Dual-function anti-PD-1 / IL-7 molecules

Abstract

This invention relates to a bifunctional molecule containing an anti-PD-1 antibody and IL-7, and its use. [Solution] The present inventors provide a bifunctional molecule containing an anti-hPD-1 antibody and human IL-7, which is promising for numerous therapeutic applications, particularly in the treatment of cancer.

Description

[Technical Field]

[0001] This invention relates to the field of immunotherapy. The invention provides a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof. [Background technology]

[0002] The method of targeting T-cell inhibitory checkpoints with therapeutic antibodies for disinhibition is a field of active research (see Pardoll, Nat Rev Cancer., 2012; Vol. 12: pp. 253-264 for a review). Targeting immune checkpoints in adaptive immunity has shown excellent therapeutic efficacy in treating a wide range of cancers, though in a limited proportion of patients. Combinations of immune checkpoint therapy with other immunotherapy strategies have shown excellent efficiency in preclinical models, but remain challenging in clinical practice.

[0003] Immune cell activation is regulated by integrating the balance between costimulatory and coinhibitory signals. T cell receptor (TCR)-mediated T cell activation is modulated by both costimulatory and coinhibitory signals. A second antigen-independent signal modifies the first signal, provided by the interaction between the antigen peptide-MHC complex and the TCR, which confers specificity to the response. T cell costimulatory and coinhibitory pathways have a broad range of immunomodulatory functions, regulating effector T cells, memory T cells, regulatory T cells, and naive T cells. Therapeutic modulation of these pathways is bridging the gap to effective new strategies for treating cancer (see review article: Schildberg et al., vol. 44(5), Immunity, 2016). Ongoing research into the regulation of immune responses is leading to the identification of multiple immunological pathways that could be targeted for the development of cancer therapies. Such molecules are referred to herein as immune checkpoint co-activators or co-inhibitors (see reviews Sharma et al., Cell, Vol. 161 (No. 2), 2015 and Pardoll, Nature Reviews Cancer, Vol. 12 (No. 4), 2012).

[0004] Programmed cell death protein 1 (PD-1, also known as CD279) is a cell surface protein molecule belonging to the immunoglobulin superfamily. Programmed cell death protein 1 is expressed on T lymphocytes, B lymphocytes, and macrophages, and plays a role in cell fate and differentiation. Two ligands for PD-1, PD-L1 and PD-L2, have been identified that have been shown to downregulate T cell activation when bound to PD-1 (Freeman et al. (2000) J Exp Med vol. 192: pp. 1027-1034; Latchman et al. (2001) Nat Immunol vol. 2: pp. 261-268; Carter et al. (2002) Eur J Immunol vol. 32: pp. 634-643). The interaction between PD-1 and its ligands results in a reduction of tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells. In particular, PD1 ligation inhibits the T cell response by reducing signaling downstream of TCR stimulation to T cells, resulting in decreased activation and cytokine production.

[0005] While PD-1 / PD-L1 therapy is approved by the FDA as a first- and second-line treatment for a very broad range of hematological and solid tumors, objective responses based on a tumor size reduction of more than 30%, as defined by RECIST criteria, vary considerably among cancer subtypes.

[0006] High response rates were observed in refractory Hodgkin lymphoma (65-85%) (Borcherding N et al., J Mol Biol., July 6, 2018; Vol. 430 (No. 14): pp. 2014-2029), high microsatellite instability colorectal cancer (MSI-H, 25-80%), or Merkel cell carcinoma (56%).

[0007] In melanoma patients (24–44%) and non-small cell lung cancer patients (12.8–43.7%) using anti-PD-1 therapy as first-line treatment, moderate objective response rates are observed. Although only a portion of patients benefit from the therapy, PD-1 / PD-L1 treatment improved overall survival compared to the older standard of treatment, chemotherapy.

[0008] In some solid tumors, particularly pancreatic cancer, non-MSI colorectal cancer, gastric cancer, and some breast cancers, the clinical response was low or not observed at all (Borcherding N et al., J Mol Biol., July 6, 2018; Vol. 430 (No. 14): pp. 2014-2029).

[0009] Multiple mechanisms have been described that can explain the differences in efficacy and resistance to PD-1 / PD-L1 checkpoint therapy, some of which relate to T cell biology, including (1) impaired memory T cell formation, (2) impaired T cell infiltration, (3) insufficient tumor-specific T cell generation, (4) inadequate T cell function, and (5) an immunosuppressive microenvironment induced by regulatory T cells. Combination with IL-7 signaling-targeted therapies may be a good strategy for overcoming anti-PD-1 resistant patients by stimulating T cell infiltration, sustaining T cell effector capacity, and promoting a long-lasting memory T cell response without stimulating the expansion, proliferation, and survival of regulatory T cells.

[0010] Interleukin-7 is an immunostimulant cytokine member of the IL-2 superfamily, playing a crucial role in the adaptive immune system and promoting B and T cell-mediated immune responses. This cytokine activates immune function by promoting the survival and differentiation of T and B cells, the survival of lymphocytes, and the stimulation of natural killer (NK) cell activity. IL-7 also regulates lymph node development via lymphoid tissue inducer (LTi) cells and promotes the survival and division of naive or memory T cells. Furthermore, IL-7 enhances the human immune response by promoting the secretion of IL-2 and interferon-γ. The IL-7 receptor is a heterodimer consisting of IL-7Rα (CD127) and a common γ chain (CD132). While the γ chain is expressed in all hematopoietic cell types, IL-7Rα is primarily expressed in lymphocytes, including B and T lymphocyte progenitor cells, naive T cells, and memory T cells. IL-7Rα expression in regulatory T cells has been observed to be lower compared to effector / naive T cells that express higher levels; therefore, CD127 is used as a surface marker to distinguish these two populations. IL-7Rα is also expressed in innate lymphoid cells as NK cells and intestinal lymphoid tissue (GALT)-derived T cells. The IL-7Rα (CD127) chain is shared with TSLP (tumor stromal lymphopoietin), and CD132 is shared with IL-2, IL-4, IL-9, IL-15, and interleukin-21. Two major signaling pathways: (1) the Janus kinase / STAT pathway (i.e., Jak-Stat-3 and 5) and (2) the phosphatidyl-inositol-3 kinase pathway (i.e., PI3K-Akt) are induced by CD127 / CD132. IL-7 administration is well tolerated by patients and leads to increased proliferation of CD8 and CD4 cells, as well as a relative decrease in CD4+ T regulatory cells. Recombinant naked IL-7 or IL-7 fused to the N-terminal domain of the Fc of an antibody is being tested clinically. The latter is based on the rationale that fusion with the Fc domain extends the half-life of IL-7, thereby enhancing the long-term effectiveness of the treatment.Since IL-2 acts on both Treg and T effector cells, while IL-7 selectively activates T effector cells, targeting IL-7 signaling should be more promising than targeting IL-2 signaling.

[0011] The development of combination therapies targeting IL-7 signaling to increase the efficacy of anti-PD-1 immunotherapy and overcome patients' potential anti-PD-1 resistance may be a good strategy to stimulate T cell infiltration, sustain T cell effector capacity, and promote long-lasting memory T cell responses without stimulating the expansion, proliferation, and survival of regulatory T cells. In fact, anti-PD-1 therapy increases CD127 expression in exhausted T cells, thereby increasing their ability to respond to IL-7 and improving the co-production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) (Pauken et al., Science. December 2, 2016; Vol. 354 (No. 6316): pp. 1160-1165; Shi et al., Nat Commun. August 8, 2016; Vol. 7: pp. 12335). [Prior art documents] [Patent Documents]

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[0014] However, the validation and development of combination immunotherapies are significantly limited by the cost of biotherapy and the limited opportunities for such immunotherapies. Therefore, there remains a critical need in the art for novel and improved substances for safe immunotherapies, particularly against cancer, that target T cells and have effective and positive effects on adaptive immune responses, especially T-cell immune responses. The inventors have made significant progress with the invention disclosed herein. [Means for solving the problem]

[0015] The inventors provide a bifunctional molecule comprising an anti-hPD-1 antibody and human IL-7, which has promising applications in numerous therapies, particularly in the treatment of cancer. The present invention is based on the development of an antibody specifically targeting human PD-1 that exhibits high binding affinity to PD-1 and strongly competes with its ligands PD-L1 and PD-L2. Remarkably, by fusing the N-terminus of IL-7 to the C-terminus of the Fc region of the anti-hPD-1 antibody, its high affinity for CD127 (IL-7 receptor) can be preserved to a degree comparable to endogenous IL-7, suggesting potent IL-7R activation. Furthermore, fusing the Fc domain to IL-7 extends the product half-life. Moreover, the bifunctional anti-PD1 / IL-7 molecule disclosed herein enables the accumulation of IL-7 in PD-1+ T cell infiltrations and the relocalization of IL-7 on PD-1+ T cells. In particular, anti-PD-1 / IL-7 bifunctional molecules induce proliferation and activation of unsensitized, partially exhausted, and fully exhausted T cell subsets, as reflected by cytokine (e.g., IFNγ) secretion and integrin (e.g., alpha-4, beta-7, and LFA-1) expression. Such anti-hPD-1 / IL-7 bifunctional molecules have the ability to overcome associated resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

[0016] In the first aspect, the present invention is (a) (i) Heavy chain variable domains (VH) including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains (VLs) including LCDR1, LCDR2, and LCDR3 Anti-human PD-1 antibodies or their antigen-binding fragments, including; and (b) Human interleukin 7 (IL-7) or fragment thereof A bifunctional molecule containing, The present invention relates to a bifunctional molecule in which an antibody or fragment thereof is covalently linked to human IL-7 or a fragment thereof as a fusion protein, preferably by a peptide linker.

[0017] In particular, the N-terminus of human IL-7 or its fragment is attached to the C-terminus of the heavy chain, light chain, or both of the anti-human PD-1 antibody or its antigen-binding fragment.

[0018] In one embodiment, the antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or a human antibody.

[0019] In certain embodiments, the present invention (i) Heavy chain variable domains (VH) including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains (VLs) including LCDR1, LCDR2, and LCDR3 A bifunctional molecule comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof, which includes or consists of - Heavy chain CDR1 (HCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 1. - Heavy chain CDR2 (HCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 2. - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 3, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E. - Light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 12, where X is G or T. - Light chain CDR2 (LCDR2) contains or consists of the amino acid sequence of SEQ ID NO: 15. - Light chain CDR3 (LCDR3) contains or consists of the amino acid sequence of SEQ ID NO: 16. Regarding bifunctional molecules.

[0020] In particular, the anti-human PD-1 antibody or its antigen-binding fragment comprises (a) VH, which includes or comprises the amino acid sequence of SEQ ID NO: 17, where X1 is D or E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E; and (b) VL, which includes or comprises the amino acid sequence of SEQ ID NO: 26, where X is G or T.

[0021] More specifically, the present invention relates to a bifunctional molecule comprising an anti-human PD-1 antibody or its antigen-binding fragment, wherein the anti-human PD-1 antibody or its antigen-binding fragment is (i) A heavy chain variable region (VH) containing or consisting of the amino acid sequence of SEQ ID NO: 24; and (ii) A light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO: 28. This relates to a bifunctional molecule containing or consisting of [a specific substance].

[0022] Alternatively, the anti-PD1 antibody may be selected from the group consisting of pembrolizumab, nivolumab, pizilizumab, semiprimab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4.

[0023] In particular, IL-7 or its variants contain or consist of an amino acid sequence having at least 75% identity with wild-type human IL-7 (wth-IL-7). In certain embodiments, IL-7 contains or consists of the amino acid sequence shown in SEQ ID NO: 51.

[0024] Alternatively, IL-7 is an IL-7 variant that exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7), comprising or consisting of the amino acid sequence shown in SEQ ID NO: 51, wherein the variant includes at least one amino acid mutation that i) reduces the affinity of the IL-7 variant to the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 to IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wth-IL-7.

[0025] In particular, at least one mutation may be an amino acid substitution or a group of amino acid substitutions selected from the group consisting of (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E, D74Q, or D74N, iv) Q11E, Y12F, M17L, Q22E, and / or K81R; or any combination thereof.

[0026] In one embodiment, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S.

[0027] In another embodiment, the IL-7 variant includes an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y.

[0028] In another embodiment, the IL-7 variant includes an amino acid substitution selected from the group consisting of D74E, D74Q, and D74N.

[0029] Preferably, the IL-7 variant contains or comprises the amino acid sequence shown in SEQ ID NOs. 53 to 66. More preferably, the IL-7 variant contains or comprises the amino acid sequence shown in SEQ ID NOs. 54, 56, or 63.

[0030] In certain embodiments, the antibody or its antigen-binding fragment comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, preferably from an IgG1 or IgG4 heavy chain constant domain.

[0031] In a more specific embodiment, the antibody or its antigen-binding fragment comprises a light chain constant domain derived from the human kappa light chain constant domain, and optionally T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S23 The heavy chain constant domain derived from a human IgG1 heavy chain constant domain comprises a substitution or combination of substitutions selected from the group consisting of 9D / I332E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably N297A combined with M252Y / S254T / T256E, and L234A / L235A.

[0032] In another, more specific embodiment, the antibody or its antigen-binding fragment comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E, and K444A.

[0033] Optionally, the antibody or fragment thereof is linked to IL-7 or a variant thereof by a linker sequence selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, more preferably by (GGGGS)3 or (GGGS)3.

[0034] In a very specific embodiment, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N, and the antibody or its antigen-binding fragment includes a light chain constant domain derived from the human kappa light chain constant domain, and optionally T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E33 The antibody or fragment thereof contains a heavy chain constant domain derived from a human IgG1 heavy chain constant domain, having substitutions or combinations of substitutions selected from the group consisting of 3A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I332E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably having substitutions or combinations of substitutions selected from the group consisting of N297A combined with M252Y / S254T / T256E and L234A / L235A, and the antibody or fragment thereof is linked to the IL-7 variant by linker (GGGGS)3.

[0035] In another embodiment, the present invention relates to isolated nucleic acid sequences or groups of isolated nucleic acid molecules encoding the bifunctional molecules disclosed herein, vectors comprising the nucleic acids or groups of nucleic acid molecules disclosed herein, and / or host cells comprising vectors comprising the nucleic acids or groups of nucleic acid molecules disclosed herein.

[0036] In another embodiment, the present invention relates to a method for producing a bifunctional molecule, comprising the steps of culturing host cells as disclosed herein and optionally isolating a bifunctional molecule.

[0037] In another aspect, the present invention relates to a pharmaceutical composition comprising a bifunctional molecule, nucleic acid or group of nucleic acid molecules, a vector or host cell, and a pharmaceutically acceptable carrier as disclosed herein.

[0038] Optionally, the pharmaceutical composition may further include additional therapeutic agents selected from the group consisting of: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), activators of the cell death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (Bi-Specific T cell Engager) antibodies, antibody-drug conjugates, bioreaction modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapies, immunological agents, apoptosis protein inhibitors (IAP) inhibitors, and intercalating antibiotics. Antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian targets of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, polyvalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly-ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small molecule inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, etc., epitopes or neoepitopes derived from tumor antigens, and one or more combinations of such substances.

[0039] In particular, pharmaceutical compositions, bifunctional molecules, nucleic acids or groups of nucleic acid molecules, vectors, or host cells are intended for use as pharmaceuticals.

[0040] Finally, the present invention relates to pharmaceutical compositions, bifunctional molecules, nucleic acids or groups of nucleic acid molecules, vectors, or host cells as disclosed herein, for use as pharmaceuticals, preferably for use in the treatment of cancer, preferably cancer selected from the group consisting of: hematological malignancies or solid tumors with PD-1 and / or PD-L1 expression, e.g., cancers selected from the group consisting of hematological lymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia; cancers induced by viruses or associated with immunodeficiency, e.g., Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); cervical cancer, anal cancer, penile cancer, and vulvar squamous cell carcinoma, and oropharyngeal cancer (e.g., associated with human papillomavirus); B-cell non-Hodgkin's lymphoma, including diffuse large B-cell lymphoma. Cancers selected from the group consisting of lymphoma (NHL), Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, primary HHV-8 exudative lymphoma, classical Hodgkin lymphoma, and lymphoproliferative disorders (e.g., associated with Epstein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus); hepatocellular carcinoma (e.g., associated with hepatitis B and / or C virus); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancers associated with human immunodeficiency virus infection (HIV) infection; as well as cancers selected from the group consisting of metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer, and cervical cancer.

[0041] Optionally, bifunctional molecules, pharmaceutical compositions, isolated nucleic acid molecules or groups of isolated nucleic acid molecules, vectors, or host cells are intended for use in combination with radiotherapy or, preferably, additional therapeutic agents selected from the following group: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), activators of cell death pathways, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell induction) antibodies, antibody-drug conjugates, bioreaction modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy Methods, immunological agents, inhibitors of apoptotic protein (IAP), intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian targets of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, polyvalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly-ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small molecule inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, etc., epitopes or neoepitopes derived from tumor antigens, and one or more combinations of such substances.

[0042] The pharmaceutical compositions, bifunctional molecules, nucleic acids or groups of nucleic acid molecules, vectors, or host cells or uses disclosed herein are intended for use to inhibit the inhibitory activity of T regulatory cells, to activate T effector cells, and / or to stimulate the proliferation of unsensitized, partially exhausted and fully exhausted T cells.

[0043] Furthermore, the pharmaceutical compositions, bifunctional molecules, nucleic acids or groups of nucleic acid molecules, vectors, or host cells or uses disclosed herein may be for the treatment of infectious diseases, preferably chronic infectious diseases, and more preferably chronic viral infections. Preferably, the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis viruses, herpes viruses, adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotaviruses, measles viruses, rubella viruses, parvoviruses, vaccinia viruses, HTLV viruses, dengue viruses, papillomaviruses, molluscum contagiosum viruses, polioviruses, rabies viruses, JC viruses, and arbovirus encephalitis viruses. [Brief explanation of the drawing]

[0044] [Figure 1]PD-1-binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Chromogenic development was performed using peroxidase-bound anti-human Fc antibody. Colorimetric analysis was performed using TMB substrate at 450 nm. (A) Anti-PD1 (■), anti-PD1VL-IL7 (o), and anti-PD1VH-IL7 (●) were compared for their binding to recombinant PD1 (rPD1). (B) Comparison of chimeric Bicki (●) derived from anti-PD1 antibodies fused to IL7 on their heavy and / or light chains versus humanized Bicki (■): anti-PD1VH-IL7 (left graph), anti-PD1VL-IL7 (center graph), or anti-PD1VH and VL-IL7 (right graph). Molecules were added to plates coated with human recombinant PD1 protein at different concentrations. (C) The PD-1 binding of Bicki anti-PD-1 / IL-7 constructed with Keytruda (●) and Opdivo (■) skeletons was tested at different concentrations on plates coated with human PD1 recombinant protein. [Figure 2] Antagonist activity of the Bicki anti-PD1-IL7 molecule in blocking PD-1 / PD-L1 and PD1 / PDL2 interactions. (A) ELISA assay: PD-L1 was immobilized on a Maxisorp plate, and the complex antibody + biotinylated recombinant human PD-1 was added. This complex was generated with a fixed concentration of PD1 (0.6 μg / mL), and tested with anti-PD1 (■), anti-PD1VH-IL7 (●), or anti-PD1VL-IL7 (o) antibodies at different concentrations. (B) Affinity evaluation of PD-1 recombinant protein on human PD-L2 recombinant protein pre-incubated with anti-PD1 antibody, anti-PD1VH-IL7, or anti-PD1VL-IL7 antibody using Biacore. Human recombinant PD-L2 was immobilized on a CM5 biochip, and the complex antibody (200 nM) + recombinant human PD-1 (100 nM) was added. The data is expressed as the relative response of the interaction measured by via core, with %:100% = PD-1 relative response. [Figure 3]The Bicki anti-PD1-IL7 molecule stimulates the IL-7R signaling pathway, as measured by ex vivo STAT5 phosphorylation in human PBMCs. PBMCs isolated from the peripheral blood of healthy volunteers were incubated with recombinant IL-7 (rIL7) (gray ●)+ / - anti-PD1 (gray ▽), anti-PD1VH-IL7 (■), or anti-PD1VL-IL7 (●) for 15 minutes. The cells were then fixed, permeabilized, and stained with AF647-labeled anti-pSTAT5 (clone 47 / Stat5 (pY694)). Data were obtained by calculating the MFI pSTAT*%pSTAT5+ population, adjusted to a baseline (100% = rIL-7 57.5 nM), and represent the mean of three different donors in two independent experiments. [Figure 4]Bicki anti-PD1-IL7 enhances T cell activation in vitro. (A) Discover'x PD-1 Path Hunter bioassay: Jurkat T cells stably express modified PD-1 receptor (ED) fused to a beta-gal fragment and modified SHP1 (EA) fused to a complementary beta-gal fragment. Addition of an anti-PD1 antagonist antibody blocks PD-1 signaling, leading to loss of bioluminescence signal (RLU). Anti-PD1 (■) or anti-PD1VH-IL7 (●) were tested at different molar concentrations. Data are expressed as RLU (relative luminescence signal). (B) Promega PD-1 / PD-L1 bioassay: (1) Effector T cells (Jarcat cells stably expressing PD-1 and NFAT-induced luciferase) and (2) Activation target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to activate cognitive TCRs in an antigen-independent manner) were co-cultured. After adding BioGlo® luciferin, luminescence was quantified to reflect T cell activation. Continuous molar concentrations of anti-PD1 antibody + / - recombinant IL-7 (rIL-7) or Bicki anti-PD1VH-IL7 or anti-PD1VL-IL7 antibody were tested. Each dot represents the EC50 of a single experiment. (C) Ability of Bicki anti-PD-1 / IL-7 constructed with a Keytruda or Opdivo scaffold to stimulate NFAT. T cell activation was also tested using the Promega PD-1 / PD-L1 bioassay. Keytruda alone or Opdivo alone (●) was tested against pembrolizumab VH IL-7 or nivolumab VH IL-7 (○) at different concentrations. [Figure 5] The Bicki anti-PD1-IL7 molecule enhances IFNg secretion. T cells isolated from the peripheral blood of healthy volunteers were stimulated with OKT3 / PDL1-coated plates (2 and 5 μg / mL, respectively) in the presence of isotype control, anti-PD1+ / -rIL7, anti-PD1VH-IL7, anti-PD1VL-IL7, and isotype VH-IL7, at a final antibody concentration of 5 μg / mL. Five days after stimulation, the volume of secreted IFNγ was determined by sandwich ELISA. The results are representative of four donors (n=2 experiment). [Figure 6] Bicki anti-PD1-IL7 stimulates T cell proliferation to a similar extent as recombinant lytic IL-7. PBMC cells isolated from the peripheral blood of healthy volunteers were stimulated with anti-CD3 / CD28. 24 hours after stimulation, PBMCs were harvested and re-stimulated on OKT3 / PDL1 coated plates (2 and 5 μg / mL, respectively) in the presence of rIL-7 or Bicki anti-PD1VH-IL7. (A) Fixed dose (29 nM BiCKI or 3.2 nM rIL-7) or (B) Multiple doses of rIL-7 (□), anti-PD1 alone, anti-PD1VH-IL7 (●), anti-PD1VL-IL7 (■), or isotype VH IL-7 (▲) were tested. Five days after stimulation, T cell proliferation was evaluated by 3H-thymidine uptake. The data is adjusted to a baseline (100% = rIL7 10nM) and represents the average obtained from three different donors. EC50(pM) refers to the concentration required to reach 50% of T cell proliferation. [Figure 7] Bicki anti-PD1VH-IL7 stimulates integrin expression on the surface of T cells. Human PBMCs were incubated for 3 days with no molecule (gray histogram) or with rIL-7 / rIL-2 or rIL-7 (50 ng / mL) or anti-PD-1 or anti-PD1VH-IL7 (5 μg / mL). (A) Alpha-4 and beta-7 integrin cell surface expression analysis. FACS was analyzed by LSR, and the data were expressed as doubling changes for each donor, regulated to 1, which corresponds to the mean fluorescence of the control (untreated) cells. (B) LFA-1 cell surface expression analysis (CD11a and CD18) by FACS using LSR. Results are expressed as mean fluorescence. Each dot represents one donor from three independent experiments. [Figure 8]Moderation of chronic antigen stimulation of T cells, which leads to exhausted T cells. Human PBMCs were repeatedly stimulated every 3 days on CD3 CD28 coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibodies). (A) 24 hours after stimulation, T cells were stained for PD-1, Lag3, and Tim3 inhibitory receptors. Expression was analyzed by flow cytometry and FACS LSRII using fluorescently labeled antibodies. Data are expressed as the percentage of positive cells for three donors (one donor = one curve). (B) T cell proliferation capacity was determined by thymidine 3H uptake 5 days after each stimulation. (C) IFNg secretion from the supernatant was analyzed by ELISA (pg / ml) 24 hours after each stimulation. [Figure 9] Activation of the IL7 pathway in exhausted T cells: Response of exhausted T cells to 15-minute incubation of cells with rIL-7 or Bicki anti-PD1VH-IL7, by measuring STAT5 phosphorylation 48 hours after each stimulus. Cells were then fixed, permeabilized, and stained with AF647-labeled anti-pSTAT5 (clone 47 / Stat5 (pY694)). (A) Gray histograms represent cells treated with rIL7, and black histograms represent cells treated with Bicki anti-PD1VH-IL7. Data are representative of four different donors, adapted to a baseline (MFI pSTAT*%pSTAT5 population). (B) ED50 of pSTAT5 was determined in pM for each stimulus, indicating the concentration of rIL-7 (■gray) or Bicki anti-PD1VH-IL7 (■black) required to achieve 50% pSTAT5 activation. [Figure 10A]Proliferation of exhausted T cells upon IL-7 stimulation. Human PBMCs were repeatedly stimulated on CD3-CD28 coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibodies). 24 hours after each stimulation, T cells were re-stimulated on OKT3 coated plates (2 μg / mL) in the presence of anti-PD1, rIL-7, or Bicki anti-PD1-IL7 (anti-PD1VH-IL7 or anti-PD1VL-IL7). H3 uptake assays were performed on day 5 to determine T cell proliferation. Untreated proliferation data from one donor after stimulation (STIM) 3, 4, and 5 are shown (H3 uptake (cpm)). [Figure 10B] Proliferation of exhausted T cells upon IL-7 stimulation. Human PBMCs were repeatedly stimulated with CD3-CD28 coated plates (3 μg / mL OKT3 and 3 μg / mL CD28.2 antibodies). T cells stimulated three times were either left unstimulated or re-stimulated on anti-CD3 (StimOKT3), anti-CD3+ recombinant PDL1 (StimOKT3 / PDL1), or anti-CD3+ recombinant PDL2 (StimOKT3 / PDL2) coated plates (2 and 5 μg / mL, respectively). H3 uptake assays were performed on day 5, and data were adjusted to baseline (1 = H3 uptake with isotype antibody). n = 4 donors and 2 different experiments. [Figure 11]Treg inhibitory activity on CD8 effector T cell proliferation. CD8+ effector T cells and CD4+CD25 high CD127 low Treg cells were isolated from the peripheral blood of healthy donors and stained with cell proliferation dye (CPDe450 for CD8+ T cells). Next, Treg / CD8+ Teff cells were co-cultured in a 1:1 ratio on OKT3 coated plates (2 μg / mL) for 5 days in the presence or absence of rIL-7, anti-PD-1, anti-PD-1+rIL-7, and anti-PD1VH-IL-7. Effector T cell proliferation was analyzed by cell fluorescence assay. (A) Data represent the percentage of + / - SEM of proliferating Teff cells alone (black histogram) or Teff cells co-cultured with Treg cells (gray histogram) based on the loss of CPD markers in the CD8 T effector cell population (n=4 donors in 4 different experiments). (B) Treg proliferation was evaluated using CPD proliferation dye after incubation with different equimolar doses of IL-7 (▲), IL-2 (●), IL-15 (■), or anti-PD1VH-IL7 (▼). [Figure 12] In vivo efficacy of Bicki anti-PD1-IL7 antibody in a humanized mouse model. Human PBMCs were intraperitoneally injected into mice. They were treated twice a week with either anti-PD1 antibody alone or Bicki anti-PD1VH-IL7 (5 mg / kg). Blood was collected 16 days after injection, and the mice were sacrificed. (A) Percentage of peripheral human CD3 T cells was analyzed by flow cytometry in a human CD45+ cell population. Each dot represents one mouse. (B) Dosage of human IFNg was determined in plasma by ELISA. Each dot represents one mouse. (C) Infiltration of human CD3+ cells was quantified in the colon, liver, and lung by immunohistofluorescence. The proximal and distal colon, liver, and lung were embedded in Tissue Tek® OCT and stained for Dapi and human CD3. Each dot represents one mouse, and for the large intestine, each dot represents the average of the CD3+ counts from three sections. [Figure 13]Immunophenotypes of human tumor-infiltrating lymphocytes. T cells were extracted from renal cancer (△)(▽), metastatic colorectal cancer (□), pancreatic cancer (○), and hepatocellular carcinoma (●)(◇), and stained for CD3, CD4, CD8, PD-1, CD127, and CD132. Immunofluorescence was analyzed by FACS LSRII. Data are presented for CD4+CD3+ or CD8+CD3+ populations. [Figure 14] STAT5 activation of intratumoral regulatory T cells or effector T cells in ex vivo tumors. Cells were extracted from tumors of patients with Schwannoma (▼), renal cancer (○), hepatocellular carcinoma (□)(■), metastatic colorectal cancer (●), or pancreatic cancer (▲), and treated with rIL-7 or Bicki anti-PD1VH-IL7 (29nM) for 15 minutes. The cells were then fixed, permeabilized, and stained for pSTAT5 (clone 47 / Stat5(pY694)), CD3, and Foxp3. The histograms represent the average pSTAT5 fluorescence in the CD3+FoxP3+ population (regulatory T cells) or CD3+FoxP3- (effector T cells). [Figure 15] In vitro study of IFNγ secretion from human cancer biopsies treated with Bicki anti-PD1-IL7: Human tumor biopsies were crushed in complete medium and cells were separated. The cells were resuspended in complete medium with 5 μg / mL of isotype control, anti-PD1, B12-IL7 isotype control antibody (isotype-VH IL-7), anti-PD-1 + recombinant IL-7, or anti-Bicki anti-PD1VH-IL7. After 48 hours, the supernatant was collected and IFNγ secretion was analyzed using MSD Technology (Mesoscale Discovery). A. Results for colorectal cancer cells, B. Results for individual tumors (CC: colorectal cancer biopsy, HCC: hepatocellular carcinoma biopsy, KC: renal cancer). [Figure 16]STAT5 activation of intratumoral regulatory T cells or effector T cells after treatment with Bicki anti-PD1VH-IL7. (A) Percentage of intratumoral FoxP3 Treg cells that have invaded tumors of colorectal cancer, schwannoma, renal cancer, or hepatocellular carcinoma. (B) STAT5 activation in FoxP3-CD3+ effector T cells versus FoxP3-CD3+ Treg cells was analyzed after treatment of cells derived from colorectal cancer (●), schwannoma (○), and pancreatic cancer (□) with rIL7 or anti-PD1VH-IL7 (29nM) (incubation for 15 minutes). Next, the cells were fixed, permeabilized, and stained for pSTAT5 (clone 47 / Stat5 (pY694)), CD3, and Foxp3. [Figure 17] PD-1 binding ELISA assay of bicki IL-7 mutants. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Chromogenic development was performed with peroxidase-conjugated anti-human Fc antibody. Colorimetric analysis was performed using TMB substrate at 450 nm. (A) PD-1 binding of anti-PD1 antibody and bifunctional molecules containing IL-7 mutated at amino acids D74, Q22, Y12F, M17, Q11, and K81. (B) PD-1 binding of bifunctional molecules containing IL-7 mutated at amino acid W142. (C) PD-1 binding of bifunctional molecules with disulfide bond mutations in IL-7 (SS1, SS2, and SS3 mutations). All molecules tested in this figure were constructed using the IgG4m isotype and the GGGGSGGGGSGGGGS linker between Fc and the IL-7 domain. [Figure 18]ELISA assay of IgG fused to mutated IL-7 with CD127. Recombinant PD-1 protein was immobilized on a plate, and then a bifunctional anti-PD-1 IL-7 molecule was pre-incubated with recombinant CD127 protein (histidine tag, Sino inquiry 10975-H08H) and added to the wells. Chromogenic development was performed using a mixture of biotin-bound anti-histidine antibody and peroxidase-bound streptavidin. Colorimetric analysis was determined at 450 nm using TMB substrate. (A) CD127-bound bifunctional molecule containing IL-7 mutated at amino acids D74, Q22, M17, Q11, Y12F, and K81. (B) CD127-bound bifunctional molecule containing IL-7 mutated at amino acid W142. [Figure 19] IL-7-7R signaling pathways of different bifunctional molecules as measured by STAT5 phosphorylation. Human PBMCs isolated from peripheral blood of healthy volunteers were incubated with bifunctional anti-PD-1 IL-7 molecules for 15 minutes. The cells were then fixed, permeabilized, and stained with AF647-labeled anti-pSTAT5 (clone 47 / Stat5(pY694)). Data were obtained by calculating MFI pSTAT5 in CD3 T cells. (A) pSTAT5 activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutated at amino acids D74, Q22, M17, Y12F, Q11, and K81. (B) pSTAT activation of anti-PD-1 IL-7 bifunctional molecules containing IL-7 mutated at amino acid W142. (C) pSTAT5 activation of anti-PD-1 IL-7 bifunctional molecules, including IL-7 mutations in the disulfide bonds of IL-7, SS2 (● black), and SS3 (▲), compared with anti-PD-1 IL-7 WT (● gray). All molecules tested in this figure were constructed with the IgG4m isotype and the GGGGSGGGGSGGGGS linker between Fc and the IL-7 domain. [Figure 20]Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules in mice. Mice were intravenously injected with a single dose of IgG fused to wild-type or mutant IL-7. Serum concentrations of the molecules were evaluated at multiple time points after injection by ELISA. (A) Injection of IgG4-G4S3 IL7 WT (■ gray); IgG4-G4S3 IL7 D74E (● black). (B) Injection of IgG4-G4S3 IL7 WT (■ gray) or IgG4-G4S3 IL7 W142H (● black). (C) Injection of IgG4-G4S3 IL7 WT (■ gray); IgG4-G4S3 IL7 SS2 (●) or IgG4-G4S3 IL7 SS3 (▲). (D) Correlation between area under the curve (AUC) calculated from PK vs. ED50 pSTAT5 (nM) for each molecule. All molecules tested in this figure were constructed using the IgG4m isotype and the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. [Figure 21] The addition of a disulfide bond between anti-PD-1 and IL-7 reduces pSTAT5 activation while increasing drug exposure in vivo. (A) IL7R signaling measured by pSTAT5 activation in human PBMCs after treatment with anti-PD-1 IL-7 bifunctional molecule WT (gray ●) or anti-PD-1 IL-7 bifunctional molecule with an additional disulfide bond (black ●). (B) Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecule WT (gray ●) or anti-PD-1 IL-7 bifunctional molecule with an additional disulfide bond (black ●) in mice. Mice were intravenously injected with a single dose containing the anti-PD-1 IL-7 bifunctional molecule. Serum molecular concentrations were evaluated by ELISA at multiple time points after injection. All molecules tested in this figure were constructed with the IgG4m isotype and the GGGGSGGGGSGGGGS linker between Fc and the IL-7 domain. [Figure 22]PD-1 binding ELISA assay. Human recombinant PD-1 (rPD1) protein was immobilized, and antibodies were added at different concentrations. Chromogenic development was performed using an anti-human Fc antibody conjugated to peroxidase. Colorimetric analysis was performed using a TMB substrate at 450 nm. (A) PD-1 binding of anti-PD-1 IL-7 WT bifunctional molecules containing IgG4m (● gray), anti-PD-1 IL-7 WT bifunctional molecules containing IgG1m (▲ black), anti-PD-1 IL-7 D74E bifunctional molecules containing the IgG1m isotype (■), or anti-PD-1 IL-7 W142H bifunctional molecules containing IgG1m (◇). (B) In another experiment, PD-1 binding of anti-PD-1 IL-7 SS2 bifunctional molecules containing the IgG4m isotype (■) or anti-PD-1 IL-7 SS2 bifunctional molecules containing IgG1m (▲) was tested. [Figure 23]CD127-conjugated ELISA assay of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotype. Recombinant proteins targeted by the antibody backbone were immobilized, and then antibodies fused to IL-7 were pre-incubated with CD127 recombinant protein (histidine tag, Sino query 10975-H08H). Chromogenic development was performed using a mixture of biotin-conjugated anti-histidine antibody and peroxidase-conjugated streptavidin. Colorimetric analysis was performed using TMB substrate at 450 nm. (A) CD127 conjugation of anti-PD-1 IL-7 W142H bifunctional molecule containing IgG4m isotype (● gray), anti-PD-1 IL-7 W142H bifunctional molecule containing IgG1m (▲ black), or anti-PD-1 IL-7 WT bifunctional molecule containing IgG1m isotype (● black). (B) CD127 binding of anti-PD-1 IL-7 SS2 bifunctional molecule containing IgG4m isotype (●gray), anti-PD-1 IL-7 SS2 bifunctional molecule containing IgG1m (▲black), or anti-PD-1 IL-7 WT bifunctional molecule containing IgG1m (●black). (C) CD127 binding of anti-PD-1 IL-7 SS3 bifunctional molecule containing IgG4m isotype (●gray), anti-PD-1 IL-7 SS3 bifunctional molecule containing IgG1m (▲black), or anti-PD-1 IL-7 WT bifunctional molecule IgG1m (●black). (D) CD127 binding of anti-PD-1 W142H bifunctional molecule containing isotype IgG1m (●black) or isotype IgG1m+YTE (●gray). CD127 binding of anti-PD-1 D74E bifunctional molecules containing isotype IgG1m (▲black) or isotype IgG1m + YTE (▲gray) was also tested. All molecules tested in this figure were constructed with the GGGGSGGGGSGGGGS linker between the Fc and IL-7 domains. [Figure 24]Analysis of IL-7R signaling of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1N298A or IgG4 isotype. Human PBMCs or Jarcut PD1+CD127+ cells were incubated with anti-PD-1 IL-7 bifunctional molecules for 15 minutes. The cells were then fixed, permeabilized, and stained with AF647-labeled anti-pSTAT5 (clone 47 / Stat5(pY694)). Data were obtained by calculating the percentage of pSTAT5 in CD3 T cells. (A) pSTAT5 signaling on human PBMCs after treatment with the bifunctional molecule anti-PD-1 IL-7 having mutation D74E containing IgG4m isotype (● gray) or IgG1m isotype (▲ black). (B) pSTAT5 signaling on human PBMCs after treatment with anti-PD-1 IL-7 SS2 containing IgG4m isotype (● gray) or anti-PD-1 IL-7 SS2 containing IgG1m (▲ black). (C) pSTAT5 signaling on human PBMCs after treatment with anti-PD-1 IL-7 SS3 (●gray) containing IgG4m isotype or IgG1m (▲black). (D) (Left panel) pSTAT5 signaling in Jarcut PD1+CD127+ cells after treatment with anti-PD-1 IL-7 WT constructed with IgG4m (●gray) or IgG1m (▲black) isotype. (Right panel) pSTAT5 signaling after treatment with anti-PD-1 IL-7 SS2 (●gray) containing IgG4m isotype or anti-PD-1 IL-7 SS2 (▲black) containing IgG1m. [Figure 25]The anti-PD-1 IL-7 mutant bifunctional molecule enhances T cell activation in vitro. Promega PD-1 / PD-L1 bioassay: (1) Effector T cells (Jarcat cells stably expressing PD-1 and NFAT-induced luciferase) and (2) Activation target cells (CHO K1 cells stably expressing PDL1 and surface proteins designed to activate cognitive TCRs in an antigen-independent manner) were co-cultured. After adding BioGlo® luciferin, luminescence was quantified to represent T cell activation. Continuous molar concentrations of anti-PD1 antibody + / - recombinant IL-7 (rIL-7) or anti-PD1 IL-7 bifunctional molecule were tested. Each dot represents the EC50 of a single experiment. (A) NFAT activation of anti-PD-1 IL-7 WT bifunctional molecule containing IgG4m isotype (● gray) or anti-PD-1 (▲) or anti-PD-1 + rIL-7 (○). (B) NFAT activation of anti-PD-1 IL-7 D74E IgG4m (●), PD-1 IL-7 D74E IgG1m (▲ dotted line), and anti-PD-1 alone (black ▲). (C) NFAT activation of anti-PD-1 IL-7 W142H bifunctional molecule containing IgG4m (●), PD-1 IL-7 W142H bifunctional molecule containing IgG1m (▲ dotted line), and anti-PD-1 alone (black ▲). (D) NFAT activation of anti-PD-1 IL-7 SS2 bifunctional molecule containing IgG4m (●), and anti-PD-1 alone (black ▲). [Figure 26]Pharmacokinetics of anti-PD-1 IL-7 bifunctional molecules constructed with IgG1m or IgG4m isotypes. Mice were intravenously injected with a single dose containing IgG fused to wild-type or mutant IL-7. Serum drug concentrations were evaluated by ELISA at multiple time points after injection. (A) Pharmacokinetics of anti-PD-1 IL-7 WT bifunctional molecules containing IgG4m (● gray flat line), anti-PD-1 IL-7 WT bifunctional molecules containing IgG1m (● gray dashed line), anti-PD-1 IL-7 D74E bifunctional molecule containing IgG1m (▲ black dashed line), anti-PD-1 IL-7 W142H bifunctional molecule containing IgG4m (○ black flat line), anti-PD-1 IL-7 W142H bifunctional molecule containing IgG1m (○ dashed black flat line), anti-PD-1 IL-7 SS3 containing IgG4 (■ flat line), and anti-PD-1 IL-7 SS3 containing IgG1m (■ dashed line). (B) Pharmacokinetics of anti-PD-1 IL-7 D74E, D74Q, W142H, and D74E+W142H mutant bifunctional molecules containing IgG1m. [Figure 27] Pharmacokinetics of a bifunctional anti-PD-1 IL-7 molecule constructed with the IgG1 N298A+K444A isotype. Mice were intravenously injected with a single dose of the anti-PD-1 IL-7 D74E bifunctional molecule containing either the IgG1N298A isotype (■) or the IgG1m+K444A mutant isotype (●). Antibody concentrations were evaluated by ELISA at multiple time points after injection. [Figure 28]Linker length does not significantly affect pharmacokinetics but reduces stimulation of IL-7R signaling. (A) Pharmacokinetics of anti-PD-1 IL-7 WT bifunctional molecules constructed with different linkers (GGGGS), (GGGGS)2, and (GGGGS)3). (B) Pharmacokinetics of anti-PD-1 IL-7 D74 bifunctional molecules constructed with different linkers (GGGGS), (GGGGS)2, and (GGGGS)3). (C) Pharmacokinetics of anti-PD-1 IL-7 W142H bifunctional molecules constructed with different linkers ((GGGGS)2 and (GGGGS)3). Mice were intravenously injected with a single dose containing IgG fused to wild-type or mutant IL-7. The concentration of IgG fused to IL-7 was evaluated by ELISA at multiple time points after injection. (D) pSTAT5 signaling of anti-PD-1 IL-7 bifunctional molecules constructed without a linker or with GGGGS, (GGGGS)2, or (GGGGS)3 linkers. [Figure 29] Anti-PD-1 IL-7 mutants selectively target PD-1+CD127+ cells rather than PD-1-CD127+ cells. Jurcut cells expressing CD127+ or co-expressing CD127+ and PD-1+ were stained with 45 nM anti-PD-1 IL-7 bifunctional molecule and identified using anti-IgG-PE (Biolegend, clone HP6017). The data represent the ratio of average fluorescence on PD-1+CD127+ Jurcut cells to average fluorescence obtained on PD1-cell CD127+ Jurcut cells. In this assay, the following bifunctional molecules were tested: anti-PD-1 IL-7 WT IgG1m, anti-PD-1 IL-7 D74E IgG1m, anti-PD-1 IL-7 W142H IgG1m, anti-PD-1 IL-7 SS2 IgG4m, and anti-PD-1 IL-7 SS3 IgG1m. [Figure 30-1]The anti-PD-1 IL-7 molecule enhances T cell proliferation and demonstrates preclinical safety in cynomolgus monkeys. It targets PD-1+CD127+ cells rather than PD-1-CD127+ cells. Cynomolgus monkeys were intravenously injected with a single dose of bicki anti-PD-1 IL-7 WT (6.87 nM / kg (n=2)) or 34.35 nM (n=1). Blood analysis was performed up to 15 days or 4 hours after injection. (A) Lymphocyte counts were evaluated in peripheral blood at multiple time points after injection of Bicki anti-PD-1 IL-7 WT at 6.87 nM / kg (n=2). (B) CD4 / CD8 or B cell proliferation was evaluated by flow cytometry using Ki67 / CD4 / CD8 and CD19 markers after injection of Bicki anti-PD-1 IL-7WT at 6.87 nM / kg (n=2). (C) pSTAT5 in CD3+ T cells was analyzed by FACS at multiple time points after injection of Bicki anti-PD-1 IL-7WT at 6.87 nM / kg (n=2). (D / E / F / G / H) Biochemical and cellular / hemorrhagic analyses were evaluated at multiple time points. [Figure 30-2] See Figure 30-1. [Figure 30-3] See Figure 30-1. [Figure 30-4] See Figure 30-1. [Figure 30-5] See Figure 30-1. [Figure 30-6] See Figure 30-1. [Figure 31] Explanation of the mechanism of action of Bicki anti-PD1-IL-7 according to the present invention. [Modes for carrying out the invention]

[0045] Introduction The antibody of the present invention is bifunctional because it combines a specific anti-PD-1 effect with the effect of human interleukin 7 fused to the anti-PD-1 antibody. In fact, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody and IL-7, wherein the interleukin is covalently linked to either the light chain or heavy chain, or both, or a fragment thereof, of the polypeptide chain of the anti-PD-1 antibody. The anti-PD-1 antibody or fragment thereof chain and IL-7 are prepared as a fusion protein. In this particular embodiment, the N-terminus of IL-7 is optionally linked to the C-terminus of the anti-PD-1 antibody or fragment thereof chain via a peptide linker.

[0046] As is well known to those skilled in the art, tumor cells may not be adequately eliminated by T cells due to a phenomenon called T cell exhaustion, which is observed in many cancers. For example, as described by Jiang, Y., Li, Y., and Zhu, B (Cell Death Dis 6, e1792 (2015)), exhausted T cells in the tumor microenvironment may lead to overexpression of inhibitory receptors, decreased effector cytokine production and cytolytic activity, resulting in failure to eliminate cancer and, in general, immune evasion of cancer. Therefore, the recovery of exhausted T cells is a clinical strategy conceived for cancer treatment.

[0047] PD-1 is a major inhibitory receptor that modulates T cell exhaustion. In fact, T cells exhibiting high PD-1 expression show reduced ability to eliminate cancer cells. Anti-PD1 therapeutic compounds, particularly anti-PD1 antibodies, are clinically used in cancer treatment to inhibit the PD1-PDL1 interaction (PD1 on T cells and PDL1 on tumor cells) and prevent T cell exhaustion. However, anti-PD1 antibodies are not always efficient enough to enable the "re"activation of exhausted T cells.

[0048] The applicant hereby demonstrates that the bifunctional anti-PD1-IL-7 molecule according to the present invention enhances the activation of T cells, particularly exhausted T cells (NFAT-mediated activation), compared to anti-PD-1 alone. In particular, the anti-PD1-IL-7 bifunctional molecule induces proliferation and activation of unsensitized, partially exhausted and fully exhausted T cell subsets, as reflected by cytokine (e.g., IFNγ) secretion. Such an anti-PD1-IL-7 bifunctional molecule has the ability to overcome associated resistance mechanisms and improve the efficacy of anti-PD-1 immunotherapy.

[0049] The applicant demonstrates that the interaction between the anti-PD1-IL-7 bifunctional molecule and a single T cell expressing i) PD1 and ii) IL-7 receptors leads to unexpected activation of the NFAT pathway (TCR signaling), exhibiting a positive effect on T cell activation, particularly on exhausted T cells, and enhancing the T cell's ability to eliminate tumor cells.

[0050] This means that, on the one hand, the IL-7 portion of the bifunctional molecule of the present invention targets the IL-7 receptor and activates the PSTAT5 pathway, and on the other hand, the anti-PD1 portion of the bifunctional molecule inhibits the PD-1 / PD-L1 interaction. The BICKI molecule targets both IL-7 and PD-1 on the same cell. This results in synergistic activation of TCR (NFAT) signaling. This is never observed when the combination of anti-PD1 antibody and IL-7 is used separately (as two separate compounds). This activation cannot be provided by a bifunctional molecule that targets PD-L1. In fact, it is known in the art that PD-L1 is expressed on tumor cells and not on immune cells such as T cells.

[0051] In addition, the bifunctional anti-PD1 / IL-7 molecule enables the accumulation of IL-7 in PD-1+ T cell infiltrations and the relocalization of IL-7 on PD-1+ T cells. This accumulation of IL-7 near PD-1+ T cells is particularly interesting in the context of exhausted T cells that require high doses of IL-7 to activate or reactivate these T cells.

[0052] The synergistic effect on T cell activation has been observed not only with the specific anti-PD-1 antibody of the present invention, but also with two other reference anti-PD-1 antibodies, namely Opdivo and Keytruda.

[0053] In addition, the bifunctional anti-PD1 / IL-7 molecule has the ability to promote T-cell infiltration into tumors. Today, given that the lack of T-cell infiltration at tumor sites is a major impediment to the effectiveness of anti-PD1 antibody therapy, this ability is advantageous for optimizing anti-PD-1 antibody therapy.

[0054] Furthermore, the bifunctional anti-PD1 / IL-7 molecule inhibits Treg-mediated inhibitory effects. Therefore, the bifunctional molecule can specifically activate T effector cells rather than Treg cells, whereas the anti-PD1 antibody cannot inhibit Treg-suppressive activity against T effector cells. Accordingly, the inventors have shown that the bifunctional anti-PD1-IL-7 molecule favors T cell effectors in the T regulatory immune balance by stimulating the proliferation and survival of effector T cells while preserving regulatory T cells.

[0055] In addition, the IL7 anti-PD-1 bifunctional molecule has other advantages.

[0056] Furthermore, the inventors demonstrate that the IL7 anti-PD-1 bifunctional molecule activates T effector cells (Teff) more favorably than T regulatory cells (Treg). The IL7 anti-PD-1 bifunctional molecule has the advantages of not promoting Treg proliferation, inducing Treg inactivation, and inducing the activation of T cells, particularly exhausted T cells.

[0057] The IL7 anti-PD-1 bifunctional molecule allows for highly advantageous dosages and exhibits a favorable therapeutic index (ratio of lethal dose (DL50) to therapeutically effective dose). In particular, IL7 can typically be used in patients in the range of 10-1500 μg / kg, and more favorably, 200-1200 μg / kg, and even high doses such as 1200 μg / kg are well tolerable to patients. Therefore, the IL7 anti-PD-1 bifunctional molecule makes it possible to produce therapeutic compounds in a way that ensures appropriate dosages of the component compounds and the final product. In fact, a well-tolerated high dose of IL7 (for example, approximately 1.2 mg / kg in the case of IL7) corresponds to an antibody of about 2 mg / kg, which is a sufficient dose to administer to a patient.

[0058] The IL7 anti-PD-1 bifunctional molecule has the advantage of being able to essentially target partially exhausted T cell progenitor cells, which are an important target for meeting the medical needs mentioned above. In addition, chronic activation is important in the case of viral infections associated with similar T cell exhaustion, and therefore viral pathology is included in the scope of pathologies targeted by the novel product of this application.

[0059] Finally, in certain embodiments, the inventors have designed bifunctional molecules containing IL-7 variants or variants. IL-7 variants or variants are characterized by i) reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity for wild-type IL-7, and ii) improved pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wild-type IL-7. Firstly, the use of IL-7 variants in bifunctional molecules is important for increasing the in vivo pharmacokinetics of the bifunctional molecule. Secondly, by reducing the affinity of the IL-7 variant to its receptor, the bifunctional molecule gains increased ability not only to selectively bind to target T cells via the anti-PD-1 antibody portion of the bifunctional molecule and provide specific effects on such cells, but also to leverage the synergistic effects associated with the actions of the two portions of the bifunctional molecule on the same T cells. Bifunctional molecules containing IL-7 variants exhibit good PD-1 binding and antagonist activity. In addition, the bifunctional molecule provides a favorable equilibrium between its affinity for PD-1 and its affinity for IL-7R. Surprisingly, the inventors observed that the bifunctional molecule having an IgG1 heavy chain constant domain exhibited enhanced IL-7 variant activity (pStat5 signaling, synergistic effects, and CD127 binding) compared to the same molecule having an IgG4 heavy chain constant domain. Furthermore, the use of a linker (GGGGS) 3 between the antibody and IL-7 maximized the activity of the IL-7 variant (pStat5 signaling and CD127 binding).

[0060] The bifunctional molecule of the present invention has, in particular, one or more of the following advantages.

[0061] - The bifunctional molecule induces proliferation of unsensitized, partially exhausted, and fully exhausted T cell subsets, but does not induce proliferation of only partially exhausted T cells, as is the case with anti-PD1 / PDL1 therapy. More specifically, the bifunctional molecule has a synergistic effect on T cell activation. - The bifunctional molecule specifically localizes IL-7 near or on PD-1+ exhausted T cells that have infiltrated the tumor, enabling the targeting of cells that require higher concentrations of IL-7. In particular, the bifunctional molecule induces the accumulation of IL-7 in PD-1+ T cell infiltrations and the relocalization of IL-7 on PD-1+ T cells. - Blocking anti-PD-1 prevents the reprogramming of exhausted T cells into active memory T cells, limiting long-term tumor clearance. However, the bifunctional molecule promotes the formation, survival, and proliferation of memory T cells through the presence of IL-7. Therefore, the bifunctional molecule induces continuous anti-tumor immunity through a sustained and expanded memory T cell response. - While the efficacy of anti-PD1 / PD-L1 therapy is associated with existing T cell infiltration and T cell effector function, particularly the IFNγ signature, the bifunctional molecule synergistically increases the proliferation of effector T cells and their ability to secrete IFNγ. - The bifunctional molecule can reduce the immunosuppressive microenvironment by decreasing the Treg population and inhibiting the secretion of TGFβ (an inhibitory cytokine). More specifically, the bifunctional molecule specifically stimulates effector T cells without stimulating Tregs. - In bifunctional molecules, IL-7 may be fused to the C-terminal portion of the heavy and / or light chain, and the high affinity of IL-7 for CD127 is conserved, similar to naked / natural IL-7. Bifunctional molecules may be more potent in terms of IL-7R activation and half-life. - The bifunctional molecule is produced as a bifunctional molecule with high production yield. - The bifunctional molecule reduces the immunosuppressive activity of Treg cells that have entered the tumor microenvironment by decreasing the number of Tregs. More specifically, the bifunctional molecule specifically stimulates effector T cells without stimulating Tregs. - The bifunctional compounds increase the expression of integrins (i.e., alpha-4 and / or beta-7 and LFAT), promoting T cell infiltration into tissues and / or tumors compared to the anti-PD1 response alone. In particular, the bifunctional compounds promote T cell migration and tumor infiltration. - The bifunctional molecule may contain IL-7 variants or mutants as identified by the inventors in order to maximize in vivo pharmacokinetics while maintaining IL-7 activity and antagonist activity of anti-PD-1 antibodies, while maintaining a suitable affinity balance between IL-7 and IL-7R and anti-PD-1 and PD-1.

[0062] definition To facilitate understanding of the present invention, certain terms are defined below in this specification. Additional definitions are provided throughout the detailed description.

[0063] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have meanings that are generally understood by those skilled in the art. In some cases, terms that have generally understood meanings are also defined herein for clarity and / or for easy reference. Where such definitions are included herein, they should not necessarily be construed as meanings that are different from those generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and are widely used by those skilled in the art using conventional methodologies.

[0064] As used herein, the terms “interleukin-7,” “IL-7,” and “IL-7” refer to mammalian endogenous secreted glycoproteins, particularly IL-7 polypeptides, derivatives, and analogs thereof, that have substantial amino acid sequence identity with wild-type mammalian IL-7 and have substantially equivalent biological activity in, for example, a standard bioassay or assay of IL-7 receptor binding affinity. For example, IL-7 refers to i) a native or naturally occurring allele variant of an IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of a biologically active variant of an IL-7 polypeptide. IL-7 may or may not contain its peptide signal. Alternative names for this molecule are “pre-B cell growth factor” and “lympopoietin-1.” Preferably, the term “IL-7” refers to human IL-7. For example, the amino acid sequence of human IL-7 consists of approximately 152 amino acids (when the signal peptide is absent), its Genbank acceptance number is NP_000871.1, and the gene is located on chromosome 8q12-13. Human IL-7 is described in UniProtKB-P13232.

[0065] As used herein, the terms “wild-type interleukin-7,” “wt-IL-7,” and “wt-IL7” refer to mammalian endogenous secreted glycoproteins, particularly IL-7 polypeptides, derivatives, and analogs having substantial amino acid sequence identity with wild-type functional mammalian IL-7 and substantially equivalent biological activity in, for example, a standard bioassay or assay of IL-7 receptor binding affinity. For example, wt-IL-7 refers to i) a natural or naturally occurring IL-7 polypeptide, ii) a biologically active fragment of an IL-7 polypeptide, iii) a biologically active polypeptide analog of an IL-7 polypeptide, or iv) the amino acid sequence of a recombinant or non-recombinant polypeptide having the amino acid sequence of a biologically active IL-7 polypeptide. IL-7wt may or may not contain its peptide signal. Alternative names for this molecule are “pre-B cell growth factor” and “lympopoietin-1.” Preferably, the term “wt-IL-7” refers to human IL-7 (wt-IL7). For example, the amino acid sequence of human wt-IL-7 consists of approximately 152 amino acids (in the absence of the signal peptide), its Genbank acceptance number is NP_000871.1, and the gene is located on chromosome 8q12-13. Human IL-7 is described, for example, in UniProtKB-P13232.

[0066] As used herein, the terms “programmed death 1,” “programmed cell death 1,” “PD1,” “PD-1,” “PDCD1,” “PD-1 antigen,” “human PD-1,” “hPD-1,” and “hPD-1” are used synonymously and refer to the programmed death-1 receptor, also known as CD279, and include variants and isoforms of human PD-1, as well as analogs having at least one common epitope with PD-1. PD-1 is an important regulator of the threshold of immune response and peripheral immune tolerance. PD-1 is expressed on activated T cells, B cells, monocytes, and dendritic cells and binds to its ligands, PD-L1 and PD-L2. Human PD-1 is encoded by the PDCD1 gene. As an example, the amino acid sequence of human PD-1 is disclosed in GenBank acceptance number NP_005009. Four splice variants of PD1 are expressed on human peripheral blood mononuclear cells (PBMCs). Therefore, PD-1 proteins include full-length PD-1, as well as alternative splicing variants of PD-1 such as PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3,4. Unless otherwise specified, these terms include any variants and isoforms of human PD-1 expressed in nature by PBMCs or by cells transfected with the PD-1 gene.

[0067] As used herein, the term “antibody” is used in its broadest sense to describe a type of immunoglobulin molecule. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. The heavy chain constant domains corresponding to different classes of immunoglobulin are called alpha, delta, epsilon, gamma, and mu, respectively. Unless otherwise specifically indicated, the term “antibody” includes intact immunoglobulins, as well as any other modified configurations of immunoglobulin molecules containing antigen recognition sites of the required specificity, including “antibody fragments” or “antigen-binding fragments” (Fab, Fab', F(ab')2, Fv, etc.), single chains (scFv), their variants, molecules containing the antibody portion, diabodies, linear antibodies, single-chain antibodies, and glycosylated variants of antibodies, amino acid sequence variants of antibodies. Preferably, the term “antibody” refers to humanized antibodies.

[0068] As used herein, the “antigen-binding fragment” of an antibody means a molecule corresponding to a part of the antibody, i.e., a part of the antibody structure of the present invention that exhibits antigen-binding ability to PD-1, possibly in its natural form. In particular, such a fragment exhibits the same or substantially the same antigen-binding specificity to the antigen compared to the antigen-binding specificity of the corresponding quadruple-chain antibody. Advantageously, the antigen-binding fragment has a similar binding affinity to the corresponding quadruple-chain antibody. However, antigen-binding fragments having a reduced antigen-binding affinity compared to the corresponding quadruple-chain antibody are also included in the present invention. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. Such antigen-binding fragments may also be referred to as “functional fragments” of an antibody. The antigen-binding fragment of an antibody is a fragment containing a hypervariable domain, called a CDR (complementarity-determining region), or a portion thereof, which encompasses the recognition site of the extracellular domain of the antigen, i.e., PD1, thereby defining the antigen recognition specificity.

[0069] The "Fab" fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that a small number of residues, including one or more cysteines derived from the antibody hinge region, are added to the carboxyl terminus of the heavy chain CH1 domain. The F(ab') fragment is produced by cleaving the disulfide bond of the hinge cysteine ​​of the F(ab')2 pepsin digest product. Additional chemical coupling of antibody fragments is known to those skilled in the art. The Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, are removed more rapidly from the circulation of animals, and may have less nonspecific tissue binding than the intact antibody (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0070] The "Fv" fragment is the smallest fragment of an antibody that contains the complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of one heavy chain variable domain and one light chain variable domain, accompanied by a tight non-covalent bond. It is in this configuration that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. In many cases, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of the Fv containing only three target-specific CDRs) can have the ability to recognize and bind to the target, although with lower affinity than the entire binding site.

[0071] A "single-chain Fv" or "scFv" antibody-binding fragment contains the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. Generally, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form the desired structure for target binding.

[0072] A "single-domain antibody" consists of a single VH domain or VL domain that exhibits sufficient affinity for PD-1. In specific embodiments, the single-domain antibody is a camelized antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods, Vol. 231: pp. 25-38).

[0073] From a structural standpoint, antibodies may have heavy (H) and light (L) chains interconnected by disulfide bonds. Light chains are of two types: lambda (λ) and kappa (κ). Each heavy and light chain contains a constant region and a variable region (or "domain"). The light and heavy chain variable regions contain a "framework" region separated by three hypervariable regions, also called the "complementarity-determining region" or "CDR." The extent of the framework region and CDR is defined (see Kabat et al., Sequences of Proteins of Immunological Interest, and US Department of Health and Human Services, 1991; this document is incorporated herein by reference). Preferably, the CDR is defined by the Kabat method. The framework region acts to form a scaffold that ensures the CDR is positioned in the correct orientation by interchain non-covalent interactions. The CDR is primarily involved in binding to the antigen epitope. The CDRs of each chain are typically referred to as “complementarity-determining region 1” or “CDR1,” “CDR2,” and “CDR3,” and are numbered sequentially from the N-terminus. The VL and VH domains of the antibody according to the present invention may include four framework regions or “FRs,” which are referred to in the art and herein as “framework region 1” or “FR1,” “FR2,” “FR3,” and “FR4,” respectively. These framework regions and complementarity-determining regions are preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxy terminus). The term “antibody framework,” as used herein, refers to a portion of either the VL and / or VH variable domains, which serve as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0074] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in the antibody three-dimensional structure. The CDRs of the antibody heavy chain are typically referred to as "HCDR1," "HCDR2," and "HCDR3." The framework regions of the antibody heavy chain are typically referred to as "HFR1," "HFR2," "HFR3," and "HFR4."

[0075] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the antibody three-dimensional structure. κ-light chain and λ-light chain refer to the two main antibody light chain isotypes. The CDRs of the antibody light chain are typically referred to as "LCDR1," "LCDR2," and "LCDR3." The framework regions of the antibody light chain are typically referred to as "LFR1," "LFR2," "LFR3," and "LFR4."

[0076] With regard to the binding of an antibody to a target molecule, the term “binding” or “conjugate” refers to a peptide, polypeptide, protein, fusion protein, molecule, and antibody (including antibody fragments) that recognizes and comes into contact with an antigen. Preferably, this term refers to an antigen-antibody type interaction. With regard to a specific antigen (e.g., PD-1) or an epitope on a specific antigen (e.g., PD-1), the terms “specific binding,” “specific to,” “selectively binding to,” and “selective to” mean that the antibody recognizes and binds to a specific antigen, but substantially does not recognize or bind to other molecules in the sample. For example, an antibody that specifically (or preferentially) binds to PD-1 or a PD-1 epitope is an antibody that binds to this PD-1 epitope more easily and / or for a longer period of time, for example, with higher affinity, binding strength, than to other PD-1 epitopes or non-PD-1 epitopes. Preferably, the term “specific binding” is 10 -7 This refers to contact between an antibody and an antigen having a binding affinity equal to or lower than M. In certain embodiments, the antibody is 10 -8 M, 10 -9 M, or 10 -10It binds with an affinity equal to or lower than that of M.

[0077] As used herein, “PD-1 antibody,” “anti-PD-1 antibody,” “PD-1 Ab,” “PD-1 specific antibody,” and “anti-PD-1 Ab” are used synonymously and refer to antibodies as described herein that specifically bind to PD-1, particularly human PD-1. In some embodiments, the antibody binds to the extracellular domain of PD-1. In particular, anti-PD-1 antibodies are antibodies capable of binding to the PD-1 antigen, inhibiting the PD-1-mediated signaling pathway and thereby enhancing immune responses such as T cell activation.

[0078] As used herein, the terms “bifunctional molecule,” “bifunctional compound,” “bifunctional protein,” “Bicki,” “Bicki antibody,” “bifunctional antibody,” and “bifunctional checkpoint inhibitor molecule” have the same meaning and can be used synonymously. These terms refer to an antibody that recognizes a single antigen by having at least one region specific to that antigen (e.g., derived from the variable region of the antibody) and at least a second region that is a polypeptide. More specifically, a bifunctional molecule is a fusion protein of an antibody or a portion thereof, preferably its antigen-binding fragment, and another polypeptide or its polypeptide fragment.

[0079] As used herein, the term "chimeric antibody" means an antibody or antigen-binding fragment in which a heavy chain and / or light chain portion originates from one species and the rest of the heavy chain and / or light chain originates from a different species. In exemplary examples, a chimeric antibody may include a constant region derived from humans and a variable region derived from a non-human species such as mouse.

[0080] As used herein, the term "humanized antibody" is intended to refer to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted into a human framework sequence (e.g., a chimeric antibody containing a minimal sequence derived from a non-human antibody). Furthermore, "humanized antibody," e.g., a non-human antibody, refers to an antibody that has undergone humanization. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which one or more residues derived from CDRs are replaced by residues derived from at least one CDR of a non-human antibody (donor antibody), while maintaining the desired specificity, affinity, and volume of the original antibody. The donor antibody may be any suitable non-human antibody, such as a mouse antibody, rat antibody, rabbit antibody, chicken antibody, or non-human primate antibody, having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by framework region residues derived from the donor antibody. Alternatively, selected framework region residues of the donor antibody are replaced by framework region residues derived from a human antibody or humanized antibody. Additional framework region modifications may be present within the human framework sequence. Therefore, humanized antibodies may contain residues not found in either the recipient antibody or the donor antibody. Such amino acid modifications can further refine antibody function and / or enhance the humanization process. "Amino acid change" or "amino acid modification" as used herein means a change in the amino acid sequence of a polypeptide. Examples of "amino acid modifications" include substitutions, insertions, and / or deletions in the polypeptide sequence. "Amino acid substitution" or "substitution" as used herein means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid. "Amino acid insertion" or "insertion" means adding an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" means removing an amino acid at a specific position in the parent polypeptide sequence. Amino acid substitutions may be conservative. A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") with similar chemical properties (e.g., charge, bulk, and / or hydrophobicity).As used herein, “amino acid position” or “amino acid position number” are used synonymously and refer to the position of a particular amino acid in an amino acid sequence, generally designated by a single-letter code for the amino acid. The first amino acid in an amino acid sequence (i.e., starting from the N-terminus) should be considered to be position 1.

[0081] A conservative substitution is the replacement of a given amino acid residue with another residue having a side chain ("R group") with similar chemical properties (e.g., charge, bulk, and / or hydrophobicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of a protein. Conservative substitutions and their corresponding rules are well documented in the state of the art. For example, conservative substitutions can be defined by substitutions within the group of amino acids reflected in the table below.

[0082] [Table 1]

[0083] [Table 2]

[0084] [Table 3]

[0085] As used herein, “isolated antibody” is an antibody separated and / or recovered from components of its natural environment. Since isolated antibodies lack at least one component of the antibody’s natural environment, they include antibodies in situ within recombinant cells. In some embodiments, the antibody is purified to homogeneity and / or to a purity greater than 90%, 95%, or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC) under reducing or non-reducing conditions.

[0086] The terms “derive from” and “derived from,” as used herein, refer to a compound having a structure derived from the structure of a parent compound or parent protein, whose structure is sufficiently similar to that disclosed herein, and which, based on its similarity, would be expected by those skilled in the art to exhibit the same or similar properties, activities, and utility as the claimed compound. For example, a humanized antibody derived from a mouse antibody refers to an antibody or antibody fragment that shares similar properties to the mouse antibody, and for example, shares similar VH and VL with modification residues that recognize the same epitopes and are involved in and / or enhance the humanization of the antibody.

[0087] The term “treatment” refers to any action aimed at improving a patient’s health, including the therapy, prevention, and mitigation of a disease or its symptoms. This term encompasses both curative and / or preventive treatments of a disease. Curative treatment is defined as treatment that results in a cure or treatment that reduces, improves, and / or eliminates, diminishes, and / or stabilizes the disease or its symptoms or the suffering it directly or indirectly causes. Preventive treatment includes both treatments that result in the prevention of disease, and treatments that reduce and / or delay the progression and / or onset of disease or the risk of its occurrence. In certain embodiments, such term refers to the improvement or eradication of a disease, disorder, infection, or associated symptoms. In other embodiments, this term refers to minimizing the spread or worsening of cancer. Treatments according to the present invention do not necessarily imply 100% or complete cure. Rather, there are varying degrees of treatment that a person skilled in the art would recognize as having potential benefits or therapeutic effects. Preferably, the term “treatment” refers to applying or administering a composition comprising one or more active substances to a subject having, for example, a disorder / disease associated with a PD-1-mediated signaling pathway.

[0088] As used herein, the terms “disorder” or “disease” refer to a failure of an organ, part, structure, or system of the body to function properly due to a genetic or developmental error, infection, toxin, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors. Preferably, these terms refer to a health disorder or disease, such as a disease that interferes with normal physical or mental functioning. More preferably, the term “disorder” refers to an immune disorder and / or inflammatory disease affecting animals and / or humans, such as cancer.

[0089] The term "immune disease," as used herein, refers to a condition of an object characterized by damage to cells, tissues, and / or organs caused by an immunological response of the object to its own cells, tissues, and / or organs. The term "inflammatory disease" refers to a condition of an object characterized by inflammation, such as chronic inflammation. Autoimmune disorders may or may not be associated with inflammation. Furthermore, inflammation may or may not be caused by autoimmune disorders.

[0090] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells may spread locally or to other parts of the body via the bloodstream and lymphatic system.

[0091] As used herein, the terms “PD-1-related or associated disease,” “PD-1-positive cancer,” or “PD-1-positive infectious disease” are intended to refer to any cancer or infection (e.g., caused by viruses and / or bacteria) that is caused by, exacerbated by, or otherwise associated with, the symptoms / characteristics of PD-1 expression, i.e., an increase or decrease in PD-1 expression or activity.

[0092] As used herein, the terms “subject,” “host,” “individual,” or “patient” refer to human beings, including adults and children.

[0093] Where used herein, “pharmaceutical composition” refers to one or more preparations of an active substance, including those comprising the bifunctional molecule according to the present invention together with other optional chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the active substance to a living organism. The compositions of the present invention may be in any conventional route of administration or in a form suitable for use. In one embodiment, “composition” is typically intended to be a combination of an active substance, e.g., a compound or composition, and a pharmaceutically acceptable carrier, such as an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, or an inert (e.g., a detectable substance or label) or active carrier, whether naturally occurring or not. Where used herein, “acceptable vehicle” or “acceptable carrier” is any known compound or combination of compounds that is known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0094] When used herein, “effective dose” or “therapeutic effective dose” refers to the amount of an active substance required to impart a therapeutic effect to a subject, either alone or in combination with one or more other active substances, for example, the amount of an active substance required to treat a target disease or disorder or to produce a desired effect. “Effective dose” will vary depending on the substance, the disease and its severity, the characteristics of the subject to be treated, including age, physical condition, size, sex, and weight, the duration of treatment, the nature of the combination therapy (if applicable), the specific route of administration, and similar factors within the scope of the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed through routine experimental work. Generally, it is preferable to use the maximum dose of each individual component or combination thereof, i.e., the highest safe dose based on sound medical judgment.

[0095] As used herein, the term “pharmaceutical” refers to any substance or composition having therapeutic or preventive properties for a disorder or disease.

[0096] As used herein, the term “combined” refers to the use of more than one therapy (e.g., prophylactic and / or therapeutic agents). The use of the term “combined” does not limit the order in which the therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or disorder.

[0097] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid sequence” are equivalent and refer to polymeric forms of nucleotides of any length, such as RNA or DNA or analogs thereof. The nucleic acids of the present invention (e.g., components or portions of nucleic acids) may be naturally occurring, modified, genetically engineered, isolated, and / or non-natural. Examples of genetically engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids.

[0098] "Isolated nucleic acid encoding an anti-PD1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell. As used herein, the terms "nucleic acid construct," "plasmid," and "vector" are equivalent and refer to nucleic acid molecules that serve to transfer a passenger nucleic acid sequence, such as DNA or RNA, into a host cell.

[0099] As used herein, the term “host cell” is intended to include any individual cell or cell culture that may or may be a recipient of the vector, exogenous nucleic acid molecule, and polynucleotide encoding the antibody construct of the present invention, and / or the antibody construct itself. The introduction of each substance into a cell can be carried out by transformation and transfection, etc. Furthermore, the term “host cell” is intended to include the offspring or potential offspring of a single cell. Examples of host cells include bacterial cells, microbial cells, plant cells, and animal cells.

[0100] As used herein, “immune cells” refers to cells involved in innate and adaptive immunity, such as leukocytes (white blood cells), lymphocytes (T cells, B cells, natural killer (NK) cells, and natural killer T cells (NKT)), and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, and dendritic cells), derived from hematopoietic stem cells (HSCs) produced in the bone marrow. In particular, immune cells can be selected from a non-exclusive list including B cells, T cells, especially CD4+ T cells and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. As used herein, “T cells” include, for example, CD4+ T cells, CD8+ T cells, T helper 1 T cells, T helper 2 T cells, T helper 17 T cells, and inhibitory T cells.

[0101] As used herein, the terms “T effector cells,” “Teff,” or “effector cells” describe a group of immune cells, including several T cell types, that respond to stimuli such as co-stimulation. This term includes, in particular, T cells that function to eliminate antigens (e.g., by producing cytokines that modulate the activation of other cells, or by cytotoxic activity). This term includes, in particular, CD4+ cells, CD8+ cells, Treg cells, cytotoxic T cells, and helper T cells (Th1 and Th2).

[0102] As used herein, the terms “regulatory T cells,” “Treg cells,” or “Treg” refer to a subgroup of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to originate from the same lineage as naive CD4 cells.

[0103] The term "exhausted T cells" refers to a population of T cells in a dysfunctional (i.e., "exhausted") state. T cell exhaustion is characterized by progressive loss of function, altered transcriptional profiles, and sustained expression of inhibitory receptors. Exhausted T cells lose their ability to produce cytokines, proliferate, and cytotoxicize, ultimately leading to their elimination. Exhausted T cells typically exhibit lower expression of CD62L and CD127, along with higher levels of CD43, CD69, and inhibitory receptors.

[0104] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, resulting in selective damage, destruction, or elimination from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0105] As used herein, the term "antagonist" refers to a substance that inhibits or reduces the activity or functionality of another substance. In particular, the term refers to an antibody that binds to a cell receptor (e.g., PD-1) as a reference substance (e.g., PD-L1 and / or PD-L2) and prevents it from producing all or part of its normal biological effect (e.g., the creation of an immunosuppressive microenvironment). The antagonist activity of antibodies according to the present invention can be evaluated by competitive ELISA.

[0106] As used herein, the term “isolated” indicates that a substance described (e.g., antibodies, polypeptides, nucleic acids, etc.) has been substantially separated from or concentrated compared to other substances that naturally coexist with it. In particular, an “isolated” antibody is one that has been identified, separated, and / or recovered from components of its natural environment. For example, an isolated antibody may be purified (1) to a level higher than 75% by mass of the antibody as determined by the Lowry method, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions. Since an isolated antibody will be absent from at least one component of the antibody’s natural environment, it may contain the antibody in situ within recombinant cells. However, typically, an isolated antibody will be prepared by at least one purification step.

[0107] When used herein, the term "and / or" should be interpreted as a specific disclosure of each of the two designated features or components, with or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were presented individually.

[0108] The terms "a" or "an" can refer to one or more of the elements they modify, unless the context makes it clear that either one or more of the elements are being described (for example, "reagent" can mean one or more reagents).

[0109] When used herein in relation to any value (including the lower and upper limits of a numerical range), the term "approximately" means any value that has an acceptable deviation of up to + / -10% (e.g., + / -0.5%, + / -1%, + / -1.5%, + / -2%, + / -2.5%, + / -3%, + / -3.5%, + / -4%, + / -4.5%, + / -5%, + / -5.5%, + / -6%, + / -6.5%, + / -7%, + / -7.5%, + / -8%, + / -8.5%, + / -9%, + / -9.5%). The use of the term "approximately" at the beginning of a string of values ​​qualifies each of the values ​​(i.e., "approximately 1, 2, and 3" refers to approximately 1, approximately 2, and approximately 3). Furthermore, if a list of values ​​is provided herein (for example, about 50%, 60%, 70%, 80%, 85%, or 86%), the list shall include all of its intermediate and decimal values ​​(for example, 54%, 85.4%).

[0110] Anti-PD-1 antibody The bifunctional molecule according to the present invention comprises a first entity containing an anti-hPD-1 antibody or an antigen-binding fragment thereof.

[0111] This specification provides antibodies that bind, in particular, to human PD-1. In some embodiments, the antibody specifically binds to human PD-1, preferably to the extracellular domain of human PD-1. In some embodiments, the antibody selectively binds to one or more of full-length human PD-1, PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3,4.

[0112] In some embodiments, the anti-PD1 antibody is an isolated antibody, particularly a non-natural isolated antibody. Such an isolated anti-PD1 antibody can be prepared by at least one purification step. In some embodiments, the isolated anti-PD1 antibody is purified to at least 80% by mass, 85% by mass, 90% by mass, 95% by mass, or 99% by mass. In some embodiments, the isolated anti-PD1 antibody is provided as a solution containing at least 85% by mass, 90% by mass, 95% by mass, 98% by mass, 99% by mass to 100% by mass of the antibody, with the remaining mass being the mass of other solutes dissolved in the solvent.

[0113] Preferably, such an antibody has the ability to block or inhibit the interaction between PD-1 and at least one of its ligands (e.g., PD-L1 and / or PD-L2). The ability to “block binding,” “block interaction,” or “inhibit interaction,” as used herein, refers to the ability of an antibody or antigen-binding fragment to prevent the binding interaction between two molecules (e.g., PD-1 and its ligand PD-L1 and / or PD-L2) to any detectable degree.

[0114] Preferably, the anti-PD1 antibody or its antigen-binding fragment is an antagonist for the binding of human PD-L1 and / or PD-L2 to human PD-1, more preferably for the binding of human PD-L1 and PD-L2 to human PD-1.

[0115] In certain embodiments, the anti-hPD1 antibody or antigen-binding fragment inhibits the binding interaction between PD-1 and at least one of its ligands (e.g., PD-L1 and / or PD-L2, preferably PD-L1 and PD-L2) by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0116] The anti-hPD1 antibody according to the present invention may contain an immunoglobulin chain or fragment thereof (Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding partial sequences of the antibody) containing a minimal sequence derived from any class of immunoglobulin such as immunoglobulin, IgD, IgE, IgG, IgA, or IgM (or its subclasses), or a non-human (e.g., mouse) immunoglobulin targeting human PD-1. Preferably, the anti-hPD-1 antibody according to the present invention is derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG4 or IgG1.

[0117] In one embodiment, an antigen-binding fragment of an antibody comprises a heavy chain comprising a variable domain of the heavy chain including HCDR1, HCDR2, and HCDR3, a light chain comprising a variable domain including LDCR1, LDCR2, and LDCR3, and a fragment of the heavy chain constant domain. Thus, it should be understood that by the fragment of the heavy chain constant domain, the antigen-binding fragment comprises at least a portion of the complete heavy chain constant domain. As an example, the heavy chain constant domain may comprise at least the C H 1 domain of the heavy chain, or at least the C H 1 and C H 2 domains of the heavy chain, or at least the C H 1, C H 2, and C H 3 domains of the heavy chain, and may comprise or consist of the same. The fragment of the heavy chain constant domain can also be defined as comprising at least a portion of the Fc domain of the heavy chain. Thus, the antigen-binding fragment of an antibody encompasses the Fab portion of a complete antibody, the F(ab')2 portion of a complete antibody, and the Fab' portion of a complete antibody. Also, the heavy chain constant domain may comprise or consist of, for example, the complete heavy chain constant domains exemplified herein, and several complete heavy chain constant domains are described herein. In certain embodiments of the present invention, and when the antigen-binding fragment of an antibody comprises a fragment of the heavy chain constant domain that comprises or consists of a portion of the complete heavy chain constant domain, the heavy chain constant domain fragment may consist of at least 10 amino acid residues, or may consist of 10 to 300 amino acid residues, particularly 210 amino acid residues.

[0118] Preferably, the antibody against human PD-1 is a monoclonal antibody. As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitope. Preferably, such monoclonal antibodies (mAbs) are derived from mammals such as mice, rodents, rabbits, goats, primates, non-human primates, or humans. Techniques for preparing such monoclonal antibodies can be found, for example, in Stites et al. (eds.), BASIC AND CLINICAL IMMUNOLOGY (4th edition), Lange Medical Publications, Los Alamos, California, USA, and the references cited therein; Harlow and Lane (1988), ANTIBODIES: A LABORATORY MANUAL, CSH Press; and Goding (1986), MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2nd edition), Academic Press, New York City, New York, USA.

[0119] In certain embodiments, the anti-hPD1 antibodies provided herein are chimeric antibodies. In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass has changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0120] In certain embodiments, the anti-hPD1 antibody is a humanized antibody. A humanized antibody typically contains one or more variable domains in which the CDR (or portions thereof) are derived from a non-human antibody and the FR (or portions thereof) are derived from a human antibody sequence or a humanized antibody sequence. Alternatively, some FR residues may be substituted to restore or enhance the specificity, affinity, and / or humanization of the antibody. The humanized antibody will also optionally contain a human constant region (Fc) or at least a portion of a humanized constant region (Fc).Methods for antibody humanization are well known in this field; see, for example, the following publications: Winter and Milstein, Nature, 1991, Vol. 349: pp. 293-299; Riechmann et al., Nature, Vol. 332, p. 323 (1988); Verhoeyen et al., Science, Vol. 239, p. 1534 (1988); Rader et al., Proc. Nat. Acad. Sci. USA, 1998, Vol. 95: pp. 8910-8915; Steinberger et al., J. Biol. Chem., 2000, Vol. 275: pp. 36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, Vol. 86: pp. 10029-10033; Almagro, JC and Fransson, J., Front. Biosci. Volume 13 (2008), pp. 1619-1633; Kashmiri, SV et al., Methods Volume 36 (2005), pp. 25-34 (SDR (a-CDR) transplantation is described); Padlan, EA, Mol. Immunol. Volume 28 (1991), pp. 489-498 ("Resurfacing" is described); Dall'Acqua, WF et al., Methods Volume 36 (2005), pp. 43-60 ("FR shuffling" is described); and Osbourn, J. et al., Methods Volume 36 (2005), pp. 61-68, and Klimka, A. et al., Br. J. Cancer Volume 83 (2000), pp. 252-260 ("Guided selection" for FR shuffling). The selection method is described in U.S. Patents No. 5,585,089, No. 5,693,761, No. 5,693,762, No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; and No. 6,180,370. Preferably, the humanized antibody against human PD-1 is a monoclonal antibody.

[0121] In particular, the humanized antibody has a T20 humanness score of at least 80% or at least 85%, more preferably at least 88%, and even more preferably at least 90%, most preferably 85% to 95%, and more preferably 88% to 92%.

[0122] "Humanity" is generally measured using a T20 score analyzer to quantify the humanity of the variable region of a monoclonal antibody, as described in Gao SH, Huang K, Tu H, Adler A S., BMC Biotechnology. 2013:13:55. The T20 humanity score is a widely used parameter in the field of antibody humanization, first disclosed by Gao et al. (BMC Biotechnol., 2013, 13, 55). The T20 humanity score is typically used in patent applications to define humanized antibodies (e.g., International Publication Nos. 15161311, 17127664, 18136626, 18190719, 19060750, or 19170677).

[0123] A web-based tool for calculating the T20 score of antibody sequences is provided using the T20 Cutoff Human Database (http: / / abAnalyzer.lakepharma.com). The T20 score calculation first assigns a Kabat number to the input VH, VK, or VL variable region protein sequence and identifies CDR residues. The full-length sequence or framework-only sequence (with CDR residues removed) is compared to all sequences in the respective antibody database using the blastp protein-protein BLAST algorithm. Sequence identity between each pairwise comparison is extracted, and after analyzing all sequences in the database, the sequences are sorted from highest to lowest based on their sequence identity to the input sequence. The T20 score is obtained by averaging the identity percentages of the top 20 matching sequences.

[0124] For each chain type (VH, VK, VL) and sequence length (full length or framework only) of the "All Human Databases," each antibody sequence was scored in its corresponding database using a T20 score analyzer. After excluding the input sequences themselves, the T20 scores of the top 20 matching sequences were obtained (sequence 1 was always the input antibody itself, so the identity percentages of sequences 2-21 were averaged). The T20 scores for each group were sorted from highest to lowest. The decrease in score was almost linear for most sequences, but the T20 scores of the bottom approximately 15% of antibodies began to decrease sharply. Therefore, the bottom 15 percent of sequences were removed, and the remaining sequences formed the T20 cutoff human database. The T20 score cutoff indicates the lowest T20 score of the sequences in the new database.

[0125] Therefore, the humanized anti-PD1 antibody contained in the bifunctional molecule according to the present invention has a T20 humanity score of at least 80% or at least 85%, more preferably at least 88%, and even more preferably at least 90%, most preferably 85% to 95%, and most preferably 88% to 92%.

[0126] In one embodiment, the anti-PD1 antibody can be selected from the following group: pembrolizumab (also known as keytruda lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pizilizumab (CT-011), semiprimab (Libtayo), camrelizumab, AUNP12 AMP-224, AGEN-2034, BGB-A317 (Chithlaizumab), PDR001 (Spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, Genolimuzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), MEDI0608, JS001 (Si-Yang Liu et al., J. Hematol. See Oncol. Vol. 10: p. 136 (2017), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (Armo), STI-1110 (see International Publication No. 2014 / 194302), AGEN2034 (see International Publication No. 201 Monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 as described in International Publication No. 7 / 040790, MGA012 (see International Publication No. 2017 / 19846), or IBI308 (see International Publication Nos. 2017 / 024465, 2017 / 025016, 2017 / 132825, and 2017 / 133540), International Publication No. 2006 / 121168. Other bifunctional or bispecific molecules that target PD-1 include RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda), and XmAb23104 (Xencor).

[0127] In certain embodiments, the anti-PD1 antibody may be pembrolizumab (also known as keytruda lambrolizumab, MK-3475) or nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538).

[0128] Specific examples of humanized anti-hPD1 antibodies are described below in this specification, including their CDR, framework region, Fc region, and hinge region.

[0129] CDR In this technology, the "complementarity-determining region" or "CDR" is known to refer to a non-adjacent sequence of amino acids within the antibody variable region that confers antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be easily determined using one of several well-known methods, including those described in the following literature: Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition (1991), "Kabat" numbering system); Al-Lazikani et al., 1997, J. Mol. Biol, vol. 273: pp. 927-948 ("Chothia" numbering system); MacCallum et al., 1996, J. Mol. Biol., vol. 262: pp. 732-745 ("Contact" numbering system); Lefranc et al., Dev. Comp. Immunol., 2003, vol. 27: pp. 55-77 ("IMGT" numbering system); and Honegge and Pluckthun, J. Mol. Biol, 2001, vol. 309: pp. 657-70 ("AHo" numbering system). Unless otherwise specified, the numbering scheme used in this specification to identify a particular CDR is the Kabat numbering scheme.

[0130] In one embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or its antigen-binding fragment. The CDR region of the humanized antibody may be derived from a mouse antibody, and is configured to i) provide a safe humanized antibody with a very high level of humanization (greater than 85%) and stability, and ii) have a binding affinity (KD) of 10 to human PD-1. -7 Less than M, preferably 10-8 The antibody may be optimized to increase antibody properties, more specifically, higher manufacturability when produced in mammalian cells, and higher production yields in mammalian cells such as COS cells and HCO cells, while preserving antagonist activity (i.e., inhibition of human PD-L1 binding to human PD-1), so that the M value is less than M.

[0131] In very specific embodiments, the bifunctional molecule is an anti-human PD-1 antibody or its antigen-binding fragment, preferably (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 A humanized anti-human PD-1 antibody or its antigen-binding fragment is included, - Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 1, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1. - Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2. - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 3, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 3. - Light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 12, where X is G or T, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 12. - The light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, - The light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16.

[0132] In one aspect, the bifunctional molecule (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 comprises a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, - The heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1, - The heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2, - The heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, wherein in the sequence, X1 is D, X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably selected from the group consisting of H, A, Y, N, E, or X1 is E, X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably selected from the group consisting of H, A, Y, N, E, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 3, - The light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 12, wherein in the sequence, X is G or T, and optionally has one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 12. - The light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 15, and optionally has one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof. - The light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 16, and optionally has one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16.

[0133] In another embodiment, the bifunctional molecule (i) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3, and (ii) a light chain variable domain comprising LCDR1, LCDR2, and LCDR3 comprising a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, - The heavy chain CDR1 (HCDR1) comprises or consists of the amino acid sequence of SEQ ID NO: 1, and optionally has one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof at any position other than position 3 of SEQ ID NO: 1. - The heavy chain CDR2 (HCDR2) comprises or consists of the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2. - The heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11, and optionally has one, two, or three modifications selected from substitution, addition, deletion, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11. - Light chain CDR1 (LCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 13 or SEQ ID NO: 14. - Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 15 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. - Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16.

[0134] In another embodiment, the bifunctional molecule is (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 A humanized anti-hPD-1 antibody or its antigen-binding fragment is included, (a) Light chain CDR1 (LCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 13, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 13, (b) Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 15 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. (c) Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16. (d) Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 1 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1. (e) Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2, and (f) Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 4 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 4, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 5, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 5, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 6, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 6, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 7, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 7, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 8, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 8, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 9, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 9, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 10, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 10, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 11 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 11.

[0135] In another embodiment, the bifunctional molecule is (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 A humanized anti-hPD-1 antibody or its antigen-binding fragment is included, (a) Light chain CDR1 (LCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 14 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 14, (b) Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 15 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. (c) Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16. (d) Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 1 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1. (e) Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2, and (f) Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 4 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 4, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 5, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 5, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 6, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 6, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 7, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 7, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 8, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 8, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 9, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 9, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 10, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 10, or - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 11 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 11.

[0136] In certain aspects, modification is a substitution, particularly a conservative substitution.

[0137] In one embodiment, the anti-human PD-1 antibody or its antigen-binding fragment comprises (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein X1 is D or E in the sequence, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E; and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X in the sequence is G or T.

[0138] In one embodiment, the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein in the sequence, X1 is D, X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably selected from the group consisting of H, A, Y, N, and E, or X1 is E, X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably selected from the group consisting of H, A, Y, N, E, and S, and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X is G or T in the sequence.

[0139] In one embodiment, the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein in the sequence, X1 is D, X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably selected from the group consisting of H, A, Y, N, and E, or X1 is E, X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably selected from the group consisting of H, A, Y, N, E, and S, and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X is G or T in the sequence.

[0140] In one embodiment, the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, wherein in the sequence, X1 is E, X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably selected from the group consisting of H, A, Y, N, E, and S, and (ii) a light chain comprising CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16, wherein X is G or T in the sequence.

[0141] In another embodiment, the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises or consists essentially of (i) a heavy chain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, or 11, and (ii) a light chain comprising CDR1 of SEQ ID NO: 13 or SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16.

[0142] In another embodiment, an anti-human PD-1 antibody or its antigen-binding fragment is (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 7, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 8, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 9, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 10, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 11, and (ii) a light chain containing CDR1 of SEQ ID NO: 13, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16. It includes or essentially consists of.

[0143] In another embodiment, an anti-human PD-1 antibody or its antigen-binding fragment is (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 4, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 5, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 6, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 7, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 8, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 9, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 10, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; or (i) a heavy chain containing CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 11, and (ii) a light chain containing CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16. It includes or essentially consists of.

[0144] framework In one embodiment, the anti-PD1 antibody or antigen-binding fragment according to the present invention includes framework regions, particularly heavy chain variable region framework regions (HFR) HFR1, HFR2, HFR3, and HFR4, and light chain variable region framework regions (LFR) LFR1, LFR2, LFR3, and LFR4.

[0145] Preferably, the anti-PD1 antibody or antigen-binding fragment according to the present invention includes a human framework region or a humanized framework region. For the purposes of this specification, "human acceptor framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework, as defined below. The human acceptor framework derived from a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence. The "Human Consensus Framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences.

[0146] In particular, the anti-PD1 antibody or antigen-binding fragment each comprises heavy chain variable region framework (HFR) HFR1, HFR2, HFR3, and HFR4, each containing the amino acid sequences of SEQ ID NOs. 41, 42, 43, and 44, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 27, 29, and 32 of SEQ ID NO. 43, specifically HFR1 of SEQ ID NO. 41, HFR2 of SEQ ID NO. 42, HFR3 of SEQ ID NO. 43, and HFR4 of SEQ ID NO. 44.

[0147] Alternatively, the anti-PD1 antibody or antigen-binding fragment may each include light chain variable region framework (LFR) LFR1, LFR2, LFR3, and LFR4 comprising the amino acid sequences of SEQ ID NOs. 45, 46, 47, and 48, and optionally have one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. Preferably, the humanized anti-PD1 antibody or antigen-binding fragment includes LFR1 of SEQ ID NOs. 45, LFR2 of SEQ ID NOs. 46, LFR3 of SEQ ID NOs. 47, and LFR4 of SEQ ID NOs. 48.

[0148] VH-VL The VL domain and VH domain of the anti-hPD1 antibody contained in the bifunctional molecule according to the present invention may preferably include four framework regions separated by three complementarity-determining regions, which are operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxyl terminus).

[0149] In the first embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0150] In the second embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) comprising or consisting of the amino acid sequence of Sequence ID No. 17, wherein X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, E, preferably from the group consisting of H, A, Y, N, E, or X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, E, and S. , optionally, a heavy chain variable region (VH) having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of sequence number 17; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0151] In the third embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, where X1 is D, and X2 is selected from the group consisting of T, H, A, Y, N, and E, preferably from the group consisting of H, A, Y, N, and E, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0152] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, wherein X1 is E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, E, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Includes.

[0153] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) Heavy chain variable regions (VH) comprising or consisting of the amino acid sequences of SEQ ID NOs. 18, 19, 20, 21, 22, 23, 24, or 25, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 22, 23, 24, or 25, respectively; (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27 or SEQ ID NO: 28. Includes.

[0154] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 18, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 18. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 19, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 19. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 20, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 20. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 21, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 21. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 22, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 22. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 23, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 23. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 24, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 24. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 25, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 25. (b) a heavy chain variable region (VH) having, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 18, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 18. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 19, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 19. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 20, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 20. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 21, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 21. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 22, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 22. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 23, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 23. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 24, wherein, at any option, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 24. (b) a heavy chain variable region (VH) and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28; or (a) A sequence containing or comprising the amino acid sequence of SEQ ID NO: 25, wherein, at any choice, one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof are made at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 25. (b) a heavy chain variable region (VH) having decorations, and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 28. Includes.

[0155] In certain aspects, modification is a substitution, particularly a conservative substitution.

[0156] CH-CL In one embodiment, the heavy chain (CH) and the light chain (CL) include the VL sequence and the VH sequence as described above in this specification.

[0157] In certain embodiments, the anti-human PD-1 antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) Heavy chains comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, each having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, or 36, respectively, and (b) A light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37 or SEQ ID NO: 38. Includes.

[0158] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule is (a) Consists of or includes an amino acid sequence selected from the group consisting of SEQ ID NO: 29, and optionally includes one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 29 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 30, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 30 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 31, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 31 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 32, and optionally one of substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 32 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 33, which optionally includes one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 33 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 34, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 34 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 35, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 35 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 36, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 36 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 37; or (a) Consists of or includes an amino acid sequence selected from the group consisting of SEQ ID NO: 29, and optionally includes one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 29 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 30, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 30 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 31, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 31 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 32, and optionally one of substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 32 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 33, which optionally includes one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 33 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 34, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 34 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 35, and optionally comprising one substitution, addition, deletion, or any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 35 (b) a heavy chain having two or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38; or (a) Consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, and optionally includes substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 36. (b) a heavy chain having one, two, or three modifications, and (b) a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 38. Includes.

[0159] Preferably, the modification is a substitution, particularly a conservative substitution.

[0160] Fc and hinge region Several studies to develop therapeutic antibodies have led to the optimization of antibody properties through genetic manipulation of the Fc region, enabling the creation of molecules better suited to their required pharmacological activity. The Fc region of an antibody mediates its serum half-life and effector functions such as complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent phagocytosis (ADCP). Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L and M252Y / S254T / T256E+H433K / N434F, have been shown to increase binding affinity to FcRn and the in vivo half-life of IgG1. However, there is not always a direct relationship between increased FcRn binding and improved half-life. One approach to improving the efficacy of therapeutic antibodies is to increase their serum persistence, thereby enabling higher circulating levels, lower administration frequency, and reduced doses. Genetic engineering of the Fc region may be desirable to either reduce or increase the effector function of an antibody. For antibodies targeting cell surface molecules, particularly those on immune cells, it is necessary to disable effector function. Conversely, for antibodies intended for oncological use, increasing effector function can improve therapeutic activity. The four human IgG isotypes bind to activated Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa), inhibitory FcγRIIb receptor, and complement component 1 (C1q) with different affinities, resulting in very different effector functions. IgG binding to FcγR or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are crucial for FcγR and C1q binding and have unique sequences in IgG2 and IgG4.

[0161] The antibodies according to the present invention optionally comprise at least a portion of the immunoglobulin constant region (Fc), typically, at least a portion of the immunoglobulin constant region (Fc) of mammalian immunoglobulin, and more preferably, at least a portion of the immunoglobulin constant region (Fc) of human immunoglobulin or humanized immunoglobulin. Preferably, the Fc region is part of the anti-hPD-1 antibody described herein. The anti-hPD1 antibody or its antigen-binding fragment contained in the bifunctional molecule of the present invention may comprise the immunoglobulin constant region, or a fragment, analog, variant, mutant, or derivative of the constant region. As is well known to those skilled in the art, the selection of the IgG isotype of the heavy chain constant domain is a central concern for whether a specific function is required and for the need for a suitable in vivo half-life. For example, antibodies designed for the selective eradication of cancer cells typically require an active isotype that enables complement activation and effector-mediated cytotoxicity by antibody-dependent cell-mediated cytotoxicity. Both human IgG1 and IgG3 (shorter half-life) isotypes, and especially human IgG1 isotypes (wild type and variant), meet these criteria. In particular, depending on the IgG isotype of the heavy chain constant domain (especially the human wild-type and variant IgG1 isotype), the anti-hPD1 antibody of the present invention may be cytotoxic to PD-1-expressing cells via CDC, ADCC, and / or ADCP mechanisms. Indeed, the fragment crystallizable (Fc) region interacts with various accessory molecules to mediate indirect effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and complement-dependent cell-mediated cytotoxicity (CDC).

[0162] In preferred embodiments, the constant region is derived from a human immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, and other classes. In further embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. Preferably, the anti-PD1 antibody includes an IgG1 Fc region or an IgG4 Fc region. Even more preferably, the anti-hPD1 antibody includes an IgG4 Fc region having S228P for stabilizing IgG4.

[0163] In one embodiment, the anti-PD1 antibody comprises a truncated Fc region or a fragment of a truncated Fc region. In one embodiment, the constant region comprises a CH2 domain. In another embodiment, the constant region comprises a CH2 domain and a CH3 domain, or a hinge-CH2-CH3 domain. Alternatively, the constant region may comprise all or part of the hinge region, the CH2 domain, and / or the CH3 domain. In a preferred embodiment, the constant region contains a CH2 domain and / or a CH3 domain derived from a human IgG quadruple chain. In some embodiments, the constant region contains a CH2 domain and / or a CH3 domain derived from a human IgG quadruple chain.

[0164] In another embodiment, the constant region includes at least a portion of the CH2 domain and the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable classes, with or without mutation. More preferably, the hinge region is derived from a human IgG1 heavy chain. In one embodiment, the constant region includes a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a particular embodiment, the CH2 domain is derived from a human IgG2 or IgG4 heavy chain, and the hinge region is derived from a modified human IgG1 heavy chain.

[0165] In one embodiment, the constant region contains a mutation that reduces affinity for the Fc receptor or reduces Fc effector function. For example, the constant region may contain a mutation that eliminates a glycosylation site within the constant region of the IgG heavy chain.

[0166] In another embodiment, the constant region includes the CH2 domain and at least a portion of the hinge region. The hinge region may be derived from an immunoglobulin heavy chain, e.g., IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other preferred classes. The IgG1 hinge region has three cysteines, two of which are involved in the disulfide bond between the two heavy chains of the immunoglobulin. These same cysteines enable efficient and consistent disulfide bond formation between the Fc portions. Therefore, the preferred hinge region of the present invention is derived from IgG1, more preferably human IgG1. In some embodiments, the first cysteine ​​in the human IgG1 hinge region is mutated to another amino acid, preferably serine. The IgG2 isotype hinge region has four disulfide bonds, which tend to promote oligomerization and the possibility of erroneous disulfide bond formation during secretion in recombinant systems. A preferred hinge region may be derived from the IgG2 hinge, where the first two cysteine ​​molecules are preferably mutated to other amino acids. The IgG4 hinge region is known to be inefficient in the formation of interchain disulfide bonds. However, a preferred hinge region of the present invention may be derived from the IgG4 hinge region, preferably containing mutations that enhance the correct formation of disulfide bonds between heavy chain-derived portions (Angal S et al. (1993) Mol. Immunol., Vol. 30: pp. 105-108). More preferably, the hinge region is derived from the human IgG4 heavy chain.

[0167] In one embodiment, the constant region includes a CH2 domain derived from a first antibody isotype and a hinge region derived from a second antibody isotype. In a particular embodiment, the CH2 domain is derived from a human IgG quadruple chain, and the hinge region is derived from a modified human IgG monocoque.

[0168] According to the present invention, the constant region may contain CH2 and / or CH3 domains and hinge regions derived from different antibody isotypes, i.e., a hybrid constant region. For example, in one embodiment, the constant region contains CH2 and / or CH3 domains derived from IgG2 or IgG4 and a mutant hinge region derived from IgG1. Alternatively, the hybrid constant region may use a mutant hinge region derived from another IgG subclass. For example, a mutant form of the IgG4 hinge that enables efficient disulfide bonding between two heavy chains may be used. The mutant hinge may also be derived from an IgG2 hinge in which the first two cysteines are each mutated to different amino acids. An assembly of such a hybrid constant region is described in U.S. Patent Application Publication No. 20030044423, the disclosure of which is incorporated herein by reference.

[0169] In one embodiment, the steady region may contain CH2 and / or CH3 having one of the mutations listed in Table D (Table 4) below, or any combination thereof.

[0170] [Table 4]

[0171] In certain embodiments, the bifunctional molecule, preferably the binding moiety, comprises a human IgG1 heavy chain constant domain or an IgG1 Fc domain, and optionally includes T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I332 The substitutions or combinations of substitutions are selected from the group consisting of E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably N297A combined with M252Y / S254T / T256E, and selected from the group consisting of L234A / L235A.

[0172] In another embodiment, the binding moiety comprises a human IgG4 heavy chain constant domain or a human IgG4 Fc domain and optionally has substitutions or combinations of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E, and K444A. More preferably, a bifunctional molecule, preferably, the binding moiety comprises an IgG4 Fc region having S228P to stabilize IgG4.

[0173] In certain embodiments, amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. In certain embodiments, the Fc region variants possess some, but not all, effector functions. Such antibodies may be useful in applications where, for example, the half-life of the antibody in vivo is important, but certain effector functions are unnecessary or detrimental. Examples of effector functions include complement-dependent cell-mediated cytotoxicity (CDC) and antibody-mediated complement-mediated cytotoxicity (ADCC). Numerous substitutions or deletions that alter effector functions are known in the art.

[0174] In one embodiment, the constant region contains a mutation that reduces affinity for the Fc receptor or reduces Fc effector function. For example, the constant region may contain a mutation that eliminates a glycosylation site within the constant region of the IgG heavy chain. Preferably, the CH2 domain contains a mutation that eliminates a glycosylation site within the CH2 domain.

[0175] In one embodiment, the anti-hPD1 according to the present invention has a heavy chain steady domain of SEQ ID NO: 39 or 52 and / or a light chain steady domain of SEQ ID NO: 40, in particular a heavy chain steady domain of SEQ ID NO: 39 or 52 and a light chain steady domain of SEQ ID NO: 40.

[0176] In another embodiment, the anti-hPD1 according to the present invention has the heavy chain steady domain of SEQ ID NO: 52 and / or the light chain steady domain of SEQ ID NO: 40, in particular the heavy chain steady domain of SEQ ID NO: 52 and the light chain steady domain of SEQ ID NO: 40.

[0177] [Table 5]

[0178] Modifications to amino acids near the junction of the Fc and non-Fc portions can dramatically increase the serum half-life of the Fc fusion protein (International Publication No. 01 / 58957). Therefore, the junction region of the protein or polypeptide of the present invention may preferably contain modifications within about 10 amino acids of the junction compared to the naturally occurring sequences of the immunoglobulin heavy chain and erythropoietin. Such amino acid changes may result in increased hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which the C-terminal lysine residue is replaced. Preferably, the C-terminal lysine of the IgG sequence is replaced with a non-lysine amino acid such as alanine or leucine to further increase the serum half-life.

[0179] All subclasses of human IgG have a C-terminal lysine residue (K444) in the antibody heavy chain that is cleaved in circulation. This cleavage in the blood can impair the biological activity of the bifunctional molecule by releasing IL-7. To avoid this problem, the K444 amino acid in the IgG1 or IgG4 domain can be substituted with alanine to reduce proteolytic cleavage. This mutation is widely used in antibodies. Therefore, in one embodiment, an anti-PD1 antibody includes at least one further amino acid substitution consisting of K444A.

[0180] In one embodiment, the anti-PD1 antibody contains an additional cysteine ​​residue in the C-terminal domain of IgG to generate an additional disulfide bond and potentially limit the flexibility of the bifunctional molecule.

[0181] In certain embodiments, the antibody may be modified to increase, decrease, or eliminate the degree to which it is glycosylated.

[0182] Checkpoint inhibitors The inventors hereby demonstrate that the bifunctional molecule according to the present invention combines the effect of an IL-7 variant or mutant on the IL-7 receptor with the blockade of the inhibitory effect of PD-1, and is suitable for optimizing the effects of checkpoint inhibitors such as anti-PD-1 antibodies. In particular, a synergistic effect on the activation of T cells, especially exhausted T cells, and more specifically on TCR signaling, is demonstrated. The inventors particularly demonstrate activation on the same cell, provided by the binding of the anti-PD-1 antibody and IL-7 contained in the bifunctional molecule on the same immune cell. This synergistic effect is never observed when the IL-7 antibody and the anti-PD-1 antibody are used as separate compounds. Therefore, it can be conceivable that any molecule other than PD-1, particularly exhaustion factors, expressed on immune cells expressing IL-7R, can be induced by the bifunctional construct according to the present invention. Accordingly, in embodiments, the bifunctional molecule includes an antibody or its antigen-binding fragment against a target other than PD-1 expressed on an immune cell. For example, the target may be a receptor expressed on the surface of an immune cell, especially a T cell. The receptor may be an inhibitor receptor. Alternatively, the receptor may be an activating receptor.

[0183] As used herein, the term “target” refers to a peptide, polypeptide, protein, antigen, or epitope expressed on the external surface of an immune cell. With respect to the expression of a target on the surface of an immune cell, the term “expressed” refers to a target that is present on or presented on the outer surface of the cell. The term “specifically expressed” means that the target is expressed on immune cells but substantially not on other cell types, particularly tumor cells, etc.

[0184] In one embodiment, the target is specifically expressed by immune cells in a healthy subject or in a subject suffering from a disease, particularly cancer. This means that the target exhibits a higher expression level in immune cells than in other cells, or that the ratio of immune cells expressing the target to all immune cells is higher than the ratio of other cells expressing the target to all other cells. Preferably, the expression level or ratio is 2, 5, 10, 20, 50, or 100 times higher. The expression level or ratio can be determined more specifically for a particular type of immune cell, such as T cells, more specifically CD8+ T cells, effector T cells, or exhausted T cells, or in specific circumstances, for a subject suffering from a disease such as cancer or an infectious disease.

[0185] In one embodiment, the target is an immune checkpoint. Preferably, the target is selected from the group consisting of PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8. Such targets are described in detail in Table F (Table 6) below.

[0186] [Table 6A]

[0187] [Table 6B]

[0188] Therefore, in this embodiment, the antibody or antigen fragment contained in the bifunctional molecule according to the present invention binds to a target selected from the group consisting of CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8.

[0189] In a preferred embodiment, the antibody or antigen-binding fragment contained in the bifunctional molecule according to the present invention is selected from the group consisting of CTLA-4, BTLA, TIGIT, LAG3, and TIM3.

[0190] Furthermore, antibodies against TIM3 and bifunctional or bispecific molecules targeting TIM3 are known, such as Sym023, TSR-022, MBG453, LY3321367, INCAGN02390, BGTB-A425, LY3321367, and RG7769 (Roche). In some embodiments, TFM-3 antibodies are as described in International Publication No. 2013006490, International Publication No. 2016 / 161270, International Publication No. 2018 / 085469, or International Publication No. 2018 / 129553, International Publication No. 2011 / 155607, U.S. Patent No. 8,552,156, European Patent No. 2581113, and U.S. Patent Application No. 2014 / 044728.

[0191] Furthermore, antibodies against CTLA-4 and bifunctional or bispecific molecules that target CTLA-4 are known, including ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), and MEDI5752 (AstraZeneca). Anti-CTLA-4 antibodies are listed in International Publication Nos. 18025178, 19179388, 19179391, 19174603, 19148444, 19120232, 19056281, 19023482, 18209701, 18165895, 18160536, 18156250, 18106862, and 18 It is also disclosed in International Publication No. 106864, International Publication No. 18068182, International Publication No. 18035710, International Publication No. 18025178, International Publication No. 17194265, International Publication No. 17106372, International Publication No. 17084078, International Publication No. 17087588, International Publication No. 16196237, International Publication No. 16130898, International Publication No. 16015675, International Publication No. 12120125, International Publication No. 09100140, and International Publication No. 07008463.

[0192] Furthermore, antibodies against LAG-3 and bifunctional or bispecific molecules targeting LAG-3 are known, such as BMS-986016, IMP701, MGD012, or MGD013 (bispecific PD-1 and LAG-3 antibodies). Anti-LAG-3 antibodies are also disclosed in International Publication No. 2008132601, European Patent No. 2320940, and International Publication No. 19152574.

[0193] In this technical field, antibodies against BTLA such as hu Mab8D5, hu Mab8A3, hu Mab21H6, hu Mab19A7, or hu Mab4C7 are also known. The antibody against BTLA, TAB004, is currently undergoing clinical trials in subjects with advanced malignancies. Anti-BTLA antibodies are also disclosed in International Publication No. 08076560, International Publication No. 10106051 (e.g., BTLA8.2), International Publication No. 11014438 (e.g., 4C7), International Publication No. 17096017, and International Publication No. 17144668 (e.g., 629.3).

[0194] In this technical field, as disclosed in International Publication No. 19232484, BMS-986207 or AB154, BMS-986207 CPA. 9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CHA.9.536 .1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.560.5, CHA.9. Antibodies against TIGIT, such as 560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8, are also known. Anti-TIGIT antibodies are listed in International Publication Nos. 16028656, 16106302, 16191643, 17030823, 17037707, 17053748, 17152088, 18033798, 18102536, 18102746, and 18160704. It is also disclosed in International Publication Nos. 18200430, 18204363, 19023504, 19062832, 19129221, 19129261, 19137548, 19152574, 19154415, 19168382, and 19215728.

[0195] In the art, antibodies against CD160 are also known, such as CL1-R2 CNCM I-3204 as disclosed in International Publication No. 06015886, or others as disclosed in International Publication Nos. 10006071, 10084158, and 18077926.

[0196] In certain embodiments, the bifunctional molecule according to the present invention comprises an anti-CTLA-4 antibody or its antigen-binding fragment, preferably a human, humanized, or chimeric anti-CTLA-4 antibody or its antigen-binding fragment. Preferably, the antibody is a CTLA-4 antagonist. Thus, the bifunctional molecule combines the effects of IL-7wt, its variants, or mutants on the IL-7 receptor with the blocking of the inhibitory effect of CTLA-4, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more specifically, on the activation of TCR signaling.

[0197] In another specific embodiment, the bifunctional molecule according to the present invention comprises an anti-BTLA antibody or its antigen-binding fragment, preferably a human, humanized, or chimeric anti-BTLA antibody or its antigen-binding fragment. Preferably, the antibody is a BTLA antagonist. Thus, the bifunctional molecule combines the effects of IL-7wt, its variants, or mutants on the IL-7 receptor with the blocking of the inhibitory effect of BTLA, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more specifically, on the activation of TCR signaling.

[0198] In another specific embodiment, the bifunctional molecule according to the present invention comprises an anti-TIGIT antibody or its antigen-binding fragment, preferably a human, humanized, or chimeric anti-TIGIT antibody or its antigen-binding fragment. Preferably, the antibody is a TIGIT antagonist. Thus, the bifunctional molecule combines the effects of IL-7wt, its variants, or mutants on the IL-7 receptor with the blocking of the inhibitory effect of TIGIT, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more specifically, on the activation of TCR signaling.

[0199] In another specific embodiment, the bifunctional molecule according to the present invention comprises an anti-LAG-3 antibody or its antigen-binding fragment, preferably a human, humanized, or chimeric anti-LAG-3 antibody or its antigen-binding fragment. Preferably, the antibody is an antagonist of LAG-3. Thus, the bifunctional molecule combines the effects of IL-7wt, its variants, or mutants on the IL-7 receptor with the blocking of the inhibitory effect of LAG-3, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more specifically, on the activation of TCR signaling.

[0200] In another specific embodiment, the bifunctional molecule according to the present invention comprises an anti-TIM3 antibody or its antigen-binding fragment, preferably a human, humanized, or chimeric anti-TIM3 antibody or its antigen-binding fragment. Preferably, the antibody is a TIM3 antagonist. Thus, the bifunctional molecule combines the effect of an IL-7 variant or mutant on the IL-7 receptor with the blockade of the inhibitory effect of TIM3, and can exhibit a synergistic effect on T cells, particularly exhausted T cells, and more specifically, on the activation of TCR signaling.

[0201] Peptide linker The present invention comprises a bifunctional molecule which may contain a peptide linker between an anti-PD-1 antibody or a fragment thereof and IL-7. The peptide linker typically has sufficient length and flexibility to ensure that the two protein elements connected by the linker have sufficient spatial freedom to perform their functions and to avoid the effects of α-helix and β-fold formation on the stability of the recombinant bifunctional molecule.

[0202] In aspects of this disclosure, the anti-hPD1 antibody is preferably linked to IL-7 by a peptide linker. In other words, the present invention relates to a bifunctional molecule comprising an anti-PD1 antibody or its antigen-binding fragment as detailed herein, having a chain, e.g., a light chain or a heavy chain or a fragment thereof, preferably a heavy chain or a fragment thereof, and linked to IL-7 via a peptide linker. As used herein, the term “linker” refers to a sequence of at least one amino acid that links IL-7 and the anti-PD-1 immunoglobulin sequence portion. Such a linker may be useful in preventing steric hindrance. The linker is typically 3 to 44 amino acid residues in length. Preferably, the linker has 3 to 30 amino acid residues. In some embodiments, the linker has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues.

[0203] In embodiments, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or an antigen-binding fragment thereof as defined above and IL-7, wherein the antibody chain, for example, a light chain or a heavy chain, preferably a heavy chain, and more preferably the C-terminus of the heavy chain or light chain, is linked to IL-7, preferably the N-terminus of IL-7, by a peptide linker.

[0204] In certain embodiments, the present invention relates to a bifunctional molecule comprising an anti-hPD-1 antibody or an antigen-binding fragment thereof as defined above, wherein IL-7 is preferably linked to the C-terminus of the heavy chain of the antibody (e.g., the C-terminus of the heavy chain constant domain) by a peptide linker.

[0205] In embodiments, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or an antigen-binding fragment thereof as defined above, wherein IL-7 is preferably linked to the C-terminus of the light chain of the antibody (e.g., the C-terminus of the constant domain of the light chain) by a peptide linker.

[0206] The linker sequence may be a naturally occurring sequence or a sequence that does not exist in nature. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to which the bifunctional molecule is administered. One useful group of linker sequences is those derived from the hinge region of heavy chain antibodies, as described in International Publications 96 / 34103 and 94 / 04678. Another example is polyalanine linker sequences. A more preferred example of linker sequences is Gly / Ser linkers of various lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, particularly (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3.

[0207] In one embodiment, the linker contained in the bifunctional molecule is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, and is preferably (Gly4Ser)3. Preferably, the linker is selected from the group consisting of (Gly4Ser)4, (Gly4Ser)3, and (Gly3Ser2)3. Even more preferably, the linker is (GGGGS)3.

[0208] In embodiments, the present invention relates to a bifunctional molecule comprising an anti-PD-1 antibody or a fragment thereof as defined above, wherein the antibody or fragment thereof is preferably linked to IL-7 by a linker sequence selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and more preferably by (GGGGS)3. Preferably, the linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, and (GGGS)3.

[0209] Preferably, the C-terminus of the heavy chain, preferably the heavy chain of the anti-PD-1 antibody, is genetically fused with the N-terminus of IL-7 via a flexible (Gly4Ser)3 linker. At the fusion junction, the C-terminal lysine residue of the antibody heavy chain can be mutated to alanine to reduce proteolytic cleavage.

[0210] Preferably, the C-terminus of the heavy chain, preferably the light chain, of the anti-PD-1 antibody is genetically fused to the N-terminus of IL-7 via a flexible (Gly4Ser)3 linker. At the fusion junction, the C-terminal lysine residue of the antibody light chain can be mutated to alanine to reduce proteolytic cleavage.

[0211] IL-7 The bifunctional molecule according to the present invention comprises an additional or second entity containing interleukin 7, or a variant or fragment thereof.

[0212] Preferably, the IL-7 protein is human IL-7 or a variant thereof. Therefore, IL-7 or its variant has an amino acid sequence that is at least 75% identical to wild-type IL-7 and, in particular, to the protein of SEQ ID NO: 51.

[0213] In one embodiment, the bifunctional molecule contains a typical wild-type human IL-7 protein (SEQ ID NO: 51) consisting of 152 amino acids. Preferably, the IL-7 protein is the protein of SEQ ID NO: 51. The IL-7 protein may or may not contain its peptide signal.

[0214] A “variant” of the IL-7 protein is defined as an amino acid sequence in which one or more amino acids are modified. Variants may have “conservative” or “non-conservative” modifications. Such modifications include amino acid substitutions, deletions, and / or insertions. Guidance in determining which and how many amino acid residues can be substituted, inserted, or deleted without impairing biological properties (e.g., activity, binding ability, and / or structure) can be found using computer programs well known in the art, such as molecular modeling or alignment software. In certain embodiments, variant IL-7 proteins included in the present invention include IL-7 proteins that, in particular, retain substantially equivalent biological IL-7 properties compared to wild-type IL-7. In alternative embodiments, variant IL-7 proteins included in the present invention include IL-7 proteins that, in particular, do not retain substantially equivalent biological properties (e.g., activity, binding ability, and / or structure) compared to wild-type IL-7. Furthermore, IL-7 variants include modified polypeptide sequences of IL-7 (e.g., oxidized, reduced, deaminated, or truncated forms). In particular, truncated forms or fragments of IL-7 that retain biological properties equivalent to those of the full-length IL-7 protein are included within the scope of the present invention. In one embodiment, interleukin 7 is any biologically active fragment thereof. More preferably, IL-7 variants include native allele variants resulting from native genetic polymorphisms, such as SNPs and splicing variants.

[0215] The biological activity of the IL-7 protein can be measured using an in vitro cell proliferation assay. Preferably, the IL-7 variant according to the present invention maintains at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, and 60% of the biological activity compared to wild-type human IL-7, and preferably at least 80%, 90%, 95%, and more preferably 99% compared to wild-type IL-7.

[0216] Furthermore, examples of mutant IL-7 proteins include polypeptides that have at least approximately 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with wild-type IL-7, particularly the protein of sequence number 51.

[0217] A preferred IL-7 according to the present invention is a human IL-7 polypeptide comprising or comprising the amino acid sequence as described in Sequence ID No. 51, European Patent No. 314415, or International Publication No. 2004 / 018681A2, as well as any natural variants and homologs thereof.

[0218] In one embodiment, the IL-7 polypeptide used in the present invention is recombinant IL-7. The term "recombinant," as used herein, means that the polypeptide is obtained or derived from a recombinant expression system, i.e., from a culture of host cells (e.g., microorganisms, insects, plants, or mammals), or from a transgenic plant or animal that has been genetically engineered to contain a nucleic acid molecule encoding the IL-7 polypeptide. Preferably, the recombinant IL-7 is human recombinant IL-7 (e.g., human IL-7 produced in a recombinant expression system).

[0219] Furthermore, the present invention provides a bifunctional molecule containing an IL-7 protein with enhanced biological activity compared to wild-type IL-7 protein. For example, as described in U.S. Patent No. 7,960,514, IL-7 proteins having disulfide bond patterns of Cys2-Cys92, Cys34-Cys129, and Cys47-141 are more active in vivo than wild-type recombinant IL-7 protein. As described in European Patent No. 1,904,635, etc., highly glycosylated IL-7 proteins, such as those in which Asn116 is not glycosylated but Asn70 and Asn91 are glycosylated, improve the biological activity of IL-7.

[0220] Alternatively, the present invention provides a bifunctional molecule comprising an IL-7 protein in which immunogenicity is reduced compared to wild-type IL-7 protein by removing a T cell epitope within IL-7 that can stimulate an immune response. An example of such IL-7 is described in International Publication No. 2006061219.

[0221] In certain embodiments, the Disclosure also provides a bifunctional molecule comprising an IL-7 variant or mutant. The terms “interleukin-7 mutant,” “mutant IL-7,” “IL-7 variant,” “IL-7 variant,” “IL-7m,” or “IL-7v” are used synonymously herein.

[0222] In this context, IL-7 variants or mutants do not retain substantially equivalent biological properties (e.g., activity, binding ability, and / or structure) compared to wild-type IL-7. IL-7 mutants or variants contain at least one mutation. In particular, at least one mutation reduces the affinity of the IL-7 variant or mutant for the IL-7 receptor (IL-7R), but does not lead to a loss of IL-7R recognition. Therefore, IL-7 mutants or variants retain the ability to activate IL-7R, as disclosed, for example, by pStat5 signaling, as disclosed in Bitar et al., Front. Immunol., 2019, Vol. 10, etc. The biological activity of the IL-7 protein can be measured using in vitro cell proliferation assays or by measuring P-Stat5 in T cells by ELISA or FACS. Preferably, the IL-7 variant according to the present invention reduces the biological properties (e.g., activity, binding ability, and / or structure) by at least 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 250, 1 / 500, 1 / 750, 1 / 1000, 1 / 2500, 1 / 5000, or 1 / 8000 compared to wild-type IL-7, preferably with-IL7. More preferably, the IL-7 variant exhibits reduced binding to the IL-7 receptor but retains the ability to activate IL-7R. For example, binding to the IL-7 receptor may be reduced by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% compared to wild-type IL-7, while retaining at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the ability to activate IL-7R compared to wild-type IL-7.

[0223] In one embodiment, an IL-7 variant or mutant has at least one amino acid mutation that differs from wt-IL-7, i) reducing the affinity of the IL-7 variant for the IL-7 receptor (IL-7R) compared to the affinity of wt-IL-7 for IL-7R, and ii) improving the pharmacokinetics of the IL-7 variant compared to wt-IL-7. More specifically, the IL-7 variant or mutant further retains the ability to activate IL-7R, particularly via pStat5 signaling.

[0224] In another embodiment, a bifunctional molecule containing an IL-7 variant or mutant has at least one amino acid mutation that differs from wt-IL-7, i) reducing the affinity of the bifunctional molecule to the IL-7 receptor (IL-7R) compared to the affinity of a bifunctional molecule containing wt-IL-7 to IL-7R, and ii) improving the pharmacokinetics of the bifunctional molecule containing the IL-7 variant or mutant compared to a bifunctional molecule containing wt-IL-7. More specifically, the bifunctional molecule containing the IL-7 variant or mutant further retains the ability to activate IL-7R, particularly via pStat5 signaling. For example, the binding of a bifunctional molecule containing an IL-7 variant or mutant to the IL-7 receptor may be reduced by at least 10%, 20%, 30%, 40%, 50%, or 60% compared to a bifunctional molecule containing wild-type IL-7, while retaining at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, or 20% of the ability to activate IL-7R compared to a bifunctional molecule containing wild-type IL-7.

[0225] In certain embodiments, IL-7 variants or mutants exhibit reduced affinity for the IL-7 receptor (IL-7R) compared to the affinity of with-IL-7 for IL-7R. In particular, IL-7 variants or mutants exhibit reduced affinity for CD127 and / or CD132 compared to the affinity of with-IL-7 for CD127 and / or CD132, respectively. Preferably, IL-7 variants or mutants exhibit reduced affinity for CD127 compared to the affinity of with-IL-7 for CD127.

[0226] Preferably, at least one amino acid mutation reduces the affinity of an IL-7 variant or mutant to IL-7R, particularly to CD132 or CD127, to at least 1 / 10, 1 / 10

[0227] Preferably, at least one amino acid mutation reduces the affinity of the IL-7 variant or mutant to IL-7R, but does not reduce the biological activity of the IL-7 variant or mutant compared to IL-7 wt, particularly as measured by the pStat5 signaling pathway.

[0228] Alternatively, at least one amino acid mutation reduces the affinity of an IL-7 variant or mutant to IL-7R, but does not significantly reduce the biological activity of IL-7m compared to IL-7 wt, particularly as measured by the pStat5 signaling pathway.

[0229] In addition to or instead of this, IL-7 variants or mutants improve the pharmacokinetics of bifunctional molecules containing IL-7 variants or mutants compared to bifunctional molecules containing wild-type IL-7. In particular, the IL-7 variants or mutants according to the present invention improve the pharmacokinetics of bifunctional molecules containing IL-7 variants or mutants by at least 10-fold, 100-fold, or 1000-fold compared to bifunctional molecules containing with-IL-7. Pharmacokinetic profile comparisons can be performed by any method known to those skilled in the art, such as injecting the drug in vivo and performing dose ELISA of the serum drug at multiple time points, as shown in Example 9.

[0230] As used herein, the terms “pharmacokinetics” and “PK” are used synonymously and refer to the fate of a compound, substance, or drug administered to a living organism. Pharmacokinetics includes, in particular, ADME or LADME schemes representing release (i.e., release of the substance from a composition), absorption (i.e., entry of the substance into the bloodstream), distribution (i.e., dispersion or diffusion of the substance throughout the body), metabolism (i.e., transformation or breakdown of the substance), and excretion (i.e., removal or clearance of the substance from the organism). The two stages of metabolism and excretion can also be grouped together under the heading of elimination. Those skilled in the art can monitor various pharmacokinetic parameters, such as elimination half-life, elimination constant rate, clearance (i.e., volume of plasma from which the drug is cleared per unit time), Cmax (maximum serum concentration), and drug exposure (determined by the area under the curve) (Scheff et al., Pharm Res., 2011, Vol. 28, pp. 1081-1089).

[0231] Therefore, the pharmacokinetic improvement due to the use of IL-7 variants or mutants refers to an improvement in at least one of the parameters mentioned above. Preferably, the improvement refers to an improvement in the elimination half-life of the bifunctional molecule, i.e., an increase in half-life duration or Cmax.

[0232] In certain embodiments, at least one mutation in the IL-7 variant or mutant improves the elimination half-life of the bifunctional molecule containing the IL-7 variant or mutant compared to the bifunctional molecule containing IL-7 wt.

[0233] In one embodiment, the IL-7 variant or mutant exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with the 152-amino acid wild-type human IL-7 (wth-IL-7) protein, such as that disclosed in SEQ ID NO: 51. Preferably, the IL-7 variant or mutant exhibits at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 51.

[0234] In particular, at least one mutation occurs at amino acid positions 74 and / or 142 of IL-7. In addition or instead, at least one mutation occurs at amino acid positions 2 and 141, 34 and 129, and / or 47 and 92. These positions refer to the amino acid positions shown in SEQ ID NO: 51.

[0235] In particular, at least one mutation is an amino acid substitution or group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, Q11E, Y12F, M17L, Q22E, K81R, D74E, D74Q, and D74N, or any combination thereof. Such mutations refer to the amino acid positions shown in SEQ ID NO: 51. For example, the mutation W142H represents the substitution of tryptophan in wth-IL7 with histidine to obtain IL-7m having histidine at amino acid position 142. Such a variant is described, for example, in SEQ ID NO: 56.

[0236] In one embodiment, the IL-7 variant or mutant includes a set of substitutions to break disulfide bonds between C2 and C141, between C47 and C92, and between C34-C129. In particular, the IL-7 variant or mutant includes two sets of substitutions to break disulfide bonds between C2 and C141 and between C47 and C92; between C2 and C141 and between C34-C129; or between C47 and C92 and between C34-C129. For example, cysteine ​​residues may be substituted with serine to prevent disulfide bond formation. Therefore, amino acid substitutions can be selected from the group consisting of C2S-C141S and C47S-C92S (referred to as "SS2"), C2S-C141S and C34S-C129S (referred to as "SS1"), and C47S-C92S and C34S-C129S (referred to as "SS3"). These mutations refer to the amino acid positions shown in SEQ ID NO: 51. Such IL-7 variants or mutants are specifically described in the sequences shown in SEQ ID NOs: 53-55 (SS1, SS2, and SS3, respectively). Preferably, the IL-7 variants or mutants include the amino acid substitutions C2S-C141S and C47S-C92S. More preferably, the IL-7 variants or mutants represent the sequence shown in SEQ ID NO: 54.

[0237] In another embodiment, the IL-7 variant or variant comprises at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7 variants or variants are specifically described in the sequences shown in SEQ ID NOs. 57-58, respectively. Preferably, the IL-7 variant or variant comprises the W142H mutation. More preferably, the IL-7 variant or variant represents the sequence shown in SEQ ID NO. 56.

[0238] In another embodiment, the IL-7 variant or variant comprises at least one mutation selected from the group consisting of D74E, D74Q, and D74N, preferably D74E and D74Q. Such IL-7 variants or variants are specifically described in the sequences shown in SEQ ID NOs. 63-65, respectively. Preferably, the IL-7 variant or variant comprises the D74E mutation. More preferably, the IL-7 variant or variant represents the sequence shown in SEQ ID NO. 63.

[0239] In another embodiment, the IL-7 variant or variant comprises at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and / or K81R. Such mutations point to the amino acid positions shown in SEQ ID NO: 51. Such IL-7 variants or variants are specifically described in the sequences shown in SEQ ID NOs: 59, 60, 61, 62, and 66, respectively.

[0240] In one embodiment, the IL-7 variant or mutant includes i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or iii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0241] In one embodiment, the IL-7 variant or mutant comprises a W142H substitution and i) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or ii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0242] In one embodiment, the IL-7 variant or mutant comprises a D74E substitution and i) W142H, W142F, or W142Y, and / or ii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0243] In one embodiment, the IL-7 variant or mutant comprises the mutations C2S-C141S and C47S-C92S and at least one substitution consisting of i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q.

[0244] In one embodiment, the IL-7 variant or mutant includes i) D74E and W142H substitutions, and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0245] IL-7 variants or mutants may or may not contain their peptide signal.

[0246] In one embodiment, the bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequences shown in SEQ ID NOs. 53-58 or SEQ ID NOs. More preferably, the bifunctional molecule according to the present invention comprises an IL-7 variant comprising or consisting of the amino acid sequences shown in SEQ ID NOs. 54, 56, or 63.

[0247] Bifunctional molecule or "Bicki" The present invention provides, in particular, a bifunctional molecule comprising or comprising an anti-hPD1 antibody or an antibody fragment thereof and IL-7, wherein the anti-hPD1 antibody or antibody fragment thereof is covalently linked to IL-7, preferably by a peptide linker as disclosed above herein, and particularly as a fusion protein.

[0248] In particular, the bifunctional molecule according to the present invention comprises two entities: a first entity comprising or essentially comprising an anti-hPD1 antibody or a fragment thereof; and a second entity comprising or essentially comprising interleukin 7 (IL-7), preferably human IL-7, wherein these two entities are optionally linked by a peptide linker.

[0249] In particular, the bifunctional molecule according to the present invention contains one, two, three, or four IL-7 molecules. Specifically, the bifunctional molecule may contain only one IL-7 molecule linked to only one of the light or heavy chains of the anti-PD-1 antibody. Alternatively, the bifunctional molecule may contain two IL-7 molecules linked to either the light or heavy chain of the anti-PD-1 antibody. Furthermore, the bifunctional molecule may contain two IL-7 molecules, with the first molecule linked to the light chain of the anti-PD-1 antibody and the second molecule linked to the heavy chain of the anti-PD-1 antibody. Alternatively, the bifunctional molecule may contain three IL-7 molecules, two of which are linked to either the light or heavy chain of the anti-PD-1 antibody, and the last one is linked to the other chain of the anti-PD-1 antibody. Finally, the bifunctional molecule may contain four IL-7 molecules, two of which are linked to the light chain of the anti-PD-1 antibody, and two of which are linked to the heavy chain of the anti-PD-1 antibody. Thus, the bifunctional molecule contains one to four immunotherapy molecules as disclosed herein.

[0250] In one embodiment, the immunotherapy may involve one light chain containing only one IL-7 molecule (e.g., a bifunctional molecule contains one IL-7 molecule), one heavy chain containing only one IL-7 molecule (e.g., a bifunctional molecule contains one IL-7 molecule), each light chain containing one IL-7 molecule (e.g., a bifunctional molecule contains two IL-7 molecules), each heavy chain containing one IL-7 molecule (e.g., a bifunctional molecule contains two IL-7 molecules), or one light chain containing only one IL-7 molecule and one heavy chain containing only one IL-7 molecule. The molecule may contain a compound molecule (for example, a bifunctional molecule contains two IL7 molecules), each light chain may contain one IL7 molecule and only one heavy chain may contain one IL7 molecule (for example, a bifunctional molecule contains three IL7 molecules), each heavy chain may contain one IL7 molecule and only one light chain may contain one IL7 molecule (for example, a bifunctional molecule contains three IL7 molecules), or both the light and heavy chains may contain one IL-7 molecule (for example, a bifunctional molecule contains four IL7 molecules).

[0251] In one embodiment, the bifunctional molecule according to the present invention is (a) an anti-human PD-1 antibody or its antigen-binding fragment, comprising (i) a heavy chain and (ii) a light chain, and (b) Human interleukin 7 (IL-7) or its fragments or variants Includes or consists of The antibody heavy chain and / or light chain, or fragments thereof, are preferably covalently linked to IL-7 by a peptide linker as a fusion protein.

[0252] Preferably, the bifunctional molecule according to the present invention is (a) a humanized anti-human PD-1 antibody or its antigen-binding fragment, comprising (i) a heavy chain and (ii) a light chain, and (b) Human interleukin 7 (IL-7) or its variant or fragment Includes or consists of The antibody heavy chain or light chain, or fragments thereof, are preferably covalently linked to IL-7 by a peptide linker as a fusion protein.

[0253] Preferably, such a bifunctional molecule comprises at least one peptide linker connecting the N-terminus of IL-7 to the C-terminus of the heavy chain, light chain, or both of the C-terminus of an anti-human PD-1 antibody, wherein the peptide linker is preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and more preferably (GGGGS)3.

[0254] Preferably, the N-terminus of IL-7 is connected to the C-terminus of the heavy chain, light chain, or both of the anti-human PD-1 antibody via at least one peptide linker. Alternatively, the C-terminus of IL-7 is connected to the N-terminus of the heavy chain, light chain, or both of the anti-human PD-1 antibody via at least one peptide linker.

[0255] In one embodiment, the bifunctional molecule according to the present invention is (a) an anti-human PD-1 antibody or its antigen-binding fragment, comprising (i) a heavy chain and (ii) a light chain. (b) Human interleukin 7 (IL-7) or its variants or fragments, (c) A peptide linker that connects the N-terminus of IL-7 to the C-terminus of the heavy chain, light chain, or both of the C-terminus of an anti-human PD-1 antibody, preferably selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and more preferably (GGGGS)3. It includes or consists of.

[0256] In certain embodiments, the bifunctional molecule according to the present invention is (a) (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, - Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 1, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1. - Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2. - Heavy chain CDR3 (HCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 3, where X1 is either D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 3. - Light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 12, where X is G or T, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 12. - Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 15 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. - Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16. Anti-human PD-1 antibody or its antigen-binding fragment; and (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. Includes or consists of The antibody heavy chain and / or light chain, or fragments thereof, are covalently linked to IL-7 as a fusion protein, preferably by a peptide linker.

[0257] In another embodiment, the bifunctional molecule according to the present invention is (a) (i) Heavy chain variable domains including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains including LCDR1, LCDR2, and LCDR3 Includes, - Heavy chain CDR1 (HCDR1) contains or comprises the amino acid sequence of SEQ ID NO: 1, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1. - Heavy chain CDR2 (HCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 2, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2. - Heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of SEQ ID NO: 3, wherein X1 is D and X2 is selected from the group consisting of T, H, A, Y, N, E, preferably from the group consisting of H, A, Y, N, E, or X1 is E and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, E, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO: 3. - Light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 12, where X is G or T, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 12. - Light chain CDR2 (LCDR2) contains or comprises the amino acid sequence of SEQ ID NO: 15 and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. - Light chain CDR3 (LCDR3) contains or comprises the amino acid sequence of SEQ ID NO: 16, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16. Anti-human PD-1 antibody or its antigen-binding fragment; and (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. Includes or consists of The antibody heavy chain, light chain, or both, or fragments thereof, are covalently linked to IL-7 as a fusion protein, preferably by a peptide linker.

[0258] In another embodiment, the bifunctional molecule according to the present invention is (a) - Heavy chain CDR1 (HCDR1) containing or comprising the amino acid sequence of SEQ ID NO: 1, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than position 3 of SEQ ID NO: 1, - Heavy chain CDR2 (HCDR2) containing or comprising the amino acid sequence of SEQ ID NO: 2, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 13, 14, and 16 of SEQ ID NO: 2, - Heavy chain CDR3 (HCDR3) containing or comprising the amino acid sequence of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, or 11. - Light chain CDR1 (LCDR1) containing or comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO: 13 or SEQ ID NO: 14, - Light chain CDR2 (LCDR2) containing or comprising the amino acid sequence of SEQ ID NO: 15, and having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof, - Light chain CDR3 (LCDR3) containing or comprising the amino acid sequence of SEQ ID NO: 16, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 1, 4, and 6 of SEQ ID NO: 16. Humanized anti-human PD-1 antibody or its antigen-binding fragment containing; and (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. Includes or consists of The antibody heavy chain or light chain, or fragments thereof, are covalently linked to IL-7 as a fusion protein, preferably by a peptide linker.

[0259] Preferably, the peptide linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and more preferably (GGGGS)3.

[0260] In another embodiment, the present invention is (a) (i) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Humanized anti-hPD1 antibodies including; and (b) Human interleukin-7 of Sequence ID No. 51 or its variants or fragments; (c) A peptide linker between the light and / or heavy chain of an anti-hPD1 antibody and human IL-7 or a variant or fragment thereof, selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and more preferably (GGGGS)3. This relates to bifunctional molecules, including those containing bifunctional molecules.

[0261] Preferably, the N-terminus of IL-7 is connected to the C-terminus of the heavy chain, light chain, or both of the anti-human PD-1 antibody via at least one peptide linker. Alternatively, the C-terminus of IL-7 is connected to the N-terminus of the heavy chain, light chain, or both of the anti-human PD-1 antibody via at least one peptide linker.

[0262] In another embodiment, the present invention is a) (i) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Humanized anti-hPD1 antibodies including; and (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. A bifunctional molecule comprising or consisting of This invention relates to a bifunctional molecule in which the C-terminuses of the heavy and / or light chains of an antibody or its antigen-binding fragment are preferably covalently linked to the N-terminus of IL-7 by a (GGGGS)3 peptide linker.

[0263] In another embodiment, the present invention is (a) (i) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NOs. 18, 19, 20, 21, 22, 23, 24, or 25, each optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NOs. 18, 19, 20, 21, 22, 23, 24, or 25, respectively. (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 27 or SEQ ID NO: 28. Humanized anti-hPD1 antibodies including; and (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. A bifunctional molecule comprising or consisting of This invention relates to a bifunctional molecule in which the C-terminuses of the heavy and / or light chains of an antibody or its antigen-binding fragment are covalently linked to the N-terminus of IL7, preferably by a (GGGGS)3 peptide linker, to form a fusion protein.

[0264] In a preferred embodiment, the C-terminus of the heavy chain of the antibody or its antigen-binding fragment is covalently linked to the N-terminus of IL-7 to form a fusion protein. Preferably, only the heavy chain of the antibody or its antigen-binding fragment is covalently linked to IL-7.

[0265] In another embodiment, the present invention is a) (i) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 17, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally having one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95, 96, 97, 98, 100, 101, 105, 106, and 112 of SEQ ID NO: 17, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, and S, and optionally has one, two, or three modifications selected from substitutions, additions, deletions, and any combination thereof. (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 26, where X is G or T, and optionally having one, two or three modifications selected from substitutions, additions, deletions, and any combination thereof at any position other than positions 3, 4, 7, 14, 17, 18, 28, 29, 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of SEQ ID NO: 26. Humanized anti-hPD1 antibodies including; and (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. A bifunctional molecule comprising or consisting of This invention relates to a bifunctional molecule in which the C-terminus of the heavy chain of an antibody or its antigen-binding fragment is covalently linked to the N-terminus of IL7 by a (GGGGS)3 peptide linker, preferably to form a fusion protein.

[0266] In another embodiment, the present invention is a) (i) A heavy chain variable region (VH) comprising or including the amino acid sequence of SEQ ID NO: 24, (ii) Light chain variable region (VL) containing or consisting of the amino acid sequence of SEQ ID NO. 28 Humanized anti-hPD1 antibody containing; (b) Human interleukin-7 of Sequence ID No. 51 or its variant or fragment. A bifunctional molecule comprising or consisting of This invention relates to a bifunctional molecule in which the C-terminus of the heavy chain of an antibody or its antigen-binding fragment is covalently linked to the N-terminus of IL7 by a (GGGGS)3 peptide linker, preferably to form a fusion protein.

[0267] Preferably, the antibody or antibody fragment has an IgG1 domain or an IgG4Fc domain.

[0268] In one embodiment, the antibody or antibody fragment is optionally T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I The IgG1 Fc domain has a substitution or combination of substitutions selected from the group consisting of 332E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably N297A combined with M252Y / S254T / T256E by optional choice, and L234A / L235. More preferably, the IgG1 Fc domain has a mutant N297A, such as those described above.

[0269] In another embodiment, the antibody or antibody fragment thereof has an IgG4 Fc domain having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E, and K444A, and more preferably the IgG4 Fc domain has a mutant S228P, such as those described above.

[0270] Optionally, in any of the embodiments specified above, IL-7 is an IL-7 variant or mutant.

[0271] More specifically, the IL-7 variant or mutant exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7), comprising or consisting of the amino acid sequence shown in SEQ ID NO: 51, and such IL-7 variant includes at least one amino acid mutation that i) reduces the affinity of the IL-7 variant to the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 to IL-7R, ii) retains the ability to activate IL-7R, and iii) improves the pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wth-IL-7. More preferably, such a variant i) reduces the affinity of the IL-7 variant to the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 to IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wth-IL-7.

[0272] More specifically, an IL-7 variant or mutant may exhibit at least 75% identity with wild-type human IL-7 (wth-IL-7) comprising or consisting of the amino acid sequence shown in Sequence ID No. 51, and such an IL-7 variant includes at least one mutation selected from the group consisting of (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E, D74Q, or D74N, preferably D74E or D74Q, iv) Q11E, Y12F, M17L, Q22E, and / or K81R, or any combination thereof.

[0273] IL-7 variants or mutants may contain at least one set of substitutions selected from the group consisting of C2S-C141S and C47S-C92S (referred to as "SS2"), C2S-C141S and C34S-C129S (referred to as "SS1"), and C47S-C92S and C34S-C129S (referred to as "SS3"). Such mutations point to the amino acid positions shown in SEQ ID NO: 51. Such IL-7 variants or mutants are specifically described in the sequences shown in SEQ ID NOs: 53-55 (SS1, SS2, and SS3, respectively). Preferably, the IL-7 variants or mutants contain the amino acid substitutions C2S-C141S and C47S-C92S. More preferably, the IL-7 variants or mutants represent the sequence shown in SEQ ID NO: 54.

[0274] IL-7 variants or mutants may contain at least one mutation selected from the group consisting of W142H, W142F, and W142Y. Such IL-7 variants or mutants are specifically described in the sequences shown in SEQ ID NOs. 57-58. Preferably, the IL-7 variant or mutant contains the W142H mutation. More preferably, the IL-7 variant or mutant represents the sequence shown in SEQ ID NO. 56.

[0275] IL-7 variants or mutants may contain at least one mutation selected from the group consisting of D74E, D74Q, and D74N, preferably D74E or D74Q. Such IL-7 variants or mutants are specifically described in the sequences shown in SEQ ID NOs. 63-65, respectively. Preferably, the IL-7 variant or mutant contains the D74E mutation. More preferably, the IL-7 variant or mutant represents the sequence shown in SEQ ID NO. 63.

[0276] IL-7 variants or variants may contain at least one mutation selected from the group consisting of Q11E, Y12F, M17L, Q22E, and / or K81R. Such mutations point to the amino acid positions shown in SEQ ID NO: 51. Such IL-7 variants or variants are specifically described in the sequences shown in SEQ ID NOs: 59, 60, 61, 62, and 66, respectively.

[0277] IL-7 variants or mutants may include i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or iii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0278] An IL-7 variant or mutant may include a W142H substitution and i) D74E, D74Q, or D74N, preferably D74E or D74Q, and / or ii) at least one mutation consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0279] An IL-7 variant or mutant may include a D74E substitution and at least one mutation consisting of i) W142H, W142F, or W142Y, and / or ii) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0280] IL-7 variants or mutants may include mutations C2S-C141S and C47S-C92S, and at least one substitution consisting of i) W142H, W142F, or W142Y, and / or ii) D74E, D74Q, or D74N.

[0281] IL-7 variants or mutants may include i) D74E and W142H substitutions, and ii) mutations C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S.

[0282] IL-7 variants or mutants may contain or consist of the amino acid sequences shown in SEQ ID NOs. 53, 54, 55, 56, 57, 58, 63, 64, or 65.

[0283] In certain embodiments, IL-7 is an IL-7 variant according to the present invention, and the antibody or antibody fragment is optionally T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / The antibody has an IgG1 Fc domain having a substitution or combination of substitutions selected from the group consisting of E333S;S239D / I332E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably N297A in combination with M252Y / S254T / T256E, and L234A / L235, and more preferably the IgG1 Fc domain has a mutant N297A, such as those described above. Preferably the antibody or a fragment thereof is linked to IL-7 or a variant thereof by a linker selected from the group consisting of (GGGGS)3, (GGGGS)4, and (GGGS)3, more preferably by (GGGGS)3. Preferably, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. More preferably, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. More preferably, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, and D74E.

[0284] In certain embodiments, the bifunctional molecule according to the present invention is (a) Antibodies or antibody fragments thereof, such as those described above in this specification, which specifically bind to targets expressed on the surface of immune cells, preferably on T cells, and more preferably the targets are selected from the group consisting of PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8, preferably selected from the group consisting of PD-1, TIM3, CD244, LAG-3, BTLA, TIGIT, and CD160. (b) Human interleukin-7 of Sequence ID No. 51 or its variants or fragments, and (c) A peptide linker, optionally selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGS, GGG, GGS, and (GGGS)3, preferably (GGGGS)3. It is a fusion protein containing or consisting of [the specified elements].

[0285] All or any of the specific embodiments and models detailed above that are disclosed with respect to the bifunctional anti-PD-1 molecule can be applied to such alternative bifunctional molecules.

[0286] In certain embodiments, antibodies or antibody fragments thereof, such as those described above in this specification, specifically bind to targets expressed on the surface of immune cells, preferably on T cells, and more preferably the targets are selected from the group consisting of PD-1, CD28, CD80, CTLA-4, BTLA, TIGIT, CD160, CD40L, ICOS, CD27, OX40, 4-1BB, GITR, HVEM, Tim-1, LFA-1, TIM3, CD39, CD30, NKG2D, LAG3, B7-1, 2B4, DR3, CD101, CD44, SIRPG, CD28H, CD38, CXCR5, CD3, PDL2, CD4, and CD8, preferably from the group consisting of PD-1, TIM3, CD244, LAG-3, BTLA, TIGIT, and CD160; IL-7 is included in the present invention. The IL-7 variant is as follows, and the antibody or antibody fragment is optionally T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S23 The antibody has an IgG1 Fc domain having a substitution or combination of substitutions selected from the group consisting of 9D / I332E / G236A;N297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably N297A in combination with M252Y / S254T / T256E, and L234A / L235, and more preferably the IgG1 Fc domain has a mutant N297A, such as those described above. Preferably the antibody or a fragment thereof is linked to IL-7 or a variant thereof by a linker selected from the group consisting of (GGGGS)3, (GGGGS)4, and (GGGS)3, more preferably by (GGGGS)3. Preferably, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, C47S-C92S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N.More preferably, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, W142F, W142Y, D74E, D74Q, and D74N. Even more preferably, the IL-7 variant includes a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, W142H, and D74E.

[0287] The binding of bifunctional molecules to these specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. In each of these assays, the presence of a specific target protein-antibody complex is generally detected by using a labeling reagent (e.g., antibody) specific to the complex of interest. For example, the anti-hPD-1 antibody / IL-7 complex can be detected, for example, by using an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to IL-7 or its receptor.

[0288] In some cases, the bifunctional molecules described herein inhibit the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least half, at least one-fifth, at least one-tenth, at least one-twentieth, at least one-fiftieth, at least one-hundredth, or at least one-thousandth.

[0289] Preferably, such a bifunctional molecule has the ability to block or inhibit the interaction between PD-1 and its ligand (e.g., PD-L1 and / or PD-L2). In certain embodiments, the bifunctional molecule inhibits the binding interaction between PD-1 and its ligand (e.g., PD-L1 and / or PD-L2) by at least 50%. In certain embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0290] In some cases, the bifunctional molecules described herein inhibit the PD-1 signaling pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least half, at least one-fifth, at least one-tenth, at least one-twentieth, at least one-fiftieth, at least one-hundredth, or at least one-thousandth.

[0291] In some cases, the bifunctional molecules described herein stimulate IFN-gamma secretion and / or alpha-4 and beta-7.

[0292] In another example, the bifunctional molecules described herein promote T cell infiltration in tumors.

[0293] In some cases, the bifunctional molecules described herein stimulate the IL-7R signaling pathway by at least 10%, at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times.

[0294] In other embodiments, the bifunctional molecules described herein retain substantially equivalent biological IL-7 properties compared to wild-type IL-7. For example, they retain biological properties equivalent to full-length IL-7 protein. The biological activity of IL-7 protein can be measured using an in vitro cell proliferation assay or by measuring P-Stat5 of T cells by ELISA or FACS. Preferably, the bifunctional IL-7 molecules described herein maintain at least 10%, 20%, 30%, 40%, 50%, or 60% of the biological activity compared to wild-type human IL-7, and preferably at least 80%, 90%, 95%, and more preferably 99% compared to wild-type IL-7. For example, biological activity can be evaluated by measuring the ability of the bifunctional molecules described herein to bind to IL-7R and / or to compete with wild-type IL-7 for binding to IL-7R.

[0295] In another example, the bifunctional molecules described herein induce cytokine secretion and / or proliferation of unsensitized, partially exhausted and / or fully exhausted T cell subsets.

[0296] Preparation of bifunctional molecules - nucleic acid molecules encoding bifunctional molecules, recombinant expression vectors containing them, and host cells To generate the bifunctional molecule of the present invention, the anti-hPD1 antibody of the present invention is functionally linked to IL-7 or a variant thereof.

[0297] Both entities of a bifunctional molecule are encoded by the same vector and produced as a fusion protein. Accordingly, nucleic acids encoding any of the bifunctional molecules described herein, vectors such as expression vectors or recombinant viruses containing such nucleic acids, and host cells containing nucleic acids and / or vectors are also disclosed herein.

[0298] To produce the bifunctional fusion protein according to the present invention, which is secreted in a stable form by mammalian cells, the nucleic acid sequence encoding the bifunctional molecule is subcloned into an expression vector commonly used to transfect mammalian cells. The basic techniques for producing molecules containing antibody sequences are described in Coligan et al. (eds.), Current protocols in immunology, pp. 10.19.1-10.19.11 (Wiley Interscience, 1992) (the contents of this document are incorporated herein by reference), and in WH Freeman and Company's "Antibody engineering: a practical guide" (1992), where commentaries related to molecule production are scattered throughout the corresponding text.

[0299] Generally, such methods are (1) A step of transfecting or transforming a suitable host cell with a polynucleotide or a variant thereof encoding the recombinant bifunctional molecule of the present invention or a vector containing the polynucleotide, (2) A step of culturing host cells in an appropriate medium, and (3) Optionally, a step of isolating or purifying the protein from the culture medium or host cells. Includes.

[0300] The present invention further relates to nucleic acids encoding a bifunctional molecule as disclosed above, vectors comprising the nucleic acids of the present invention, preferably expression vectors, genetically modified host cells directly transformed with the vectors of the present invention or with sequences encoding recombinant bifunctional molecules, and methods for producing the proteins of the present invention by recombinant techniques.

[0301] Nucleic acids, vectors, and host cells are described in more detail below in this specification.

[0302] nucleic acid sequence Furthermore, the present invention relates to nucleic acid molecules encoding a bifunctional molecule as defined above, or to a group of nucleic acid molecules encoding a bifunctional molecule as defined above.

[0303] The antibody DNA sequence may be amplified, for example, from RNA of a cell that synthesizes immunoglobulins, synthesized using PCR with cloned immunoglobulins, or synthesized using oligonucleotides that encode a known signal peptide amino acid sequence.

[0304] Preferably, the peptide signal comprises or consists of the amino acid sequence of SEQ ID NO: 49 for VH and / or CH, and the amino acid sequence of SEQ ID NO: 50 for / or VL and / or CL. In particular, the peptide signal is located at the N-terminus of CH, VH, CL, and / or VL.

[0305] Such nucleic acids can encode the amino acid sequence containing the VL and / or VH of an antibody (e.g., the light chain and / or heavy chain of the antibody). Such nucleic acids can be readily isolated and sequenced using conventional procedures.

[0306] In particular, nucleic acid molecules encoding a bifunctional molecule as defined above, - A first nucleic acid molecule encoding the variable heavy chain domain of the anti-hPD-1 antibody as disclosed herein, and optionally having the peptide signal of SEQ ID NO: 49, and - A second nucleic acid molecule encoding the variable light chain domain of the anti-hPD-1 antibody as disclosed herein, and optionally having the peptide signal of SEQ ID NO: 50, and - A third nucleic acid operably linked to either or both of the first or second nucleic acids via a nucleic acid encoding IL-7 or a variant thereof, preferably human IL-7 or a variant thereof, and optionally encoding a peptide linker. Includes.

[0307] Preferably, the nucleic acid molecule encoding the bifunctional molecule as defined above is - A first nucleic acid molecule encoding the variable heavy chain domain of SEQ ID NO: 17, in which X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E, and optionally having the peptide signal of SEQ ID NO: 49, and - A second nucleic acid molecule encoding the variable light chain domain of SEQ ID NO: 26, where X is G or T in the sequence, and optionally having the peptide signal of SEQ ID NO: 50, and - A third nucleic acid molecule operably linked to either or both of the first and second nucleic acids via a nucleic acid encoding human IL-7 or a variant or fragment thereof of sequence number 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63, and optionally encoding a peptide linker. Includes.

[0308] Preferably, the nucleic acid molecule encoding the bifunctional molecule as defined above is - A first nucleic acid molecule encoding a variable heavy chain domain of the amino acid sequence shown in SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, or 25, and optionally having the peptide signal of SEQ ID NO: 49, and - A second nucleic acid molecule that encodes a variable light chain domain of the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28, and optionally has the peptide signal of SEQ ID NO: 50, and - A third nucleic acid molecule operably linked to either or both of the first and second nucleic acids via a nucleic acid encoding human IL-7 or a variant thereof of SEQ ID NO: 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63, and optionally encoding a peptide linker. Includes.

[0309] In a very specific embodiment, the nucleic acid molecule encoding the variable heavy chain domain has the sequence shown in SEQ ID NO: 73, and / or the nucleic acid molecule encoding the variable light chain domain has the sequence shown in SEQ ID NO: 74.

[0310] "Operationally linked" means that the nucleic acid is intended to encode a protein fusion comprising a variable heavy or light chain domain, optionally a peptide linker, and IL-7. Preferably, the linker is selected from the group consisting of (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and more preferably (GGGGS)3.

[0311] In one embodiment, the nucleic acid molecule is an isolated, particularly non-natural, nucleic acid molecule.

[0312] The nucleic acid molecule or group of nucleic acid molecules encoding the bifunctional molecule according to the present invention is preferably included in a vector or group of vectors.

[0313] vector In another embodiment, the present invention relates to a vector comprising a nucleic acid molecule or a group of nucleic acid molecules as defined above.

[0314] As used herein, “vector” refers to a nucleic acid molecule used as a vehicle for transferring genetic material into cells. The term “vector” encompasses plasmids, viruses, cosmids, and artificial chromosomes. Generally, genetically engineered vectors include an origin of replication, multiple cloning sites, and selectable markers. A vector is generally a nucleotide sequence, typically a DNA sequence, containing an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Modern vectors may also include other additional features of the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, target-directed sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically designed to express a transgene in target cells and generally contain regulatory sequences.

[0315] In one embodiment, both the heavy chain coding sequence and the light chain coding sequence, and / or the constant region of the anti-PD1 antibody, are contained in a single expression vector. The heavy chain coding sequence and the light chain coding sequence may each be operably linked to a suitable promoter, and the heavy chain and / or light chain may be operably linked to the immunotherapy agent according to the present invention. Alternatively, the expression of both the heavy chain and the light chain may be driven by the same promoter. In another embodiment, the heavy chain and light chain of the antibody are each cloned into separate vectors, and one or both of the heavy chain and / or light chain, or the heavy chain and / or light chain, may be operably linked to the immunotherapy agent according to the present invention. In the latter case, the expression vectors encoding the heavy chain and the light chain can be co-transfected into a single host cell for the expression of both chains, and the two chains can be assembled to form an intact antibody either in vivo or in vitro. Alternatively, to express the heavy chain and the light chain respectively, the expression vectors encoding the heavy chain and the expression vectors encoding the light chain may be introduced into different host cells, and then they may be purified and assembled to form an intact antibody in vitro.

[0316] Those skilled in the art can clone a nucleic acid molecule encoding a humanized anti-PD-1 antibody or a fragment thereof into a vector and then transform it into host cells. Accordingly, the present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the anti-PD-1 antibody or a fragment thereof. In one preferred embodiment, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally, at least one drug resistance gene for screening.

[0317] A suitable expression vector typically contains (1) a prokaryotic DNA element encoding a bacterial origin of replication and an antibiotic resistance marker to provide growth and selection of the expression vector in the bacterial host; (2) a eukaryotic DNA element controlling transcription initiation, such as a promoter; and (3) a DNA element controlling transcript processing, such as a transcription termination / polyadenylation sequence.

[0318] An expression vector containing the nucleic acid sequence of the bifunctional molecule described herein and appropriate regulatory components for transcription / translation can be constructed using methods known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant techniques. The DNA sequence is efficiently ligated to an appropriate promoter in the expression vector for directing mRNA synthesis. The expression vector may further include a ribosome binding site for initiating translation and a transcription terminator, etc.

[0319] Expression vectors can be introduced into host cells using various techniques, including calcium phosphate transfection, liposome-mediated transfection, and electroporation. Preferably, the expression vector is used to select and proliferate transfected cells that are stably integrated into the host cell genome to produce stable transformants. Techniques for introducing vectors into eukaryotic cells and techniques for selecting stable transformants using dominant selection markers are described in Sambrook, Ausubel, and Bebbington, "Expression of Antibody Genes in Nonlymphoid Mammalian Cells" in 2 METHODS: A companion to methods in enzymology, Vol. 136 (1991), and in Murray (ed.), Gene transfer and expression protocols (Humana Press, 1991). Suitable cloning vectors are described in Sambrook et al. (eds.), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (Cold Spring Harbor Press, 1989) (hereafter referred to as "Sambrook"); Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience, 1987) (hereafter referred to as "Ausubel"); and Brown (ed.), MOLECULAR BIOLOGY LABFAX (Academic Press, 1991).

[0320] host cell In another embodiment, the present invention relates, for example, to a host cell comprising a vector or nucleic acid molecule or group of nucleic acid molecules as defined above, for the purpose of producing a bifunctional molecule.

[0321] As used herein, the term “host cell” is intended to include any individual cell or cell culture that may or may be a recipient of the vector, exogenous nucleic acid molecule, and polynucleotide encoding the antibody construct of the present invention; and / or the antibody construct or the bifunctional molecule itself. The introduction of each substance into a cell can be carried out by transformation and transfection, etc. The term “host cell” is also intended to include the offspring or potential offspring of a single cell. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, as well as bacterial, yeast, fungal, plant cells, and insect and mammalian cells, such as animal cells from mice, rats, rabbits, macaques, or humans.

[0322] In one embodiment, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence containing the VL of the antibody and / or an amino acid sequence containing the VH of the antibody and / or a constant region of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence containing the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence containing the VH of the antibody (e.g., transformed).

[0323] In another embodiment, the host cell comprises a vector (e.g., transformed) containing both entities of the bifunctional molecule. Preferably, the host cell comprises a vector (e.g., transformed) containing a first nucleic acid molecule encoding the variable heavy chain domain of the anti-hPD-1 antibody as disclosed herein and a second nucleic acid molecule encoding the variable light chain domain of the anti-hPD-1 antibody as disclosed herein, operably linked to a third nucleic acid encoding IL-7 or a variant or mutant thereof, preferably human IL-7 or a variant thereof.

[0324] Furthermore, a method for producing a humanized anti-PD1 antibody is provided herein. This method comprises the steps of culturing host cells containing the nucleic acid encoding the antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium). In particular, for recombinant production of a humanized anti-PD1 antibody, for example, the nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in host cells.

[0325] The bifunctional molecules of the present invention are preferably expressed in eukaryotic cells such as mammalian cells, plant cells, insect cells, or yeast cells. Mammalian cells are particularly preferred eukaryotic hosts because they provide suitable post-translational modifications such as glycosylation. Preferably, such preferred eukaryotic host cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; insect cells such as Mythimna separate; plant cells such as tobacco; and mammalian cells such as BHK cells, 293 cells, CHO cells, NSO cells, and COS cells. Other examples of useful mammalian host cell lines include CV-1 in Origin with SV40 genes cells (COS cells), SV40-transformed monkey kidney CV1 cell line (COS-7); human fetal kidney cell line (e.g., 293 or 293 cells as described by Graham, FL et al., J. Gen Virol., Vol. 36 (1977), pp. 59-74); baby hamster kidney cells (BHK); and mouse Sertoli cells (e.g., Mather, JP, Biol.). TM4 cells as described in Reprod., Vol. 23 (1980), pp. 243-252; human epithelial kidney cells (HEK cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor cells (MMT060562); for example, TRI cells as described by Mather, JP et al., Annals NY Acad. Sci., Vol. 383 (1982), pp. 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR" CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. Examples include Chinese hamster ovary (CHO) cells (USA, Vol. 77 (1980), pp. 4216-4220), as well as myeloma cell lines such as Y0, NSO, and Sp2 / 0.For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, New Jersey, USA (2004), pp. 255-268. For example, mammalian cell lines suitable for growth in suspension may be useful.

[0326] In particular, the host cells of the present invention are selected from the group consisting of CHO cells, COS cells, NSO cells, and HEK cells.

[0327] In mammalian hosts, the transcriptional and translational regulatory signals of the expression vector may originate from a viral source such as adenovirus, bovine papillomavirus, or simian virus, and these regulatory signals are associated with specific genes exhibiting high levels of expression. Furthermore, suitable transcriptional and translational regulatory sequences can be obtained from mammalian genes such as actin genes, collagen genes, myosin genes, and metallothionein genes.

[0328] Stable transformants producing the bifunctional molecules according to the present invention can be identified using a variety of methods. After identifying the molecule-producing cells, the host cells are cultured under conditions (e.g., temperature, culture medium) suitable for their growth and expression of the bifunctional molecules. The bifunctional molecules are then isolated and / or purified by any method known in the art. Such methods include, but are not limited to, conventional regeneration treatments, treatment with protein precipitants (e.g., salt precipitation), centrifugation, osmotic cell lysis, sonication, ultracentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography, and combinations thereof. For example, as described by Coligan, bifunctional molecule isolation techniques include affinity chromatography with protein A Sepharose, size exclusion chromatography, and ion exchange chromatography. Protein A is preferably used for the isolation of the bifunctional molecules of the present invention.

[0329] Pharmaceutical composition and method of administering the same Furthermore, the present invention relates to a pharmaceutical composition comprising, preferably as an active ingredient or compound, any of the bifunctional molecules described herein, nucleic acid molecules as disclosed above herein, a group of nucleic acid molecules, vectors, and / or host cells. The formulations can be sterilized and, if desired, can be mixed with pharmaceutically acceptable carriers and excipients, etc., that do not have adverse interactions with the bifunctional molecules, nucleic acids, vectors, and / or host cells of the present invention. Optionally, the pharmaceutical composition may further comprise additional therapeutic agents as detailed below.

[0330] Preferably, the pharmaceutical compositions of the present invention may contain the bifunctional molecules as described herein, nucleic acid molecules, groups of nucleic acid molecules, vectors, and / or host cells as described above herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, excipients, salts, and antioxidants as described below herein. Preferably, a pharmaceutically acceptable form is used that does not adversely affect the desired immunoenhancing effect of the bifunctional molecules according to the present invention. To facilitate administration, the bifunctional molecules as described herein can be prepared into pharmaceutical compositions for in vivo administration. Means for preparing such compositions are described in the art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)).

[0331] In particular, the pharmaceutical compositions according to the present invention can be formulated for any conventional route of administration, including topical administration, enteral administration, oral administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intraocular administration. Preferably, the pharmaceutical compositions according to the present invention are formulated for enteral or parenteral administration routes. Compositions and formulations for parenteral administration may include a sterile aqueous solution containing buffers, diluents, and other suitable additives, but not limited to, osmotic enhancers, carder compounds, and other pharmaceutically acceptable carriers or excipients.

[0332] Pharmaceutical compositions can be prepared in the form of lyophilized formulations or aqueous solutions by mixing a substance of desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); and low molecular weight (less than approximately 10 residues) polypeptides. Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0333] A solid pharmaceutically acceptable vehicle may contain one or more substances that can also act as flavoring agents, lubricants, solubilizers, suspending agents, colorants, fillers, flow enhancers, compression aids, inert binders, sweeteners, preservatives, colorants, coatings, or tablet disintegrants. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting-point waxes, and ion-exchange resins. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. Unless any conventional media or substance is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is intended.

[0334] The bifunctional molecules according to the present invention can be dissolved or suspended in water, organic solvents, ethanol, and pharmaceutically acceptable liquid vehicles such as polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), pharmaceutically acceptable oils or fats, or mixtures thereof, and preferred mixtures thereof. The liquid vehicle may contain other preferred pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, wetting agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Preferred examples of liquid vehicles for oral and enteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionally distilled coconut oil and peanut oil). For parenteral administration, the vehicle may be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful for sterile liquid compositions for enteral administration. Liquid vehicles for pressurized compositions may be halogenated hydrocarbons or other pharmaceutically acceptable propellants.

[0335] The pharmaceutical compositions of the present invention may further contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts. Examples of acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as those derived from non-toxic organic acids such as aliphatic mono and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Examples of base addition salts include those derived from alkali metals or alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0336] Furthermore, the pharmaceutical composition of the present invention may contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0337] To facilitate delivery, either the bifunctional molecule or its coding nucleic acid may be conjugated with the chaperone. The chaperone may be a naturally occurring substance such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or a lipid. Alternatively, the chaperone may be a recombinant or synthetic molecule such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, and polyphosphatidine. In one example, the chaperone is a micelle, liposome, nanoparticle, or microsphere. Methods for preparing such micelles, liposomes, nanoparticles, or microspheres are well known in the art. See, for example, U.S. Patents 5,108,921; 5,354,844; 5,416,016; and 5,527,5285.

[0338] Pharmaceutical compositions typically must be sterile and stable under manufacturing and storage conditions. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other regular structures suitable for high drug concentrations and / or injection. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0339] In one embodiment, the pharmaceutical composition is an injectable composition that may contain various carriers such as vegetable oil, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, and liquid polyethylene glycol, etc.). For intravenous injection, a water-soluble antibody can be administered by drip infusion, and a formulation containing the antibody and a physiologically acceptable excipient is injected. Examples of physiologically acceptable excipients include 5% dextrose, 0.9% physiological saline, Ringer's solution, or other suitable excipients. Intramuscular formulations, for example, sterile formulations in a suitable soluble salt form of the antibody, can be dissolved in a pharmaceutical excipient such as water for injection, 0.9% physiological saline, or 5% glucose solution and administered.

[0340] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having one or a combination of the components listed above, followed by sterile microfiltration as necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying (lyophilization), from which a powder of the active ingredient + any additional desired components is obtained from a previously sterile filtered solution. Long-term absorption of injectable compositions can be achieved by including absorption-delaying substances, such as monostearate and gelatin, in the composition.

[0341] The prevention of microbial presence can be ensured by both sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, long-term absorption of the injectable pharmaceutical form can be achieved by including absorption-delaying substances such as aluminum monostearate and gelatin.

[0342] Those skilled in the art will understand that the formulations of the present invention may be isotonic with human blood, that is, they have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by a vapor pressure type or ice-freezing type osmometer. The tonicity of a formulation is adjusted using a tonicity modifier. A “tonicity modifier” is a pharmaceutically acceptable inert substance that can be added to a formulation to provide the formulation with isotonicity. Suitable tonicity modifiers for the present invention include, but are not limited to, saccharides, salts, and amino acids.

[0343] The pharmaceutical composition according to the present invention may be formulated to release the active ingredient (e.g., the bifunctional molecule of the present invention) substantially immediately after administration, or at any predetermined time or period after administration. In some embodiments, the pharmaceutical composition may be equipped with timed-release, delayed-release, and sustained-release delivery systems so that the delivery of the composition occurs before and with sufficient time to induce sensitization of the site to be treated. By means known in the art, the release and absorption of the composition can be prevented or minimized until it reaches the target tissue or organ, or timed release of the composition can be ensured. Such systems can avoid repeated administration of the composition, thereby improving convenience for both the subject and the physician.

[0344] The amount of active ingredient that can be combined with a carrier to produce a single dosage form will vary depending on the target being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier to produce a single dosage form will be the amount of the composition that produces the therapeutic effect.

[0345] Subjects, regimens, and administration The present invention relates to a bifunctional molecule as disclosed herein, for use as a pharmaceutical, or for use in the treatment of a disease, or for administration to a subject, or for use as a pharmaceutical; a nucleic acid or vector, host cell, or pharmaceutical composition, nucleic acid, vector, or host cell encoding the same. The present invention also relates to the use of the pharmaceutical composition, nucleic acid, vector, or host cell of the present invention, or a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof and IL-7 or a variant thereof, in the manufacture of a pharmaceutical for the treatment of a disease of interest. Finally, the present invention relates to a method for treating a disease or disorder of interest, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition or a bifunctional molecule comprising an anti-PD1 antibody or an antibody fragment thereof and IL-7 or a variant thereof to a subject. Examples of treatments are described in more detail in the following “Methods and Uses” section of this specification.

[0346] The subjects of treatment may be humans, particularly prenatal humans, newborns, children, infants, adolescents, or adults, especially adults aged at least 30 or 40 years, preferably at least 50 years, more preferably at least 60 years, and even more preferably at least 70 years.

[0347] In particular, the subjects suffer from diseases in which the PD-1 / PD-L1 pathway may be involved, especially diseases in which at least one of the ligands of PD-1 (e.g., PD-L1 and / or PD-L2) or PD-1 is expressed, especially overexpressed. Preferably, the subjects suffer from cancer, more preferably PD1, PD-L1, and / or PD-L2-positive cancer, or PD-1-positive cancer. Examples of diseases and cancers are described in more detail in the "Methods and Uses" section below.

[0348] In certain embodiments, the subject has already received at least one treatment, preferably several treatment strategies, prior to administration of the bifunctional molecule or pharmaceutical composition according to the present invention, which comprises an anti-PD1 antibody or an antibody fragment thereof and IL-7 or a variant thereof.

[0349] Using conventional methods known to those skilled in the medical field, the bifunctional molecules or pharmaceutical compositions disclosed herein can be administered to the target area depending on the type of disease to be treated or the site of the disease. The compositions can be administered by conventional routes, for example, orally, parenterally, enterally, by inhalation spray, topically, transrectally, transnasally, buccally, transvaginally, or via an implanted reservoir. The term "parenterally," as used herein, includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intratumoral, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. When administered parenterally, the pharmaceutical compositions according to the present invention are preferably administered by an intravenous route. When administered enterally, the pharmaceutical compositions according to the present invention are preferably administered by an oral route. The compositions can also be administered topically.

[0350] The form of the pharmaceutical composition, the route of administration, and the dosage of the pharmaceutical composition or bifunctional molecule according to the present invention can be adjusted according to the type and severity of the infection, the patient, and especially the patient's age, weight, sex, and overall health condition, as can be seen by those skilled in the art. The compositions of the present invention can be administered in several forms, depending on whether topical or systemic treatment is desired.

[0351] Preferably, treatment with the bifunctional molecule or pharmaceutical composition according to the present invention is administered regularly, preferably daily, weekly, or monthly, more preferably daily to every 1, 2, 3, or 4 weeks. In certain embodiments, the treatment is administered several times a day, preferably two or three times a day.

[0352] The treatment period with the bifunctional molecule or pharmaceutical composition according to the present invention is preferably 1 day to 20 weeks, more preferably 1 day to 10 weeks, even more preferably 1 day to 4 weeks, and even more preferably 1 day to 2 weeks. Alternatively, treatment may be continued for as long as the disease persists.

[0353] The bifunctional molecules disclosed herein can be provided in an effective dose range of approximately 1 ng / kg body weight to approximately 30 mg / kg body weight, 1 μg / kg to approximately 20 mg / kg, 10 μg / kg to approximately 10 mg / kg, or 100 μg / kg to 5 mg / kg, at any choice every 1, 2, 3, or 4 weeks, preferably by parenteral or oral administration, and particularly by intravenous or subcutaneous administration.

[0354] In particular, the bifunctional molecules according to the present invention may be administered in doses less than therapeutic doses. The term "less than therapeutic dose," as used herein, refers to doses below the level of effective monotherapy doses commonly used to treat a disease, or doses not currently typically used in effective monotherapy with anti-hPD1 antibodies.

[0355] Method and Use Use in disease treatment The bifunctional molecules, nucleic acids, vectors, host cells, compositions, and methods o...

Claims

1. (a)(i) Heavy chain variable domains (VH) including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains (VLs) including LCDR1, LCDR2, and LCDR3 Anti-human PD-1 antibodies or their antigen-binding fragments, including; and (b) Human interleukin 7 (IL-7) or its fragments or variants A bifunctional molecule containing, The antibody or fragment thereof is preferably covalently linked to the human IL-7 or fragment or variant thereof as a fusion protein by a peptide linker, thereby forming a bifunctional molecule.

2. The bifunctional molecule according to claim 1, wherein the N-terminus of the human IL-7 or a fragment thereof is connected to the C-terminus of the heavy chain, light chain, or both of the anti-human PD-1 antibody or an antigen-binding fragment thereof.

3. The bifunctional molecule according to claim 1 or 2, wherein the antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or a human antibody.

4. The aforementioned anti-human PD-1 antibody or its antigen-binding fragment is (i) Heavy chain variable domains (VH) including HCDR1, HCDR2, and HCDR3, and (ii) Light chain variable domains (VLs) including LCDR1, LCDR2, and LCDR3 Includes, - The heavy chain CDR1 (HCDR1) is included in or consists of the amino acid sequence of SEQ ID NO:

1. - The heavy chain CDR2 (HCDR2) is included in or consists of the amino acid sequence of SEQ ID NO:

2. - The heavy chain CDR3 (HCDR3) comprises or consists of the amino acid sequence of Sequence ID No. 3, where X1 is D or E, and X2 is selected from the group consisting of T, H, A, Y, N, E, and S, preferably from the group consisting of H, A, Y, N, and E. - The light chain CDR1 (LCDR1) contains or consists of the amino acid sequence of SEQ ID NO: 12, where X is G or T. - The light chain CDR2 (LCDR2) contains or consists of the amino acid sequence of SEQ ID NO:

15. - The light chain CDR3 (LCDR3) contains or consists of the amino acid sequence of SEQ ID NO:

16. A bifunctional molecule according to any one of claims 1 to 3.

5. The anti-human PD-1 antibody or its antigen-binding fragment comprises (a) VH, which includes or comprises the amino acid sequence of SEQ ID NO: 17, where X1 is D or E, and X2 is VH, which is preferably selected from the group consisting of H, A, Y, N, and E, from the group consisting of T, H, A, Y, N, E, and S; and (b) VL, which includes or comprises the amino acid sequence of SEQ ID NO: 26, where X is G or T.

6. The anti-human PD-1 antibody or its antigen-binding fragment comprises (i) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO: 24; and (ii) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO: 28, as described in any one of claims 1 to 5.

7. The anti-PD1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pizilizumab, semiprimab, PDR001, and monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, as a bifunctional molecule according to any one of claims 1 to 3.

8. The bifunctional molecule according to any one of claims 1 to 7, wherein the IL-7 or its variant comprises or consists of an amino acid sequence having at least 75% identity with wild-type human IL-7 (wth-IL-7).

9. The IL-7 comprises or consists of the amino acid sequence shown in SEQ ID NO: 51, according to any one of claims 1 to 8.

10. The IL-7 is an IL-7 variant that exhibits at least 75% identity with wild-type human IL-7 (wth-IL-7), comprising or consisting of the amino acid sequence shown in SEQ ID NO: 51, wherein the variant comprises at least one amino acid mutation that i) reduces the affinity of the IL-7 variant to the IL-7 receptor (IL-7R) compared to the affinity of wth-IL-7 to IL-7R, and ii) improves the pharmacokinetics of the bifunctional molecule containing the IL-7 variant compared to the bifunctional molecule containing wth-IL-7, according to any one of claims 1 to 8.

11. The bifunctional molecule according to claim 10, wherein the at least one mutation is an amino acid substitution or a group of amino acid substitutions selected from the group consisting of (i) C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, or C47S-C92S and C34S-C129S, (ii) W142H, W142F, or W142Y, (iii) D74E, D74Q, or D74N, iv) Q11E, Y12F, M17L, Q22E, and / or K81R, or any combination thereof.

12. The bifunctional molecule according to claim 10, wherein the IL-7 variant comprises a group of amino acid substitutions selected from the group consisting of C2S-C141S and C47S-C92S, C2S-C141S and C34S-C129S, and C47S-C92S and C34S-C129S.

13. The bifunctional molecule according to claim 10, wherein the IL-7 variant comprises an amino acid substitution selected from the group consisting of W142H, W142F, and W142Y.

14. The bifunctional molecule according to claim 10, wherein the IL-7 variant comprises an amino acid substitution selected from the group consisting of D74E, D74Q, and D74N.

15. The IL-7 variant comprises or consists of the amino acid sequences shown in SEQ ID NOs. 53 to 66, according to any one of claims 1 to 8 and 10.

16. The IL-7 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 54, 56, or 63, according to any one of claims 1 to 8 and 10.

17. The antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant domain, preferably IgG1 or IgG4 heavy chain constant domain, according to any one of claims 1 to 16.

18. The antibody or its antigen-binding fragment comprises a light chain constant domain derived from the human kappa light chain constant domain, and optionally, T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I332E / G236A;N2 A bifunctional molecule according to any one of claims 1 to 17, comprising a heavy chain constant domain derived from a human IgG1 heavy chain constant domain, having a substitution or combination of substitutions selected from the group consisting of 97A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably optionally combined with M252Y / S254T / T256E, and selected from the group consisting of L234A / L235A.

19. The antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E.17, and K444A, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain, and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain having a substitution or combination of substitutions selected from the group consisting of S228P;L234A / L235A, S228P+M252Y / S254T / T256E.17, and K444A, as described in any one of claims 1 to 17.

20. The antibody or fragment thereof is preferably (GGGGS) 3 (GGGGS) 4 (GGGGS) 2 , GGGGS, GGGS, GGG, GGS, and (GGGS) 3 A linker sequence selected from the group consisting of (GGGGS) is more preferably (GGGGS) 3 A bifunctional molecule according to any one of claims 1 to 19, wherein IL-7 or a variant thereof is linked thereto.

21. The antibody or its antigen-binding fragment comprises a light chain constant domain derived from the human kappa light chain constant domain, and optionally, T250Q / M428L;M252Y / S254T / T256E+H433K / N434F;E233P / L234V / L235A / G236A+A327G / A330S / P331S;E333A;S239D / A330L / I332E;P257I / Q311;K326W / E333S;S239D / I332E / G236A;N The antibody or fragment thereof comprises a heavy chain constant domain derived from a human IgG1 heavy chain constant domain, having a substitution or combination of substitutions selected from the group consisting of 297A;L234A / L235A;N297A+M252Y / S254T / T256E;K322A; and K444A, preferably N297A in combination with M252Y / S254T / T256E, and L234A / L235A, wherein the antibody or fragment thereof is linked by a linker (GGGGS) 3 A bifunctional molecule according to any one of claims 12 to 16, which is linked to an IL-7 variant by means of...

22. An isolated nucleic acid molecule or a group of isolated nucleic acid molecules encoding a bifunctional molecule as described in any one of claims 1 to 21.

23. A vector comprising the group of nucleic acids or nucleic acid molecules described in claim 22.

24. A host cell comprising the vector according to claim 23, or the group of nucleic acids or nucleic acid molecules according to claim 22.

25. A method for producing a bifunctional molecule according to any one of claims 1 to 21, comprising the steps of culturing a host cell according to claim 24, and optionally isolating the bifunctional molecule.

26. A pharmaceutical composition comprising a bifunctional molecule according to any one of claims 1 to 21, a nucleic acid or group of nucleic acid molecules according to claim 22, a vector according to claim 23, or a host cell according to claim 24, and a pharmaceutically acceptable carrier.

27. Additional therapeutic agents, preferably alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), activators of the cell death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell induction) antibodies, antibody-drug conjugates, bioreaction modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunological agents, apoptosis protein inhibitor (IAP) inhibitors, intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors The pharmaceutical composition according to claim 26, further comprising an epitope or neoepitope derived from a tumor antigen, such as a mammalian target of rapamycin inhibitors, microRNA, mitogen-activated extracellular signal-regulated kinase inhibitors, polyvalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly-ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small molecule inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, as well as additional therapeutic agents selected from the group consisting of one or more combinations of such substances.

28. A pharmaceutical composition according to claim 26 or 27, a bifunctional molecule according to any one of claims 1 to 21, a nucleic acid or group of nucleic acid molecules according to claim 22, or a vector according to claim 23, or a host cell according to claim 24, for use as a pharmaceutical.

29. A pharmaceutical composition, a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector, or a host cell according to claim 28, for use in the treatment of a disease selected from the group consisting of cancer.

30. The aforementioned cancers include, but are not limited to, cancers selected from the group consisting of hematological malignancies or solid tumors with PD-1 and / or PD-L1 expression, e.g., hematological lymphoid neoplasms, angioimmunoblastic T-cell lymphoma, myelodysplastic syndrome, and acute myeloid leukemia; cancers induced by viruses or associated with immunodeficiency, e.g., Kaposi's sarcoma (e.g., associated with Kaposi's herpes zoster virus); cervical cancer, anal cancer, penile cancer, and vulvar squamous cell carcinoma, and oropharyngeal cancer (e.g., associated with human papillomavirus); B-cell non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary exudative lymphoma, classical Hodgkin lymphoma, and lymphoproliferative disorders (e.g., Epsilon A pharmaceutical composition, a bifunctional molecule, a group of nucleic acids or nucleic acid molecules, a vector or host cell according to claim 29, selected from the group consisting of cancers associated with Stein-Barr virus (EBV) and / or Kaposi's sarcoma herpesvirus); hepatocellular carcinoma (e.g., associated with hepatitis B and / or C virus); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and human immunodeficiency virus infection (HIV) infection, as well as cancers selected from the group consisting of metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or gastroesophageal cancer, and cervical cancer.

31. Radiotherapy, or additional therapeutic agents, preferably alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, mitotic inhibitors, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), activators of the cell death receptor pathway, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell induction) antibodies, antibody-drug conjugates, bioreaction modifiers, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVDs, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunoassayants, apoptosis protein inhibitors (IAP) inhibitors, intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian targets of rapamycin inhibitors, micro A pharmaceutical composition, a bifunctional molecule, a group of nucleic acids or nucleic acid molecules, a vector, or a host cell, for use in combination with an additional therapeutic agent selected from the group consisting of RNA, mitogen-activated extracellular signal-regulated kinase inhibitors, polyvalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), polyADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum chemotherapeutic agents, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinoid / deltoid plant alkaloids, small molecule inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, and peptide vaccines, etc., and an epitope or neoepitope derived from a tumor antigen, and one or more combinations thereof, according to any one of claims 28 to 30.

32. A pharmaceutical composition, a bifunctional molecule, a nucleic acid or a group of nucleic acid molecules, a vector, or a host cell according to claim 28, for use in the treatment of infectious diseases, preferably chronic infectious diseases, and more preferably chronic viral infections.

33. The pharmaceutical composition, bifunctional molecule, nucleic acid or group of nucleic acid molecules, vector or host cell according to claim 32, wherein the infectious disease is caused by a virus selected from the group consisting of HIV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

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