicates that the composition is used for treating a selected medical condition. The composition includes the PD-1 antibody or antigen-binding fragment thereof, immunoconjugate.

[0167] For example, any PD-1 antibody or antigen-binding fragment thereof provided herein; Any of the IL-2 variants provided in the present disclosure; Any of the immunoconjugates provided herein; A composition comprising:

[0168] Optionally, when the composition is a pharmaceutical composition, it further comprises at least one medicamentable excipient, diluent or vector.

[0169] Methods of Treatment and Pharmaceutical Use The present disclosure relates to 1) To treat cancer, 2) Preparation of drugs for treating cancer; 3) To activate cytotoxic T lymphocytes (CTLs), 4) Preparation of agents for activating cytotoxic T lymphocytes (CTLs); 5) CD4 + and / or CD8 + To promote T cell proliferation 6) CD4 + and / or CD8 + Preparation of an agent for promoting the proliferation of T cells; 7) To confer a higher cell activation efficiency to cytotoxic T lymphocytes (CTLs) with high expression of PD-1 antibody than to cytotoxic T lymphocytes (CTLs) with low or no expression of PD-1 antibody, 8) CD4 with high expression of PD-1 antibody + and / or CD8 + T, CD4 + and / or CD8 + To confer higher cell proliferation efficiency than T cells, The present invention also provides uses of the above-mentioned PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, immunoconjugates, polynucleotides, and compositions (including pharmaceutical compositions) in at least one of the following:

[0170] The present disclosure provides methods in which the above-mentioned PD-1 antibodies or antigen-binding fragments thereof, IL-2 variants, immunoconjugates, polynucleotides, and compositions (including pharmaceutical compositions) can be used to treat, ameliorate, prevent, or diagnose a disease or condition.

[0171] In some embodiments, methods are provided for ameliorating, alleviating, treating, or preventing a disease, comprising administering to a subject an ameliorating, alleviating, treating, or preventing effective amount of the above-described PD-1 antibody or antigen-binding fragment thereof, immunoconjugate, polynucleotide, or composition (including pharmaceutical composition).

[0172] In some embodiments, there is provided a use of a PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, or immunoconjugate of the present disclosure for the preparation of a medicament for ameliorating, alleviating, treating, or preventing a disease.

[0173] In some embodiments, the disease is a proliferative condition or any other disease or condition characterized by uncontrolled cell proliferation (e.g., cancer, although in this disclosure cancer and tumor may be used interchangeably), such as a related disease associated with overexpression of PD-L1 or that would benefit from treatment with a PD-1 antibody or antigen-binding fragment thereof, such as cancer.

[0174] In some embodiments, the cancer is a solid tumor or a hematological tumor.

[0175] In some embodiments, the cancer is advanced or metastatic.

[0176] In some embodiments, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell proliferation, or a combination thereof.

[0177] For example, the cancer may be selected from prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous cell and basal cell carcinoma), glioma, renal cell cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel Cells cancer, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS).

[0178] For example, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, liver tumor, intraepithelial neoplasia, kidney cancer or renal cancer, laryngeal cancer, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, The cancer is selected from adenocarcinoma, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small nonlytic cell NHL, macroma NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloblastic leukemia.

[0179] In some embodiments, the subject has a condition associated with PD-L1, hi some specific embodiments, the subject's condition includes cancers that aberrantly express PD-L1 or that do not aberrantly express PD-L1, and includes non-metastatic or non-invasive and invasive or metastatic cancers.

[0180] In some embodiments, a method of activating the immune system of a subject is provided, comprising activating cytotoxic T cells (CTLs) in a subject in vivo or in vitro, the method comprising: + and / or CD8 +provided are methods for increasing T cell proliferation in vivo or in vitro; provided are methods for activating NK cells in vivo or in vitro in a subject; provided are methods for activating γδ T cells in a subject; provided are methods for activating Th1 cells in vivo or in vitro in a subject; provided are methods for activating, reducing or eliminating the number and / or activity of at least one type of regulatory T cells (Treg) in a subject; provided are methods for increasing IFN-γ production and / or secretion of pro-inflammatory cytokines in a subject; provided are methods for blocking or inhibiting the interaction of PD-1 and PD-L1 / PD-L2 in a subject, all of which comprise administering to the subject an effective amount of the PD-1 antibody or antigen-binding fragment thereof, IL-2 variant, immunoconjugate, polynucleotide, or composition (including pharmaceutical composition) according to the present disclosure.

[0181] detection The present disclosure provides detective uses of PD-1 antibodies or antigen-binding fragments thereof, immunoconjugates, polynucleotides, and compositions. The present disclosure further provides methods, systems, or devices for detecting PD-1 in vivo or in vitro, comprising treating a sample with the binding proteins, polynucleotides, and compositions of the present disclosure.

[0182] In some embodiments, the in vitro detection method, system, or device includes, for example, (1) contacting the sample with a PD-1 antibody or antigen-binding fragment thereof, immunoconjugate, polynucleotide, or composition; (2) detecting a complex formed between the binding protein, the polynucleotide and the sample; and / or (3) contacting a reference sample (e.g., a control sample) with a binding protein, a nucleic acid, and (4) Ascertaining the extent of compound formation by comparison with a reference sample; For example, a change (e.g., a statistically significant change) in complex formation in the sample or subject compared to that in a control sample or subject indicates the presence of PD-1 in the sample.

[0183] In some embodiments, the in vivo detection method, system, or device comprises: (1) administering to a subject the binding protein or polynucleotide; (2) detecting the formation of a complex between the binding protein, the polynucleotide and the target binding; and may include:

[0184] Detection may include determining the location or time of complex formation. Detection of the binding protein or polynucleotide (e.g., PD-1) is achieved by labeling the binding protein or polynucleotide with a detectable substance and detecting the label. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Formation of a complex between the binding protein or polynucleotide and PD-1 can be detected by measuring or visualizing the substance that binds or does not bind to PD-1. For example, conventional detection assays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry can be used. For detection purposes, the binding protein or polynucleotide of the present disclosure can be labeled with a fluorophore.

[0185] In some embodiments, a reagent kit is further provided, which includes the binding protein, the polynucleotide, and may further include a diagnostic manual. The reagent kit may further include at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the antibody may be prepared as a pharmaceutical composition. [Brief description of the drawings]

[0186] [Figure 1A-1B]These are the results of detecting the activity of anti-PD-1 nanobodies using the PD-1 / PD-L1 NFAT reporter gene system. Pembrolizumab was used as a positive control and human IgG4 was used as a negative control. Figure 1A shows the results for A6_IgG4 and A17_IgG4, and Figure 1B shows the results for A17h1_IgG4, A17m09_hIgG4, A17m0901_hIgG4, A17m0902_hIgG4, A17m0903_hIgG4, and A17m0905_hIgG4. [Diagram 2] DC cells: This was to detect the degree of activation of A17m09_hIgG4 in T cells by the release of cytokines during T-cell mixed lymphocyte reaction. Pembrolizumab was used as a positive control and hIgG4 was used as a negative control. [Figure 3A-3B] 3 shows the results of an in vivo efficacy study of A17m09_hIgG4 in inhibiting the growth of mouse M38 colon cancer tumors. FIG. 3A shows the results of the mouse tumor volume, and FIG. 3B shows the mouse body weight. Pembrolizumab was used as a positive control, and hIgG4 was used as a negative control. [Figure 4]The construction of a fusion protein between an anti-PD-1 antibody and an IL-2 mutant. The molecular structure shown in Figure 4A shows that the anti-PD-1 nanobody A17m0902 is coupled to the N-terminus of the knob and hole chains via the IgG1 heavy chain hinge region, and the molecular structure shown in Figure 4B shows that two anti-PD-1 nanobodies A17m0902 are linked end-to-end with a (G4S)2 linker, and their C-terminus is coupled to the N-terminus of the knob and hole chains via the IgG1 heavy chain hinge region. The molecular structure shown in FIG. 4C is that the anti-PD-1 nanobody A17m0902 is coupled to the N-terminus of the hole chain via the IgG1 heavy chain hinge region, and the heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab is coupled to the N-terminus of the knob chain via the IgG1 heavy chain hinge region. The molecular structure shown in FIG. 4D is that two anti-PD-1 nanobodies A17m0902 are connected end-to-end via a (G4S)2 linker. and its C-terminus is coupled to the N-terminus of the hole chain via an IgG1 heavy chain hinge region, and the heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab is coupled to the N-terminus of the knob chain via an IgG1 heavy chain hinge region; the molecular structure shown in E of Figure 4 is coupled to the N-terminus of the knob and hole chains via an IgG1 heavy chain hinge region, respectively, and anti-PD-1 nanobody A17m0902 is coupled to the N-terminus of the heavy chain of the Fab fragment corresponding to pembrolizumab via a (G4S)2 linker; and the molecule F-V91T shown in F of Figure 4 is coupled to two anti-PD-1 nanobodies A17m0902 end-to-end via a (G4S)2 linker, and its C-terminus is coupled to the N-terminus of the hole chain via an IgG1 heavy chain hinge region. In molecules A to E in Figure 4, an IL-2 mutant is linked to the C-terminus of the knob chain of Fc via a (G4S)3 linker, and in molecule F, IL-2v(V91T) is coupled to the N-terminus of the knob chain via a G4S linker. [Figure 5A-5B]Figure 5 shows the results of detecting the proliferation activity of different forms of PD-1-IL-2v(V91T) fusion proteins in M07e and M07e-hPD-1 cells, where Figure 5A shows the results of the proliferation activity of different forms of PD-1-IL-2v(V91T) fusion proteins in M07e-hPD-1 cells, and Figure 5B shows the results of the proliferation activity of different forms of PD-1-IL-2v(V91T) fusion proteins in M07e cells. [Figure 6A-6B] Figure 6 shows the results of detecting the proliferation activity of M07e and M07e-hPD-1 cell lines by the same type of PD-1-IL-2v fusion protein with different IL-2 variants, where Figure 6A shows the result of proliferation activity of PD-1-IL-2v fusion protein in M07e-hPD-1 cells, and Figure 6B shows the result of proliferation activity of PD-1-IL-2v fusion protein in M07e cells. [Figure 7] 1 shows the results of detecting the proliferation-promoting activity of PD-1-IL-2v or IgG-IL-2v on M07e-hPD-1 cells. [Figure 8A-8B] Figure 8 shows the results of detecting the proliferation activity of unstimulated T cells by PD-1-IL-2v with different IL-2 variants, where Figure 8A shows the proliferation results of CD4+ T cells and Figure 8B shows the proliferation results of CD8+ T cells. [Figure 9A-9B] Figure 9 shows the results of detecting the proliferation activity of T cells stimulated with anti-CD3 antibody by PD-1-IL-2v carrying different IL-2 variants, where Figure 9A shows the proliferation results of CD4+ T cells and Figure 9B shows the proliferation results of CD8+ T cells. [Figure 10] Figure 1 shows the results of detecting the proliferation activity of PD-1-IL-2v carrying different anti-PD-1 nanobody variants on the M07e-hPD-1 cell line. [Figure 11] Figure 1 shows the inhibition of tumor growth in a mouse CT26 tumor model by A-N88D, with A17m0902_hIgG4 as a control. [Figure 12] 1 shows the change in body weight of a mouse CT26 tumor model after administration of A-N88D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0187] Definition of Terms In order that this disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0188] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0189] As used herein, the term "immunoconjugate" refers to a fusion polypeptide molecule comprising an effector moiety and at least one antigen-binding moiety, optionally comprising an Fc region. In some embodiments, the immune complex comprises an effector moiety, an antigen-binding moiety and an Fc region. In some embodiments, the immune complex comprises an effector moiety, two antigen-binding moieties and two Fc regions. In some embodiments, the immune complex comprises an effector moiety, four antigen-binding moieties and two Fc regions. In some embodiments, the immune complex comprises an effector moiety, an antigen-binding moiety and two Fc regions. In some embodiments, the immune complex comprises an effector moiety, three antigen-binding moieties and two Fc regions.

[0190] In a specific embodiment, the immunoconjugates described herein are fusion proteins, i.e., the components of the immunoconjugate are linked to each other directly by peptide bonds or via linkers.

[0191] In a specific embodiment herein, the antigen-binding moiety in said "immunoconjugate" is a PD-1 binding domain and said effector moiety is IL-2 or a variant thereof.

[0192] "Programmed death 1", "cellular programmed death 1", "protein PD-1", "PD-1", "PDCD1" and "hPD-1" may be used interchangeably and include variants, isotypes, species homologs, and analogs of human PD-1 that share at least one common epitope with PD-1. The complete PD-1 sequence can be found at GenBank Accession No. U64863. "Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, "PD-L1" includes human PD-L1 (hPD-L1), variants, isotypes and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found at GenBank Accession No. Q9NZQ7.

[0193] As used herein, the terms "PD-1 binding domain" or "PD-1 binding protein" may be used interchangeably and refer to any protein capable of specifically binding to PD-1, or any molecule comprising said protein. The PD-1 binding protein may comprise an antibody, antigen-binding fragment thereof or complex thereof as defined in this disclosure against PD-1. The PD-1 binding protein further covers an immunoglobulin superfamily antibody (IgSF) or a CDR-grafted molecule. The "PD-1 binding protein" according to the present disclosure may comprise at least one immunoglobulin single variable domain (e.g., VHH) that binds to PD-1. In some embodiments, the "PD-1 binding protein" may comprise two, three, four or more immunoglobulin single variable domains (e.g., VHH) that bind to PD-1. A PD-1 binding protein according to the present disclosure comprises an immunoglobulin single variable domain of PD-1 and may also comprise a moiety with effector function, such as a linker and / or a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, a "PD-1 binding protein" according to the present disclosure also encompasses immunoglobulins that bind different antigens (e.g., bi / multispecific antibodies comprising a first antibody that binds a first antigen (e.g., PD-1) and a second antibody that binds a second antigen (e.g., 4-1BB), optionally comprising a third antibody that binds a third antigen, and optionally comprising a fourth antibody that binds a fourth antigen).

[0194] A "PD-1 binding protein" or "PD-1 antibody or antigen-binding fragment thereof" of the present disclosure can include one or more effector molecules, e.g., by way of conjugation. Such "effector molecules" include, for example, anti-tumor agents, drugs, toxins, biologically active proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides, particularly radioactive iodides, radioisotopes, chelated metals, nanoparticles, and reporter groups, e.g., fluorescent compounds, or compounds detectable by NMR or ESR spectroscopy. In some embodiments, the effector molecule is a cytokine (e.g., IL-2, IL-15, or variants thereof). In another embodiment, the effector molecule is a polymer, which may generally be a synthetic or naturally occurring polymer, specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, and specific naturally occurring polymers include lactose, amylose, glucan, glycogen or derivatives thereof. In some other embodiments, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. Conjugation of the polymer to the PD-1 binding protein or PD-1 antibody can be accomplished by conventional methods.

[0195] "Cytokine" is a general term for proteins released from one cell population that act on other cells as intercellular substances. Examples of such cytokines include lymphokines, monokines, chemokines, and traditional polypeptide hormones. Exemplary cytokines include human IL-2, IL-15, IFN-γ, IL-6, TNFα, IL-17, and IL-5 and variants thereof.

[0196] "Interleukin-2" or "IL-2" refers to any naturally occurring IL-2 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term covers unprocessed IL-2 and any cell-derived processed form of IL-2. The term also covers naturally occurring IL-2 variants, such as splice variants and allelic variants. An exemplary wild-type human IL-2 amino acid sequence is shown in SEQ ID NO:87. Unprocessed human IL-2 further includes an N-terminal 20 amino acid signal peptide (e.g., as shown in SEQ ID NO:272 in WO2012107417), which is absent in the mature IL-2 molecule.

[0197] "Mutated IL-2" or "IL-2 variant" refers to a polypeptide that differs from the wild-type IL-2 amino acid sequence by virtue of a mutation (eg, substitution, deletion, addition) at at least one amino acid position.

[0198] In this disclosure, when referring to IL-2 mutants, single amino acid substitutions are described as [original amino acid residue / position / substituted amino acid residue]. For example, a phenylalanine to alanine substitution at position 42 can be represented as F42A. Combination mutations at multiple given positions can be indicated by connecting each single amino acid substitution with a " / ". For example, a combination mutation at positions F42A, L72G, and N88R can be represented as F42A / L72G / N88R, which means that the mutant contains F42A, L72G, and N88R mutations.

[0199] In this disclosure, references to amino acid positions in an IL-2 protein or IL-2 sequence segment are determined by reference to SEQ ID NO: 87, the amino acid sequence of wild-type human IL-2 protein (also referred to as wt IL-2). Corresponding amino acid positions in other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by alignment of the amino acid sequences with SEQ ID NO: 87. Thus, in this disclosure, unless otherwise stated, an amino acid position in an IL-2 protein or polypeptide is an amino acid position numbered according to SEQ ID NO: 87. For example, reference to "F42" refers to the phenylalanine residue F at position 42 of SEQ ID NO: 40, or the amino acid residue at the corresponding position in other IL-2 polypeptide sequences by alignment. Sequence alignments performed to determine amino acid positions can be performed with default parameters using the Basic Local Alignment Search Tool available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0200] "CD25" or "α subunit of the IL-2 receptor" refers to any native CD25 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes "full-length" unprocessed CD25 and any cell-derived processed form of CD25, as well as naturally occurring CD25 variants, such as splice variants and allelic variants. In one embodiment, CD25 is human CD25.

[0201] "High affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, consisting of a receptor gamma subunit (also known as the common cytokine receptor gamma subunit, gammac or CD132), a receptor beta subunit (also known as CD122 or p70) and a receptor alpha subunit (also known as CD25 or p55). In contrast, "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only the gamma and beta subunits, but lacks the alpha subunit (see, e.g., Olejniczak & Kasprzak, MedSci Monit 14, RA179-189 (2008)).

[0202] "Antibody" is used in the broadest sense to cover various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies and antibody fragments (or antigen-binding fragments or antigen-binding portions). Antibody may also refer to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in amino acid composition and sequence of the heavy chain constant region, and therefore in their antigenicity. Thus, immunoglobulins can be divided into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Ig of the same type can be further divided into different subclasses depending on the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains and λ chains depending on the difference in the constant region. Each of the five types of Ig may have a κ chain or a λ chain. In the antibody heavy and light chains, the sequence of about 110 amino acids near the N-terminus is significantly changed and becomes a variable region (V region), and the remaining amino acid sequence near the C-terminus is relatively stable and becomes a constant region (C region). The variable region includes three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conservative. The three hypervariable regions determine the specificity of the antibody and are also called complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0203] "Antigen-binding fragment" covers single chain antibodies (i.e. full length heavy and light chains), Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope-binding fragments of any one of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3),209-217). In some embodiments, the antigen-binding fragment is a VHH. Methods for producing and preparing these antigen-binding fragments are known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide bond stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. Antigen-binding fragments according to the present disclosure further include the Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171. Multivalent antibodies may be multispecific, e.g. bispecific, or monospecific (see, e.g., WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) as described in WO92 / 22583.

[0204] "Fc domain" or "Fc region" is to define a C-terminal region comprising at least a portion of the constant region in an immunoglobulin heavy chain. It includes native sequence Fc regions and variant Fc regions. Although the boundaries of an IgG heavy chain Fc region may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced from host cells may be post-translationally cleaved to remove one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced from a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or may comprise a truncated variant of the full-length heavy chain (also referred to in the present disclosure as a "truncated variant heavy chain"). This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, Kabat EU index numbering). Thus, the C-terminal lysine (K447) of the Fc region, or the C-terminal glycine (G446) and lysine (K447) may or may not be present. Unless otherwise indicated, the amino acid sequence comprising the heavy chain of the Fc region (or a subunit of the Fc region as defined in this disclosure) is depicted in this disclosure without the C-terminal glycine-lysine dipeptide. In one embodiment, the heavy chain of one subunit of the Fc region comprised in the immune complex comprises the other C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). In one embodiment, the heavy chain of one subunit of the Fc region comprised in the immune complex comprises the other C-terminal glycine residue (G446, numbering according to the EU index of Kabat). A composition (e.g., a pharmaceutical composition) according to the present disclosure comprises a population of immune complexes according to the present disclosure. The population of immune complexes may comprise molecules having full-length heavy chains and molecules having truncated mutant heavy chains.The population of immune complexes may be comprised of a mixture of molecules with full-length heavy chains and molecules with truncated mutant heavy chains, of which at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immune complexes have truncated mutant heavy chains, which may be the C-terminal lysine (K447) of the Fc region or may be absent the C-terminal glycine (G446) and lysine (K447). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, e.g., as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0205] A "subunit" of an Fc region refers to one of the two polypeptides that form the dimeric Fc region, i.e., a polypeptide that comprises a C-terminal constant region capable of stabilizing self-association in an immunoglobulin heavy chain. For example, a subunit of an IgG Fc region comprises the IgG CH2 and IgG CH3 constant domains. In this disclosure, the use of "first subunit" or Fc1 and "second subunit" or Fc2 is used for ease of distinction and not to confer any particular order or orientation.

[0206] A "modification (or mutation) that promotes binding between a first subunit and a second subunit of an Fc domain" is a peptide backbone manipulation or post-translational modification of an Fc domain subunit that reduces or prevents a polypeptide comprising an Fc domain subunit from binding to the same polypeptide such that homodimers are formed. As used herein, specifically, a modification that promotes binding includes individual modifications to each of the two Fc domain subunits to be bound (i.e., the first subunit and the second subunit of the Fc domain), where the modifications are complementary to each other and thus promote the binding of the two Fc domain subunits. For example, a modification that promotes binding can change the structure or charge of one or two Fc domain subunits, thereby promoting their binding sterically or electrostatically, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be similar in the sense that another component (e.g., an antigen-binding moiety) fused to each subunit is different. In some embodiments, binding-enhancing modifications include amino acid mutations, specifically amino acid substitutions, in the Fc domain.

[0207] "Complementarity" refers to the combination of interactions that affect heavy / light chain pairing, for example, at the CH1 and CL (or CH3 and CH3) interface of an antigen-binding protein described herein. "Spatial complementarity" or "conformational complementarity" refers to the compatibility of three-dimensional structures, for example, at the interaction surface of CH1 and CL (or CH3 and CH3). "Electrostatic complementarity" refers to the compatibility of negatively and / or positively charged atoms, for example, at the interaction surface of CH1 and CL (or CH3 and CH3). Methods for measuring electrostatic complementarity at protein / protein interfaces are known in the art and are described, for example, in McCoy et al., (1997) J Mol Biol 268, 570-584, Lee et al., (2001) Protein Sci. 10, 362-377, and Chau et al., (1994) J Comp Mol Des 8, 51325. Methods for measuring spatial complementarity at protein / protein interfaces are known in the art and are described, for example, in Lawrence et al., (1993) J Mol Biol 234, 946-950, Walls et al., (1992) J Mol Biol 228, 277-297, and Schueler-Furman et al., (2005) Proteins 60, 187-194.

[0208] "Effector function" refers to biological activities that can be attributed to the antibody Fc region and vary with the antibody isotype when referring to the use of antibodies. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor (FcR) binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell antigen uptake, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. ADCC is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or its derivative containing an Fc region is specifically bound, typically via the protein moiety at the N-terminus of the Fc region. "Reduced ADCC" is defined as a reduction in the number of target cells lysed in a given time period by the ADCC mechanism defined above, utilizing a given concentration of antibody in the medium surrounding the target cells, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced from the same type of host cell, but not yet engineered, by the same standard manufacturing, purification, preparation and storage methods known to those skilled in the art. For example, the reduction in ADCC mediated by an antibody containing an amino acid substitution in its Fc domain that reduces ADCC is relative to the ADCC mediated by the same antibody without that amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are known in the art (see, for example, WO2006 / 082515 or WO2012 / 130831).

[0209] An "activating Fc receptor" is an Fc receptor that, upon binding of the Fc domain of an antibody, initiates a signaling event that stimulates the receptor-bearing cell to perform an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0210] "Immune complex" refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody. As described herein, the IL-2 molecule can be linked to the antibody by various interactions and various structures. In a particular embodiment, the IL-2 molecule is fused to the antibody via a peptide linker. A particular immune complex according to the invention consists essentially of one IL-2 molecule and an antibody linked by one or more linker sequences.

[0211] "Reduced affinity" or "reduced binding", e.g., reduced binding to IL-2Rα, refers to a reduction in the affinity of the corresponding interaction, e.g., as measured by SPR. The term further includes affinity being reduced to zero, or below the detection limit of the analytical method, i.e., the interaction being completely eliminated. In contrast, "reduced affinity" or "improved binding" refers to an increase in the binding affinity of the corresponding interaction.

[0212] The determination or definition of CDRs can be achieved by solving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby allowing for accurate delineation of the CDRs and identification of the residues that comprise the antibody's binding site. This can be achieved by any one of a variety of techniques known to those skilled in the art, for example, X-ray crystallography. Various analytical methods can be used to identify CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0213] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs from the Oxford Molecular Group that models antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272, "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the base sequence (see, e.g., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). The contact definition is based on the analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make an enthalpic contribution to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).It should be noted that the definition of the boundaries of other CDRs may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they can be shortened or extended due to predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is well known to those skilled in the art, and is illustratively shown in Table 1 below.

[0214] [Table 1]

[0215] The CDR amino acid residues of the VL and VH regions of the antibodies of this disclosure conform in number and position to the known Kabat numbering system.

[0216] The antibodies of the present disclosure may be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic or modified forms thereof, of which monoclonal antibodies are particularly applicable in some embodiments. Generally, the antibodies of the present disclosure are recombinant antibodies. As used herein, "recombinant" refers broadly to a product, such as a cell or a nucleic acid, protein or vector, that has already been modified by introducing a heterologous nucleic acid or protein or by modifying a naturally occurring nucleic acid or protein, or that the cell is derived from a cell that has been so modified. For example, recombinant cells express genes that are not present in the native (non-recombinant) cellular form, or express naturally occurring genes that are aberrantly expressed, under-expressed or not expressed at all.

[0217] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of the protein, and in many cases can be added, removed, or transferred to other proteins without losing function of the other parts and / or domains of the protein. "Immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional tetrapeptide chain structure antibody or a chain of a heavy chain antibody) or a polypeptide substantially composed of such a globular region. Immunoglobulin domains maintain the immunoglobulin fold characteristic of antibody molecules and are characterized by being composed of a two-layer sandwich of about seven antiparallel β-strands arranged in two β-sheets and optionally stabilized by conservative disulfide bonds.

[0218] An "immunoglobulin variable domain" refers to an immunoglobulin domain that is substantially composed of four "framework regions" referred to in the art and below as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, of which said framework regions are separated by three "complementarity determining regions" or "CDRs", referred to in the art and below as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. The general structure or sequence of an immunoglobulin variable domain may therefore be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain contains the antigen binding site and thus confers specificity for an antigen.

[0219] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain and includes a VH, VHH or VL domain) that is capable of forming a functional antigen-binding site when it does not interact with other variable domains (e.g. in the absence of the necessary VH / VL interactions between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, such as camelized human VH), IgNAR, domains, antibodies as or derived from a VH domain (single domain) (e.g. dAbsTM) and antibodies as or derived from a VL domain (single domain) (e.g. dAbsTM). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g. VH or VHH domains) are generally preferred. One specific example of an immunoglobulin single variable domain is a "VHH domain" (or abbreviated as "VHH"), defined as follows:

[0220] A "VHH domain" is also called a heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, and is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an antibody lacking a light chain) (Hamers-Casterman C, Atharhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains", Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the above variable domain from the heavy chain variable domain (referred to as "VH domain" in this disclosure) and light chain variable domain (referred to as "VL domain" in this disclosure) present in a conventional tetrapeptide chain structure antibody. A VHH domain specifically binds an epitope without the need for another antigen-binding domain (this is the opposite of the VH or VL domain in a conventional tetrapeptide chain structure antibody, where the epitope is recognized by both the VL and VH domains). A VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) may be used interchangeably. "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, and may be humanized antibodies produced by camelids or screened by phage display technology. In some embodiments, a VHH domain comprises three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In some embodiments, the VHH domain may be truncated at the N-terminus or C-terminus to comprise only a portion of FR1 and / or FR4, or may lack one or two of these framework regions, as long as the VHH essentially retains its antigen binding and specificity.The total number of amino acid residues in a VHH domain typically ranges from 110 to 120, and often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

[0221] As is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions based on the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows). This generally means that the numbers based on Kabat may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or numbering conventions include Chothia, IMGT, AbM.

[0222] A "humanized antibody", also called a CDR-grafted antibody, refers to an antibody produced by grafting a non-human CDR sequence onto a human antibody variable region framework. This can overcome the strong immune response induced by a chimeric antibody having a large amount of non-human protein components. In order to avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing minimal back mutations on the fully human antibody variable region. An example of "humanization" includes a case where a VHH domain derived from a camelid can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a normal human tetrapeptide chain structure antibody (also called "sequence optimization" in the present disclosure, which may include other modifications made to the sequence by one or more mutations that confer improved properties of the VHH, such as removal of potential post-translational modification sites, in addition to humanization). The humanized VHH domain may comprise one or more fully human framework region sequences, and in some specific embodiments may comprise human framework region sequences of IGHV3. Humanization methods include, for example, protein surface amino acid resurfacing and antibody humanization universal framework grafting (CDR grafting to a universal framework), in which CDRs are "grafted" onto other "stents" (including, but not limited to, human stents or non-immunoglobulin stents). Suitable stents and techniques for CDR grafting are known in the art. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies of the present disclosure also include humanized antibodies after affinity maturation of the CDRs by phage display.Furthermore, in order to avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing minimal back mutations or reverse mutations on the framework sequences of the variable regions of the human antibody.

[0223] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) that improve the affinity of the antibody for its antigen, compared to a parent antibody that does not possess such alterations. For example, an "affinity matured" PD-1 antibody has one or more changes in one or more CDRs that increase its affinity for antigen, compared to the parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example as described in Marks et al., 1992, Biotechnology 10:779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813., Shier et al., 1995, Gene 169:147-155, Yelton et al., 1995, Immunol. 155:1994-2004, Jackson et al., 1995, J. Immunol. 154(7):3310-9, and Hawkins et al., 1992, J. MoI. Biol. 226(3):889896, KS Johnson & RE Hawkins, "Affinity maturation of antibodies using phage display", Oxford University Press 1996.

[0224] Typically, the PD-1 antibodies, immunoconjugates of the disclosure have a cytotoxicity of about 10%, preferably about 10%, as measured in a Biacore or KinExA or Fortibio assay. -7 ~10 -10 moles / liter (M), more preferably 10 -8 ~10 -10 moles / liter, more preferably 10 -9 ~10 -10 or less, and / or a dissociation constant (KD) of at least 10 -7M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 It binds to the antigen (i.e., PD-1) with an association constant (KA) of M. 10 -4 Any K value greater than M is generally considered to indicate non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable method known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) as described in this disclosure.

[0225] An "antigen" is a molecule with which an immunocompetent vertebrate is immunized so that antibodies that recognize the antigen can be produced or an expression library (e.g., phage, yeast, or ribosome display libraries, among others) can be screened. In this disclosure, antigen is defined more broadly and includes the target molecule that is specifically recognized by an antibody, and includes portions or mimetics of the molecule used in the immunization process to produce antibodies or in library screening to select antibodies. For example, with respect to the human PD-1 binding antibodies of the present disclosure, monomers and multimers (e.g., dimers, trimers, etc.) of human PD-1, and deletion and other mutants of human PD-1 are all referred to as PD-1 antigens.

[0226] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. Epitopes may be formed from adjacent amino acids or from non-adjacent amino acids in parallel by tertiary folding of a protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically contain at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include Western blotting and immunoprecipitation detection assays. Methods for determining the spatial conformation of epitopes include techniques in the art and described in this disclosure, such as X-ray crystallography and 2D nuclear magnetic resonance.

[0227] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a given antigen. For example, when human PD-1 or an epitope thereof is used as an analyte and an antibody is used as a ligand, the antibody binds to an antigen at a specific binding site by surface plasmon resonance (SPR) technology in an instrument, and the antibody binds to an epitope on a specific antigen at a specific binding site of about 10 -7 The equilibrium dissociation constant (K D ) to a given antigen or epitope thereof, and the binding affinity for the given antigen or epitope thereof is at least twice its binding affinity for a non-specific antigen other than the given antigen (or epitope thereof) or a closely related antigen (e.g., BSA, etc.). An "antibody that recognizes an antigen" may be used interchangeably with an "antibody that specifically binds" in this disclosure.

[0228] "Binding affinity" or "affinity" is used in this disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or a portion thereof and an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined, for example, by measuring the kinetics of complex formation and dissociation with surface plasmon resonance (SPR) techniques (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. K D is K D The dissociation constants are related to ka and kd by the equation: ka = kd / ka. Values ​​of the dissociation constants can be determined directly by well-known methods, and can also be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, the K can be calculated using a double filtration nitrocellulose filter binding assay as disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other standard assays to evaluate the binding ability of an antibody to a target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, and other assays listed elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TM The K of each antibody / antigen complex may be evaluated by the KinExA system. D By comparing the K values, one can compare the binding affinities associated with interactions with different molecules, for example, comparing the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by comparing the K of an interaction of interest (e.g., a specific interaction between an antibody and an antigen). D value and the K of a non-target interaction (e.g., a known control antibody that does not bind CD40). D It can be evaluated by determining and comparing values.

[0229] "Conservative substitution" refers to substitution of an amino acid residue with another amino acid residue having similar properties to the original amino acid residue. For example, lysine, arginine and histidine have similar properties in that they have a basic side chain, and aspartic acid and glutamic acid have similar properties in that they have an acidic side chain. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine ​​and tryptophan have similar properties in that they have a polar side chain that is uncharged, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have a non-polar side chain. Furthermore, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have an aromatic side chain. Therefore, it is clear to those skilled in the art that even if an amino acid residue in a group showing similar properties as described above is substituted, it does not show a specific change in properties.

[0230] "Homology", "identity" or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If the positions in the two compared sequences are occupied by the same nucleotide or amino acid monomer, for example, if the positions of each of the two DNA molecules are occupied by the same nucleotide, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, if 6 out of 10 positions in the two sequences are matched or homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0231] "Nucleic acid molecule", "nucleic acid" or "polynucleotide" may be used interchangeably and refer to DNA and RNA. A nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if the promoter or enhancer affects the transcription of the coding sequence.

[0232] "Vector" refers to a construct capable of delivering, and in some embodiments expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and eukaryotic cells such as producer cells.

[0233] A "host cell" includes any cell or cell culture that may be or has been a recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and due to natural, accidental, or deliberate mutations, the progeny are not necessarily completely identical (in morphology or genomic DNA complement) to the original parent cell. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide according to the present disclosure. "Cells," "cell lines," and "cell cultures" may be used interchangeably, and all such designations include their progeny. It should also be understood that due to deliberate or unintended mutations, all progeny may not be precisely identical in DNA content. Mutant progeny that have the same function or biological activity as screened from the originally transformed cell are included.

[0234] By "pharmaceutical composition" is meant a mixture of one or more of the antibodies described herein or their physiologically / pharmaceutical acceptable salts or prodrugs with other chemical components, and other components such as physiologically / pharmaceutical acceptable vectors and excipients, etc. The pharmaceutical composition is intended to facilitate administration to a living body and contribute to the absorption of the active ingredients to further exert biological activity.

[0235] A "pharmaceutically acceptable vector" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical vector, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such vectors are prepared by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and R Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0236] "Inhibition" or "blocking" may be used interchangeably and covers both partial and complete inhibition / blocking. "Inhibition of growth" (e.g., of a cell) is intended to include any measurable decrease in cell growth.

[0237] "Proliferative disease" refers to a medical condition associated with some degree of abnormal cell proliferation. In one embodiment, the proliferative condition refers to cancer.

[0238] "Cancer" generally refers to or describes a physiological disorder in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, black lung cancer, and ovarian cancer. These include, but are not limited to, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), and phakomatoses, abnormal blood vessel proliferation associated with edema (e.g., associated with brain tumors) and Meigs syndrome, brain tumors and cancers, and head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment with the PD-1 binding proteins of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Further, in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (e.g., triple negative breast cancer), including metastatic forms of these cancers.

[0239] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0240] "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0241] "Cancer prevention" means delaying, inhibiting, or preventing the onset of cancer in a subject, where the onset of cancer or tumor formation in said subject has not been confirmed, but a susceptibility to cancer has been identified, e.g., as determined by genetic screening or other methods. It further includes treating a subject with a pre-cancerous condition, whereby the progression of said pre-cancerous condition to a malignant tumor is halted or regression is caused.

[0242] "Giving", "administration" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refer to contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid, e.g., therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving", "administration" and "treatment" also refer to treating, e.g., cells, ex vivo and in vitro, with a reagent, diagnostic, binding composition, or through another cell. When applied to a human, veterinary or research subject, refer to therapeutic treatment, preventative or prophylactic measures, research and diagnostic uses.

[0243] "Treatment" refers to administering to a subject an internal or external therapeutic agent comprising any one of the antibodies or pharmaceutical compositions thereof disclosed herein as a therapeutic agent, the subject already suffering from, at risk of suffering from, or prone to suffering from one or more proliferative diseases or symptoms thereof, the known therapeutic agent having a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the subject or population being treated in an amount that effectively relieves one or more disease symptoms, whether by inducing regression of such symptoms or by inhibiting such symptoms so that they do not progress to any clinically measurable extent. The amount of therapeutic agent that effectively relieves any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, such as the disease state, age and weight of the subject, and the ability of the agent to produce the required therapeutic effect in the subject. Whether the disease symptoms have been reduced can be evaluated by any clinical detection method commonly used by a physician or other professional health care provider to evaluate the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or product) may be ineffective in alleviating a target disease symptom in a subject, but should alleviate the target disease symptom in a statistically significant number of subjects, as determined by any statistical testing method known in the art, such as, for example, Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0244] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or pathology of a medical condition. Effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as the condition being treated, the overall health of the subject, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

[0245] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but need not necessarily occur, and the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur. "And / or" should be considered as specifically indicating that each of the two specified features or components may or may not have the other. Thus, the term "and / or," as used in the phrase "A and / or B" in this disclosure, includes "A and B," "A or B," "A" (alone) and "B" (alone). Unless the context clearly indicates otherwise, throughout the specification and claims, words such as "comprise," "have," "contain," and the like, should be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning.

[0246] In this disclosure, unless otherwise defined, the use of, for example, "first," "second," and "third" is not intended to confer a particular order or orientation, but rather to facilitate distinction when there is, for example, one or more modules of the same type.

[0247] In the present disclosure, when " / " is used for a mutation, it means that the above mutations exist simultaneously in the same IL-2 mutant or the same Fc or the same immune complex, for example, "F42A / L72G" means that F42A and L72A mutations exist simultaneously in the same IL-2 mutant.

[0248] In this disclosure, "subject" and "patient" refer to mammals, particularly primates, and especially humans.

[0249] Unless otherwise stated, all amino acid position numbers for IL-2 or variants thereof in this disclosure are relative to wild-type IL-2 (eg, as set forth in SEQ ID NO:87).

[0250] Working Example The present disclosure will be further described below with reference to examples, but these examples are not intended to limit the scope of the present disclosure.

[0251] Experimental methods for which specific conditions are not specified in the Examples or Test Examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which a specific source is not specified are conventional reagents that are commercially available.

[0252] Example 1. Screening and preparation of anti-PD-1 nanobodies In the present disclosure, His-tagged human PD-1 was used as the immunizing antigen, and biotin-labeled human PD-1 and cynomolgus PD-1 were used as the screening antigens, all of which were purchased from AcroBiosystems.

[0253] 1. Sequence and preparation of immunization antigens and screening antigens [Table 2]

[0254] The sequence of human PD-1 protein (NP_005009.2) is as follows, where the underlined part is the extracellular domain sequence and the start and end amino acids are Leu25-Gln 167:

[0255] > Human PD-1 amino acid sequence [ka] 2. Alpaca Immunization, Nanobody Yeast Display Library Construction, and Antibody Screening 1) Alpaca immunity Two healthy alpacas were immunized. On day 0, the first immunization, 0.25 mg of human PD-1 antigen was mixed homogeneously with 1 mL of complete Freund's adjuvant (CFA) and then injected subcutaneously. On days 21, 42, and 63, 0.125 mg of human PD-1 antigen was mixed homogeneously with 1 mL of incomplete Freund's adjuvant (IFA) and then injected subcutaneously. 10 mL, 50 mL, and 50 mL of blood were collected on days 28, 49, and 70, respectively. The serum titers were detected, and alpaca peripheral lymphocytes that met the library construction criteria were used for the construction of a nanobody yeast display library.

[0256] 2) Construction of yeast library After the third and fourth immunizations, 50 mL of peripheral blood was collected from each of the two alpacas, PBMCs were isolated from the peripheral blood, and RNA in the PBMCs was extracted and reverse transcribed to obtain total cDNA. The cDNA was used as a reaction template for the first round of nested PCR. Two rounds of nested PCR amplification were performed, and the nested PCR product fragments from the second round were ligated to a yeast library vector (yeast library vector pYDN3), and the ligated product was electroporated into yeast competent cells. The insertion rate and library diversity were verified using colony PCR. Analysis and sequencing were performed based on the number of library transformants, library insertion rate, and library diversity, and the library capacity of one alpaca library was 9.28 × 10 7 The library capacity of the other alpaca library is 1.54 × 10 8 It was found to be the case.

[0257] 3) Nanobody (VHH) screening Antibodies were screened using this yeast display system by flow cytometry (FACS) selection and library screening and identification. Selection was performed in two steps: the first step was enrichment with biotin-labeled human PD-1 protein (Avitag-His Tag) magnetic beads, and the second step was selection by flow cytometry with biotin-labeled monkey PD-1 protein (His, Avitag) (flow antibody: Mouse anti-HA tag Dylight 488, streptavidin Dylight 650). After the second step, 18 unique VHH sequences cross-linked to human and monkey PD-1 antigens were obtained by monoclonal identification, sequencing, and sequence analysis. The A6 and A17 sequences are shown below.

[0258] >A6 variable region [ka] >A17 variable region [ka] [Table 3]

[0259] The above sequences were each linked to a human IgG4 Fc (including hinge region and with S228P according to the EU numbering system) fragment to construct a VHH-Fc antibody. The plasmid was constructed, transiently transfected into 293 cells, expressed, purified by protein A column, washed with PBS, eluted with 0.1 M glycine buffer, pH 2.5, and dialyzed into PBS pH 7.4 buffer. Here, the human IgG4 Fc (including hinge region) sequence is as follows: >hIgG4 [ka] >A6_hIgG4 [ka] >A17_ hIgG4 [ka]

[0260] Example 2. Identification of the affinity of anti-PD-1 nanobodies to the antigen PD-1 1. ELISA Human PD-1 protein (his tag) was dissolved at 1 μg / mL in PBS and added to a 96-well plate at 100 μL / well, and the antigen was coated overnight at 4°C. The plate was washed three times with PBST (PBS + 0.05% Tween 20) solution. PBST / 1% BSA solution was added, incubated at 37°C for 1 h, and blocked. After washing three times with PBST solution, different concentrations of PD-1 candidate VHH-Fc antibodies (diluted with PBST / 1% BSA solution) were added and incubated at 37°C for 1.5 h. The plate was washed three times with PBST solution. 100 μL of a secondary antibody (Thermo) solution of anti-human IgG4 Fc coupled to HRP was added and incubated at 37°C for 1 h. After washing three times with PBST solution, 100 μL / well of TMB solution was added, reacted at room temperature for 5 min, and then 50 μL of stop solution was added, and the 450 nM value was read on a plate reader, and the EC 50 Calculate As a result, as shown in Table 4, both A6_hIgG4 and A17_hIgG4 have strong binding affinity to the PD-1 antigen.

[0261] [Table 4]

[0262] 2.FACS To detect the binding ability of anti-PD-1 nanobodies to cell membrane-expressed PD-1, different concentrations of candidate anti-PD-1 VHH-Fc antibodies were added to the cells at 10 5The cells were added to Jurkat cells (Cat:J1250, Promega) overexpressing human PD-1 at 100 μL / well, incubated at room temperature for 20 min, washed twice with PBS, and then 100 μL of anti-human IgG Fc fluorescent secondary antibody (Biolegend) was added and incubated at room temperature for 20 min, washed twice with PBS, resuspended in 250 μL of PBS, and the fluorescent signal was detected by FACS. EC 50 was calculated.

[0263] Pembrolizumab (purchased from Biointron) was used as a control, and the results are shown in Table 5. In addition, the EC 50 The value was 0.3568 nM, which is significantly superior to other antibodies obtained in synchronous screening (results not shown).

[0264] [Table 5]

[0265] Example 3. Sequence modifications of anti-PD-1 nanobodies 1. Humanization and backmutation of anti-PD-1 nanobodies The antibody humanization was performed by CDR-grafting method. The parent PD-1 antibody was aligned with the complete human germline genes in the IMGT or NCBI / igblast databases by IMGT or NCBI website, and the human germline gene IGHV3-23 (IGHV3-23*01, IGHV3-23*04) with high homology to PD-1 nanobody was selected as the humanization template, and CDR was grafted, leaving four key residues (Y37, E44, R45, L47) in FR2 of the nanobody. The key core residues close to the CDR region and the residues at the Vernier positions interacting with the CDR were backmutated. The humanized antibodies A17h1, A17h2, A17h3, and A6h1 were obtained.

[0266] >A17h1 variable region [ka] >A17h2 variable region [ka] >A17h3 variable region [ka] >A6h1 variable region [ka]

[0267] 2. Modification by removing TCE (T cell epitope), reducing antibody deamidation, and reducing antibody isomerization To reduce the potential immunogenicity risk of the antibody, the antibody was modified by removing TCE. To improve the druggability of the antibody, the antibody was deamidated and modified at the antibody isomerization site. The following sequences were obtained: >A17m01 variable region [ka] >A17m02 variable region [ka] >A17m03 variable region [ka] >A17m04 variable region [ka] >A17m05 variable region [ka] >A17m06 variable region [ka] >A17m07 variable region [ka] >A17m08 variable region [ka] >A17m09 variable region [ka] >A17m10 variable region [ka] >A17m11 variable region [ka] >A17m12 variable region [ka] >A17m13 variable region [ka] >A17m14 variable region [ka] >A17m15 variable region [ka] In addition, to improve the degree of humanization, A17m09 was further selected and germline modifications were made to the antibody sequence to obtain the following sequence: >A17m0901 variable region [ka] >A17m0902 variable region [ka] >A17m0903 variable region [ka] >A17m0905 variable region [ka] That is, the CDR region of A17 according to the present disclosure has a sequence represented by the following general formula: CDR1: DYSMS (SEQ ID NO: 7) CDR2: IISGSGX1X2X3HYVDSVKG, where X1 is selected from V or G, X2 is selected from I or S, and X3 is selected from T or A (SEQ ID NO: 36) CDR3: VSDWX4X5Y, where X4 is selected from D or E, and X5 is selected from D or E (SEQ ID NO: 37) Specifically, CDR2 is [ka] [ka] [ka] [ka] [ka] may be CDR3 is [ka] [ka] [ka] , may be also possible.

[0268] Example 4. Detection of blocking of anti-PD-1 nanobodies to PD-1 by PD-1 / PD-L1 reporter gene system The PD-1 / PD-L1 NFAT gene reporter system (Cat:J1250, Promega) can detect the biological activity function of anti-PD-1 antibody at the cellular level. The system consists of two genetically engineered cell lines: effector cells Jurkat (containing NFAT reporter gene luciferase) overexpressing PD-1, and antigen-presenting cells CHO-K1 cells overexpressing PD-L1. When the two types of cells are co-cultured, PD-1 / PD-L1 interaction will inhibit TCR-mediated NFAT activation and further inhibit the expression of NFAT reporter gene luciferase. When PD-1 / PD-L1 interaction is disrupted, TCR activation will induce the expression of luciferase via the NFAT pathway, and the degree of effector cell activation and the biological activity of the antibody can be measured by adding Bio-Glo reagent and performing fluorometric detection using a photometer.

[0269] 4 × 10 CHO-K1 cells overexpressing PD-L1 5 100 μL was added to each well of a 96-well plate and cultured overnight. After aspirating the medium, 1.25 × 10 6 40 μL of PD-1 Jurkat cells at 100 μg / mL and 40 μL of anti-PD-1 nanobody solution at different concentrations (diluted with 1640+2% FBS solution) were quickly added. After incubation at 37°C for 6 hours and returning to room temperature, 40 μL of Bright-glo reagent (Cat: E2620, Promega) was added to each well, shaken at 350 rpm in the dark for 5 min, and then left to stand at room temperature in the dark for 5 min. The fluorescence value was read using a plate reader, and the IC was calculated. 50 was calculated.

[0270] Pembrolizumab and hIgG4 isotype were used as controls. As shown in Figure 1A and Table 6, the biological functional activity IC 50The value was 1.199 nM, significantly superior to other antibodies screened simultaneously in this disclosure (e.g., 7.62 for A3_hIgG4 and 3.208 for A13_hIgG4, sequences of A3_hIgG4 and A13_hIgG4 not shown).

[0271] [Table 6]

[0272] Using the above method, the function of the humanized anti-PD-1 nanobody was verified. As shown in Figure 1B and Table 7, the IC 50 The activity is similar to that of the positive antibody pembrolizumab. In the modified candidate molecules, the biological activity IC of A17m0901_hIgG4 and A17m0902_hIgG4 antibodies 50 The values ​​were 0.5307 nM and 0.4352 nM, respectively, which is superior to pembrolizumab.

[0273] [Table 7]

[0274] Example 5. Identification of the binding affinity of engineered anti-PD-1 nanobodies to the antigen PD-1 The affinity of anti-PD-1 nanobody to PD-1 antigen was detected by surface plasmon resonance (SPR) technology, and Biacore 8k (GE Healthcare) was used. In the experiment, CM5 sensor chip was selected, and HBS-EP+buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was adopted as the mobile phase. Anti-human IgG (Fc) antibody was prepared to a 30 μg / mL solution in 10 mM sodium acetate buffer (pH 5.0), and the Immobilization program was selected to automatically perform amino coupling fixation of the anti-human IgG (Fc) antibody channel. Each antibody waiting to be measured was prepared as a ligand using HBS-EP+buffer solution, and captured by anti-human IgG (Fc) antibody in the chip channel. Human or cynomolgus monkey PD-1 antigen protein (Sino Biological, 10377-H08H, 90311-C08H) was used as the analyte, which was prepared in HBS-EP+ buffer solution. The analyte was diluted 2-fold in gradient and flowed through the experimental and reference channels at a flow rate of 30 μL / min, bound for 1 min, and dissociated for 15 min. The regeneration buffer, 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54), was run at a flow rate of 10 μL / min for 30 s to measure the binding rate Ka, dissociation rate Kd, and dissociation constant (i.e., affinity K D ) was calculated. See Tables 8 and 9 for the results.

[0275] [Table 8] [Table 9]

[0276] As evidenced by the binding to the human PD-1 antigen, the humanized antibody A17h1_hIgG4 and the subsequently modified and optimized antibodies A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 had K DA17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 all have slower dissociation times than pembrolizumab. The A17h1_hIgG4, A17m09_hIgG4, A17m0901_hIgG4, and A17m0902_hIgG4 antibodies cross-bind to monkey PD-1, and the antibodies bind to human and monkey PD-1 with similar affinity.

[0277] Example 6. Validation of in vitro efficacy of engineered anti-PD-1 nanobodies by DC:T mixed lymphocyte reaction (MLR) By co-culturing mature dendritic cells (DCs) with allogeneic T cells, T cells can be activated and cytokine secretion by T cells can be promoted. This process is inhibited by PD-1 / PD-L1 signaling, and the inhibition can be removed by using PD-1 antibodies to promote T cell activation. Therefore, the in vitro efficacy of PD-1 antibodies can be verified using DC:T mixed lymphocyte reaction experiments.

[0278] Monocytes were sorted from fresh peripheral blood (PBMC) of one healthy human using a sorting reagent kit (cat:19359, stemcell). After 7 days of culture, monocytes were induced to differentiate into mature DC cells using a DC cell induction reagent kit (cat:10985, stemcell). Then, allogeneic T cells were sorted from fresh PBMC of another healthy human using a sorting reagent kit (cat:17951, stemcell). 5000 mature DC cells were co-cultured with 50000 allogeneic T cells, and the corresponding concentration of candidate antibodies was added, and after 6 days of co-culture, the cell supernatant was aspirated and the concentration of IFN-γ was detected using a Cisbio-HTRF IFN-γ detection reagent kit (Cat:62HIFNGPEH, Perkinelmer) to obtain ECs. 50 was calculated.

[0279] As shown in Figure 2, A17m09 hIgG4 showed in vitro efficacy almost identical to that of pembrolizumab in the MLR experiment. Here, the EC50 The EC of pembrolizumab was 0.2931 nM. 50 is 0.3271nM.

[0280] Example 7. Anti-PD-1 nanobodies inhibit tumor growth in a mouse colon cancer model This example examined the in vivo antitumor efficacy of PD-1 antibodies in the MC38 tumor model in humanized PD-1 C57BL / 6 mice.

[0281] MC38 mouse colon cancer cell line was cultured in DMEM medium (10% FBS) at 5 × 10 5 MC38 cells were inoculated subcutaneously into humanized PD-1 C57BL / 6 female mice (Shui Sui Yakang Co., Ltd.), and the average tumor volume in the mice was 80 mm 3 When the tumors reached a certain level, they were randomly divided into groups of 8 mice each and administered antibody or control by intraperitoneal injection, and tumor volume was measured, body weight was weighed, and data was recorded twice a week. See Table 10 for experimental grouping and dosing schedule. Considering that the molecular weight of A17m09_hIgG4 is about 75 kDa, which is only half the molecular weight of normal IgG, the dosage was adjusted to half that of pembrolizumab to achieve an equimolar dose.

[0282] As shown in Figures 3A and 3B, at equimolar doses of A17m09_hIgG4 and pembrolizumab, the in vivo antitumor efficacy of A17m09_hIgG4 is consistent with that of pembrolizumab. As shown in Table 11, at equimolar doses, the tumor inhibition rate of A17m09_hIgG4 is slightly superior to that of pembrolizumab.

[0283] [Table 10] [Table 11]

[0284] Example 8. Construction of a fusion protein of anti-PD-1 antibody and IL-2 mutant (PD-1-IL-2v) Wild-type IL-2 inhibits NK and CD8+ IL-2 preferentially activates regulatory T cells (Treg) rather than T cells, due to the high expression of the high affinity receptor for IL-2, IL-2Rαβγ, in Treg. To reduce the preferential activation of Treg by IL-2, it is common to introduce mutations such as F42A and F72G into IL-2 so that the binding of IL-2 to IL-2Rα is eliminated. However, such an IL-2 mutant, IL-2v (F42A, L72G), still activates NK cells and CD8 + T cells can be systemically activated to produce toxicity. To increase the specificity of IL-2, we introduced the mutation V91T, reducing the affinity of IL-2 for IL-2Rβγ, thereby targeting NK and CD8 + The activity of IL-2 on T cells was reduced. Furthermore, the inventors coupled the above IL-2 mutant with an anti-PD-1 antibody to deliver the IL-2 mutant to PD-1-positive NK and T cells, thereby specifically activating these cells.

[0285] Fusion proteins of anti-PD-1 antibodies and IL-2 mutants were constructed according to the molecular morphology diagram shown in Figure 4. The Fc of each molecule in Figure 4 uses Knob-in-Hole (KIH) technology, that is, the following mutations are introduced into the two Fc: S354C, T366W (Eu numbering system) (constituting the knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting the hole chain), respectively, to promote the formation of heterodimers and facilitate purification: H435R, Y436F (Eu numbering system) are additionally introduced into the hole chain; furthermore, L234A, L235A, and P329G mutations are introduced into both the knob and hole chains to eliminate the ADCC effect; and the heavy chain that is not paired with the light chain and the hinge region are mutations of C220S to eliminate free, unpaired cysteines. For IL-2, we used the mutant IL-2v (V91T), which contains: 1) T3A to remove glycosylation modifications that may be present when the protein is expressed in mammalian cells; 2) R38E and F42A to remove binding of IL-2 to CD25 (i.e., IL-2Rα); 3) V91T to reduce binding of IL-2 to the medium affinity IL-2 receptor IL-2Rβγ; and 4) C125A to remove a free cysteine ​​in IL-2 and avoid the formation of intermolecular disulfide bonds by IL-2.

[0286] Molecule A-V91T shown in FIG. 4A is composed of anti-PD-1 nanobody A17m0902 coupled to the N-terminus of the knob and hole chains via the hinge region of an IgG1 heavy chain, and molecule B-V91T shown in FIG. 4B is composed of two anti-PD-1 nanobodies A17m0902 linked end-to-end via a (G4S)2 linker, and their C-termini are coupled to the N-terminus of the knob and hole chains via the hinge region of an IgG1 heavy chain. The molecule C-V91T shown in FIG. 4C has anti-PD-1 nanobody A17m0902 coupled to the N-terminus of the hole chain via the IgG1 heavy chain hinge region, and the heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab coupled to the N-terminus of the knob chain via the IgG1 heavy chain hinge region. The molecule D-V91T shown in FIG. 4D has two anti-PD-1 nanobodies A17m0902 linked to each other via a (G4S)2 linker. The heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab is coupled to the N-terminus of the Knob chain via the IgG1 heavy chain hinge region, and the C-terminus of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab is coupled to the N-terminus of the Knob chain via the IgG1 heavy chain hinge region. The molecule E-V91T shown in FIG. 4E has a heavy chain of the Fab fragment corresponding to the anti-PD-1 antibody pembrolizumab coupled to the Knob and Hole chains via the IgG1 heavy chain hinge region. In the molecule F-V91T shown in FIG. 4F, two anti-PD-1 nanobodies A17m0902 are linked end-to-end by a (G4S)2 linker, and their C-terminus is coupled to the N-terminus of the hole chain via the IgG1 heavy chain hinge region. In the molecules A to E in FIG. 4, IL-2v(V91T) is linked to the C-terminus of the Fc knob chain by a (G4S)3 linker, and in the molecule F, IL-2v(V91T) is coupled to the N-terminus of the knob chain by a G4S linker.

[0287] The sequence of each molecule is as follows, with the underlined part being the Fc and the italics being the linker.

[0288] >A-V91T Knob Chain [ka] >A-V91T Hole Chain [ka] >B-V91T knob chain [ka] >B-V91T Hole Chain [ka] >C-V91T knob chain [ka] The hole strand of C-V91T is sequence number 44.

[0289] >C-V91T light chain [ka] For D-V91T, the knob chain is SEQ ID NO: 47, the hole chain is SEQ ID NO: 46, and the light chain is SEQ ID NO: 48.

[0290] >E-V91T Knob Chain [ka] >E-V91T Hole Chain [ka] The light chain of E-V91T is sequence number 48.

[0291] >F-V91T Knob Chain [ka] The hole strand of F-V91T is sequence number 46.

[0292] The sequence of pembrolizumab above is as follows: >Pembrolizumab Fab heavy chain [ka] >Pembrolizumab Fab light chain [ka] > Pembrolizumab HCDR1 [ka] > Pembrolizumab HCDR2 [ka] > Pembrolizumab HCDR3 [ka] > LCDR1 of pembrolizumab [ka] > LCDR2 of pembrolizumab [ka] > LCDR3 of pembrolizumab [ka] The sequence of IL-2v(V91T) in the above PD-1-IL-2v is as follows: [ka] That is, IL-2v(V91T) contains T3A / R38E / F42A / V91T / C125A.

[0293] The above fusion protein was expressed in HEK293 cells and purified with Protein A. The molecular weight of the purified product was confirmed to be as expected by SDS-PAGE, proving that the target protein had been prepared.

[0294] Example 9. Detection of M07e cell proliferation promoting activity by PD-1-IL-2v M07e is a human cell line that expresses only IL-2Rβ and IL-2Rγ, but not IL-2Rα. To detect the activity of the different forms of PD-1-IL-2v(V91T) fusion protein and its dependency on PD-1 expression, we overexpressed human PD-1 (SEQ ID NO: 1) in the M07e cell line to obtain the M07e-hPD-1 cell line, and then detected the effects of the fusion protein on cell proliferation in the M07e and M07e-hPD-1 cell lines, respectively.

[0295] Complete medium: RPMI1640 + 2mM L-glutamine + 1mM sodium pyruvate + 10% fetal bovine serum + 15ng / mL GM-CSF, Basal medium: RPMI1640 + 2mM L-glutamine + 1mM sodium pyruvate + 10% fetal bovine serum.

[0296] M07e cell proliferation experiment: M07e cells or M07e-hPD1 were cultured in complete culture medium at 37°C and 5% CO2, and the cells were passaged. After 1-2 days, the cells were collected by centrifugation, washed three times with PBS, and resuspended in basal culture medium at 6.0 × 10 cells per mL. 5 A cell suspension containing cells was prepared and placed in a 96-well plate with a volume of 50 μL per well. Test drugs prepared in 50 μL of basal culture medium with the corresponding concentration of 2× were added, and the cells were cultured for 72 hours. Then, 100 μL of CELLTITER-Glo reagent (Promega), a dissolving solution, was added to each well and mixed uniformly. The absorbance was measured by a plate reader at a wavelength of 570 nm with a reference wavelength of 630 nm.

[0297] The proliferation activity data of M07e and M07e-hPD-1 cells by different forms of PD-1-IL-2v(V91T) fusion protein are shown in Table 12 and Figure 5A and Figure 5B. A PEGylated IL-2 mutant (PEG-IL-2v) was used as a positive control, in which the IL-2 mutant has T3A, F42A, L72G, N26Q, N29S, N71Q and C125A mutations, and does not bind to IL-2Rα but can bind to the affinity receptor IL-2Rβγ in IL-2.

[0298] As a result, the activity of PEG-IL-2v on M07e and M07e-hPD-1 cell proliferation was comparable, and the activity of various different forms of PD-1-IL-2(V91T) on M07e-hPD-1 was obviously stronger than that on M07e cell proliferation, indicating that anti-PD-1 antibodies can increase the selectivity of IL-2 mutants for PD-1 positive cells. In addition, when comparing various different forms of PD-1-IL-2(V91T), their activities on M07e proliferation were comparable, but the activity of C-V91T on M07e-hPD-1 proliferation was slightly stronger than that of A-V91T, and A-V91T was even stronger than B-V91T and E-V91T.

[0299] [Table 12]

[0300] Example 10. Construction of more PD-1-IL-2v Although PD-1-IL-2v(V91T) was less active than PEG-IL-2v in M07e, which lacks PD-1 expression, it was still able to stimulate cell proliferation, IL-2v(V91T) was still able to strongly bind to the IL-2 intermediate affinity receptor IL-2Rβγ, and PD-1-IL-2(V91T) still stimulated NK and CD8 +It has been shown that PD-1 can activate T cells systemically and cause toxicity. To further reduce the toxicity of PD-1-IL-2v so that the activity of IL-2 is more dependent on the expression of PD-1, we constructed PD-1-IL-2v with different IL-2 mutants based on the A-V91T molecular form. Both IL-2 mutants have F42A and L72G mutations to eliminate the binding of IL-2 to IL-2Rα, and N26Q, N29S and N71Q were added to the IL-2 mutants to eliminate possible deamidation modification in IL-2 and increase the stability of IL-2 for stability consideration. And they further have different unique mutations as shown in Table 13, each of which reduces the affinity of IL-2 to the medium affinity receptor IL-2Rβγ to various degrees.

[0301] [Table 13] >IL-2 sequence in A-PC [ka] >IL-2 sequence in A-V91T-02 [ka] >IL-2 sequence in A-D20A [ka] >IL-2 sequence in A-D20N [ka] >IL-2 sequence in A-N88D [ka] >IL-2 sequence in A-N88R [ka] >IL-2 sequence in A-Q126D [ka] IL-2 sequence in A-H16A / D84S [ka] To eliminate the ADCC effect, L234A and L235A mutations were introduced into Fc, and to promote the formation of heterodimers, S354C, T366W (constituting the Knob chain) and Y349C, T366S, L368A, Y407V (Eu numbering system) (constituting the Hole chain) mutations were introduced, and a C220S mutation was introduced into the hinge region. To facilitate purification, the antibody may contain H435R, Y436F (Eu numbering system) mutations in the Hole chain. In addition, the Hole chain of the Fc region of these antibodies may be D356, L358 or E356, M358 (Eu numbering system) at amino acids 356 and 358, respectively, which appear in different alleles and do not affect the function of the antibody.

[0302] >Fc knob chain (D356, L358) [ka] >Fc hole chain (D356, L358) [ka] >Fc knob chain (E356, M358) [ka] >Fc hole chain (E356, M358) [ka] As an example, the full-length sequences of A-D20A and A-N88D are shown, with the underlined part being the Fc and the italics being the linker.

[0303] > Knob chain of A-D20A (D356, L358) [ka] >A-D20A Hole Strand (D356, L358) [ka] > Knob chain of A-D20A (E356, M358) [ka] >A-D20A Hole Chain (E356, M358) [ka] > Knob chain of A-N88D (D356, L358) [ka] >Hole chain of A-N88D (D356, L358) [ka] >A-N88D Knob chain (E356, M358) [ka] >Hole chain of A-N88D (E356, M358) [ka] In addition, to better demonstrate the dependency of PD-1-IL-2v on PD-1, the inventors replaced the anti-PD-1 antibody portion of some PD-1-IL-2v with an antibody targeting an unrelated antigen while retaining the mutations in the IL-2 portion to obtain IgG-PC, corresponding IgG-V91T containing V91T in IL-2, corresponding IgG-D20A containing D20A in IL-2, and corresponding IgG-N88D containing N88D in IL-2. IgG-PC and IgG-V91T, IgG-D20A, and IgG-N88D are different only in that the IL-2 portion is different. The sequences are as follows: > IgG-PC Knob chain [ka] > IgG-V91T Knob chain [ka] > Knob chain of IgG-N88D [ka] > IgG-D20A Knob Chain [ka] >Hole chain of IgG-PC, IgG-V91T, IgG-D20A or IgG-N88D [ka] > IgG-PC, IgG-V91T, IgG-D20A or IgG-N88D light chain [ka] The above fusion protein was expressed in HEK293 cells and purified with Protein A. The purified product was confirmed to have the expected molecular weight by SDS-PAGE, proving that the target protein was obtained.

[0304] Additionally, the wild-type IL-2 sequence of the present disclosure is as follows: >wtIL-2 [ka]

[0305] Example 11. Detection of M07e cell proliferation-promoting activity by more PD-1-IL-2v To detect the activity of the PD-1-IL-2v fusion proteins with the above different IL-2 variants and their dependency on PD-1 expression, the present inventors detected the effects of the fusion proteins in Example 10 on cell proliferation in M07e and M07e-hPD-1 cell lines. For the experimental method, see Example 9. The relative cell proliferation activity of PD-1-IL-2v was calculated by the following method:

number

number

number

number

number

[0306] The experimental results are shown in Table 14 and Figures 6A and 6B. The results are as follows: 1) In M07e cells, A-PC has the activity of PEG-IL-2v, suggesting that in the absence of PD-1 overexpression, the IL-2 bound to the antibody is consistent with the activity of IL-2 coupled with PEG; the activities of A-V91T and IgG-V91T are comparable and weaker than A-PC, suggesting that V91T indeed reduces the activity of IL-2, but can still stimulate the proliferation of M07e; other variants, including D20A, D20N, N88R, N88D and Q126D, essentially do not activate M07e cells, and only A-N88D has a slight activity at a concentration of 1000nM. 2) In M07e-hPD-1 cells, where PD-1 was overexpressed in the cells, the activity of each PD-1-IL-2v at low concentrations was far greater than that of IgG-V91T and PEG-IL-2v, suggesting that the anti-PD-1 antibody significantly enhanced the dependency of the cell proliferation-promoting activity of PD-1-IL-2v on PD-1 expression. Compared with A-PC and A-V91T-02, the maximum activity of the other PD-1-IL-2v in promoting M07e-hPD-1 cell proliferation was slightly lower. 3) Comparing the activity of each PD-1-IL-2v in the M07e and M07e-hPD-1 cell proliferation experiments, PD-1-IL-2v with D20A, D20N, N88R, N88D or Q126D mutations essentially do not activate M07e cells, but can strongly stimulate the proliferation of M07e-hPD-1 cells. It has been shown that PD-1-IL-2v maintains selective activation of PD-1-positive cells and reduces activation of PD-1-negative cells, whereas A-PC and A-V91T-02 have selective activation of PD-1-positive cells but can also activate PD-1-negative cells.

[0307] To better demonstrate the dependence of PD-1-IL-2v(N88D) and PD-1-IL-2v(D20A) on PD-1, we compared the effects of A-N88D and IgG-N88D, and A-D20A and IgG-D20 on cell proliferation in the M07e-hPD-1 cell line.

[0308] The experimental results are shown in Table 15 and Figure 7. The results show that even at a relatively high concentration (1000 nM), IgG-N88D and IgG-D20A cannot activate the proliferation of M07e-hPD-1 cells, while A-N88D and A-D20A can activate the proliferation of M07e-hPD-1 cells at a relatively low concentration.

[0309] [Table 14-1] [Table 14-2] [Table 15]

[0310] Example 12. Detection of T cell proliferation promoting activity of PD-1-IL-2v Unstimulated T cells do not express or express PD-1 at a low level, whereas T cells stimulated with CD3 antibody express PD-1 at a high level. To detect whether the PD-1-IL-2v fusion proteins with different IL-2 variants described above can activate T cells in human peripheral blood cells (PBMCs) with low and high PD-1 expression, the present inventors isolated T cells in PBMCs, stimulated the isolated T cells with CD3 antibody, and detected the effects of the fusion proteins on the proliferation of unstimulated T cells and stimulated T cells.

[0311] CD3 antibody stimulated T cell proliferation experiment: After resuscitating cryopreserved human PBMCs, T cells were isolated using a Pan T Cell Isolation Kit (Miltenyi, 130-096-535) to obtain T cells. CD3 antibody was diluted to 0.1 μg / mL in PBS, placed in a 10 cm culture dish, and incubated at 37 °C for 2 hours to coat, then washed with PBS before use. T cells were plated at 2 × 10 6The stimulated T cells were harvested, washed, counted, and the cell density was adjusted to 5 × 10 5 The cells were adjusted to 100 cells / mL and seeded at 200 μL / well in a 96-well plate. Samples to be measured were serially diluted with culture medium to prepare a 5× working solution, and 50 μL of each concentration of samples to be measured was added to stimulated T cells, mixed uniformly, and cultured for 4 days. The treated cells were washed twice with FACS buffer (PBS + 2% FBS), labeled for cell viability with Fixable Viability Dye (eBioscience, 65-0865-14), stained with flow antibodies for CD3 (BD, 565491), CD4 (BD, 558116), CD8 (BD, 565310), and Ki-67 (eBioscience, 12-5698-82), and then the cell proliferation status was detected using a FACS instrument.

[0312] Unstimulated T cell proliferation experiments: After counting isolated T cells, 5 × 10 5 The cells were seeded at 10 ...

[0313] The results of the experiment are shown in Table 16, Table 17 and Figures 8A, 8B, 9A and 9B. As a result, 1) in unstimulated T cells, A-PC showed activity to activate T proliferation when the concentration was higher than 1 nM (CD8 + T cell proliferation EC 50 =21.46nM, CD4 + T cell proliferation EC 50=8.61nM), the activity of A-V91T-02 was weaker than A-PC, suggesting that V91T indeed reduces the activity of IL-2 but can still promote the proliferation of unstimulated T cells, and other mutants including D20A, D20N, N88R, N88D and Q126D did not activate T cells at a concentration of 100nM. 2) In T cells stimulated with CD3 antibody, each PD-1-IL-2v showed significant T cell proliferation-promoting activity at a relatively low concentration (0.1nM). Compared with A-PC and A-V91T-02, PD-1-IL-2v with other mutations had a slightly lower maximum activity on pan T cell proliferation stimulated with CD3 antibody. 3) Comparing the activity of each fusion protein in proliferation experiments of unstimulated T cells and T cells stimulated with CD3 antibody, PD-1-IL-2v with the D20A, D20N, N88R, N88D, or Q126D mutations did not activate the proliferation of unstimulated T cells, but could relatively strongly stimulate the proliferation of T cells stimulated with CD3 antibody, whereas A-PC and A-V91T-02 could more strongly activate the proliferation of T cells stimulated with CD3 antibody, but could also strongly activate T cells not stimulated with CD3 antibody.

[0314] [Table 16] [Table 17-1] [Table 17-2]

[0315] Example 13. Identification of the binding affinity of PD-1-IL-2v to antigens PD-1 and IL-2Rα or IL-2Rβ / γ The binding affinity of the PD-1-IL-2v fusion proteins with different IL-2 variants to PD-1 and human IL-2Rα or IL-2Rβ / γ was detected by surface plasmon resonance (SPR) technology. The experiment was performed using a Biacore 8K (GE Healthcare) instrument. A CM5 sensor chip was selected, and HBS-EP+ buffer solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) was used as the mobile phase. Anti-human IgG (Fc) antibody was prepared in 10 mM sodium acetate buffer (pH 5.0) to a concentration of 25 μg / mL, and the Immobilization program was selected to automatically perform amino coupling immobilization of the anti-human IgG (Fc) antibody channel. Each PD-1-IL-2v waiting for measurement was prepared as a ligand using HBS-EP+ buffer solution, and captured by the anti-human IgG (Fc) antibody in the chip channel. Human PD-1 antigen protein (Sino Biological, 10377-H08H), human IL-2Rα receptor protein (Sino Biological, 10165-H08H) or human IL-2Rβ / γ receptor heterodimer protein (Acrosystem, ILG-H5283) was used as the analyte, which was prepared with HBS-EP+ buffer solution, and the analyte was serially diluted 2-fold from 200 nM to a total of seven concentration points, and the experimental and reference channels were run at a flow rate of 30 μL / min, with a binding time of 140 s and a dissociation time of 600 s. The regeneration buffer, 10 mM Glycine pH 1.5 (GE Healthcare, 29238268-AA), was run at a flow rate of 10 μL / min for 30 s. The binding rate Ka and dissociation rate Kd, as well as the dissociation constant (i.e., affinity K D The results are shown in Tables 18 and 19.

[0316] [Table 18] [Table 19] [Table 20]

[0317] As shown by the results of binding to human PD-1 antigen, A-PC, A-N88D and A-D20A have similar binding affinity to human PD-1, and the binding affinity to human PD-1 is similar to that of A17m0902_hIgG4 (Table 8). As shown by the results of binding to human IL-2Rα, none of A-PC, A-N88D and A-D20A bind to human IL-2Rα. As shown by the results of binding to human IL-2Rβ / γ, A-PC has the strongest affinity to human IL-2Rβ / γ, and A-N88D and A-D20A have reduced affinity to human IL-2Rβ / γ by approximately 13-fold and approximately 63-fold, respectively.

[0318] Example 14. Construction and affinity identification of anti-PD-1 nanobody and IL-2 variant fusion proteins (PD-1-IL-2v) with different PD-1 binding affinities 1. Construction of fusion protein of anti-PD-1 nanobody mutant and IL-2 mutant (PD-1-IL-2v) By introducing mutations into anti-PD-1 nanobodies, nanobodies with different affinities for PD-1 can be obtained. We constructed various A-N88D mutants by introducing mutations into the anti-PD-1 nanobody of A-N88D, while keeping the IL-2 mutant sequence of A-N88D intact. The anti-PD-1 nanobody mutations introduced into A-N88D are shown in Table 21, and the mutated anti-PD-1 nanobody sequence is as follows (underlined CDRs): >A17m0902M08 [ka] >A17m0902M15 [ka] >A17m0902M26 [ka] >A17m0902M27 [ka] >A17m0902M28 [ka] [Table 21]

[0319] The CDR sequences of the anti-PD-1 nanobody variants are shown in the table below: [Table 22]

[0320] Thus, antibodies related to A17m09 have a sequence according to the general formula: CDR1 is X6YSMS, where X6 is selected from D or S (SEQ ID NO: 97); CDR2 is IISGSGX1ITHYX7DSVKG, in which X1 is selected from V or G, and X7 is selected from V or A (SEQ ID NO: 98); CDR3 is VSDWX8DY, where X8 is selected from Q or E (SEQ ID NO: 99).

[0321] Specifically, CDR1 is [ka] [ka] may be selected from.

[0322] CDR2 is [ka] [ka] [ka] may be selected from.

[0323] CDR3 is [ka] [ka] may be selected from.

[0324] Additionally, A17 of the present disclosure has a sequence according to the general formula: CDR1: X6YSMS (SEQ ID NO: 97) CDR2: IISGSGX1X2X3HYX7DSVKG, in which X1 is selected from V or G, X2 is selected from I or S, X3 is selected from T or A, and X7 is selected from V or A (SEQ ID NO: 100) CDR3:VSDWX9X5Y, of which X9 is Q、 D or E, and X5 is selected from D or E (SEQ ID NO: 101) Specifically, CDR1 is [ka] [ka] may be selected from.

[0325] CDR2 is [ka] [ka] [ka] [ka] [ka] [ka] may be selected from.

[0326] CDR3 is [ka] [ka] [ka] [ka] may be also possible.

[0327] The fusion proteins of anti-PD-1 Nanobody variants and IL-2 variants (PD-1-IL-2v) are A-N88D-M08, A-N88D-M15, A-N88D-M26, A-N88D-M27, and A-N88D-M28, respectively. For illustrative purposes, the full-length sequence of A-N88D-M08 is shown, with the underlined part being the Fc and the italics being the linker.

[0328] >A-N88D-M08 knob chain [ka] >A-N88D-M08 Hole Chain [ka] 2. Identification of the binding affinity of fusion proteins of anti-PD-1 antibodies and IL-2 mutants (PD-1-IL-2v), including anti-PD-1 nanobody mutants, to human PD-1 antigen The binding affinity of A-N88D with different anti-PD-1 nanobody variants to PD-1 antigen was detected by SPR, and the experimental method was the same as in Example 13. The detection results are shown in Table 23.

[0329] [Table 23-1] [Table 23-2]

[0330] As shown by the results in Table 23, all mutants can achieve effective binding to human PD-1.

[0331] Example 15. Detection of M07e cell proliferation-promoting activity of fusion proteins (PD-1-IL-2v) of anti-PD-1 antibodies with different PD-1 binding affinities and IL-2 variants To detect the activity of PD-1-IL-2v fusion proteins with different PD-1 binding affinities and their dependence on PD-1 binding affinity, the present inventors detected the effects of fusion proteins of PD-1 antibody mutants in Example 14 and IL-2v on cell proliferation in M07e-hPD-1 cell line. Please refer to Example 11 for the experimental method and the calculation method of the relative cell proliferation activity of PD-1-IL-2v.

[0332] The experimental results are shown in Table 24 and Figure 10. As a result, it can be seen that A-N88D-M08, A-N88D-M15, A-N88D-M26, A-N88D-M27 and A-N88D-M28 all have M07e-hPD-1 cell proliferation promoting activity. The M07e-hPD-1 cell proliferation promoting activity of the PD-1-IL-2v fusion protein tends to be almost consistent with the binding affinity to the PD-1 antigen (Table 23), suggesting that the strength of the cell proliferation promoting activity of the PD-1-IL-2v fusion protein depends on the strength of the binding affinity of PD-1-IL-2v to PD-1.

[0333] [Table 24]

[0334] Example 16. A-N88D can inhibit the growth of mouse CT26 tumors This example verified the in vivo antitumor efficacy of A-N88D constructed in Example 10 in a CT26 tumor model of humanized PD-1 Balb / c mice. CT26 mouse colon cancer cell line was cultured in DMEM medium (10% FBS) and 5 × 10 6 CT26 cells were inoculated subcutaneously into humanized PD-1 Balb / c female mice (purchased from Chubu Pharmaceutical Co., Ltd.), and the average tumor volume of the mice was 90 mm 3 When the tumors reached a certain level, they were randomly assigned to groups of 6 and administered antibody or control by intraperitoneal injection, and tumor volumes were measured, body weights were weighed, and data were recorded twice a week. See Table 25 for experimental groupings and dosing schedules. Considering that the molecular weight of A-N88D is approximately 92 kDa and that of A17m0902_hIgG4 is approximately 76 kDa, the dose of A-N88D was adjusted to 120% of A17m0902_hIgG4 to achieve an equimolar dose.

[0335] As shown in Figure 11, compared to the control group, A-N88D exhibited stronger antitumor efficacy, and at equimolar doses, A-N88D had significantly stronger in vivo antitumor efficacy than A17m0902_hIgG4 (*P<0.05, **P<0.01 in the figure). In addition, in terms of mouse toxicity, A-N88D did not cause any changes in mouse body weight (Figure 12), indicating a relatively good safety profile.

[0336] [Table 25]

[0337] Example 17. A-N88D can inhibit the growth of B16F10 tumors in mice This example examined the in vivo antitumor efficacy of A-N88D in a B16F10 tumor model in humanized PD-1 C57BL / 6 mice. B16F10 mouse skin melanoma cell line was cultured in DMEM medium (10% FBS) at 1 × 10 5 B16F10 cells were inoculated subcutaneously into humanized PD-1 C57BL / 6 female mice (purchased from Biocytogen), and the average tumor volume in the mice was 75 mm 3 When the mice reached the age of 10, they were randomly divided into groups of 8 mice each and administered PBS or A-N88D (24 mg / kg) by intraperitoneal injection. Every 2 days, the tumor volume was measured, the body weight was weighed, and the data was recorded. The results showed that on the 10th day, the tumor inhibition rate of A-N88D was 39.9%, significantly higher than that of the PBS control group (p, 0.0007), indicating that A-N88D has a relatively strong antitumor effect. At the same time, A-N88D did not cause any change in mouse body weight, indicating a relatively good safety profile.

Claims

1. An immune complex comprising a PD-1 antibody or an antigen-binding fragment thereof and an IL-2 mutant, the IL-2 mutant having any one or any combination of the following mutations (i) and (ii) to (iii): (i) a mutation that confers on the IL-2 mutant a reduced affinity for IL-2Rβ / γ compared to wild-type IL-2; (ii) a mutation that confers on the IL-2 mutant a reduced affinity for IL-2Rα compared to wild-type IL-2, or that does not bind IL-2Rα; (iii) a mutation that confers improved stability, expression level and / or purity to the IL-2 variant; Including, Preferably, (i) the mutations conferring to the IL-2 variant reduced affinity for IL-2Rβ / γ compared to wild-type IL-2 are at any one or any combination selected from positions 12, 15, 16, 18, 19, 20, 23, 43, 69, 84, 87, 88, 91, 92, 95, 123, 126, 127, 129, and 130; (ii) the mutations conferring to the IL-2 variant a reduced affinity for IL-2Rα compared to wild-type IL-2 or no binding to IL-2Rα are at any one or any combination selected from positions 38, 42, 45, 62, 65, 68, and 72; (iii) the mutation that confers improved stability, expression level and / or purity to the IL-2 variant compared to wild-type IL-2 is at any one or any combination selected from positions 11, 26, 27, 29, 30, 70, 71, 78, 82 and 132; The positions are numbered according to the wild-type IL-2 amino acid sequence set forth in SEQ ID NO:

87. immune complex.

2. The mutation (i) is L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G, D84A, D84T, and / or comprising any one or any combination of the following mutations: D84P, N88D, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K and S127Q. The mutation (ii) is R38A, R38D, R38E, E62Q, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, P65R, and / or comprising any one or any combination of the following mutations: P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K; and / or The mutation (iii) comprises at least one of the following mutations: N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, and G27C / F78C; Preferably, said mutation (i) comprises at least one of the following mutations: N88D, N88R, D20A, D20N, V91T, Q126D and H16A / D84S; and / or The mutation (ii) comprises at least one of the following mutations: F42A, Y45A, L72G, R38E / F42A, F42A / L72G, F42A / Y45A, Y45A / L72G and F42A / Y45A / L72G; and / or The mutation (iii) comprises at least one of the following mutations: N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, and N26Q / N30S / G27C / F78C; The immune complex of claim 1.

3. the IL-2 variant further comprises a mutation at position 3 and / or 125, the mutation at position 3 being preferably T3A and the mutation at position 125 being preferably C125A or C125S; The immune complex according to claim 1 or 2.

4. The IL-2 mutant is F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / R38E / V91T, F42A / L72G / Q126D, and It contains any one of the following mutations: F42A / L72G / H16A / D84S; Preferably, the IL-2 mutant comprises F42A / L72G / N88D / N26Q / N29S / N71Q, F42A / L72G / N88R / N26Q / N29S / N71Q, F42A / L72G / D20A / N26Q / N29S / N71Q, F42A / L72G / D20N / N26Q / N29S / N71Q, F42A / L72G / V91T / N26Q / N29S / N71Q, F42A / L72G / Q126D / N26Q / N29S / N71Q, and Contains any one of the following mutations: F42A / L72G / H16A / D84S / N26Q / N29S / N71Q; More preferably, the IL-2 mutant is T3A / F42A / R38E / V91T / C125A, T3A / F42A / L72G / N88D / N26Q / N29S / N71Q / C125A, T3A / F42A / L72G / N88R / N26Q / N29S / N71Q / C125A, T3A / F42A / L72G / D20A / N26Q / N29S / N71Q / C125A, T3A / F42A / L72G / D20N / N26Q / N29S / N71Q / C125A, T3A / F42A / L72G / V91T / N26Q / N29S / N71Q / C125A, T3A / F42A / L72G / Q126D / N26Q / N29S / N71Q / C125A, and Contains any one of the following mutations: T3A / F42A / L72G / H16A / D84S / N26Q / N29S / N71Q / C125A; The immune complex according to any one of claims 1 to 3.

5. The IL-2 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 60 to 68 or a sequence having at least 90% identity thereto; The immune complex according to any one of claims 1 to 4.

6. The PD-1 antibody, or antigen-binding fragment thereof, comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprising: 1) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 97, 100, 101, or 2) comprising CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6; Preferably, 1-1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 36 and 37; 1-2) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 97, 98, or 99, or 1-3) comprising a CDR1 represented by SEQ ID NO: 7 or 93, a CDR2 represented by any one of SEQ ID NOs: 8, 38 to 40, and 94 to 95, and a CDR3 represented by any one of SEQ ID NOs: 9, 41, 42, and 96; Preferably, the immunoglobulin single variable domain is selected from 1) to 19), i.e. 1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 41; 2) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 96; 3) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 94 and 41; 4) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 94 and 41; 5) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 94 and 96; 6) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 95 and 96; 7) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 9; 8) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 42; 9) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 6; 10) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 9; 11) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 6; 12) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 41; 13) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 42; 14) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 41; 15) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 42; 16) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 41; 17) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 42; 18) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 41, and 19) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 42; and Preferably, said immunoglobulin single variable domain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 13-15, 17-35, 88-92, an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 16, or an amino acid sequence having at least 90% sequence identity to said any one of said sequences. The immune complex according to any one of claims 1 to 5.

7. the PD-1 antibody or antigen-binding fragment thereof comprises a PD-1-binding domain 1 and / or a PD-1-binding domain 2, the PD-1-binding domain 1 comprising at least one of the immunoglobulin single variable domains, and the PD-1-binding domain 2 comprising a heavy chain variable region (VH) and a light chain variable region (VL); Preferably, the PD-1 binding domain 2 is selected from Fab, Fab', Fv, or ScFv, more preferably Fab; Preferably, the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 54 to 56, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 57 to 59. More preferably, the VH comprises a heavy chain variable region in a Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the VL comprises a light chain variable region in a Fab light chain of the amino acid sequence shown in SEQ ID NO:

53. The immune complex according to any one of claims 1 to 6.

8. An immune complex comprising a PD-1 antibody or an antigen-binding fragment thereof and IL-2 or a variant thereof, The PD-1 antibody, or antigen-binding fragment thereof, comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable domain comprising: 1) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 97, 100, 101, or 2) comprising CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6; Preferably, 1-1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 36 and 37; 1-2) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 97, 98, or 99, or 1-3) comprising a CDR1 represented by SEQ ID NO: 7 or 93, a CDR2 represented by any one of SEQ ID NOs: 8, 38 to 40, and 94 to 95, and a CDR3 represented by any one of SEQ ID NOs: 9, 41, 42, and 96; immune complex.

9. The immunoglobulin single variable domains are selected from 1) to 19), i.e. 1) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 41; 2) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 96; 3) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 94 and 41; 4) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 94 and 41; 5) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 94 and 96; 6) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 93, 95 and 96; 7) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 9; 8) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 42; 9) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 6; 10) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 9; 11) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 6; 12) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 41; 13) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 8 and 42; 14) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 41; 15) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 38 and 42; 16) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 41; 17) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 39 and 42; 18) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 41, and 19) CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 7, 40 and 42; and The immune complex of claim 8.

10. the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reducing antibody deamidation and / or reducing antibody isomerization, Preferably, the heavy chain framework region of the human germline template used in the humanization modification process is IGHV3-23*01 or IGHV3-23*04. The immune complex according to claim 8 or 9.

11. The immunoglobulin single variable domain comprises: An amino acid sequence represented by any one of SEQ ID NOs: 3, 13 to 15, 17 to 35, and 88 to 92; An amino acid sequence shown in any one of SEQ ID NOs: 2 and 16, or The present invention relates to a method for producing a nucleic acid sequence comprising the steps of: The immune complex according to any one of claims 8 to 10.

12. the PD-1 antibody or antigen-binding fragment thereof comprises a PD-1-binding domain 1 and / or a PD-1-binding domain 2, the PD-1-binding domain 1 comprising at least one of the immunoglobulin single variable domains, and the PD-1-binding domain 2 comprising a heavy chain variable region (VH) and a light chain variable region (VL); Preferably, the PD-1 binding domain 2 is selected from Fab, Fab', Fv, or ScFv, more preferably Fab; Preferably, the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 54 to 56, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 57 to 59. More preferably, the VH comprises a heavy chain variable region in a Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the VL comprises a light chain variable region in a Fab light chain of the amino acid sequence shown in SEQ ID NO:

53. The immune complex according to any one of claims 8 to 11.

13. The IL-2 mutant has the following mutations (i) to (iii): (i) a mutation that confers on the IL-2 mutant a reduced affinity for IL-2Rβ / γ compared to wild-type IL-2; (ii) a mutation that confers on the IL-2 mutant a reduced affinity for IL-2Rα compared to wild-type IL-2, or that does not bind IL-2Rα; (iii) a mutation that confers improved stability, expression level and / or purity to the IL-2 variant; any one or any combination of the following mutations: Preferably, (i) the mutations conferring to the IL-2 variant reduced affinity for IL-2Rβ / γ compared to wild-type IL-2 are at any one or any combination selected from positions 12, 15, 16, 18, 19, 20, 23, 43, 69, 84, 87, 88, 91, 92, 95, 123, 126, 127, 129, and 130; (ii) the mutations conferring to the IL-2 variant a reduced affinity for IL-2Rα compared to wild-type IL-2 or no binding to IL-2Rα are at any one or any combination selected from positions 38, 42, 45, 62, 65, 68, and 72; (iii) the mutation that confers improved stability, expression level and / or purity to the IL-2 variant compared to wild-type IL-2 is at any one or any combination selected from positions 11, 26, 27, 29, 30, 70, 71, 78, 82 and 132; The positions are numbered according to the wild-type IL-2 amino acid sequence set forth in SEQ ID NO:

87. The immune complex according to any one of claims 8 to 12.

14. The mutation (i) is L12R, L12K, L12E, L12Q, E15Q, E15R, E15A, E15S, H16N, H16A, H16E, H16D, H16G, H16S, H16T, H16V, H16P, D20A, D20H, D20Y, D20N, M23A, M23R, M23K, M23G, M23S, M23T, M23V, M23P, S87K, S87A, D84S, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, D84G, D84A, D any one or any combination of the following mutations: 84T, D84P, N88D, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, E95Q, E95G, E95T, E95V, E95P, E95H, E95N, T123A, T123E, T123K, T123Q, Q126D, Q126L, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K, and S127Q; Preferably, said mutation (i) is At least one of the following mutations: N88D, N88R, D20A, D20N, V91T, Q126D, and H16A / D84S; The immune complex of claim 13.

15. The mutation (ii) is R38A, R38D, R38E, E62Q, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, P6 any one or any combination of the following mutations: 5R, P65E, P65K, P65H, P65Y, P65Q, P65D, P65N, E68A, E68Q, E68K, E68R, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K; Preferably, said mutation (ii) is At least one of the following mutations is included: F42A, Y45A, L72G, R38E / F42A, F42A / L72G, F42A / Y45A, Y45A / L72G, and F42A / Y45A / L72G; 15. The immune complex of claim 13 or 14.

16. the mutation (iii) comprises the mutations N26Q, N29S, N30S, N71Q, Q11C / L132C, L70C / P82C, and G27C / F78C; Preferably, said mutation (iii) is at least one of the following mutations: N26Q, N29S, N30S, N26Q / N29S / N71Q, N26Q / N29S, N26Q / N30S, N26Q / N30S / Q11C / L132C, N26Q / N29S / L70C / P82C, and N26Q / N30S / G27C / F78C; The immune complex according to any one of claims 13 to 15.

17. The IL-2 mutant is F42A / L72G / N88D, F42A / L72G / N88R, F42A / L72G / D20A, F42A / L72G / D20N, F42A / L72G / V91T, F42A / R38E / V91T, F42A / L72G / Q126D, and It contains any one of the following mutations: F42A / L72G / H16A / D84S; Preferably, the IL-2 mutant comprises F42A / L72G / N88D / N26Q / N29S / N71Q, F42A / L72G / N88R / N26Q / N29S / N71Q, F42A / L72G / D20A / N26Q / N29S / N71Q, F42A / L72G / D20N / N26Q / N29S / N71Q, F42A / L72G / V91T / N26Q / N29S / N71Q, F42A / L72G / Q126D / N26Q / N29S / N71Q, and Contains any one of the following mutations: F42A / L72G / H16A / D84S / N26Q / N29S / N71Q; The immune complex according to any one of claims 13 to 16.

18. the IL-2 variant further comprises a mutation at position 3 and / or 125, the mutation at position 3 being preferably T3A and the mutation at position 125 being preferably C125A or C125S; The immune complex according to any one of claims 13 to 17.

19. The IL-2 variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 60 to 68 or a sequence having at least 90% identity thereto; The immune complex according to any one of claims 13 to 18.

20. further comprising an immunoglobulin Fc region, Preferably, the Fc region is a human IgG1 or IgG4 Fc region. An immune complex according to any one of the preceding claims.

21. the human IgG1 Fc region comprises one or more amino acid mutations that reduce binding to an Fc receptor (FcR) and / or reduce effector function; Preferably, the Fc region of said human IgG1 comprises one or more amino acid mutations that reduce binding to Fcγ receptors (FcγR) and / or reduce ADCC effect; More preferably, the human IgG1 Fc region comprises any one of the following mutations: L234A, L235A, P329G, or any combination of these mutations; The immune complex of claim 20.

22. the Fc region comprises a first subunit and a second subunit, the Fc region comprising a knob-into-hole modification that promotes binding between the first subunit and the second subunit; Preferably, the first subunit of the Fc region comprises a knob modification and the second subunit of the Fc region comprises a hole modification.

22. The immune complex of claim 20 or 21.

23. the first subunit of the Fc region comprises a T366 mutation, and the second subunit comprises a mutation selected from T366, L368, and Y407, or any combination thereof; a first subunit of the Fc region comprises an S354 or E356 mutation and a second subunit comprises a Y349 mutation; or a first subunit of the Fc region comprises S354 and T366 mutations, and a second subunit comprises Y349, T366, L368 and Y407 mutations; Preferably, the first subunit of the Fc region comprises a T366W mutation, and the second subunit comprises a mutation selected from T366S, L368A, and Y407V, or any combination thereof; a first subunit of the Fc region comprises an S354C or E356C mutation and a second subunit comprises a Y349C mutation; or The first subunit of the Fc region comprises a S354C / T366W mutation and the second subunit comprises a Y349C / T366S / L368A / Y407V mutation; 23. The immune complex of claim 22.

24. The second subunit of the Fc region further comprises a H435 and / or Y436 mutation, preferably a H435R / Y436F, H435R or H435K mutation; 24. The immune complex of claim 22 or 23.

25. The first subunit and / or the second subunit of the Fc region further comprises a C220 mutation, preferably a C220S mutation. The immune complex according to any one of claims 22 to 24.

26. the human IgG1 Fc region comprises a mutation that enhances FcRn-mediated recycling and / or extends half-life; Preferably, the human IgG1 Fc region comprises a mutation selected from M252, S254, T256, M428, or any combination thereof; Preferably, the human IgG1 Fc region comprises the following mutations: M252Y / M428V, M252Y / M428L, M252Y / S254T / T256E; The immune complex according to any one of claims 20 to 25.

27. the PD-1 antibody or antigen-binding fragment thereof is at the N-terminus of the Fc region, and the IL-2 or variant thereof is at the N-terminus or C-terminus of the Fc region; Preferably, the PD-1 antibody or antigen-binding fragment thereof is at the N-terminus of the Fc region and the IL-2 or variant thereof is at the C-terminus of the Fc region. An immune complex according to any one of the preceding claims.

28. said IL-2 or variant thereof is in a polypeptide chain in which the first subunit of the Fc region is present, and preferably said IL-2 or variant thereof is linked directly or via a linker to the first subunit of the Fc region; The immune complex according to any one of claims 22 to 27.

29. The valency ratio of the PD-1 binding domain to IL-2 or a variant thereof in the PD-1 antibody or antigen-binding fragment thereof is between 4:1 and 1:2, preferably 4:1, 3:1, 2:1, or 1:

1. An immune complex according to any one of the preceding claims.

30. Further comprising a linker, Preferably, the linker is (G ｘ S) ｙ a linker, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6; More preferably, x is 4 and y is 1, 2 or 3. An immune complex according to any one of the preceding claims.

31. From the N-terminus to the C-terminus, the following I) to VI) are respectively: I) First polypeptide chain: [PD-1 binding domain 1]-[first subunit of Fc region]-[linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[second subunit of Fc region]; II) First polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[first subunit of Fc region]-[linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[second subunit of the Fc region]; III) First polypeptide chain: [Fab heavy chain of PD-1 binding domain 2]-[first subunit of Fc region]-[linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[second subunit of Fc region]; Third polypeptide chain: [Fab light chain of PD-1 binding domain 2], IV) first polypeptide chain: [Fab heavy chain of PD-1 binding domain 2]-[first subunit of Fc region]-[linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[second subunit of the Fc region]; Third polypeptide chain: [Fab light chain of PD-1 binding domain 2], V) First polypeptide chain: [PD-1 binding domain 1]-[Linker 2]-[Fab heavy chain of PD-1 binding domain 2]-[First subunit of Fc region]-[Linker 1]-[IL-2 or a mutant thereof]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[Fab light chain of PD-1 binding domain 2]-[second subunit of Fc region]; Third polypeptide chain: [Fab light chain of PD-1 binding domain 2], VI) First polypeptide chain: [IL-2 or a variant thereof]-[Linker 3]-[First subunit of Fc region]; Second polypeptide chain: [PD-1 binding domain 1]-[linker 2]-[PD-1 binding domain 1]-[second subunit of the Fc region]; The present invention relates to a combination of polypeptide chains represented by any one of the following: In the formula, - represents a peptide bond, the linker is a polypeptide capable of performing a linking function, and any two of the linker 1, the linker 2, and the linker 3 may be the same or different. Preferably, the linker is (G ｘ S) ｙ linker, wherein x is selected from an integer of 1 to 5 and y is selected from an integer of 1 to 6, more preferably linker 1 is (G ４ S) ３ and linker 2 is (G ４ S) ２ and linker 3 is G ４ S, Any two of the [PD-1-binding domains 1] may be the same or different and are any PD-1-binding domain selected from claims 1 to 3; Preferably, [PD-1-binding domain 2] comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 54 to 56, and the VL comprises LCDR1, LCDR2, and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 57 to 59, and preferably, the VH comprises a heavy chain variable region in a Fab heavy chain of the amino acid sequence shown in SEQ ID NO: 52, and the VL comprises a light chain variable region in a Fab light chain of the amino acid sequence shown in SEQ ID NO: 53, An immune complex according to any one of the preceding claims.

32. SEQ ID NOs: 73 and 74, SEQ ID NOs: 75 and 76, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47, 44 and 48, SEQ ID NOs: 47, 46 and 48, SEQ ID NOs: 49, 50 and 48, or SEQ ID NOs: 51 and 46, SEQ ID NOs: 102 and 103, The combination of polypeptide chains represented by the amino acid sequence An immune complex according to any one of the preceding claims.

33. A nucleic acid molecule encoding the immunoconjugate of any one of claims 1 to 32.

34. A host cell comprising the nucleic acid molecule of claim 33.

35. A method for preparing an immune complex according to any one of claims 1 to 32, comprising culturing a host cell according to claim 34 and expressing said immune complex, and optionally isolating or purifying said immune complex. method.

36. 33. A method for the preparation of a composition comprising administering to a patient an immunoconjugate according to any one of claims 1 to 32, and one or more pharma- ceutically acceptable vectors, diluents or excipients. Pharmaceutical compositions.

37. The following (1) to (5), namely: (1) For the treatment of cancer or for the preparation of a drug for treating cancer, (2) For the activation of cytotoxic T lymphocytes (CTLs) or for the preparation of a drug for activating cytotoxic T lymphocytes (CTLs), (3) CD4 ＋ and / or CD8 ＋ Stimulation of T cell proliferation, or CD4 ＋ and / or CD8 ＋ For the preparation of a medicament for promoting T cell proliferation, (4) To confer a higher cell activation efficiency to cytotoxic T lymphocytes (CTLs) with high expression of PD-1 antibody than to cytotoxic T lymphocytes (CTLs) with low or no expression of PD-1 antibody; and (5) CD4 with high expression of PD-1 antibody ＋ and / or CD8 ＋ T, low or no expression of PD-1 antibody, CD4 ＋ and / or CD8 ＋ To confer higher cell proliferation efficiency than T cells, Use of an immunoconjugate according to any one of claims 1 to 32, a nucleic acid molecule according to claim 33 or a pharmaceutical composition according to claim 36 in at least one of the following ways: Preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and blood system cancer; use.

38. A method for treating or ameliorating a disease or condition, comprising administering to a subject in need thereof an immunoconjugate according to any one of claims 1 to 32, or a nucleic acid molecule according to claim 33, or a pharmaceutical composition according to claim 36, Preferably, the disease is cancer; More preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, and blood system cancer; method.

39. A method for activating cytotoxic T lymphocytes (CTLs), comprising administering to a subject in need thereof an immunoconjugate according to any one of claims 1 to 32, or a nucleic acid molecule according to claim 33, or a pharmaceutical composition according to claim 36, Preferably, the subject is suffering from cancer; More preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, blood system cancer; Preferably, the cytotoxic T cells (CTLs) specifically express PD-1. method.

40. CD4 ＋ and / or CD8 ＋ A method for promoting T cell proliferation, comprising administering to a subject in need thereof an immunoconjugate according to any one of claims 1 to 32, or a nucleic acid molecule according to claim 33, or a pharmaceutical composition according to claim 36, Preferably, the subject is suffering from cancer; More preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, renal cancer, squamous cell carcinoma, blood system cancer; Preferably, the CD4 ＋ and / or CD8 ＋ T cells specifically express PD-1. method.

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