Bifunctional molecules against human pd-1

Abstract

The present invention provides bifunctional molecules comprising a humanized anti-hPD-1 antibody or antigen binding fragment thereof linked to an immunotherapeutic agent capable of specifically enhancing an immune response and uses thereof.

Description

Invention Field

[0001] This invention belongs to the field of immunotherapy. This invention provides a bifunctional molecule comprising a humanized anti-PD-1 antibody or an antibody-binding fragment thereof linked to an immunotherapeutic agent, and its uses. Background of the Invention

[0003] Targeting T-cell suppressor checkpoints to desuppress inhibition using therapeutic antibodies is an area of ​​extensive research (see Pardoll, Nat Rev Cancer, 2012; 12:253-264 for commentary). Immune checkpoints targeting adaptive immunity have shown significant therapeutic efficacy against a variety of cancers, but only in a limited proportion of patients. Immune checkpoints on innate bone marrow cells (macrophages, dendritic cells, MDSCs, PMNs) remain poorly studied, despite these cells representing the most abundant immune cell types in many solid tumors and frequently being associated with poor outcomes. Combined immune checkpoint therapies targeting both innate (bone marrow cell-mediated) and adaptive (T-cell-mediated) immune responses have shown high efficiency in preclinical models, but remain a challenge in clinical practice.

[0004] Immune cell activation is controlled by the integration of balanced co-stimulatory and co-inhibitory signals. T cell receptor (TCR)-mediated T cell activation is regulated by both co-stimulatory and co-inhibitory signals. An antigen-independent second signal modifies the first signal, provided by the interaction of the antigen peptide-MHC complex with the TCR, thereby conferring specificity to the response. The T cell co-stimulatory and co-inhibitory pathway has broad immunomodulatory functions, controlling effectors, memory and regulatory T cells, as well as naive T cells. T cells. Therapeutic modulation of those pathways is translating into effective new strategies for treating cancer (see Schildberg et al., 44(5), Immunity, 2016 for the review). Ongoing research into the modulation of immune responses has identified multiple immune pathways that can be targeted to develop cancer therapies. Those molecules are referred to in this paper as immune checkpoint coactivators or co-inhibitors (see reviews Sharma et al., Cell, 161(2), 2015 and Pardoll, Nature Reviews Cancer, 12(4), 2012).

[0005] Programmed cell death protein 1 (PD-1, also known as CD279) is a cell surface protein molecule belonging to the immunoglobulin superfamily. PD-1 is expressed on T and B lymphocytes and macrophages and plays a role in cell fate and differentiation. In particular, PD-1 acts as an immune checkpoint, playing a crucial role in downregulating the immune system by blocking T cell activation, thereby reducing autoimmunity and promoting self-tolerance. Two ligands of PD-1, PD-L1 and PD-L2, have been identified and have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al., (2000) J Exp Med 192:1027-34; Latchman et al., (2001) Nat Immunol 2:261-8; Carter et al., (2002) Eur J Immunol 32:634-43). The interaction between PD-1 and its ligands leads to a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells. In particular, PD1 linkage reduces downstream signaling to TCR stimulation on T cells, inhibiting T cell responses and leading to reduced activation and cytokine production.

[0006] Both strategies of using anti-PD1 and anti-PDL1 inhibitors to disrupt their interaction have been successful in cancer treatment (Brahmer et al., N Eng J Med, 366(26), 2012; Powles et al., Nature, 515(7528), 2014; Topalian et al., N Eng J Med, 366(26), 2012; Ansell, Curr Opin Hematol, 22(4), 2015). However, the accumulation of immunosuppressive and hypostimulatory bone marrow cells in the tumor microenvironment limits the efficiency of T cell responses and the efficacy of immunotherapies, especially those targeting immune checkpoints (e.g., PD-1 / PD-L1). Meanwhile, immunotherapies targeting innate immune checkpoints alone show limited efficacy because T cell responses are still primarily blocked by the absence of co-stimulatory mechanisms and / or the binding of co-inhibitory molecules to ligands expressed by tumor cells or antigen-presenting cells in the tumor microenvironment. Combination immunotherapy targeting both adaptive (T-cell) and innate (bone marrow cell) immune checkpoints has shown preclinical efficacy.

[0007] However, the validation and development of combination immunotherapies are severely limited by the cost of biotherapies and the limited availability of such immunotherapies. Therefore, there remains a great need in the art for novel, improved agents for safe immunotherapies, particularly those targeting cancer, innate bone marrow immune cells, and possessing a potent and positive effect on adaptive immune responses, especially T-cell immune responses. The inventors have taken an important step forward with the inventions disclosed herein. Summary of the Invention

[0008] The inventors have provided a bifunctional molecule comprising a humanized anti-hPD-1 antibody and an immunotherapeutic agent, which holds promise for a variety of therapeutic applications, particularly for cancer treatment. This invention is based on the development of a humanized antibody that specifically targets human PD-1, exhibiting high binding affinity for PD-1 and strong competition with its ligands PDL-1 and / or PD-L2. Surprisingly, this humanized antibody exhibits a high humanization score and allows for high yields when produced as a bifunctional molecule that binds to an immunotherapeutic agent. In fact, this humanized antibody has been engineered to exhibit high prepareability in mammalian cell-based production systems, particularly when fused to an active protein domain at the C-terminus of its heavy or light chain, a condition typically associated with poor prepareability.

[0009] In the first aspect, the bifunctional molecule consists of the following:

[0010] (a) A humanized anti-human PD-1 antibody or its antigen-binding fragment, comprising:

[0011] (i) Heavy chain variable domains (VH) containing HCDR1, HCDR2, and HCDR3, and

[0012] (ii) Light chain variable structural domains (VL) comprising LCDR1, LCDR2 and LCDR3,

[0013] in:

[0014] - The heavy chain CDR1 (HCDR1) contains or is composed of the amino acid sequence of SEQ ID NO:1;

[0015] - The heavy chain CDR2 (HCDR2) contains or is composed of the amino acid sequence of SEQ ID NO:2;

[0016] - The heavy chain CDR3 (HCDR3) comprises or is composed of the amino acid sequence of SEQ ID NO:3, wherein X1 is D or E, and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N and E;

[0017] - The light chain CDR1 (LCDR1) comprises or is composed of the amino acid sequence of SEQ ID NO:12, wherein X is G or T;

[0018] - The light chain CDR2 (LCDR2) comprises or is composed of the amino acid sequence of SEQ ID NO:15.

[0019] - The light chain CDR3 (LCDR3) comprises or is composed of the amino acid sequence of SEQ ID NO:16.

[0020] and

[0021] (b) Immunotherapy agents or fragments thereof

[0022] The C-terminus of the heavy chain and / or light chain of the antibody or its antigen-binding fragment is preferably covalently linked to the N-terminus of the immunotherapeutic agent via a peptide linker to form a fusion protein.

[0023] Specifically, the humanized anti-human PD-1 antibody or its antigen-binding fragment comprises (a) a VH comprising the amino acid sequence of SEQ ID NO:17 or composed of the amino acid sequence of SEQ ID NO:17, wherein X1 is D or E, and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N and E; and (b) a VL comprising the amino acid sequence of SEQ ID NO:26 or composed of the amino acid sequence of SEQ ID NO:26, wherein X is G or T.

[0024] Preferably, the antibody or its antigen-binding fragment is an antagonist of human PDL-1 and / or PD-L2 binding to human PD1.

[0025] Preferably, the immunotherapeutic agent or its fragments are selected from tumor-targeting peptides, cytokines, cytokine receptors, chemokines, chemokine receptors, co-stimulatory molecules, inhibitory or co-inhibitory molecules, enzymes, molecular chaperone inhibitors, and type I or type II human transmembrane immune proteins, preferably their extracellular domains.

[0026] Specifically, the size of the immunotherapeutic agent fragments is between 10 kDa and 50 kDa.

[0027] In one particular aspect, the immunotherapeutic agent is a type I human transmembrane immune protein or a fragment thereof, preferably selected from ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, connexin-1, connexin-2, connexin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, IL6RA, IL-1R1, IL-1RAcP, IL6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD1 55. CD113, VISTA, CD244, OX40, SIRPα, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R and GITR.

[0028] In another aspect, the immunotherapeutic agent is a type II human transmembrane immune protein or a fragment thereof, preferably selected from CD40L, OX40L, FasL, TRAIL, TNF, LIGHT, APRIL, GITRL, CD30, CD70, CD40, CD27, CD30, CD153, RANK, CD96, CLEC1, CLEC2 / CLE1B, CLEC3A, CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-α, LT-β, ​​4-1BBL, and MINCLE, preferably a member of the TNF family.

[0029] In another aspect, the immunotherapeutic agent is a cytokine or fragment thereof selected from TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL-12B, IL-15, IL-21 and IL-18.

[0030] In a very particular aspect, the immunotherapeutic agent is human IL-2 or a mutant thereof, particularly an IL-2 mutant having the sequence shown in SEQ ID NO:58, having the following substitutions: F42A, Y45A and L72G, preferably T3A, F42A, Y45A, L72G and C125A.

[0031] In one particular aspect, the antibody or its antigen-binding fragment comprises a light chain constant domain derived from the human κ light chain constant domain and a heavy chain constant domain derived from the human IgG1, IgG2, IgG3 or IgG4 heavy chain constant domain.

[0032] In a more specific aspect, the antibody or its antigen-binding fragment comprises a light chain constant domain derived from the human κ light chain constant domain and a heavy chain constant domain derived from the human IgG1 heavy chain constant domain, optionally having substitutions or combinations thereof selected from: T250Q / M428L; M252Y / S254T / T256E+H433K / N434F; E233P / L234V / L235A / G236A+A327G / A330S / P331 S; E333A; S239D / A330L / I332E; P257I / Q311; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K444A and K322A, preferably selected from the following: N297A, which is optionally combined with M252Y / S254T / T256E, and L234A / L235A.

[0033] In another, more specific aspect, the antibody or its antigen-binding fragment comprises a light chain constant domain derived from the human κ light chain constant domain and a heavy chain constant domain derived from the human IgG4 heavy chain constant domain, optionally having a substitution or combination of substitutions selected from: S228P; L234A / L235A, S228P+M252Y / S254T / T256E and K444A.

[0034] The present invention also relates to an isolated nucleic acid sequence or isolated nucleic acid molecule encoding a bifunctional molecule as disclosed herein; a vector comprising the nucleic acid or nucleic acid molecule; and a host cell comprising a vector, or nucleic acid or nucleic acid molecule as disclosed herein.

[0035] In one aspect, the present invention relates to a method for producing the bifunctional molecule, comprising the steps of culturing host cells as disclosed herein and optionally isolating the bifunctional molecule.

[0036] In another aspect, the present invention relates to a pharmaceutical composition comprising the bifunctional molecule, the nucleic acid or group of nucleic acid molecules, a carrier as disclosed herein, and a pharmaceutically acceptable carrier.

[0037] Optionally, the pharmaceutical composition further comprises additional therapeutic agents, preferably selected from the following: alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell conjugate) antibodies, antibody-drug conjugates, biological response modulators, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVD, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors. Preparations, histone deacetylase (HDAC) inhibitors, hormone therapy, immunotherapies, inhibitors of apoptosis protein inhibitors (IAP), intercalating antibiotics, kinase inhibitors, kinase inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly(adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapy agents, polo-like kinase (Plk) inhibitors, phosphoinositol-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinol / vitamin D-like plant alkaloids (deltoids), small inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc., epitopes or neoepitaxes derived from tumor antigens, and combinations of one or more of these agents.

[0038] The invention ultimately relates to a pharmaceutical composition, bifunctional molecule, nucleic acid or nucleic acid molecule group, carrier, or host cell as disclosed herein, which is used as a medicine.

[0039] In one particular aspect, pharmaceutical compositions, bifunctional molecules, nucleic acids or groups of nucleic acid molecules, vectors, or host cells, as disclosed herein, are used to treat cancer. Preferably, the cancer is selected from: hematologic malignancies or solid tumors expressing PD-1 and / or PD-L1, such as cancers selected from: lymphohematopoietic tumors, angioimmunoblastic T-cell lymphomas, myelodysplastic syndromes, and acute myeloid leukemia; virus-induced or immunodeficiency-related cancers, such as cancers selected from: Kaposi's sarcoma (e.g., Kaposi's sarcoma herpesvirus-associated); cervical cancer, anal cancer, penile cancer, and vulvar squamous cell carcinoma and oropharyngeal cancer (e.g., human papillomavirus-associated); B-cell non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HH... V-8 Primary exudative lymphoma, classical Hodgkin's 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 hepatitis C virus); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancer associated with human immunodeficiency virus infection (HIV); and cancers selected from: metastatic or non-metastatic, melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric or esophageal cancer, and cervical cancer.

[0040] In another particular aspect, pharmaceutical compositions, bifunctional molecules, nucleic acids or groups of nucleic acid molecules, vectors, or host cells disclosed herein are used to treat infectious diseases, preferably chronic infectious diseases, and even more preferably chronic viral infections, said infectious diseases preferably caused by viruses selected from: HIV, hepatitis viruses, herpesviruses, adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, Coxsackieviruses, coronaviruses, 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.

[0041] Optionally, the bifunctional molecule, the pharmaceutical composition, the isolated nucleic acid molecule or group of nucleic acid molecules, the carrier, or the host cell is used in combination with radiotherapy or other therapeutic agents, wherein the other therapeutic agents are preferably selected from alkylating agents, angiogenesis inhibitors, antibodies, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell conjugate) antibodies, antibody-drug conjugates, biological response modulators, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVD, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, and histone deacetylases (HDACs). Inhibitors, hormone therapy, immunotherapies, inhibitors of apoptosis protein inhibitors (IAP), intercalating antibiotics, kinase inhibitors, kinase inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly(adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapy agents, polo-like kinase (Plk) inhibitors, phosphoinositol-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinol / vitamin D alkaloids, small inhibitory ribonucleic acid (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc., epitopes or neoepitaxes derived from tumor antigens, and combinations of one or more of these agents. Attached Figure Description

[0042] Figure 1The yield of humanized anti-PD-1 bifunctional antibodies was compared to chimeric conjugates fused to type I Ig-like, type II TNF family, or cytokine proteins at the C-terminus of the heavy chain (A), light chain (B), or both heavy and light chains (C). HEK free-form antibodies were transiently transfected with lipofectamine and a humanized or chimeric form of DNA plasmid encoding the bifunctional antibody. On day 3 post-transfection, the supernatant containing the antibody was collected, and the concentration was measured by a sandwich ELISA using anti-human IgG Fc as a capture antibody and anti-human IgK as the detection antibody. Statistical analysis was performed using the Mann-Whitney test (p < 0.05). For example, in this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A corresponding to CD86 and protein B corresponding to CD80 are type I protein fusions; protein C corresponds to SIRPa (i.e., SIRPα); and protein A corresponding to OX40L and protein B corresponding to 4-1BBL are type II protein fusions. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:19 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0043] Figure 2 Yield of bifunctional proteins with chimeric, humanized, or other anti-PD-1 backbones. HEK free-range or adherent CHO cells were transiently transfected with a humanized or chimeric form of DNA plasmid encoding the anti-PD-1 antibody described herein, or with a nivol2 mab or pembrolizumab anti-PD-1 sequence fused to the C-terminus of the heavy chain with a cytokine or type I protein. Shake flasks or 12-well plates were used for HEK or CHO production, respectively. On day 3 post-transfection, the supernatant containing the antibody was collected and the concentration was measured by a sandwich ELISA using anti-human IgG Fc as a capture antibody and anti-human IgK as the detection antibody. Figure 2 A: Yield of bifunctional anti-PD-1 fused with IL-7 cytokine. Figure 2 B: Yield of bifunctional anti-PD-1 fused with CD80 protein I fusion protein. Figure 2 C: Yield of a bifunctional anti-PD-1 fusion protein fused with SIRPa protein I. In this experiment, the bifunctional molecule comprises a humanized anti-PD-1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:24 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0044] Figure 3Yields of bifunctional proteins with a humanized anti-PD-1 backbone and those with other non-anti-PD-1 backbones were compared. HEK free-form proteins were transiently transfected using DNA plasmids encoding various bifunctional proteins with or without a humanized anti-PD-1 backbone. Antibody-containing supernatants were collected on day 3 post-transfection, and concentrations were measured using a sandwich ELISA employing anti-human IgG Fc as a capture antibody and anti-human IgK as the detection antibody. In this experiment, the bifunctional molecule contained a humanized anti-PD-1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:19, 22, or 24 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0045] Figure 4 ELISA assay for PD-1. Recombinant human PD-1 protein was immobilized and different concentrations of anti-PD-1 bifunctional antibodies were added. Revelation was performed using an anti-human Fc antibody conjugated with peroxidase. A colorimetric assay was performed at 450 nm using TMB substrate. (A) Data for anti-PD-1 chimeric antibodies with unfused (■) or fused (●) to three type I Ig-like family proteins A, B, or C in the VL domain (o) or VH domain (●); (B) Data for anti-PD-1 chimeric antibodies with unfused (■) or fused (●) to two type II TNF family proteins A, B, or C in the VL domain (o) or VH domain (●); (C) Data for anti-PD-1 chimeric antibodies with unfused (■) or fused (●) to cytokine family protein A in the VL domain (o) or VH domain (●). In this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A corresponding to CD86 and protein B corresponding to CD80 are fused with type I proteins; protein C corresponds to SIRPa; protein A corresponding to OX40L and protein B corresponding to 4-1BBL are fused with type II proteins. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:19 and a light chain as disclosed in SEQ ID NO:28.

[0046] Figure 5ELISA assay comparing the binding of heavy chain chimeras fused to type I (A), type II (B), and cytokine (C) proteins to humanized bifunctional anti-PD-1 antibodies. Recombinant human PD-1 protein was immobilized, and different concentrations of anti-PD-1 bifunctional antibodies were added. Revelation was performed using an anti-human Fc antibody conjugated to peroxidase. A colorimetric assay at 450 nm was used with TMB substrate. (A) Data for anti-PD-1 chimeras fused to three type I Ig-like family proteins A, B, or C (●) compared to humanized (■) antibodies; (B) Data for anti-PD-1 chimeras fused to type II TNF family proteins A or B (●) compared to humanized (■) antibodies; (C) Data for anti-PD-1 chimeras fused to cytokine family protein A (●) compared to humanized (■) antibodies. In this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A corresponding to CD86 and protein B corresponding to CD80 are fused with type I proteins; protein C corresponds to SIRPa; and protein A corresponding to OX40L and protein B corresponding to 4-1BBL are fused with type II proteins. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:19 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0047] Figure 6 ELISA assay comparing the binding of chimeric light chains fused with type I (A), type II (B), and cytokine (C) proteins to humanized bifunctional anti-PD-1 antibodies. Recombinant human PD-1 protein was immobilized, and different concentrations of anti-PD-1 bifunctional antibodies were added. Revelation was performed using an anti-human Fc antibody conjugated to peroxidase. A colorimetric assay at 450 nm was used with TMB substrate. (A) Data for anti-PD-1 chimeric antibodies (●) fused with three type I Ig-like family proteins A, B, or C compared to humanized (■) antibodies; (B) Data for anti-PD-1 chimeric antibodies (●) fused with type II TNF family proteins A or B compared to humanized (■) antibodies; (C) Data for anti-PD-1 chimeric antibodies (●) fused with cytokine family protein A compared to humanized (■) antibodies. In this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A corresponding to CD86 and protein B corresponding to CD80 are fused with type I proteins; protein C corresponds to SIRPα; protein A corresponding to OX40L and protein B corresponding to 4-1BBL are fused with type II proteins. In this experiment, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:19 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0048] Figure 7The chimeric combination of both heavy and light chains fused with cytokine protein A was compared to the binding of a humanized bifunctional anti-PD-1 antibody to PD-1 by an ELISA assay. Human recombinant PD-1 protein was immobilized, and chimeric combinations (▲) of both heavy and light chains fused with cytokine A (IL-7) were added at different concentrations compared to a humanized (Δ) anti-PD-1 Ab. Revelation was performed using an anti-human Fc antibody conjugated to peroxidase. A colorimetric assay at 450 nm was used to determine the composition using a TMB substrate. In this experiment, the bifunctional molecule comprised a humanized anti-PD-1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:19 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0049] Figure 8 Competitive PD-1 / PD-L1 or PD-L2 ELISA assay. A: PD-1 / PD-L1 antagonist activity: PD-L1 was immobilized, and a complex antibody was added + biotinylated recombinant human PD-1. Different concentrations of anti-PD-1 antibody were tested, and 0.6 μg / mL of recombinant biotinylated PD-1 protein was added. PD-1 molecules were detected by streptavidin peroxidase and revealed by colorimetric assay at 450 nm using TMB substrate. (A) Data of anti-PD-1 antibodies fused with three type I Ig-like family proteins A or C; (B) Data of anti-PD-1 antibodies fused with type II TNF family proteins A or B; (C) Data of anti-PD-1 antibodies fused with cytokine family protein A. In this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A fused with the type I protein corresponds to CD86; protein C corresponds to SIRPα; protein A fused with type II protein A corresponds to OX40L, and protein B corresponds to 4-1BBL. (D): PD-1 / PD-L2 antagonist activity. PD-L2 was immobilized and compounded with biotinylated recombinant human PD-1 + anti-PD-1VH IL-7 (●) or anti-PD-1 VH CD80 (■). A similar protocol to the PD-L1 / PD-1 assay was used for revelation. In this assay, the bifunctional molecule comprises a humanized anti-PD1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:24 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0050] Figure 9Bridging ELISA binding assay. PD1-His recombinant protein was immobilized in (A) and (B) and bifunctional anti-PD-1 antibodies (type I or II proteins fused with the VL domain (o) and VH domain (●)) were added serially. A soluble recombinant receptor ligand for protein C, A, or B was then added at 1 μg / mL. Detection was performed using receptor-specific mouse antibody + peroxidase-conjugated anti-IgG mouse antibody. The ELISA was revealed using a 450 nm colorimetric assay with TMB substrate. The histogram represents recombinant protein A, B, or C immobilized on the plate and serves as a positive control for the ELISA. In this histogram, protein A corresponds to OX40L, protein B corresponds to 4-1BBL, and protein C corresponds to SIRPα.

[0051] Figure 10 T cell proliferation stimulated by bifunctional anti-PD-1 molecules. CD3 / PDL1-coated pre-activated CD3 / CD28 T cells were restimulated on CD3 / PDL1-coated plates in the presence of bifunctional anti-PD-1 antibodies fused with type I (A), type II (B), or (C) cytokine proteins (10 μg / mL). Unfused anti-PD-1 antibodies or allotype VH-fused antibodies served as controls. T cell proliferation was assessed by H3 thymidine incorporation on day 6. Data are expressed as fold changes, with allotype treatment serving as a control. Each point represents data from one donor. In this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A fused with type I proteins corresponds to CD86, protein B to CD80; protein C to SIRPa; and protein A fused with type II proteins to OX40L and protein B to 4-1BBL.

[0052] Figure 11 IFNγ secretion from T cells treated with an anti-PD-1 bifunctional antibody. CD3 / PDL1-coated pre-activated CD3 / CD28 T cells were restimulated on CD3 / PDL1-coated plates in the presence of an anti-PD-1 bifunctional antibody fused with a (A) type I, (B) type II, or (C) cytokine protein (10 μg / mL). Unfused anti-PD-1 antibodies or antibodies fused with the same type VH were used as controls. IFNγ secretion was assessed by ELISA in the supernatant collected on day 5. Data are expressed as fold changes, with unfused anti-PD-1 treatment used as a control. Statistical analysis was performed using the Mann-Whitney test *p < 0.05. In this figure, the cytokine fused with the anti-PD-1 antibody is IL-7; protein A (type I fusion) corresponds to CD80, protein B corresponds to CD86; protein C corresponds to SIRPa; protein A (type II fusion) corresponds to OX40L, and protein B corresponds to 4-1BBL.

[0053] Figure 12Pharmacokinetics of bifunctional humanized PD-1 antibody in mice after a single injection. Balb / C mice were intravenously injected with a humanized variant of the bifunctional antibody containing either the IgG4 S228P isotype (●) or the IgG1 N298A isotype (○). Plasma drug concentrations were determined by ELISA using a fixed anti-human light chain antibody (clone NaM76-5F3) after adding diluted serum containing the anti-PD-1 antibody. Detection was performed using peroxidase-labeled donkey anti-human IgG. In this experiment, the bifunctional molecule comprised a humanized anti-PD-1 antibody having a heavy chain variable domain as disclosed in SEQ ID NO:24 and a light chain variable domain as disclosed in SEQ ID NO:28.

[0054] Detailed embodiments of the present invention

[0055] Introduction

[0056] The humanized antibodies described in this invention are bifunctional because they combine specific anti-PD-1 activity with the activity of an immunotherapeutic agent grafted onto a humanized anti-PD-1 antibody. In fact, this invention relates to bifunctional molecules comprising a specific humanized anti-PD-1 antibody and an immunotherapeutic agent. More particularly, it relates to bifunctional molecules comprising a humanized anti-PD-1 antibody and an immunotherapeutic agent, wherein the immunotherapeutic agent is covalently linked to a polypeptide chain of the humanized anti-PD-1 antibody (a light chain or heavy chain of the antibody or a fragment thereof, or both). More specifically, the chain of the humanized anti-PD-1 antibody or a fragment thereof and the immunotherapeutic agent are prepared as a fusion protein. In this particular aspect, the N-terminus of the immunotherapeutic agent is optionally linked to the C-terminus of the chain of the humanized anti-PD-1 antibody or a fragment thereof via a peptide linker.

[0057] The bifunctional molecule described in this invention has one or more of the following advantages:

[0058] - It exhibits higher prepareability and yield compared to chimeric antibodies when produced in mammalian cells (e.g., COS, CHO). Furthermore, no other anti-PD1 antibody offers the same improved yield. Figures 1 to 3As shown, the specific humanized anti-PD-1 bifunctional molecule described in this invention exhibits surprisingly better production compared to chimeric antibodies or two clinically approved reference anti-PD1 antibodies (i.e., pembrolizumab and nivolumab). Furthermore, this improved production has been demonstrated with three different types of immunotherapeutic agents and six different immunotherapeutic agents (i.e., cytokines (i.e., IL-7), type I proteins (i.e., CD80, CD86, and SIRPα), and type II proteins (i.e., OX40L and 4-1BBL)). In addition, the inventors have observed that when the immunotherapeutic agent is a type I transmembrane protein, it retains its functionality upon grafting to the C-terminus of the humanized anti-hPD1 antibody. This is both surprising and highly interesting because type I transmembrane proteins are characterized by an N-terminus influencing the extracellular domain; therefore, N-terminal grafting of type I transmembrane proteins has generally not rendered such proteins functional until now. Furthermore, improved yields were observed when the immunotherapeutic agent was fused to the C-terminus of either the heavy chain or the light chain. Furthermore, improved yield was observed if the immunotherapeutic agent was fused to the C-terminus of both the heavy chain and the light chain. Figure 1 Therefore, better production is closely related to the humanized anti-PD1 antibody described in this invention. This characteristic is quite surprising and unexpected.

[0059] - It possesses full functionality and activates both innate and adaptive immune responses. In fact, as... Figure 4-7 As shown, the binding of the bifunctional molecule to PD-1 remains essentially unchanged. Figure 8-9 As shown, the bifunctional molecule essentially retains the same antagonistic activity. It has been shown that 1) three different types of immunotherapeutic agents and six different immunotherapeutic agents, namely cytokines (i.e., IL-7), type I proteins (i.e., CD80, CD86, and SIRPα), and type II proteins (i.e., OX40L and 4-1BBL); and 2) whether the immunotherapeutic agent is fused to the C-terminus of the heavy chain or the light chain, or to both the light and heavy chains. Finally, the bifunctional molecule induces T cell proliferation at least as well as, and even better than, anti-PD1 antibodies alone. Figure 10 It showed better T cell activation efficiency than anti-PD1 antibody alone. Figure 11 Given the slight loss of binding to the PD-1 ligand, PD-L1, this ability to activate T cells better than anti-PD1 antibodies alone is a surprising property of the bifunctional molecule described in this invention.

[0060] - Due to its targeting action, the bifunctional molecule avoids hematologic toxicity caused by restricted PD-1 expression (non-binding to human erythrocytes (RBCs) and platelets);

[0061] -It reduces tumor growth and alters the tumor microenvironment;

[0062] - It enables human T-cell immune responses and is a selective antagonist of PD-1 / PD-L1 interaction and / or PD-1 / PD-L2 interaction;

[0063] - It can exhibit additional or synergistic effects by combining anti-PD-1 and immunotherapeutic agents into a single molecule (particularly in the examples concerning IFNγ secretion and T cell proliferation).

[0064] The humanized anti-PD-1 antibody described in this invention has a CDR sequence with extremely low similarity to other anti-PD-1 antibodies, including pembrolizumab (also known as Keytruda) and nivolumab (also known as Opdivo). Therefore, unexpectedly, and possibly related to this difference in the CDR sequence, the applicant has successfully obtained a bifunctional antibody exhibiting the beneficial effects described in the application. The humanized anti-PD-1 antibody using the bifunctional molecule has an unexpectedly high capacity for production, regardless of the immunotherapeutic agent conjugated to it. This then allows for the industrial-scale production of various bifunctional molecular scaffolds. The production volume is compatible with drug development and reproducibility, which greatly facilitates regulated and safe processes.

[0065] definition

[0066] To facilitate understanding of the invention, certain terms are defined below. Further definitions are set forth throughout the detailed description.

[0067] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, for clarity and / or ease of reference, terms with generally understood meanings are defined herein, and such definitions contained herein should not be construed as representing a difference from the common understanding in the art. The techniques and procedures described or referenced herein are generally well understood and commonly adopted by those skilled in the art using conventional methodologies.

[0068] As used herein, the terms “programmed cell death 1,” “programmed cell death 1,” “sequence PD1,” “PD-1,” “PDCD1,” “PD-1 antigen,” “human PD-1,” “hPD-1,” and “hPD1” are used interchangeably to refer to the programmed cell death 1 receptor, also known as CD279, and include variants and subtypes of human PD-1, as well as analogs that share at least one common epitope with PD-1. PD-1 is a key regulator of immune response thresholds and peripheral immune tolerance. It 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. For example, the amino acid sequence of human PD-1 is disclosed under GenBank accession number NP_005009. PD1 has four splice variants expressed on human peripheral blood mononuclear cells (PBMCs). Therefore, the PD-1 protein includes full-length PD-1, as well as alternative splice variants of PD-1 such as PD-1Aex2, PD-1Aex3, PD-1Aex2,3, and PD-1Aex2,3,4. Unless otherwise stated, the terminology includes any variant and subtype of human PD-1 naturally expressed by PBMCs or expressed by cells transfected with the PD-1 gene.

[0069] As used herein, the term "antibody" describes a type of immunoglobulin molecule and is used in its broadest sense. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. Unless specifically indicated, the term "antibody" includes intact immunoglobulins as well as "antibody fragments" or antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv), mutants thereof, molecules containing antibody moieties, dimers, linear antibodies, single-chain antibodies, and any other modified conformation of immunoglobulin molecules containing antigen recognition sites with desired specificity, including glycosylated variants of antibodies and amino acid sequence variants of antibodies. Preferably, the term "antibody" refers to humanized antibodies, and even more preferably bifunctional humanized antibodies.

[0070] As used herein, an "antigen-binding fragment" of an antibody refers to a portion of an antibody exhibiting antigen-binding ability against PD-1, i.e., a molecule corresponding to a portion of the structure of the antibody of the present invention, possibly in its native form; such fragments exhibit the same or substantially the same antigen-binding specificity to the antigen as the corresponding four-chain antibody. Advantageously, the antigen-binding fragment has a binding affinity similar to that of the corresponding four-chain antibody. However, antigen-binding fragments with reduced antigen-binding affinity relative to the corresponding four-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. These antigen-binding fragments may also be referred to as "functional fragments" of the antibody. An antigen-binding fragment of an antibody is a fragment containing a hypervariable domain of its termed CDR (complementarity-determining region) or a portion thereof containing an antigen recognition site (i.e., the extracellular domain of PD-1), thereby defining antigen recognition specificity.

[0071] 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 it has several residues added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. The F(ab’) fragment is produced by cleaving the disulfide bond at the hinge cysteine ​​residue of the F(ab’)2 pepsin digestion product. Other chemical conjugations of antibody fragments are well known to those skilled in the art. The Fab and F(ab’)2 fragments lack the Fc fragment of the intact antibody, are cleared from animal circulation more quickly, and may have less nonspecific tissue binding than the intact antibody (see, for example, Wahl et al., 1983, J. Nucl. Med. 24:316).

[0072] The “Fv” fragment is the smallest segment of an antibody containing both complete target recognition and binding sites. This region consists of a dimer (VH-VL dimer) consisting of a heavy-chain variable domain and a light-chain variable domain tightly bound nonvalently. 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. Typically, 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 the three target-specific CDRs) can have the ability to recognize and bind to the target, although with lower affinity than the entire binding site.

[0073] A "single-chain Fv" or "scFv" antibody-binding fragment contains the antibody's VH and VL domains, which are located within a single polypeptide chain. Typically, Fv polypeptides also include a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for target binding.

[0074] A “single-domain antibody” consists of a single VH or VL domain that exhibits sufficient affinity for PD-1. In one particular embodiment, the single-domain antibody is a camel-derived antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0075] Structurally, antibodies may have heavy chains (H) and light chains (L) interconnected by disulfide bonds. There are two types of light chains, λ (λ) and κ (κ). Each heavy and light chain contains constant and variable regions (“domains”). The variable regions of the light and heavy chains contain “framework” regions interrupted by three hypervariable regions (also called “complementarity-determining regions” or “CDRs”). The extent of the frame regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, and USDepartment of Health and Human Services, 1991, which are incorporated herein by reference). Preferably, the CDRs are defined according to the Kabat method. The frame regions act as a scaffold, positioning the CDRs in the correct orientation through non-covalent interactions between chains. The CDRs are primarily responsible for binding antigenic epitopes. The CDRs of each chain are typically referred to as “complementarity-determining region 1” or “CDR1”, “CDR2”, and “CDR3”, numbered sequentially starting from the N-terminus. The VL and VH domains of the antibody according to the invention may comprise four frame regions, or "FRs," referred to in the art and herein as "frame region 1" or "FR1," "FR2," "FR3," and "FR4," respectively. These frame 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 carboxyl terminus).

[0076] As used in this article, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody conformation. The CDRs of the antibody heavy chain are usually referred to as "HCDR1", "HCDR2", and "HCDR3". The frame regions of the antibody heavy chain are usually referred to as "HFR1", "HFR2", "HFR3", and "HFR4".

[0077] As used in this article, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in the antibody conformation, and κ and λ light chains refer to the light chain isotypes of the two major antibodies. The CDRs of antibody light chains are usually referred to as "LCDR1", "LCDR2", and "LCDR3". The frame regions of antibody light chains are usually referred to as "LFR1", "LFR2", "LFR3", and "LFR4".

[0078] Regarding antibody binding to target molecules, the term "bind" refers to peptides, polypeptides, proteins, fusion proteins, molecules, and antibodies (including antibody fragments) that recognize and contact antigens. Preferably, it refers to antigen-antibody type interactions. The terms "specific binding," "specific binding to," "specific targeting," "selective binding," and "selective targeting" of a specific antigen (e.g., PD-1) or an epitope on a specific antigen (e.g., PD-1) indicate that the antibody recognizes and binds to the 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 that PD-1 epitope, for example, with higher affinity, cohesion, ease of binding, and / or longer duration of binding than to other PD-1 epitopes or non-PD-1 epitopes. Preferably, the term "specific binding" refers to an interaction between the antibody and the antigen at a rate equal to or less than 10. -7 M binding affinity contact. In some respects, antibodies bind at a rate equal to or less than 10. -8 M, 10 -9 M or 10 -10 Affinity binding of M.

[0079] As used herein, the terms “PD-1 antibody,” “anti-PD-1 antibody,” “PD-1Ab,” “PD-1Ab,” “PD-1 specific antibody,” or “anti-PD-1Ab” or “humanized anti-PD-1 antibody” are used interchangeably and refer to antibodies that specifically bind to PD-1, preferably human PD-1, as described herein. In some embodiments, the antibody binds to the extracellular domain of PD-1. In particular, an anti-PD-1 antibody is an antibody capable of binding to the PD-1 antigen and inhibiting PD-1-mediated signal transduction pathways, thereby enhancing immune responses, such as T cell activation.

[0080] 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 are used interchangeably. These terms refer to an antibody that recognizes an antigen by containing at least one region specific to an antigen (e.g., a variable region derived from an antibody) and a second region that is at least a polypeptide. More specifically, the bifunctional molecule is a fusion protein of an antibody or a portion thereof, preferably an antigen-binding fragment thereof with another polypeptide or a polypeptide fragment thereof.

[0081] As used herein, the term "chimeric antibody" refers to an antibody or antigen-binding fragment in which a portion of its heavy chain and / or light chain is derived from one species, while the remainder of the heavy chain and / or light chain is derived from a different species. In an illustrative example, the chimeric antibody may comprise a constant region derived from a human and a variable region derived from a non-human species (e.g., a mouse).

[0082] As used herein, the term "humanized antibody" is intended to refer to an antibody in which a CDR sequence derived from another mammalian species (e.g., a mouse) has been transplanted into a human frame sequence (e.g., a chimeric antibody containing minimal sequences derived from a non-human antibody). The "humanized form" of an antibody (e.g., a non-human antibody) also refers to an antibody that has undergone humanization. Humanized antibodies are typically human immunoglobulins (receptor antibodies) in which one or more CDR residues are replaced by residues of at least one CDR from a non-human antibody (donor antibody) while retaining the desired specificity, affinity, and ability of the original antibody. The donor antibody can be any suitable non-human antibody, such as mouse, rat, rabbit, chicken, or non-human primate antibodies with the desired specificity, affinity, or biological effect. In some cases, selected frame region residues of the receptor antibody are replaced by frame region residues from the donor antibody. Alternatively, selected frame region residues of a co-antibody are replaced by frame region residues from a human or humanized antibody. Additional frame region modifications can be made within the human frame sequence. Therefore, humanized antibodies can also contain residues not found in either the receptor antibody or the donor antibody. Such amino acid modifications can be performed to further enhance antibody function and / or enhance the humanization process. In this document, “amino acid alteration” or “amino acid modification” refers to a change in the amino acid sequence of a polypeptide. “Amino acid modification” includes substitution, insertion, and / or deletion in the polypeptide sequence. In this document, “amino acid substitution” or “substitution” refers to replacing an amino acid at a specific position in the parental polypeptide sequence with another amino acid. “Amino acid insertion” or “insertion” refers to adding an amino acid at a specific position in the parental polypeptide sequence. “Amino acid deletion” or “deletion” refers to removing an amino acid at a specific position in the parental polypeptide sequence. Amino acid substitutions can be conserved. A conserved substitution is the replacement of a given amino acid residue with another residue of a side chain (“hydrophobic group”) having similar chemical properties (e.g., charge, size, and / or hydrophobicity). As used herein, “amino acid position” or “amino acid position number” can be used interchangeably and refers to the position of a specific amino acid in an amino acid sequence, typically designated by a single-letter code for the amino acid. The first amino acid in the amino acid sequence (i.e., starting from the N-terminus) should be considered to have position 1.

[0083] A conserved substitution is the replacement of a given amino acid residue with another residue in a side chain (“R-group”) having similar chemical properties (e.g., charge, size, and / or hydrophobicity). Generally, conserved amino acid substitutions will not substantially alter the functional properties of a protein. Conserved substitutions and corresponding rules are well described in the prior art. For example, a conserved substitution can be defined by substitutions within the amino acid set reflected in the table below:

[0084] Table A – Amino Acid Residues

[0085] amino acid groups amino acid residues acid residues ASP and GLU basic residues LYS, ARG and HIS Hydrophilic uncharged residues SER, THR, ASN and GLN Aliphatic uncharged residues GLY, ALA, VAL, LEU and ILE Nonpolar uncharged residues CYS, MET and PRO Aromatic residues PHE, TYR, and TRP

[0086] Table B - Group of Conserved Amino Acid Residue Substitutions

[0087] 1 Alanine (A) Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

[0088] Table C – Other alternative physical and functional classifications of amino acid residues

[0089]

[0090] As used herein, "isolated antibody" refers to an antibody isolated and / or recovered from components of its native environment. Isolated antibodies include in situ antibodies within recombinant cells, since at least one component of the antibody's native environment is absent. In some embodiments, antibodies are purified to a homogeneity and / or purity of 90%, 95%, or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAG, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC) under reducing or non-reducing conditions.

[0091] As used herein, the term "derived from" refers to a compound having a structure derived from a parent compound or protein structure, and whose structure is sufficiently similar to those disclosed herein, and based on that similarity, a person skilled in the art would expect it to exhibit the same or similar properties, activities, and utilities as the claimed compounds. For example, a humanized antibody derived from a mouse antibody refers to an antibody or antibody fragment that shares similar properties with the mouse antibody, such as recognizing the same epitopes, sharing similar VH and VL residues that participate in and / or enhance antibody humanization.

[0092] The term "treatment" refers to any action aimed at improving a patient's health condition, such as the treatment, prevention, mitigation, and delay of a disease or its symptoms. It refers to curative and / or preventative treatment of a disease. Curative treatment is defined as treatment that leads to a cure or that alleviates, improves, and / or eliminates, reduces, and / or stabilizes a disease or its symptoms or the suffering they directly or indirectly cause. Preventative treatment includes treatment that leads to disease prevention and treatment that reduces and / or delays the progression and / or occurrence of a disease or the risk of its occurrence. In some embodiments, the term refers to improving or eradicating a disease, symptom, infection, or related symptoms. In other embodiments, the term refers to minimizing the spread or worsening of cancer. Treatment according to the invention does not necessarily mean 100% or complete cure. Rather, different degrees of treatment exist, in which those skilled in the art consider to have potential benefit or therapeutic effect. Preferably, the term "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a symptom / disease, such as a symptom / disease associated with a PD-1-mediated signal transduction pathway.

[0093] As used herein, the term "symptom" or "disease" refers to an organ, part, structure, or system of the body that malfunctions due to genetic or developmental errors, infection, toxins, nutritional deficiencies or imbalances, toxicity, or adverse environmental factors. Preferably, these terms refer to a monitored symptom or disease, such as a disease that impairs normal physical or mental function. More preferably, the term symptom refers to an immune and / or inflammatory disease affecting animals and / or humans, such as cancer.

[0094] As used herein, the term "immune disease" refers to a condition in a subject characterized by damage to cells, tissues, and / or organs resulting from an immune response in the subject to their own cells, tissues, and / or organs. The term "inflammatory disease" refers to a condition in a subject characterized by inflammation (e.g., chronic inflammation). Autoimmune conditions may or may not be associated with inflammation. Accordingly, inflammation may or may not be caused by an autoimmune condition.

[0095] As used in this article, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system.

[0096] As used herein, the terms “PD-1-associated or related disease,” “PD-1-positive cancer,” or “PD-1-positive infectious disease” are intended to refer to cancer or infectious disease (e.g., caused by viruses and / or bacteria) that is caused by or has symptoms and / or features of PD-1 expression, i.e., any condition caused, aggravated, or associated with increased or decreased PD-1 expression or activity.

[0097] As used in this article, the terms “subject,” “host,” “individual,” or “patient” refer to a person, including adults or children.

[0098] As used herein, a “pharmaceutical composition” refers to a formulation of one or more active agents, such as comprising a bifunctional molecule as described in the invention, and optionally other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the active agent to a living organism. The compositions of the invention can be in forms suitable for any conventional route of administration or use. In one embodiment, “composition” generally refers to a combination of an active agent (e.g., a compound or composition) with a naturally occurring or non-naturally occurring carrier, which is inert (e.g., a detection agent or label) or active, such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc., and includes pharmaceutically acceptable carriers. As referred to herein, “acceptable carrier” or “acceptable transporter” is any known compound or combination of compounds known to those skilled in the art for use in formulating pharmaceutical compositions.

[0099] As used herein, "effective amount" or "therapeutic effective amount" refers to the amount of active agent, alone or in combination with one or more other active agents, required to impart a therapeutic effect to a subject, for example, the amount of active agent required to treat a target disease or condition or to produce the desired effect. "Effective amount" will vary depending on: one or more agents, the disease and its severity, the characteristics of the subject to be treated, including age, physical condition, physique, sex, and weight, duration of treatment, the nature of any concomitant therapies, the specific route of administration, and similar factors within the knowledge and expertise of a healthcare professional. These factors are well known to those skilled in the art and can be resolved through routine experimentation. Generally, the maximum dose of a single component or combination thereof is preferred, i.e., the highest safe dose based on reasonable medical judgment.

[0100] As used herein, the term "medicine" refers to any substance or composition that has the property of curing or preventing a symptom or disease.

[0101] As used herein, the term "combination" refers to the use of more than one therapy (e.g., prophylactic and / or therapeutic agents). The use of the term "combination" does not limit the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a subject with a disease or condition.

[0102] The terms “polynucleotide,” “nucleic acid,” and “nucleic acid sequence” are equivalent and refer to a polymer of nucleotides of any length, such as RNA or DNA or similar molecules. The nucleic acids of this invention (e.g., components or portions of nucleic acids) can be naturally occurring, modified or engineered, isolated, and / or non-natural. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. “Isolated nucleic acid encoding an anti-PD1 antibody” refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including one or more such nucleic acid molecules in a single vector or a separate vector, and one or more such nucleic acid molecules present at one or more locations within a host cell. As used herein, the terms “nucleic acid construct,” “plasmid,” and “vector” are equivalent and refer to nucleic acid molecules used to transfer passenger nucleic acid sequences, such as DNA or RNA, into host cells.

[0103] As used herein, the term "host cell" is intended to include any individual cell or cell culture that may be, or is already, a vector, exogenous nucleic acid molecule, and a polynucleotide encoding the antibody construct of the present invention; and / or a receptor for the antibody construct itself. The material may be introduced into cells via transformation, transfection, or other means. The term "host cell" is also intended to include the progeny or potential progeny of a single cell. Host cells include, for example, bacterial, microbial, plant, and animal cells.

[0104] As used herein, “immune cells” refers to cells associated with 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)) ​​derived from hematopoietic stem cells (HSCs) produced in the bone marrow, and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). Specifically, immune cells may be selected from a non-exhaustive list including B cells, T cells, particularly CD4+ and CD8+ T cells, NK cells, NKT cells, APC cells, dendritic cells, and monocytes. As used herein, “T cells” includes, for example, CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0105] As used herein, the terms “T effector cells,” “T eff,” or “effector cells” describe a group of immune cells that include several types of T cells that respond actively to stimuli (e.g., co-stimuli). In particular, this includes T cells with antigen-eliminating functions (e.g., by producing cytokines that regulate the activation of other cells or through cytotoxic activity). Specifically, this includes CD4+, CD8+, Treg cells, cytotoxic T cells, and helper T cells (Th1 and Th2).

[0106] As used herein, the terms "regulatory T cells," "Treg cells," or "Treg" refer to a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs have immunosuppressive effects, typically inhibiting or downregulating the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are believed to originate from the same lineage as naive CD4 cells.

[0107] The term "exhausted T cells" refers to a population of T cells in a state of dysfunction (i.e., "exhaustion"). T cell exhaustion is characterized by a gradual loss of function, changes in the transcriptional profile, and persistent expression of inhibitory receptors. Exhausted T cells lose their ability to produce cytokines, their high proliferative capacity, and their cytotoxic potential, ultimately leading to their absence. Exhausted T cells are typically characterized by a combination of elevated levels of CD43, CD69, and inhibitory receptors with lower expression of CD62L and CD127.

[0108] The term "immune response" refers to the selective damage, destruction, or elimination of cells or tissues from the body by factors such as lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver against invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmune or pathological inflammation.

[0109] As used herein, the term "antagonist" refers to a substance that blocks or reduces the activity or function of another substance. Specifically, the term refers to an antibody that binds to a cell receptor (e.g., PD-1) serving as a reference substance (e.g., PD-L1 and / or PD-L2), preventing it from producing all or part of its usual biological effects (e.g., establishing an immunosuppressive microenvironment). The antagonist activity of the humanized antibodies according to the invention can be assessed by competitive ELISA.

[0110] As used herein, the term "isolated" means that the material (e.g., antibody, peptide, nucleic acid, etc.) is substantially separated from or enriched relative to other materials that coexist with it in nature. Specifically, an "isolated" antibody is an antibody that has been identified and isolated and / or recovered from components of the natural environment. For example, isolated antibodies are purified (1) by a weight greater than 75% of the antibody as determined by the Lowry method, or (2) by SDS-PAGE under reducing or non-reducing conditions. Isolated antibodies include in situ antibodies from recombinant cells, as at least one component of the antibody's natural environment will be absent. However, typically isolated antibodies are prepared by at least one purification step.

[0111] As used herein, the term “and / or” will be regarded as a specific disclosure of each of two particular features or components, which may or may not contain the other. For example, “A and / or BA” will be regarded as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B as if each were listed separately.

[0112] The term “a / an” can refer to one or more of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless the context clearly describes one or more elements of the element.

[0113] As used herein, the term “about” in conjunction with any and all values ​​(including the lower and upper limits of the numerical range) refers to any value with an acceptable range of deviation 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 “about” at the beginning of a series of values ​​modifies each of those values ​​(i.e., “about 1, 2, and 3” refers to about 1, about 2, and about 3). Furthermore, when the present document describes a list of values ​​(e.g., approximately 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all its intermediate and fractional values ​​(e.g., 54%, 85.4%).

[0114] Anti-PD-1 antibody

[0115] The bifunctional molecule according to the present invention comprises a first entity, the first entity comprising a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof.

[0116] This article provides a humanized antibody that binds to human PD-1. In some aspects, the humanized antibody specifically binds to human PD-1, preferably to the extracellular domain of human PD-1. In some aspects, the humanized 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.

[0117] In some aspects, humanized anti-PD1 antibodies are isolated antibodies, particularly non-natural isolated antibodies. Such isolated humanized anti-PD1 antibodies can be prepared by at least one purification step. In some embodiments, the isolated antibody is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, the anti-PD1 isolated antibody is provided as a solution containing at least 85%, 90%, 95%, 98%, 99%, to 100% antibody by weight, the remainder of which comprises the weight of other solutes dissolved in the solvent.

[0118] Preferably, such antibodies have 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). As used herein, the ability to “block binding” or “block interaction” or “inhibit interaction” 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 ligands PD-L1 and / or PD-L2) to any detectable level.

[0119] Preferably, the anti-PD1 antibody or its antigen-binding fragment is an antagonist of human PD-L1 and / or PD-L2 binding to human PD-1, more preferably an antagonist of human PD-L1 and PD-L2 binding to PD-1.

[0120] In some embodiments, the anti-hPD1 antibody or its 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 some embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0121] The humanized form of the anti-PD1 antibody according to the present invention may comprise any class of immunoglobulin (e.g., IgD, IgE, IgG, IgA, or IgM (or subclasses thereof)), comprising a minimal sequence of an immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding sequence of the antibody) derived from a non-human (e.g., mouse) immunoglobulin targeting human PD-1. Preferably, the humanized anti-hPD-1 antibody according to the present invention is derived from IgG1, IgG2, IgG3, or IgG4, preferably from IgG4.

[0122] Humanized antibodies typically contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from a nonhuman antibody, and the FR (or a portion thereof) is derived from a human or humanized antibody sequence. Alternatively, some FR residues may be replaced to restore or improve antibody specificity, affinity, and / or humanization. Humanized antibodies may also optionally contain at least a portion of a human or humanized constant region (Fc). Methods of antibody humanization are well known in the art; see, for example, Winter and Milstein, Nature, 1991, 349:293-299; Riechmann et al., Nature, 332, p. 323 (1988); Verhoeyen et al., Science, 239, p. 1534 (1988); Rader et al., Proc. Nat. Acad. Sci. USA, 1998, 95:8910- 8915; Steinberger et al., J. Biol. Chem., 2000, 275: 36073-36078; Queen et al., Proc. Natl. Acad. Sci. USA, 1989, 86: 10029-10033; Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633; Kashmiri, SV et al., Methods 36 (2005) 25-34 (Description of SDR(a-CDR) transplantation); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (Description of “surface reshaping”); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (Description of “FR rearrangement”); and Osbourn, J. et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83(2000)252-260 (describes a “guided selection” method for FR rearrangement); and U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, 5,821,337, 7,527,791, 6,982,321, and 7,087,409; and 6,180,370.

[0123] Preferably, the humanized antibody against human PD-1 is a monoclonal antibody.

[0124] CDR

[0125] The "complementarity-determining region" or "CDR" is well-known in the art and refers to the discontinuous sequence of amino acids within the variable region of an antibody that imparts antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described in Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition (1991), “Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol, 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Pluckthun, J. Mol. Biol, 2001, 309:657-70 (“AHo” numbering scheme). Unless otherwise stated, the numbering scheme used to identify a particular CDR in this document is the Kabat numbering scheme.

[0126] The CDR region of the humanized antibody is derived from a mouse antibody and has been optimized to i) provide a safe humanized antibody with a very high level of humanization (better than 85%); and ii) improve antibody properties, particularly greater prepareability when produced in mammalian cells and higher yield in mammalian cells (e.g., COS and HCO cells), while retaining antagonist activity and inhibition of human PD-L1 binding to human PD-1, as their binding affinity (KD) to human PD-1 is less than 10. - 7 M, preferably less than 10 -8 M.

[0127] In one embodiment, the antigen-binding fragment of the antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3; the light chain comprising a variable domain comprising LCDR1, LCDR2, and LCDR3; and a fragment of a heavy chain constant domain. For the fragment of the heavy chain constant domain, it should be understood that the antigen-binding fragment therefore comprises at least a portion of the complete heavy chain constant domain. For example, the heavy chain constant domain may comprise at least C0 of the heavy chain. H 1. At least C of a structural domain or heavy chain H 1 and C H 2. At least C of a structural domain or heavy chain H 1. C H 2 and C H3 structural domains, or at least C of heavy chains H 1. At least C of a structural domain or heavy chain H 1 and C H 2. At least C of a structural domain or heavy chain H 1. C H 2 and C H 3. Domain Composition. A fragment of the heavy chain constant domain can also be defined as containing at least a portion of the Fc domain of the heavy chain. Therefore, the antigen-binding fragment of the antibody contains the complete Fab portion of the antibody, the complete F(ab')2 portion of the antibody, and the complete Fab' portion of the antibody. The heavy chain constant domain may also contain or be composed of complete heavy chain constant domains, as illustrated in this specification, which describes a plurality of complete heavy chain constant domains. In a particular embodiment of the invention, and when the antigen-binding fragment of the antibody contains a fragment of a heavy chain constant domain (which contains or is composed of a portion of a complete heavy chain constant domain), the heavy chain constant domain fragment may consist of at least 10 amino acid residues; or it may consist of 10 to 300 amino acid residues, particularly 210 amino acid residues.

[0128] In one embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, comprising:

[0129] (i) Heavy chain variable structural domains containing HCDR1, HCDR2, and HCDR3, and

[0130] (ii) A light chain variable structural domain comprising LCDR1, LCDR2 and LCDR3,

[0131] in:

[0132] - 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 of one or more substitutions, one or more additions, one or more deletions and any combination thereof selected from any position other than position 3 of SEQ ID NO:1;

[0133] - 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 of one or more substitutions, one or more additions, one or more deletions and any combination thereof at any position other than positions 13, 14 and 16 of SEQ ID NO:2;

[0134] - The heavy chain CDR3 (HCDR3) comprises or is composed of the amino acid sequence of SEQ ID NO:3, wherein X1 is D or E, and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO:3;

[0135] - The light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO:12, wherein X is G or T, and optionally has one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:12, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and

[0136] - The light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO:15, optionally having one, two, or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and

[0137] - The light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO:16, optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more deletions and any combination thereof at any position other than positions 1, 4 and 6 of SEQ ID NO:16.

[0138] In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, LCDR2, and LCDR3 as specified above and: a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO:3, wherein X1 is D and X2 is selected from T, H, A, Y, N, E, and S, preferably selected from H, A, Y, N, and E; or X1 is E and X2 is selected from T, H, A, Y, N, E, and S, preferably selected from H, A, Y, N, E, and S; optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:3, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO:12, wherein X is G or T, optionally having one, two, or three modifications selected from SEQ ID NO:3, including one, two, or three amino acid sequences of SEQ ID NO:12, including one, two, or three amino acid sequences of SEQ ID NO:12, wherein X is G or T, and optionally having one, two, or three amino acid sequences selected from SEQ ID NO:12, including one, two, or three amino acid sequences of ... one amino acid sequence of SEQ ID NO:12, including one, two, or one amino acid sequence of SEQ ID NO:12, including NO:12 may be modified by one or more substitutions, one or more additions, one or more omissions, or any combination thereof at any position other than positions 5, 6, 10, 11, and 16.

[0139] In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, LCDR2, and LCDR3 as specified above and: a heavy chain CDR3 (HCDR3) comprising or consisting of the amino acid sequence of SEQ ID NO:4, 5, 6, 7, 8, 9, 10, or 11, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:4, 5, 6, 7, 8, 9, 10, or 11, including one or more substitutions, one or more additions, one or more deletions, 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; and a light chain CDR1 (LCDR1) comprising or consisting of the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14, optionally having one, two, or three modifications selected from SEQ ID NO:13 or SEQ ID NO:14. NO:14 may be modified by one or more substitutions, one or more additions, one or more omissions, or any combination thereof at any position other than positions 5, 6, 10, 11, and 16.

[0140] In another embodiment, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, LCDR2, and LCDR3 as specified above, and:

[0141] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:4, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:4, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0142] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:5, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:5, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0143] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:6, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:6, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0144] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:7, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:7, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0145] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:8, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:8, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0146] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:9, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:9, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0147] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:10, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:10, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0148] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:11, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:11, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:13, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:13, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0149] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:4, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:4, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:14, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0150] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:5, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO:5; Light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO:14; or

[0151] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:6, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:6, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:14, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0152] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:7, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO:7; light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO:14; or

[0153] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:8, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:8, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:14, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0154] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:9, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO:9; Light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO:14;

[0155] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:10, optionally having one, two, or three modifications selected from positions 2, 3, 7, and 8 of SEQ ID NO:10, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications selected from positions 5, 6, 10, 11, and 16 of SEQ ID NO:14, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; or

[0156] - Heavy chain CDR3 (HCDR3), comprising or consisting of the amino acid sequence of SEQ ID NO:11, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 2, 3, 7, and 8 of SEQ ID NO:11; Light chain CDR1 (LCDR1), comprising or consisting of the amino acid sequence of SEQ ID NO:14, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more deletions, or any combination thereof at any position other than positions 5, 6, 10, 11, and 16 of SEQ ID NO:14.

[0157] In particular, the modification is a substitution, especially a conservative substitution.

[0158] In one embodiment, the anti-human PD-1 antibody or its antigen-binding fragment comprises or is composed of the following:

[0159] (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, and X2 is selected from T, H, A, Y, N, E, and S, preferably from H, A, Y, N, and E; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (where X is G or T), CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0160] (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, and X2 is selected from T, H, A, Y, N, and E, preferably from H, A, Y, N, and E; or wherein X1 is E, and X2 is selected from T, H, A, Y, N, E, and S, preferably from H, A, Y, N, E, and S; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (where X is G or T), CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0161] (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, and X2 is selected from T, H, A, Y, N, and E, preferably from H, A, Y, N, and E; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (where X is G or T), CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0162] (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 E and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E and S; and (ii) a light chain comprising CDR1 of SEQ ID NO:12 (where X is G or T), CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO:16.

[0163] In another embodiment, the anti-human PD-1 antibody or its antigen-binding fragment comprises or substantially comprises: (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.

[0164] In another embodiment, the anti-human PD-1 antibody or its antigen-binding fragment comprises or is substantially composed of the following:

[0165] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:4; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0166] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:5; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0167] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:6; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0168] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:7; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0169] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:8; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0170] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:9; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0171] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:10; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0172] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:11; and (ii) a light chain comprising CDR1 of SEQ ID NO:13, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0173] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:4; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0174] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:5; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0175] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:6; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0176] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:7; (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0177] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:8; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0178] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:9; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0179] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:10; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15, and CDR3 of SEQ ID NO:16; or

[0180] (i) a heavy chain comprising CDR1 of SEQ ID NO:1, CDR2 of SEQ ID NO:2 and CDR3 of SEQ ID NO:11; and (ii) a light chain comprising CDR1 of SEQ ID NO:14, CDR2 of SEQ ID NO:15 and CDR3 of SEQ ID NO:16.

[0181] frame

[0182] As used herein, the term “antibody framework” refers to a portion of the variable domain, VL and / or VH, which acts as a scaffold for the antigen-binding loop (CDR) of the variable domain.

[0183] In one embodiment, the anti-PD1 antibody or antigen-binding fragment according to the present invention comprises a frame region, particularly heavy chain variable region frame regions (HER) HFR1, HFR2, HFR3 and HFR4, and light chain variable region frame regions (LFR) LFR1, LFR2, LFR3 and LFR4.

[0184] Preferably, the anti-PD1 antibody or antigen-binding fragment according to the present invention comprises a human or humanized frame region. For purposes herein, a “human receptor frame” is a frame comprising an amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from a human immunoglobulin frame or a human common frame as defined below. Human receptor frames derived from human immunoglobulin frames or human common frames may contain the same amino acid sequence, or may contain variations in the amino acid sequence. 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 receptor human frame is sequence-equivalent to the VL human immunoglobulin frame sequence or the human common frame sequence. A “human common frame” is a frame representing the most frequently occurring amino acid residues in a series of human immunoglobulin VL or VH frame sequences.

[0185] Specifically, the anti-PD1 antibody or antigen-binding fragment comprises heavy chain variable framework regions (HFRs) HFR1, HFR2, HFR3, and HFR4, which respectively contain the amino acid sequences of SEQ ID NO:41, 42, 43, and 44, and optionally have one, two, or three modifications selected from positions 27, 29, and 32 of HFR3 (i.e., SEQ ID NO:43), including one or more substitutions, one or more additions, one or more deletions, and any combination thereof. Preferably, the anti-PD1 antibody or antigen-binding fragment comprises 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.

[0186] Alternatively, the anti-PD1 antibody or antigen-binding fragment comprises light chain variable frame regions (LFRs) LFR1, LFR2, LFR3, and LFR4, which respectively contain the amino acid sequences of SEQ ID NO:45, 46, 47, and 48, and optionally have one, two, or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof. Preferably, the humanized anti-PD1 antibody or antigen-binding fragment comprises LFR1 of SEQ ID NO:45, LFR2 of SEQ ID NO:46, LFR3 of SEQ ID NO:47, and LFR4 of SEQ ID NO:48.

[0187] VH-VL

[0188] The VL and VH domains of the anti-hPD1 antibody according to the present invention may comprise four frame regions interrupted by three complementarity-determining regions, preferably operably linked in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (from the amino terminus to the carboxyl terminus).

[0189] In one embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0190] (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 T, H, A, Y, N, E and S, preferably selected from H, A, Y, N and E; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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;

[0191] (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally has one, two or three modifications of one or more substitutions, one or more additions, one or more 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.

[0192] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0193] (a) A heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D and X2 is selected from T, H, A, Y, N, E, preferably selected from H, A, Y, N, E; or X1 is E and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E and S; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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;

[0194] (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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.

[0195] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0196] (a) Heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:17, wherein X1 is D and X2 is selected from T, H, A, Y, N, E, preferably selected from H, A, Y, N, E, and optionally has one, two, or three modifications of one or more substitutions, one or more additions, one or more 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;

[0197] (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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.

[0198] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0199] (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 T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E and S; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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;

[0200] (b) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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.

[0201] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0202] (a) A heavy chain variable region (VH) comprising or consisting of an amino acid sequence of SEQ ID NO:18, 19, 20, 21, 22, 23, 24 or 25, optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more deletions or any combination thereof at any position other than 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, 19, 20, 21, 22, 23, 24 or 25;

[0203] (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, optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more 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.

[0204] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0205] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:18, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:18, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:18, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:27 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0206] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:19, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:19, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:19, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0207] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:20, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:20, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0208] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:21, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:21, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:21, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0209] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:22, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:22, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0210] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:23, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:23, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0211] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:24, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:24, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0212] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:25, including one, two, or three modifications selected from positions SEQ ID NO:25, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:27, optionally having one, two, or three modifications selected from positions SEQ ID NO:25, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and ( Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:27; or

[0213] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:18, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:18, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0214] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:19, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:19, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0215] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:20, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:20, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:20, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0216] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:21, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:21, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0217] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:22, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:22, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0218] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:23, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:23, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:23, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0219] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:24, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:24, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0220] (a) a heavy chain variable region (VH) comprising or consisting of the amino acid sequence of SEQ ID NO:25, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions ...c) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:25, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (d) a light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:28, optionally having one, two, or three modifications selected from positions SEQ ID NO:25, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain variable region (VL) comprising or consisting of the amino acid sequence NO:28 may be modified by one or more substitutions, one or more additions, one or more omissions, or 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.

[0221] In particular, the modification is a substitution, especially a conservative substitution.

[0222] CH-CL

[0223] In one embodiment, the heavy chain (CH) and light chain (CL) comprise the VL and VH sequences as described above.

[0224] In a particular embodiment, the anti-human PD-1 antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0225] (a) a heavy chain comprising an amino acid sequence selected from SEQ ID NO:29, 30, 31, 32, 33, 34, 35 or 36, or consisting of an amino acid sequence selected from SEQ ID NO:29, 30, 31, 32, 33, 34, 35 or 36, optionally having one, two or three modifications at 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, respectively; and (b) a light chain comprising SEQ ID NO:37 or SEQ ID NO:29, 30, 31, 32, 33, 34, 35 or 36, respectively; and (b) a light chain comprising SEQ ID NO:37 or SEQ ID NO:29, 30, 31, 32, 33, 34, 35 or 36, respectively; The amino acid sequence of NO:38 or the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:38 may optionally have one, two or three modifications, including one or more substitutions, one or more additions, one or more 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.

[0226] In another embodiment, the anti-human PD-1 humanized antibody or its antigen-binding fragment contained in the bifunctional molecule comprises:

[0227] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:29, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:29, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:29, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0228] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:30, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:30, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:30, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0229] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:31, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:31, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:31, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0230] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:32, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:32, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:32, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... Modifications of one or more substitutions, one or more additions, one or more 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 NO:37;

[0231] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:33, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:33, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:33, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0232] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:34, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from positions SEQ ID NO:34, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:34, including one, two, or three modifications selected from positions 7, 16, 17, 20, 33, 38, 43, 46, 62, 63, 65, 69, 73, 76, 78, 80, 84, 85, 88, 93, 95 Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0233] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:35, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:35, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:35, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:35, including one, two, or three amino acid sequences selected from SEQ ID NO:35; Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0234] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:36, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:36, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:37, optionally having one, two, or three modifications selected from SEQ ID NO:36, including ... Modifications consisting of one or more substitutions, one or more additions, one or more 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 NO:37; or

[0235] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:29, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:29, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:29, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... NO:38 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0236] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:30, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from positions 3, 4, 7, 14, 17, 18, 28, 29, and SEQ ID NO:38 of SEQ ID NO:38. Modifications consisting of one or more substitutions, one or more additions, one or more deletions, and any combination thereof at any position other than 33, 34, 39, 42, 44, 50, 81, 88, 94, 97, 99, and 105 of IDNO:38; or

[0237] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:31, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:31, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:31, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... NO:38 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0238] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:32, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:32, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:32, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof ... NO:38 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0239] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:33, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:33, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:33, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:33, including one, two, or three amino acid sequences selected from SEQ ID NO:33; NO:38 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0240] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:34, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from positions SEQ ID NO:34, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:34, including ... NO:38 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0241] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:35, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:35, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:35, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:35, including one, two, or three modifications selected from SEQ ID NO:35, including one, two, or three modifications NO:38 may be modified by one or more substitutions, one or more additions, one or more deletions, or 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; or

[0242] (a) a heavy chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:36, optionally having one, two, or three modifications selected from 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:36, including one, two, or three modifications selected from 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; and (b) a light chain comprising or consisting of an amino acid sequence selected from SEQ ID NO:38, optionally having one, two, or three modifications selected from SEQ ID NO:36, including ... NO:38 may be modified by one or more substitutions, one or more additions, one or more omissions, or 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.

[0243] Preferably, the modification is a substitution, particularly a conservative substitution.

[0244] Fc and hinge area

[0245] Several studies developing therapeutic antibodies have led to the engineering of the Fc region to optimize antibody properties, resulting in molecules better suited to their desired pharmacological activities. The Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP). Several mutations at the interface between the CH2 and CH3 domains, such as T250Q / M428L, M252Y / S254T / T256E, and H433K / N434F, have been shown to increase affinity for FcRn binding and increase the in vivo half-life of IgG1. However, increased FcRn binding and improved half-life are not always directly correlated. One approach to improving the efficacy of therapeutic antibodies is to increase their serum persistence, thereby allowing for higher circulating levels, reduced dosing frequency, and lower doses. Engineering the Fc region may be desirable to reduce or increase the effector function of the antibody. For antibodies targeting cell surface molecules (especially those on immune cells), the effector function needs to be eliminated. Conversely, for antibodies intended for oncology use, enhancing the effector function can improve therapeutic activity. Four human IgG isotypes bind with varying affinities to activate Fcγ receptors (FcγRI, FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the first component of complement (C1q), producing vastly different effector functions. The binding of IgG to FcγR or C1q depends on residues located in the hinge region and the CH2 domain. Both regions of the CH2 domain are crucial for FcγR and C1q binding and have unique sequences in IgG2 and IgG4.

[0246] The humanized antibody according to the invention optionally comprises an immunoglobulin constant region (Fc), typically a portion of the immunoglobulin constant region of a human or humanized immunoglobulin. Preferably, the Fc region is a portion of the humanized anti-hPD-1 antibody described herein. The humanized anti-hPD1 antibody or its antigen-binding fragment contained in the bifunctional molecule of the invention may comprise an immunoglobulin constant region, or a fragment, analog, variant, mutant, or derivative of said constant region. As is well known to those skilled in the art, the selection of IgG isotypes of the heavy chain constant domain focuses on whether a specific function is required and whether a suitable in vivo half-life is required. For example, antibodies designed for the selective eradication of cancer cells typically require an active isotype that allows complement activation and effector-mediated cell killing via antibody-dependent cell-mediated cytotoxicity. Both human IgG1 and IgG3 (with shorter half-lives) isotypes meet these criteria, particularly human IgG1 isotypes (wild-type and variants). Specifically, depending on the IgG isotype of the heavy chain constant domain (particularly human wild-type and variant IgG1 isotypes), the humanized anti-hPD1 antibody described in this invention can be cytotoxic to PD-1-expressing cells via CDC, ADCC, and / or ADCP mechanisms. In fact, the crystallizable fragment (Fc) region interacts with various helper molecules to mediate indirect effector functions such as antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0247] In a preferred embodiment, the constant region is derived from the human immunoglobulin heavy chain, for example, IgG1, IgG2, IgG3, IgG4, or other classes. In a further aspect, the human constant region is selected from IgG1, IgG2, IgG3, and IgG4. Preferably, the humanized anti-PD1 antibody comprises an IgG1 or IgG4 Fc region.

[0248] In one particular aspect, the humanized anti-PD1 antibody comprises a human IgG1 Fc region, optionally having a substitution or combination of substitutions selected from: T250Q / M428L; M252Y / S254T / T256E+H433K / N434F; E233P / L234V / L235A / G236A+A327G / A330S / P331S; E333A; S239D / A330L / I332E; P257I / Q3 11; K326W / E333S; S239D / I332E / G236A; N297A; L234A / L235A; N297A+M252Y / S254T / T256E; K444A and K322A, preferably selected from the following: N297A, which is optionally combined with M252Y / S254T / T256E, and L234A / L235A.

[0249] More preferably, the humanized anti-hPD1 antibody comprises an IgG4 Fc region, optionally having a substitution or combination of substitutions selected from: S228P; L234A / L235A, S228P+M252Y / S254T / T256E, and K444A. Even more preferably, the humanized anti-hPD1 antibody contained in the bifunctional molecule according to the invention comprises an IgG4 Fc region having an S228P that stabilizes the IgG4.

[0250] In one embodiment, the anti-PD1 antibody comprises a truncated Fc region or a fragment of an Fc region. In one embodiment, the constant region comprises a CH2 domain. In another embodiment, the constant region comprises CH2 and CH3 domains or comprises a hinge-CH2-CH3 domain. Alternatively, the constant region may comprise all or part of a hinge region, a CH2 domain, and / or a CH3 domain. In a preferred embodiment, the constant region comprises a CH2 and / or CH3 domain derived from the human IgG4 heavy chain.

[0251] In another embodiment, the constant region comprises at least a portion of a CH2 domain and a hinge region. The hinge region may be derived from immunoglobulin heavy chains, such as IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable classes, whether mutated or unmutated. More preferably, the hinge region is derived from human IgG1 heavy chain. In one embodiment, the constant region comprises 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 human IgG2 or IgG4 heavy chain, while the hinge region is derived from a modified human IgG1 heavy chain.

[0252] In one implementation, the constant region contains a mutation that reduces affinity for the Fc receptor and diminishes Fc effector function. For example, the constant region may contain a mutation that eliminates glycosylation sites within the constant region of the IgG heavy chain.

[0253] In another embodiment, the constant region comprises at least a portion of the CH2 domain and the hinge region. The hinge region may be derived from the immunoglobulin heavy chain, such as IgG1, IgG2, IgG3, IgG4, or other classes. Preferably, the hinge region is derived from human IgG1, IgG2, IgG3, IgG4, or other suitable classes. The IgG1 hinge region has three cysteine ​​residues, two of which participate in the disulfide bond between the two heavy chains of the immunoglobulin. These identical cysteine ​​residues allow for the formation of efficient and consistent disulfide bonds between the Fc moieties. Therefore, the preferred hinge region of the present invention is derived from IgG1, more preferably from human IgG1. In some embodiments, the first cysteine ​​residue 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 potentially incorrect disulfide bonds during the secretion of the recombinant system. A suitable hinge region may be derived from the IgG2 hinge; the first two cysteine ​​residues are each preferably mutated to another amino acid. It is known that the hinge region of IgG4 cannot efficiently form interchain disulfide bonds. However, a suitable hinge region for the present invention may be derived from the IgG4 hinge region, preferably containing a mutation that enhances the correct formation of disulfide bonds between portions derived from the heavy chain (Angal S, et al., (1993) Mol. Immunol., 30:105-8). More preferably, the hinge region is derived from the human IgG4 heavy chain.

[0254] 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 IgG4 heavy chain, while the hinge region is derived from a modified human IgG1 heavy chain.

[0255] According to the present invention, the constant region may contain CH2 and / or CH3 domains and a hinge region derived from different antibody isotypes, i.e., hybridization constant regions. 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, a mutant hinge region from another IgG subclass may be used in the hybridization constant region. For example, a mutant form of the IgG4 hinge that allows efficient disulfide bonding between the two heavy chains may be used. The mutant hinge may also be derived from the IgG2 hinge, wherein the first two cysteine ​​residues are each mutated to another amino acid. The assembly of such hybridization constant regions has been described in U.S. Patent Publication No. 20030044423, the disclosure of which is incorporated herein by reference.

[0256] In one implementation, the constant region may contain CH2 and / or CH3 having one of the mutations described in Table D below or any combination thereof.

[0257] Table D: Suitable human-engineered Fc domains for antibodies. The residues in the heavy chain constant region are numbered based on EU numbers (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html#refs ).

[0258]

[0259]

[0260] In some embodiments, amino acid modifications can be introduced into the Fc region of the antibody provided herein to produce Fc region variants. In some embodiments, the Fc region variants possess some, but not all, effector functions. Such antibodies may be useful, for example, in applications where the in vivo half-life of the antibody is important, but some effector functions are unnecessary or detrimental. Examples of effector functions include complement-dependent cytotoxicity (CDC) and antibody-guided complement-mediated cytotoxicity (ADCC). Numerous substitutions or replacements or deletions that alter effector functions are well known in the art.

[0261] 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 glycosylation sites within the IgG heavy chain constant region. Preferably, the CH2 domain contains a mutation that eliminates glycosylation sites within the CH2 domain.

[0262] In one embodiment, the anti-hPD1 according to the present invention has a heavy chain constant structural domain of SEQ ID NO.39 and / or a light chain constant structural domain of SEQ ID NO.40, particularly the heavy chain constant structural domain of SEQ ID NO.39 and the light chain constant structural domain of SEQ ID NO.40.

[0263] In another embodiment, the anti-hPD1 according to the present invention has a heavy chain constant structural domain of SEQ ID NO:57 and / or a light chain constant structural domain of SEQ ID.40, particularly the heavy chain constant structural domain of SEQ ID NO:57 and the light chain constant structural domain of SEQ ID.40.

[0264]

[0265]

[0266] Table E: Examples of heavy chain constant domains and light chain constant domains suitable for humanized antibodies according to the present invention.

[0267] Amino acid alterations near the junction of the Fc and non-Fc regions can significantly increase the serum half-life of the Fc molecule (PCT Publication No. WO 01 / 58957). Therefore, the linker region of the protein or polypeptide of the present invention may contain alterations within approximately 10 amino acids relative to the naturally occurring sequences of immunoglobulin heavy chains and erythropoietin, preferably located at the linker site. These amino acid changes can cause an increase in hydrophobicity. In one embodiment, the constant region is derived from an IgG sequence in which a C-terminal lysine residue has been substituted. Preferably, the C-terminal lysine of the IgG sequence is substituted with a non-lysine (e.g., alanine or leucine) to further increase the serum half-life. In particular, the K444 amino acid in the IgG1 or IgG4 domain may be substituted with alanine to reduce proteolytic cleavage. Then, in one embodiment, the anti-PD1 antibody comprises at least one other amino acid substitution consisting of K444A.

[0268] In one embodiment, the anti-PD1 antibody includes additional cysteine ​​residues in the C-terminal domain of the IgG to generate additional disulfide bonds, potentially limiting the flexibility of the bifunctional molecule.

[0269] In some embodiments, the antibody can be modified to increase, decrease, or eliminate the degree of glycosylation.

[0270] Humanization

[0271] For the purposes of this invention, as described in Gao SH, Huang K, Tu H, Adler A S. BMC Biotechnology, 2013:13:55, "humanization" is measured using a T20 scoring analyzer to quantify the humanization of the variable region of a monoclonal antibody.

[0272] A web-based tool is provided to calculate the T20 score of antibody sequences using the T20 cutoff value database (http: / / abAnalyzer.lakepharma.com). In calculating the T20 score, input VH, VK, or VL variable region protein sequences are first assigned Kabat numbers, and CDR residues are identified. The blastp protein-protein BLAST algorithm is used to compare the full-length sequence or frame-only sequence (with CDR residues removed) with each sequence in the corresponding antibody database. Sequence identity is separated between each pair of comparisons, and after analyzing each sequence in the database, sequences are sorted from highest to lowest based on sequence identity with the input sequence. The average identity percentage of the top 20 matching sequences is used to obtain the T20 score.

[0273] The T20 humanization score is a commonly used parameter in the field of antibody humanization, first disclosed by Gao et al. (BMC Biotechnol, 2013, 13, 55). The T20 humanization score is frequently used to define humanized antibodies in patent applications (e.g., WO15161311, WO17127664, WO18136626, WO18190719, WO19060750, or WO19170677).

[0274] For each chain type (VH, VK, VL) and sequence length (full length or frame only) in the "All People Database," the T20 scoring analyzer was used to score each antibody sequence against the corresponding database. The T20 scores of the top 20 matching sequences were obtained after excluding the input sequence itself (since sequence 1 is always the input antibody itself, the identity percentages of sequences 2 through 21 were averaged). The T20 scores of each group were sorted from highest to lowest. For most sequences, the score reduction was roughly linear; however, the T20 scores of the last ~15% of antibodies began to drop sharply. Therefore, the last 15% of sequences were removed, and the remaining sequences were used to form the T20 cutoff values ​​in the database, where the T20 score cutoff value indicates the lowest T20 score for the sequence in the new database.

[0275] As used herein, a “humanized antibody” is an antibody having a T20 humanization score of at least 80% or at least 85%, more preferably at least 88%, or even more preferably at least 90%, most preferably between 85% and 95%, and more preferably between 88% and 92%.

[0276] Therefore, the humanized anti-PD1 antibody according to the present invention contained in the bifunctional molecule has a T20 humanization score of at least 80% or at least 85%, more preferably at least 88%, and even more preferably at least 90%, most preferably between 85% and 95%, and more preferably between 88% and 92%.

[0277] peptide linkers

[0278] This invention includes a bifunctional molecule that may contain a peptide linker between the humanized anti-PD-1 antibody or a fragment thereof and the immunotherapeutic agent. The peptide linker typically has sufficient length and flexibility to ensure that the two protein elements connected to it have sufficient spatial freedom to perform their functions and to avoid the influence of α-helical and β-sheet formation on the stability of the recombinant bifunctional molecule.

[0279] In one aspect of this disclosure, the humanized anti-hPD1 antibody is preferably linked to an immunotherapeutic agent via a peptide linker. In other words, the present invention relates to a bifunctional molecule comprising an anti-PD1 antibody or an antigen-binding fragment thereof as detailed herein, the chain of which, for example, a light chain or a heavy chain or a fragment thereof, preferably a heavy chain or a fragment thereof, is linked to an immunotherapeutic agent via a peptide linker. As used herein, the term "linker" refers to a sequence that links the immunotherapeutic agent and at least one amino acid sequence portion of the anti-PD-1 immunoglobulin. Such a linker can be used to prevent steric hindrance. The length of the linker is typically 3-44 amino acid residues. Preferably, the linker has 3-30 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.

[0280] In one embodiment, the present invention relates to a bifunctional molecule comprising a humanized anti-PD-1 antibody or an antigen-binding fragment thereof as defined above and an immunotherapeutic agent, wherein the C-terminus of the antibody chain, such as a light chain or a heavy chain, preferably a heavy chain, and even more preferably a heavy chain or a light chain, is linked to the immunotherapeutic agent via a peptide linker, preferably to the N-terminus of the immunotherapeutic agent.

[0281] In one particular aspect, the present invention relates to a bifunctional molecule comprising a humanized anti-hPD-1 antibody or an antigen-binding fragment thereof as defined above, wherein the immunotherapeutic agent is preferably linked via a peptide linker to the C-terminus of the heavy chain of the antibody (e.g., the C-terminus of the constant structural domain of the heavy chain).

[0282] In one embodiment, the present invention relates to a bifunctional molecule comprising a humanized anti-PD-1 antibody or an antigen-binding fragment thereof as defined above, wherein the immunotherapeutic agent is preferably linked to the C-terminus of the light chain of the antibody (e.g., the C-terminus of the constant structural domain of the light chain) via a peptide linker.

[0283] The adapter sequence can be a naturally occurring or non-natural sequence. If used for therapeutic purposes, the adapter is preferably non-immunogenic in subjects to whom the bifunctional molecule is administered. A useful set of adapter sequences are those derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Other examples are polyalanine adapter sequences. Other preferred examples of adapter sequences are Gly / Ser adapters of varying lengths, including (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser, and (Gly3Ser2)3, particularly (Gly4Ser)3.

[0284] In one embodiment, the connector contained in the bifunctional molecule is selected from (Gly4Ser)4, (Gly4Ser)3, (Gly4Ser)2, Gly4Ser, Gly3Ser, Gly3, Gly2ser and (Gly3Ser2)3, preferably (Gly4Ser)3.

[0285] In one embodiment, the present invention relates to a bifunctional molecule comprising a humanized anti-PD-1 antibody or a fragment thereof as defined above, wherein the antibody or fragment thereof is linked to an immunotherapeutic agent via a linker sequence preferably selected from (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, and even more preferably selected from (GGGGS)3.

[0286] Preferably, the heavy chain of the anti-PD-1 antibody, particularly the C-terminus, is genetically fused to the N-terminus of the immunotherapeutic agent 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.

[0287] Preferably, the C-terminus of the heavy chain of the anti-PD-1 antibody, and more preferably the light chain of the anti-PD-1 antibody, is genetically fused to the N-terminus of the immunotherapeutic agent 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.

[0288] Immunotherapy agents

[0289] In this invention, the bifunctional molecule comprises a humanized anti-hPD-1 antibody or a fragment thereof as described above, preferably linked to an immunotherapeutic agent via a peptide linker. Such humanized antibodies may also be referred to as "bifunctional antibodies" because they possess two therapeutic effects: a first effect resulting from the interaction between the humanized anti-hPD-1 antibody and hPD-1, and a second effect resulting from the interaction between the immunotherapeutic agent and its ligand or receptor.

[0290] As used herein, the term "immunotherapy agent" refers to an agent that has an effect on the immune system (particularly an inhibitory or stimulatory effect, preferably a stimulatory effect). The agent may be, for example, a molecule that affects the activation or inhibition of cells of the immune system. For example, the immunotherapy agent may be selected from: T-cell growth factors, particularly growth factors that increase the number and pool of naive T cells, growth factors that increase the number of dendritic cells (DCs), agonists that activate DCs and other antigen-presenting cells (APCs), adjuvants that allow and enhance cancer vaccines, agonists that activate and stimulate T cells, T-cell checkpoint blockade inhibitors, T-cell growth factors that increase the growth and survival of immune T cells, and immunosuppressive cytokines derived from cancer cells and immune cells. Preferably, the immunotherapy agent is a peptide, polypeptide, or protein. In one embodiment, the immunotherapy agent is a non-antibody entity or portion. The immunotherapy agent may also consist of one or more other binding molecules, antibody-binding mimics, receptors or their extracellular domains, receptor ligands, or fragments thereof with the same functional activity. As used herein, "antibody mimics" refer to organic compounds that, like antibodies, specifically bind to antigens but are structurally unrelated to antibodies. They are typically peptides or proteins with a molar mass of about 3 to 20 kDa. Examples of antibody mimics include affiliin, affimier, affitin, anticalin, and avimer. The immunotherapeutic agents can be mutated or altered to change their biological activity, such as increasing, decreasing, or completely inhibiting it.

[0291] In one embodiment, the immunotherapeutic agent is present in a fragment thereof. Preferably, such a fragment preserves the biological activity of the immunotherapeutic agent.

[0292] In one embodiment, the size of the immunotherapeutic agent or its fragment is at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, or at least 50 kDa. Preferably, the size of the immunotherapeutic agent or its fragment is between 10 kDa and 50 kDa, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 kDa and 20 kDa, between 20 kDa and 50 kDa, between 20 kDa and 40 kDa, or between 20 kDa and 30 kDa.

[0293] In particular, immunotherapeutic agents useful in the context of this invention are selected from immune checkpoint blockers or activators, especially adaptive immune cells (T or B lymphocytes), therapeutic vaccines (DNA, RNA, or peptide vaccines), or immunoconjugates, such as antibody-drug conjugates. In one approach, the immunotherapeutic agent is a T cell inhibitory checkpoint receptor protein on T cells (e.g., CTLA-4, BTLA, LAG-3, TIM-3, and LAIR1). In another approach, the immunotherapeutic agent is an antireceptor or ligand on antigen-presenting cells and tumor cells (which selects some of these antireceptors for its own immune evasion), such as B7-DC, HVEM, TIM-4, B7-H3, or B7-H4.

[0294] Other suitable immunotherapeutic agents according to the invention may bind to, but are not limited to, hormone receptors (e.g., estrogen, progesterone), cytokine receptors (i.e., type I, such as growth hormone receptor, prolactin, erythropoietin; type II; members of the immunoglobulin superfamily), such as interleukin-1; tumor necrosis factor receptor family, such as CD27, CD30, CD40; chemokine receptors, such as interleukin-8, CCR1, CXCR4; transforming growth factor (TGF) β receptor); cell adhesion molecules (e.g., integrins); and vascular endothelial growth factor (VEGF) receptors (e.g., neurotropic (NRP) receptors, such as NRP1, NRP2).

[0295] Preferably, the immunotherapeutic agent is derived from or originates from a human being.

[0296] Preferably, the immunotherapeutic agent is selected from tumor-targeting peptides, cytokines, cytokine receptor co-stimulatory molecules, inhibitory or co-inhibitory molecules, preferably type I or II human transmembrane immune proteins, even more preferably their extracellular domains, molecular chaperone inhibitors (e.g., HSP90 inhibitors) or microtubule inhibitors (e.g., taxanes) and / or stabilizers or DNA replication inhibitors or any anticancer agent or any derivatives and / or analogs thereof to provide additional therapeutic benefits.

[0297] In a particular embodiment of the invention, the immunotherapeutic agent is a molecule that targets an Fc receptor (e.g., human FcγRI (CD64) or human Fcα receptor (CD89)). Therefore, the invention includes bispecific molecules capable of binding to both effector cells expressing FcγR or Fcα (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)) and target cells expressing PD-1. These bifunctional molecules target PD-1-expressing cells to effector cells and trigger Fc receptor-mediated effector cell activities, such as phagocytosis by PD-1-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion production.

[0298] For example, the immunotherapeutic agents may be selected from a non-exhaustive list in Table F below, which includes ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, connexin-1, connexin-2, connexin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, IL6RA, IL-1R1, IL-1RAcP, IL6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD155, CD113, VISTA, CD244, OX40, SIRPalpha, CD80, CD24, Sig lec-10,Fas,IL15RA,SIRB1,SIRB2,LTBR,IL21R,GITR,CD40L,OX40L,FasL,TRAIL,TNF,LIGHT,APRIL,GITRL,CD30,CD70,CD40,CD27,CD30,CD153,RANK,CLEC3A,CLEC4A,CLEC4E,CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN,, LT-alpha, LT-beta, 4-1BBL, MINCLE, TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL12B, IL-15, IL-21 and IL-18.

[0299] In a very specific embodiment, the immunotherapeutic agent is interleukin-2 (IL-2), preferably human IL-2 (e.g., disclosed under UniProt accession number P60568) or a mutant or variant thereof. IL-2 can be mutated in various ways to reduce its toxicity and / or increase its efficacy. Hu et al. (Blood 101, 4853-4861 (2003), U.S. Patent Publication No. 2003 / 0124678) replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate the vascular permeability activity of IL-2. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) mutated asparagine 88 to arginine to enhance the selectivity of T cells for NK cells. Heaton et al. (Cancer Res 53,2597-602 (1993); US Patent No. 5,229,109) introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of pro-inflammatory cytokines by NK cells. Gillies et al. (US Patent Publication No. 2007 / 0036752) substituted three residues of IL-2 that promote affinity for the intermediate-affinity IL-2 receptor (Asp20Thr, Asn88Arg, and Gln126Asp) to reduce VLS. Gillies et al. (WO 2008 / 0034473) also mutated the IL-2-CD25 interface by substituting the amino acid Arg38Trp and Phe42Lys mutations to reduce interaction with CD25 and activation of Treg cells, thereby improving efficacy. In one aspect, the immunotherapeutic agent is, for example, an IL-2 mutant as described in WO2012 / 107417 or WO 2018 / 184964. Human IL-2 (hIL-2) mutants with reduced affinity for CD25 can be generated, for example, by amino acid substitutions at amino acid positions 3, 35, 38, 42, 43, 45, or 72, or combinations thereof, corresponding to residue positions in human IL-2. Preferably, the mutant IL-2 is a human IL-2 molecule comprising amino acid substitutions T3A, F42A, Y45A, L72G, and / or C125A, preferably F42A, Y45A, and L72G, more preferably T3A, F42A, Y45A, L72G, and C125A, as disclosed, for example, in WO 2018 / 184964. Even more preferably, the immunotherapeutic agent is an IL-2 mutant having the amino acid sequence shown in SEQ ID NO:58, having substitutions F42A, Y45A and L72G, preferably T3A, F42A, Y45A, L72G and C125A.

[0300] The inventors have observed that when the immunotherapeutic agent is a type I or type II transmembrane protein, the immunotherapeutic agent retains its function when it is transplanted onto the heavy and light chains of the humanized anti-hPD-1 according to the present invention.

[0301] Preferably, the bifunctional molecule comprises a humanized anti-hPD1 antibody linked to a type I or type II transmembrane protein, particularly its extracellular domain. Even more preferably, the bifunctional molecule comprises a type I or type II transmembrane protein, particularly its extracellular domain, linked to the carboxyl terminus of a humanized anti-hPD1 antibody as described herein, preferably via a peptide linker.

[0302] Type I transmembrane proteins are characterized by an N-terminus outside the cell membrane and a C-terminus inside the cell membrane. Type I transmembrane proteins typically include members of the immunoglobulin superfamily (CD4, CD8, CD28, CTLA4, CD86, etc.), receptor kinases (TGF-β receptor, EGFR, VEGFR, PDGFR, HGF receptor, etc.), and cytokine receptors (TNF receptor, RANK, IL-6 receptor, CSF1 receptor, c-kit, etc.). Upon binding to their corresponding ligands, type I transmembrane proteins can induce a variety of biological responses. There are no particular limitations on the type I transmembrane proteins used in this invention. Therefore, all biologically active type I transmembrane proteins can be used in this invention. For example, type I transmembrane proteins can be selected from a non-exhaustive list including: ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, connexin-1, connexin-2, connexin-3, NELC-5, TLT-2, LFA-3, TIM3, TIM4, LAIR1, SIRPG, IL10R, I L6RA, IL-1R1, IL-1RAcP, IL6RB, TGFBRII, CSF1R, IL22R, VEGFR1, VEGFR2, VEGFR3, CD111, CD112, CD155, CD1 13, VISTA, CD244, OX40, SIRPalpha, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R and GITR.

[0303] In a preferred embodiment, the bifunctional molecule according to the present invention comprises a type I transmembrane protein, preferably selected from CD86, ICOSL, PD-L1, PD-L2, ICOSL, RANK, VEGFR1, VEGFR2, CTLA4, TGF-pRII, B7-H3, B7-H4, HVEM, BTLA, LAG3, TIM3, VISTA, CD111, CD113, TIGIT, SIRPalpha, CD80 and / or combinations thereof.

[0304] Type II transmembrane proteins are characterized by a C-terminus outside the cell membrane and an N-terminus inside the cell membrane. Typically, type II transmembrane proteins are C-lectins or C-lectin-like receptors. The extracellular domain of these receptors includes a carbohydrate-binding domain (or C-lectin domain, CTLD). Proteins with a carbohydrate-binding domain are involved in a variety of functions, such as cell adhesion, platelet activation, pathogen immunity, and induction of apoptosis. There are no particular limitations on the type II transmembrane proteins used in this invention. Therefore, all biologically active type II transmembrane proteins can be used in this invention. For example, the type I transmembrane proteins may be selected from a non-exhaustive list including: CD40L, OX40L, FasL, TRAIL, TNF, LIGHT, APRIL, GITRL, CD30, CD70, CD40, CD27, CD30, CD153, RANK, CLEC3A, CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, and MINCLE, preferably members of the TNF family.

[0305] In a preferred embodiment, the bifunctional molecule according to the present invention comprises a type II transmembrane protein, preferably selected from OX40L, DECTIN-1, NKG2D, DC-SIGN, 4-1BBL, MINCLE, or a combination thereof.

[0306] In a particular aspect, the immunotherapeutic agent contained in the bifunctional molecule is an immune checkpoint blocker or activator. It should be understood that such immunotherapeutic agents may differ from h-PD1, PD-L1, PD-L2, or molecules that target and / or bind to h-PD1, PD-L1, and / or PDL-2.

[0307] Many immune checkpoint inhibitors or activators are known in the art. In the context of this invention, examples of immune checkpoint inhibitors or activators for adaptive immune cells (B or T lymphocytes) that may be useful are CTLA-4, CD86, CD28, CD40, CD40L, ICOS, ICOS-L, OX40L, GITR, HVEM, BTLA, CD160, LIGHT, TNFRSF25, 2B4, CD48, Tim1, Tim3, Tim4, Gal9, LAG-3, CD40, CD40L, CD70, CD27, VISTA, B7H3, B7H4 (B7x), TIGIT, CD112, HHLA2 (B7-H7), TMIGD2 (CD28H), Butyrophilin-like 2 (BTNL2), their variants and fragments, particularly CD86, OX40L, ICOSL, their variants and fragments.

[0308] Preferably, the bifunctional molecule according to the present invention comprises a humanized anti-PD-1 antibody or its antigen-binding fragment as defined above, which is preferably linked via a peptide linker to an immunotherapeutic agent selected from the group consisting of: CD86, ICOSL, PD-L1, PD-L2, ICOSL, RANK, VEGFR1, VEGFR2, CTLA4, TGF-pRII, B7-H3, B7-H4, HVEM, BTLA, LAG3, TIM3, VISTA, CD111, CD113, TIGIT, OX40L, DECTIN-1, NKG2D, DC-SIGN, and MINCLE.

[0309] In a preferred embodiment, the bifunctional molecule according to the present invention comprises cytokines as the immunotherapeutic agent, wherein the cytokines are preferably selected from TGFβ, IL-1, IL-2, IL-6, IL-10, IL-12A, IL-12B, IL-15, IL-21 and IL-18.

[0310] In one embodiment, the type I transmembrane protein, the type II protein, or the cytokine or a fragment thereof has a size of at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, or at least 50 kDa. Preferably, the immunotherapeutic agent has a size between 10 kDa and 50 kDa, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 kDa and 20 kDa, between 20 kDa and 50 kDa, between 20 kDa and 40 kDa, or between 20 kDa and 30 kDa.

[0311] In one embodiment, the immunotherapeutic agent contained in the bifunctional molecule according to the present invention is selected from Table F below.

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318] Table F. Immunotherapy agents of interest.

[0319] In particular, the bifunctional molecule described herein can be obtained by linking a functional variant or fragment of an immunotherapeutic agent (especially an immune checkpoint blocker or activator) to the humanized anti-hPD-1 antibody described herein. Preferably, the immunotherapeutic agent corresponds to the extracellular domain (ECD) of the immune checkpoint blocker or activator.

[0320] In one embodiment, the present invention relates to a humanized anti-PD-1 antibody or an antigen-binding fragment thereof as defined above, wherein the immunotherapeutic agent comprises or is composed of an extracellular domain (ECD) of a protein selected from the following proteins: CD86, PD-L1, PD-L2, ICOSL, RANK (tumor necrosis factor receptor superfamily member 11A), VEGF-R1, VEGF-R2, CTLA4, B7-H3, B7-H4, HVEM, CD40, CD111, CD112, CD155, CD113, IL1-R1, IL1-RAcP, LFA-3, CD28, ICO S, BTLA, LAG3, TIGIT, VISTA, CD244, OX40, SIRPalpha, CD80, CD24, Siglec-10, Fas, IL15RA, SIRB1, SIRB2, LTBR, ​​IL21R, CD27, TIM3, TIM4, GITR, LAIR1, CD30, OX40L, TGFRII, DECTIN-1, NKG2D, DC-SIGN, LT-alpha, LT-beta, 4-1BBL, MINCLE, CD70, CD40L, CD153, GITRL, BTL-II, and their variants and fragments. Specifically, the size of the fragments of the immunotherapeutic agents according to the invention is less than or equal to 500, 400, 300, 200, 100, or 50 amino acids.

[0321] The reference sequence of the human OX40L extracellular domain used in the embodiments of this application corresponds to the sequence associated with SEQ ID NO:51.

[0322] The reference sequence of the extracellular domain of human ICOS used in the embodiments of this application corresponds to the sequence associated with SEQ ID NO:52.

[0323] The reference sequence for the extracellular domain of human CD86 used in this embodiment corresponds to the sequence associated with SEQ ID NO:53.

[0324]

[0325] Reference sequences for ECDs of G.OX40L, ICOSL, and CD86.

[0326] The bifunctional molecules described in this invention comprise antibodies and fragments thereof, but may also comprise macromolecules, such as artificial proteins, peptides, and any compounds with antigen-binding capabilities that mimic antibodies, referred herein as “antigen-binding antibody mimics.” Such proteins include affitin and anticalin. Affitin is an artificial protein with the ability to selectively bind antigens. Structurally, it is derived from the DNA-binding protein Sac7d, found in *Sacchariformis sumarensis* (a microorganism belonging to the Archaea domain). Affitin libraries can be generated by randomizing the amino acids on the Sac7d binding surface, for example by producing variants with random substitutions of 11 residues corresponding to the Sac7d binding interface. Multiple rounds of ribosome display of the resulting protein libraries can target various targets, such as peptides, proteins, viruses, and bacteria. Affitin, an antibody mimic, is being developed as a tool in biotechnology. It is also used as a specific inhibitor of various enzymes (Krehenbrink et al., J.mol.Biol., 383:5, 2008). Those skilled in the art can use methods known in the art, particularly those disclosed in patent application WO2008068637 and the publications cited above, especially the generation and screening of phage display and / or ribosome display libraries using the antigens disclosed herein. Anticalin is an artificial protein, whether protein or small molecule, capable of binding to antigens. It is derived from antibody mimics of human lipid transport proteins, a family of naturally occurring binding proteins. Anticalin is approximately 8 times smaller, about 180 amino acids in size, and about 20 kDa in mass (Skerra, Febs J., 275:11, 2008). Anticalin phage display libraries have been developed, allowing for screening and selection, particularly for anticalins with specific binding properties. Those skilled in the art can readily develop affitins with desired binding properties using methods known in the art (particularly those disclosed in EP patent EP1270725 B1, US patent US8536307B2 (Schlehuber and Skerra, Biophys. Chem., 96:2-3, 2002) and the publications cited above, particularly the generation of phage display and / or ribosome display libraries and their screening using antigens disclosed herein). Both anticalin and affitins can be produced in many expression systems, including bacterial expression systems. Therefore, the present invention provides affitins, anicalins, and other similar antibody mimics having the characteristics of humanized antibodies described herein, particularly regarding binding to PD-1, inhibition of the interaction between PD-1 and PD-L1 and / or PD-L2, non-inhibition of T cell proliferation, and increase in T cell proliferation, all of which are considered macromolecules of the present invention.All embodiments of antibodies or their fragments disclosed herein are transpositions of macromolecules (particularly antigen-binding mimics) as described in this invention. Currently known protein drugs include growth factors, hormone proteins, enzyme proteins, cytokines, interferons, erythropoietin, and molecules. Except for molecules, other protein drugs are homogeneous proteins containing only one protein component. Although existing molecular drugs (e.g., etanercept and ilonacept) resulting from the mixing of the extracellular domain of a receptor protein with the Fc fragment of human IgG consist of two protein components, they still only function to block the binding of endogenous receptors and their corresponding ligands.

[0327] Bifunctional molecules

[0328] This invention provides a bifunctional molecule comprising, or consisting of, a humanized anti-hPD1 antibody or an antibody fragment thereof and an immunotherapeutic agent, or present in a humanized anti-hPD1 antibody or an antibody fragment thereof and an immunotherapeutic agent, wherein the humanized anti-hPD1 antibody or antibody fragment thereof is covalently linked to the immunotherapeutic agent as a fusion protein. Specifically, the bifunctional molecule according to this invention comprises two entities: a first entity comprising, or substantially consisting of, a humanized anti-hPD1 antibody or a fragment thereof; and a second entity comprising, or substantially consisting of, an immunotherapeutic agent, the two entities optionally linked by a peptide linker.

[0329] Specifically, the bifunctional molecule according to the present invention comprises one, two, three, or four molecules of the immunotherapeutic agent. Specifically, the bifunctional molecule may comprise only one molecule of the immunotherapeutic agent linked to only one light or heavy chain of the anti-PD-1 antibody. The bifunctional molecule may also comprise two molecules of the immunotherapeutic agent linked to the light or heavy chain of the anti-PD-1 antibody. The bifunctional molecule may also comprise two molecules of the immunotherapeutic agent, 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. The bifunctional molecule may also comprise three immunotherapeutic agent molecules, two of which are linked to the light or heavy chain of the anti-PD-1 antibody, and the last one linked to the other chain of the anti-PD-1 antibody. Finally, the bifunctional molecule may also comprise four immunotherapeutic agent molecules, two of which are linked to the light chain of the anti-PD-1 antibody and the other two molecules are linked to the heavy chain of the anti-PD-1 antibody. Therefore, the bifunctional molecule comprises one to four immunotherapeutic agent molecules as disclosed herein.

[0330] In one embodiment, only one light chain contains one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains one immunotherapeutic agent molecule), only one heavy chain contains one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains one immunotherapeutic agent molecule), each light chain contains one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains two immunotherapeutic agent molecules), each heavy chain contains one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains two immunotherapeutic agent molecules), only one light chain and only one heavy chain contain one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains two immunotherapeutic agent molecules), each light chain contains one immunotherapeutic agent molecule and only one heavy chain contains one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains three immunotherapeutic agent molecules), each heavy chain contains one immunotherapeutic agent molecule and only one light chain contains one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains three immunotherapeutic agent molecules), or both the light chain and the heavy chain contain one immunotherapeutic agent molecule (e.g., the bifunctional molecule contains four immunotherapeutic agent molecules).

[0331] In one embodiment, the bifunctional molecule according to the present invention comprises or is composed of the following:

[0332] (a) A humanized anti-human PD-1 antibody or its antigen-binding fragment, comprising:

[0333] (i) Heavy chain variable structural domains, which contain HCDR1, HCDR2, and HCDR3, and

[0334] (ii) Light chain variable structural domains, which include LCDR1, LCDR2, and LCDR3.

[0335] in:

[0336] - 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 of one or more substitutions, one or more additions, one or more deletions and any combination thereof selected from any position other than position 3 of SEQ ID NO:1;

[0337] - 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 of one or more substitutions, one or more additions, one or more deletions and any combination thereof at any position other than positions 13, 14 and 16 of SEQ ID NO:2;

[0338] - The heavy chain CDR3 (HCDR3) comprises or is composed of the amino acid sequence of SEQ ID NO:3, wherein X1 is D or E, and X2 is selected from T, H, A, Y, N, E and S, preferably selected from H, A, Y, N and E; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more deletions and any combination thereof at any position other than positions 2, 3, 7 and 8 of SEQ ID NO:3;

[0339] - The light chain CDR1 (LCDR1) comprises or consists of the amino acid sequence of SEQ ID NO:12, wherein X is G or T, and optionally has one, two or three modifications of one or more substitutions, one or more additions, one or more deletions and any combination thereof at any position other than positions 5, 6, 10, 11 and 16 of SEQ ID NO:12.

[0340] - The light chain CDR2 (LCDR2) comprises or consists of the amino acid sequence of SEQ ID NO:15, optionally having one, two, or three modifications selected from one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and

[0341] - The light chain CDR3 (LCDR3) comprises or consists of the amino acid sequence of SEQ ID NO:16, optionally having one, two, or three modifications at any position other than positions 1, 4, and 6 of SEQ ID NO:16, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and (b) an immunotherapeutic agent, preferably one to four immunotherapeutic agent molecules, or fragments thereof.

[0342] The antibody heavy chain or light chain or fragment thereof is preferably covalently linked to the immunotherapeutic agent as a fusion protein via a peptide linker.

[0343] More specifically, the immunotherapeutic agent may be any immunotherapeutic agent or its extracellular portion previously disclosed in the "Immunotherapeutic Agents" section, and the humanized anti-human PD-1 antibody or its antigen-binding fragment may be any humanized anti-human PD-1 antibody or its antigen-binding fragment as described in the "Anti-PD-1 Antibody" section above.

[0344] Preferably, the N-terminus of the immunotherapeutic agent is optionally linked to the C-terminus of the heavy or light chain of the humanized anti-human PD-1 antibody via a peptide linker. Optionally, such bifunctional molecules include at least one peptide linker connecting the N-terminus of the immunotherapeutic agent to the C-terminus of the heavy or light chain of the humanized anti-human PD-1 antibody, wherein the peptide linker is preferably selected from (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3, and even more preferably (GGGGS)3.

[0345] In another embodiment, the present invention relates to a bifunctional molecule comprising a humanized anti-hPD1 antibody or an antigen-binding fragment thereof, comprising or consisting of the following:

[0346] (i) 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 T, H, A, Y, N, E, and S, preferably selected from H, A, Y, N, and E; optionally having one, two, or three modifications 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, including one or more substitutions, one or more additions, one or more deletions, and any combination thereof; and

[0347] (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more 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.

[0348] The heavy chain and / or light chain are preferably linked to the immunotherapeutic agent (preferably the extracellular domain (or ECD) of the immunotherapeutic agent) at their C-terminus via a peptide linker. Such immunotherapeutic agents are preferably selected from tumor-targeting peptides, cytokines, cytokine receptors, stimulatory or co-stimulatory molecules, inhibitory or co-inhibitory molecules, and preferably immunotherapeutic agents of type I or type II (especially type I) human transmembrane immune proteins.

[0349] In another embodiment, the present invention relates to a bifunctional molecule comprising a humanized anti-hPD1 antibody or an antigen-binding fragment thereof, comprising or consisting of the following:

[0350] (i) 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 T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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;

[0351] (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more 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.

[0352] (iii) Optional peptide linker selected from (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3

[0353] The heavy chain and / or light chain are optionally linked at their C-terminus via a peptide linker to an immunotherapeutic agent selected from the group consisting of (preferably the extracellular domain (or ECD) of the immunotherapeutic agent): ICOSL, CD86, B7H4, B7H3, CD28H, PDL2, PDL1, DNAM, CTLA-4, Lag-3, TIGIT, 2B4, BTLA, HVEM, CD101, connexin-1, connexin-2, connexin-3, NEL. C-5,TLT-2,LFA-3,TIM3,TIM4,LAIR1,SIRPG,IL10R,IL6RA,IL-1R1,IL-1RAcP,IL6RB,TGFBRII,CSF1 R,IL22R,VEGFR1,VEGFR2,VEGFR3,CD111,CD112,CD155,CD113,VISTA,CD244,OX40,SIRPalpha,CD80 ,CD24,Siglec-10,Fas,IL15RA,SIRB1,SIRB2,LTBR,IL21R,GITR,CD40L,OX40L,FasL,TRAIL,TNF,LI GHT,APRIL,GITRL,CD30,CD70,CD40,CD27,CD30,CD153,RANK,CLEC1,CLEC2 / CLE1B,CLEC3A,CLEC4A, CLEC4E, CLEC4L, CLEC51, CLEC6, CLEC7A, NKG2D, BTL-II, TGFRII, DECTIN-1, DC-SIGN, LT-alpha, LT-b eta, 4-1BBL, MINCLE, TGFβ, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12A, IL12B, IL-15, IL-18 and IL-21.

[0354] In a very specific embodiment, the present invention relates to a bifunctional molecule comprising a humanized anti-hPD1 antibody or an antigen-binding fragment thereof, comprising or consisting of the following:

[0355] (i) 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 T, H, A, Y, N, E and S, preferably selected from H, A, Y, N, E; optionally having one, two or three modifications of one or more substitutions, one or more additions, one or more 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;

[0356] (ii) A light chain variable region (VL) comprising or consisting of the amino acid sequence of SEQ ID NO:26, wherein X is G or T, and optionally having one, two, or three modifications of one or more substitutions, one or more additions, one or more 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.

[0357] (iii) Optional peptide linker selected from (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS and (GGGS)3

[0358] The heavy chain and / or light chain are optionally linked to IL-2 or a mutant thereof (preferably as disclosed above, more preferably having the amino acid sequence shown in SEQ ID NO:58) at their C-terminus via a peptide linker.

[0359] The binding of the bifunctional molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blotting. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by using a labeling reagent (e.g., an antibody) that is specific to the complex of interest. For example, the humanized anti-hPD-1 antibody / immunotherapy agent complex can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the immunotherapy agent itself or a ligand of the immunotherapy agent, depending on the nature of the immunotherapy agent.

[0360] In some instances, 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 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 1000 times.

[0361] Preferably, such bifunctional molecules have the ability to block or inhibit the interaction between PD-1 and its ligands (e.g., PD-L1 and / or PD-L2). In some embodiments, the bifunctional molecule inhibits the binding interaction between PD-1 and its ligands (e.g., PD-L1 and / or PD-L2) by at least 50%. In some embodiments, this inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0362] In some embodiments, the bifunctional molecule described herein inhibits or reduces the PD-1 signal transduction pathway by at least 20%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, or at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 1000 times.

[0363] In some instances, the bifunctional molecules described in this article stimulate IFNγ secretion.

[0364] In another embodiment, the bifunctional molecule described herein enhances T cell activation, stimulates IFNg secretion from T cells, and / or stimulates the proliferation of immune cells (e.g., T cells). Preparation of the bifunctional molecule: nucleic acid molecule encoding the bifunctional molecule, recombinant expression vector containing it, and host cell.

[0365] To generate the bifunctional molecules described herein, the humanized anti-hPD1 antibody of this invention is functionally linked to an immunotherapeutic agent. Both entities of the bifunctional molecule are encoded in the same vector and generated as a fusion protein. Therefore, this document also discloses nucleic acids encoding any of the bifunctional molecules described herein, vectors (e.g., expression vectors) or recombinant viruses containing these nucleic acids, and host cells containing said nucleic acids and / or vectors.

[0366] To produce a bifunctional fusion protein secreted in a stable form by mammalian cells, according to the present invention, the nucleic acid sequence encoding the bifunctional molecule is subcloned into an expression vector commonly used for transfecting mammalian cells. General 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 which are incorporated herein by reference) and in “Antibody engineering: a practical guide” from WHFreeman and Company (1992), with comments related to molecule production scattered throughout the various texts.

[0367] Typically, this method includes the following steps:

[0368] (1) Transfect or transform suitable host cells with a polynucleotide encoding the recombinant bifunctional molecule of the present invention or a variant thereof or a vector containing the polynucleotide;

[0369] (2) The host cells are cultured in a suitable culture medium; and

[0370] (3) Optionally, the protein may be isolated or purified from the culture medium or host cells.

[0371] The present invention further relates to nucleic acids encoding the above-mentioned bifunctional molecules, vectors (preferably expression vectors) containing the nucleic acids described in the present invention, genetically engineered host cells transformed with the vectors described in the present invention or directly with sequences encoding the recombinant bifunctional molecules, and methods for producing the proteins described in the present invention by recombination technology.

[0372] The nucleic acid, the vector, and the host cell are described in more detail below.

[0373] Nucleic acid sequence

[0374] The present invention also relates to a nucleic acid molecule that encodes a bifunctional molecule as defined above; or a set of nucleic acid molecules that encodes a bifunctional molecule as defined above.

[0375] The antibody DNA sequence can be synthesized, for example, by RNA amplification from cells that synthesize immunoglobulins, by PCR using cloned immunoglobulins, or by oligonucleotides encoding a known signal peptide amino acid sequence. Preferably, for VH and / or CH, the signal peptide comprises or consists of an amino acid sequence selected from SEQ ID NO:49; and / or for VL and / or CL, the signal peptide comprises or consists of an amino acid sequence selected from SEQ ID NO:50. In particular, the signal peptide is located at the N-terminus of CH, VH, CL, and / or VL.

[0376] These nucleic acids can encode the amino acid sequence of the VL containing the antibody and / or the amino acid sequence of the VH containing the antibody (e.g., the light chain and / or heavy chain of the antibody). Such nucleic acids can be easily isolated and sequenced using standard procedures.

[0377] Specifically, the nucleic acid molecule encoding a bifunctional molecule as defined above comprises:

[0378] - A first nucleic acid molecule encoding a variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein, optionally having a peptide signal of SEQ ID NO.49, and

[0379] - A second nucleic acid molecule encoding a variable light chain domain of an anti-hPD-1 antibody as disclosed herein, optionally having a peptide signal of SEQ ID NO:50, and

[0380] - A nucleic acid encoding, optionally via a coding linker, the immunotherapeutic agent operatively linked to the first nucleic acid or the second nucleic acid or both. In one embodiment, the nucleic acid molecule encoding a bifunctional molecule as defined above comprises:

[0381] - A first nucleic acid molecule encoding the variable heavy chain domain of SEQ ID NO:17, wherein X1 is D or E, and X2 is selected from T, H, A, Y, N, E, and S, preferably from H, A, Y, N, and E; optionally having the peptide signal of SEQ ID NO:49, and

[0382] - A second nucleic acid molecule encoding the variable light chain domain of SEQ ID NO:26, wherein X is G or T; optionally having the peptide signal of SEQ ID NO:50, and

[0383] - A nucleic acid, which encodes, optionally via a coding connector, the immunotherapeutic agent operatively linked to the first nucleic acid or the second nucleic acid or both.

[0384] In another embodiment, the nucleic acid molecule encoding the bifunctional molecule as defined above includes:

[0385] - A first nucleic acid molecule encoding the variable heavy chain domain of SEQ ID NO:17, wherein X1 is D and X2 is selected from T, H, A, Y, N, E, preferably from H, A, Y, N, E, or wherein X1 is E and X2 is selected from T, H, A, Y, N, E, and S, preferably from H, A, Y, N, E, and S; optionally having the peptide signal of SEQ ID NO:49, and

[0386] - A second nucleic acid molecule encoding the variable light chain domain of SEQ ID NO:26, wherein X is G or T; optionally having the peptide signal of SEQ ID NO:50, and

[0387] - A nucleic acid, which encodes, optionally via a coding connector, the immunotherapeutic agent operatively linked to the first nucleic acid or the second nucleic acid or both.

[0388] Preferably, the nucleic acid molecule encoding the bifunctional molecule as defined above includes:

[0389] - A first nucleic acid molecule encoding a variable heavy chain domain of the amino acid sequence shown in SEQ ID NO: 18, 19, 20, 21, 22, 23, 24 or 25; optionally having a peptide signal of SEQ ID NO: 49, and

[0390] - A second nucleic acid molecule encoding a variable light chain domain of the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28; optionally having a peptide signal of SEQ ID NO.50, and

[0391] - A nucleic acid, which encodes, optionally via a coding connector, the immunotherapeutic agent operatively linked to the first nucleic acid or the second nucleic acid or both.

[0392] In a very particular embodiment, the nucleic acid molecule encoding a variable heavy chain domain has the sequence shown in SEQ ID NO:61, and / or the nucleic acid molecule encoding a variable light chain domain has the sequence shown in SEQ ID NO:62.

[0393] Operable linkage means that the nucleic acid encodes a protein fusion comprising a variable heavy or light chain domain, an optional adaptor peptide, and an immunotherapeutic agent. Preferably, the adaptor is selected from (GGGGS)3, (GGGGS)4, (GGGGS)2, GGGGS, GGGS, GGG, GGS, and (GGGS)3, and even more preferably (GGGGS)3.

[0394] In one implementation, the nucleic acid molecules are isolated, particularly non-natural, nucleic acid molecules.

[0395] The nucleic acid molecule or group of nucleic acid molecules encoding the bifunctional molecule according to the present invention is preferably contained in a vector or a vector group.

[0396] carrier

[0397] In another aspect, the present invention relates to a carrier comprising a nucleic acid molecule or a group of nucleic acid molecules as defined above.

[0398] As used herein, a "vector" is a nucleic acid molecule used as a carrier to transfer genetic material into cells. The term "vector" encompasses plasmids, viruses, kinases, and artificial chromosomes. Typically, engineered vectors contain a replication origin, a multiple cloning site, and selectability markers. The vector itself is usually a nucleotide sequence, typically a DNA sequence, containing an insert (transgenic) and a larger sequence that serves as the vector's "backbone." In addition to the transgenic insert and backbone, modern vectors may also contain other features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) are specifically designed to express transgenes in target cells and typically contain control sequences.

[0399] In one embodiment, the heavy chain coding sequence and light chain coding sequence and / or constant region of the anti-PD1 antibody are contained in an expression vector. Each of the heavy chain coding sequence and light chain coding sequence can be operatively linked to a suitable promoter, and the heavy chain and / or the light chain is operatively linked to an immunotherapeutic agent according to the invention herein. Alternatively, expression of both the heavy chain and the light chain can be driven by the same promoter. In another embodiment, each of the heavy chain and the light chain of the antibody is cloned into a separate vector, one or both of the heavy chain and the light chain, and the heavy chain and / or the light chain is operatively linked to an immunotherapeutic agent according to the invention. In the latter case, the expression vector encoding the heavy chain and the light chain can be co-transfected into a host cell to express both chains, which can be assembled in vivo or in vitro to form a complete antibody. Alternatively, the expression vector encoding the heavy chain and the light chain can be introduced into different host cells to express both the heavy chain and the light chain, and then purified and assembled in vitro to form a complete antibody.

[0400] Those skilled in the art can clone a nucleic acid molecule encoding a humanized anti-PD-1 antibody or an antibody fragment thereof into a vector and then transform it into a host cell. Therefore, the present invention also provides a recombinant vector comprising a nucleic acid molecule encoding the anti-PD-1 antibody or a fragment thereof of the present invention. In a preferred embodiment, the expression vector further comprises a promoter and a nucleic acid sequence encoding a secretion signal peptide, and optionally includes at least one drug resistance gene for screening.

[0401] Suitable expression vectors typically contain (1) prokaryotic DNA elements encoding bacterial origin of replication and antibiotic resistance markers to provide for the growth and selection of the expression vector in the bacterial host; (2) eukaryotic DNA elements controlling transcription initiation, such as promoters; and (3) DNA elements controlling transcript processing, such as transcription termination / polyadenylation sequences.

[0402] Expression vectors containing the nucleic acid sequence of the bifunctional anti-molecule described herein and suitable regulatory components for transcription / translation can be constructed using methods well-known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence is effectively linked to a suitable promoter in the expression vector to guide mRNA synthesis. The expression vector may also contain ribosome binding sites for initiating translation, transcription terminators, etc.

[0403] Various techniques can be used to introduce expression vectors into host cells, including calcium phosphate transfection, liposome-mediated transfection, electroporation, etc. Preferably, transfected cells are selected and proliferated, wherein the expression vector is 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 selectable markers are described in: Sambrook, Ausubel, Bebbington, “Expression of Antibody Genes in Nonlymphoid Mammalian Cells,” 2METHODS: A comparison to methods in enzymology 136 (1991), and Murray (ed.), Gene transfer and expression protocols (Humana Press 1991). Suitable cloning vectors are described in the following: Sambrook et al. (eds.), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (Cold Spring Harbor Press 1989) (hereinafter referred to as "Sambrook"); Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Wiley Interscience 1987) (hereinafter referred to as "Ausubel"); and Brown (ed.), MOLECULAR BIOLOGY LABFAX (Academic Press 1991).

[0404] host cells

[0405] In another aspect, the present invention relates to a host cell comprising a carrier or nucleic acid molecule or group of nucleic acid molecules as defined above, for the purpose of generating bifunctional molecules.

[0406] As used herein, the term "host cell" is intended to include any individual cell or cell culture that can be, or has been, a receptor for a vector, exogenous nucleic acid molecule, or the polynucleotide encoding the antibody construct of the present invention and / or the antibody construct or bifunctional molecule itself. The substance may be introduced into the cell by transformation, transfection, or other means. The term "host cell" is also intended to include the offspring or possible offspring of a single cell. Suitable host cells include prokaryotic or eukaryotic cells, and also include, but are not limited to, bacterial, yeast, fungal, plant, and animal cells, such as insect and mammalian cells, for example, mouse, rabbit, macaque, or human cells.

[0407] In one embodiment, the host cell comprises (e.g., transformed to have): (1) a vector containing nucleic acid encoding an amino acid sequence of a VL containing an antibody and / or an amino acid sequence of a VH containing an antibody and / or a constant region of an antibody, or (2) a first vector containing a nucleic acid encoding an amino acid sequence of a VL containing an antibody, and a second vector containing a nucleic acid encoding an amino acid sequence of a VH containing an antibody.

[0408] In another embodiment, the host cell comprises (e.g., having been transformed as described below) a vector that contains two entities of the bifunctional molecule. Preferably, the host cell comprises (e.g., having been transformed as described below) a vector containing a first nucleic acid molecule encoding a variable heavy chain domain of an anti-hPD-1 antibody as disclosed herein and a second nucleic acid molecule encoding a variable light chain domain of an anti-hPD-1 antibody as disclosed herein, the first and second nucleic acid molecules being operatively linked to a third nucleic acid encoding an immunotherapeutic agent as disclosed herein.

[0409] This article also provides a method for producing a humanized anti-PD1 antibody. The method includes culturing host cells containing nucleic acids encoding the antibody, as described above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium). Specifically, for recombinant production of a humanized anti-PD1 antibody, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells.

[0410] The bifunctional molecules described in this invention are preferably expressed in eukaryotic cells (e.g., 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 suitable eukaryotic host cells can 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-derived cells with the SV40 gene (COS cells); the monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells, as described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); human kidney epithelial 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); and human hepatocytes (Hep). G2); mouse mammary tumor (MMT060562); TRI cells, such as those described, for example, in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR"CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (eds.), Humana Press, Totowa, NJ (2004), pp. 255-268. For example, mammalian cell lines suitable for growth in suspension may be useful.

[0411] Specifically, the host cells of the present invention are selected from the following: CHO cells, COS cells, NSO cells and HEK cells.

[0412] For mammalian hosts, the transcriptional and translational regulatory signals of expression vectors can originate from viral sources, such as adenoviruses, bovine papillomaviruses, and simian viruses, with these regulatory signals associated with specific genes exhibiting high expression levels. Suitable transcriptional and translational regulatory sequences can also be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.

[0413] Stable transformants producing the bifunctional molecules according to the present invention can be identified using various methods. After identifying the cell producing the molecule, the host cell is cultured under conditions suitable for its growth and bifunctional molecule expression (e.g., temperature, culture medium). The humanized antibody is then isolated and / or purified using any method known in the art. These methods include, but are not limited to, conventional refolding treatment, protein precipitant (e.g., salt precipitation) treatment, centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography or gel chromatography, adsorption chromatography, ion exchange chromatography, HPLC, any other liquid chromatography method, and combinations thereof. As described, for example, by Colagan, bifunctional molecule separation techniques may particularly include affinity chromatography using protein A agarose, size exclusion chromatography, and ion exchange chromatography. Protein A is preferably used to separate the bifunctional molecules according to the present invention.

[0414] Methods for selecting suitable bifunctional molecules

[0415] In one aspect, the present invention relates to a method for selecting the bifunctional molecule of the present invention, comprising at least one of the following steps or consisting of at least one of the following steps:

[0416] a. Test the ability of the bifunctional molecule to bind PD-1 (e.g., according to the method described in Example X).

[0417] b. Test the ability of the bifunctional molecule (e.g., according to the method described in Example X) to inhibit the binding of human PD-L1 and / or PD-L2 to human PD-1;

[0418] c. Test (e.g., the method according to Example X) the ability of the bifunctional molecule not to inhibit T cell proliferation, preferably to increase the ability of T cells (especially regulatory T cells) to proliferate;

[0419] d. Test (e.g., the method according to Example X) the ability of the bifunctional molecule not to inhibit T cell activation, preferably to increase T cell activation;

[0420] e. Test (e.g., the method described in Example X) the ability of the bifunctional molecule to increase IFNγ secretion in human PBMCs;

[0421] f. Test (e.g., the method according to Example X) the ability of the bifunctional molecule to bind to the ligand or receptor of the immunotherapeutic agent;

[0422] And optionally include the following steps:

[0423] g. Select a bifunctional molecule that specifically binds to PD-1 and / or significantly inhibits the binding of PD-L1 and / or PD-L2 to PD-1, and / or does not significantly inhibit, preferably increases, human T cell proliferation and / or activation, and / or increases IFNγ secretion from human PBMCs, and / or is able to bind to the ligand or receptor of the immunotherapeutic agent.

[0424] As described above, the method for selectively modifying antibodies according to the present invention can be advantageously further implemented in the method for manufacturing bifunctional molecules according to the present invention.

[0425] Pharmaceutical Compositions and Methods of Administration

[0426] This invention also relates to a pharmaceutical composition comprising any of the bifunctional molecules described herein, such as nucleic acid molecules, nucleic acid molecules, carriers, and / or host cells as described above, preferably an active ingredient or compound. The formulation may be sterilized and, if necessary, mixed with adjuvants such as pharmaceutically acceptable carriers and excipients, which do not adversely interact with the bifunctional molecules, nucleic acids, carriers, and / or host cells described herein. Optionally, the pharmaceutical composition may also comprise additional therapeutic agents as described in detail below.

[0427] Preferably, the pharmaceutical compositions of the present invention may comprise bifunctional molecules as described herein, nucleic acid molecules, nucleic acid molecules, carriers and / or host cells as described above, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, excipients, salts and antioxidants as described below. Ideally, a pharmaceutically acceptable form is used that does not adversely affect the desired immunomodulatory effect of the bifunctional molecules according to the present invention. For ease of administration, the bifunctional molecules as described herein may be formulated into pharmaceutical compositions for in vivo administration. Methods for preparing such compositions have been described in the art (see, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005)).

[0428] In particular, the pharmaceutical compositions according to the present invention can be formulated for any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration. Preferably, the pharmaceutical compositions according to the present invention are formulated for enteral or parenteral administration. Compositions and formulations for parenteral administration may include sterile aqueous solutions and may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carder compounds, and other pharmaceutically acceptable carriers or excipients.

[0429] Pharmaceutical compositions can be prepared by mixing reagents of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to receptors at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (1 residue) 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. TM PLURONICS TM Or polyethylene glycol (PEG).

[0430] Pharmaceutically acceptable solid carriers may include one or more substances that can also serve as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, flow aids, compression aids, inert binders, sweeteners, preservatives, coatings, or tablet disintegrants. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low-melting-point waxes, and ion exchange resins. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. Unless any conventional media or pharmaceutical agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is contemplated.

[0431] The bifunctional molecule according to the invention can be dissolved or suspended in pharmaceutically acceptable liquid carriers, such as water, organic solvents, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and their analogues, mixtures thereof, or pharmaceutically acceptable oils or fats and suitable mixtures thereof. The liquid carrier may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, wetting agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid carriers for oral and enteral administration include water (partially containing the above-mentioned additives, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, e.g., glycols) and their derivatives), and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the carrier may also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers can be used in sterile liquid forms of compositions administered enterically. The liquid carrier used in the pressurized composition may be a halocarbon or other pharmaceutically acceptable propellant.

[0432] The pharmaceutical compositions of the present invention may further comprise one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound without producing any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts. 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, phosphoric acid, etc., and those derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkali metals or alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0433] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0434] For ease of delivery, any of the bifunctional molecules or their encoded nucleic acid can be conjugated with a chaperon agent. The chaperon agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin, low-density lipoprotein, or globulin), a carbohydrate (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or a lipid. It can also 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-ethylene glycol) 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 polyphosphazene. In one embodiment, the chaperon agent is a micelle, lipid, 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.

[0435] Pharmaceutical compositions are generally required to be sterile and stable under manufacturing and storage conditions. Pharmaceutical compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations and / or injection. A suitable mobile phase can be maintained, for example, by using coatings such as lecithin, or, in the case of dispersants, by maintaining the desired particle size and by using surfactants.

[0436] In one embodiment, the pharmaceutical composition is an injectable composition that may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by infusion, thereby infusing a pharmaceutical formulation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextran, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular formulations, such as sterile formulations in the form of suitable soluble salts of the antibody, can be dissolved and administered in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0437] Sterile injectable solutions can be prepared by incorporating the desired amount of an active compound into a suitable solvent, combining one or more of the above-mentioned components as needed, and then sterilizing and microfiltration. Typically, dispersants are prepared by incorporating the active compound into a sterile carrier containing an alkaline dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying (lyophilization) to produce a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution. Extended absorption of the injectable composition can be achieved by including delay-absorption agents such as monostearate and gelatin in the composition.

[0438] The presence of microorganisms can be prevented through sterilization procedures and the inclusion of various antimicrobial and antifungal agents, such as chlorobutanol and phenol sorbate. The inclusion of isotonic agents, such as sugars and sodium chloride, in the composition may also be desirable. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0439] Those skilled in the art will understand that the formulations of the present invention are isotonic with human blood, meaning that the formulations of the present invention have substantially the same osmotic pressure as human blood. Such isotonic formulations typically have an osmotic pressure from about 250 mSm to about 350 mSm. Isotonicity can be measured, for example, by vapor pressure or a frozen osmometer. The tension of the formulation is adjusted by using a tension modifier. A “tension modifier” is a pharmaceutically acceptable inert substance that can be added to a formulation to provide isotonicity. Tension modifiers suitable for the present invention include, but are not limited to, sugars, salts, and amino acids.

[0440] Pharmaceutical compositions according to the invention can be formulated to release the active ingredient (e.g., the bifunctional molecule described in this invention) substantially immediately after administration or at any predetermined time or period after administration. In some aspects, the pharmaceutical compositions can employ delayed-release, sustained-release, and extended-release delivery systems, such that delivery of the composition occurs prior to sensitization at the treatment site and allows sufficient time for sensitization to occur. Methods known in the art can be used to prevent or minimize the release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed release of the composition. Such systems can avoid repeated administration of the composition, thereby increasing convenience for both the subject and the physician.

[0441] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is typically the amount of the composition that produces the therapeutic effect.

[0442] Subjects, protocol and administration

[0443] This invention relates to bifunctional molecules as described herein; nucleic acids or vectors encoding them; host cells or pharmaceutical compositions; nucleic acids, vectors or host cells used as pharmaceuticals, or for treating diseases or for administration to a subject. It also relates to the use of the pharmaceutical compositions, nucleic acids, vectors or host cells of the invention, or humanized anti-PD1 antibodies or antibody fragments thereof, in the preparation of pharmaceuticals for treating diseases in a subject. Finally, it relates to methods for treating diseases or conditions in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition or humanized anti-PD1 antibody or antibody fragments thereof. Examples of treatment are described in more detail in the “Methods and Uses” section below.

[0444] The subjects to be treated can be people, especially people in the prenatal stage, newborns, children, infants, adolescents or adults, especially adults who are at least 30 years old, 40 years old, preferably adults who are at least 50 years old, even more preferably adults who are at least 60 years old, and even more preferably adults who are at least 70 years old.

[0445] Specifically, the subject suffers from a disease that may involve the PD-1 / PDL-1 pathway, particularly the expression, especially overexpression, of at least one of the PD-1 ligands (e.g., PDL-1 and / or PDL-2) or PD-1. Preferably, the subject suffers from cancer, and even more preferably from 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.

[0446] In one particular embodiment, the subject has received at least one first-line treatment, preferably several lines of treatment, prior to administration of the bifunctional molecule or pharmaceutical composition according to the invention.

[0447] The bifunctional molecule or the pharmaceutical composition disclosed herein can be administered to a subject using conventional methods known to those skilled in the art, depending on the type or site of the disease to be addressed. The composition can be administered via conventional routes, such as oral, parenteral, enteral, via inhalation spray, topical, rectal, nasal, oral, vaginal, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intratumoral, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. When administered parenterally, the pharmaceutical composition of the present invention is preferably administered via intravenous route. When administered enterally, the pharmaceutical composition of the present invention is preferably administered via oral route. The composition can also be administered topically.

[0448] The form, route of administration, and dosage of the pharmaceutical compositions or bifunctional molecules described in this invention can be adjusted by those skilled in the art according to the type and severity of the infection and the patient, particularly their age, weight, sex, and general condition. The compositions of this invention can be administered in various ways, depending on whether local or systemic treatment is required.

[0449] Preferably, the treatment with the bifunctional molecule or with the pharmaceutical composition according to the invention is administered periodically, preferably daily, weekly, or monthly, more preferably between daily and weekly, bi-weekly, bi-weekly, or bi-weekly. In a particular embodiment, the treatment is administered several times a day, preferably twice or three times a day.

[0450] The duration of treatment with the bifunctional molecule or with the pharmaceutical composition according to the invention is preferably between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, even more preferably between 1 day and 4 weeks, and even more preferably between 1 day and 2 weeks. Alternatively, treatment may continue for as long as the disease persists.

[0451] The bifunctional molecule disclosed herein can be delivered at an effective dose in the range of about 1 ng / kg body weight to about 30 mg / kg body weight, 1 μg / kg to about 20 mg / kg, 10 μg / kg to about 10 mg / kg, or from 100 μg / kg to 5 mg / kg, optionally every week, two weeks, three weeks or four weeks, preferably by parenteral or oral administration, particularly by intravenous or intradermal administration.

[0452] In particular, the bifunctional molecules described in this invention can be administered at subtherapeutic doses. As used herein, the term "subtherapeutic dose" refers to a dose below the level of an effective monotherapy dose typically used to treat a disease, or a dose not currently typically used for an effective monotherapy with anti-hPD1 antibodies.

[0453] Methods and uses

[0454] Uses in the treatment of diseases

[0455] The bifunctional molecules, nucleic acids, vectors, host cells, compositions, and methods described herein have a variety of in vitro and in vivo efficacies and applications. For example, the bifunctional molecules, nucleic acids, vectors, host cells, and / or pharmaceutical compositions described herein can be used as therapeutic agents, diagnostic agents, and in medical research. In particular, any bifunctional molecule, nucleic acid, vector, host cell, or pharmaceutical composition provided herein can be used in therapeutic methods and / or for therapeutic purposes. Specifically, the bifunctional molecules, nucleic acids, vectors, or pharmaceutical compositions provided herein can be used to treat any disease or condition, preferably involving PD-1, such as cancer, autoimmune diseases, and infections, or other diseases related to immunodeficiency, such as T-cell dysfunction.

[0456] Preferably, the present invention relates to a method for treating lesions, diseases and / or conditions that can be prevented or treated by inhibiting the binding of PD-L1 and / or PD-L2 to PD-1.

[0457] Even more preferably, the present invention relates to a method for treating in a subject with a disease and / or condition selected from cancer and infectious diseases, preferably chronic infections, said method comprising administering to said subject an effective amount of a bifunctional molecule or pharmaceutical composition as defined above. Examples of such diseases are described more specifically below.

[0458] In particular, the bifunctional molecule described in this invention is called a "bifunctional checkpoint inhibitor" because it targets PD-1 / PD-L1 / PD-L2 and another signal transduction pathway.

[0459] This invention particularly relates to bifunctional molecules, nucleic acids, nucleic acid groups or vectors encoding them, or pharmaceutical compositions containing them for the treatment of lesions, diseases and / or conditions that can be prevented or treated by inhibiting the binding of PD-L1 and / or PD-L2 to PD-1.

[0460] Therefore, this document discloses a method for treating diseases specifically associated with the PD-1 and / or PD-1 / PD-L1 and / or PD-1 / PD-L2 signaling pathways, comprising administering an effective amount of any of the bifunctional molecules or pharmaceutical compositions described herein to a subject requiring treatment. The appropriate dosage must also be determined by taking into account the patient's physiological data (e.g., age, body size, and weight) and route of administration to provide the patient with a therapeutically effective amount.

[0461] In another aspect, the bifunctional molecules described herein can be administered to a subject in vivo, for example, to enhance immunity, preferably to treat a condition and / or disease. Therefore, in one aspect, the present invention provides a method for altering the immune response of a subject, comprising administering to the subject the bifunctional molecules, nucleic acids, carriers, or pharmaceutical compositions described herein to alter the immune response in the subject. Preferably, the immune response is enhanced, increased, stimulated, or upregulated. The bifunctional molecules or pharmaceutical compositions can be used to enhance the immune response in a subject requiring treatment, such as T cell activation. Enhanced immune responses can lead to inhibition of PD-L1 and / or PD-L2 binding to PD-1, thereby reducing the immunosuppressive environment, stimulating the proliferation and / or activation of human T cells and / or IFNγ secretion by human PBMCs.

[0462] The present invention provides a method for enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of any one of the bifunctional molecules, nucleic acids, carriers, or pharmaceutical compositions comprising the thereof described herein, such that the immune response in the subject is enhanced.

[0463] In some embodiments, the amount of the bifunctional molecule described herein is effective in inhibiting PD-1 signaling (e.g., reducing PD-1 signaling by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control). In other embodiments, the amount of the anti-PD-1 antibody described herein is effective in activating an immune response (e.g., activating by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0464] In some embodiments, the amount of humanized anti-hPD-1 antibody described herein is effective in inhibiting the binding of human PD-L1 and / or PD-L2 to human PD-1 (e.g., inhibiting binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0465] In some embodiments, the amount of the humanized anti-hPD-1 antibody described herein is sufficient to have antagonistic activity for binding human PD-L1 and / or PD-L2 to human PD-1 (e.g., inhibiting binding by at least 20%, 30%, 50%, 80%, 100%, 200%, 400%, or 500% compared to a control).

[0466] This invention also relates to bifunctional molecules, nucleic acids or carriers encoding them, or pharmaceutical compositions comprising them, for use in treating conditions and / or diseases in subjects and / or as medicaments or vaccines as described herein. It further relates to the use of bifunctional molecules, nucleic acids or carriers encoding them, or pharmaceutical compositions comprising them in the preparation of medicaments for treating conditions and / or diseases in subjects. Finally, it relates to methods for treating diseases or conditions in subjects, comprising administering a therapeutically effective amount of the pharmaceutical composition or bifunctional molecule to the subject.

[0467] This document discloses a method for treating patients with diseases and / or conditions, comprising: (a) identifying patients in need of treatment; and (b) administering to said patient a therapeutically effective amount of any one of the bifunctional molecules, nucleic acids, carriers, or pharmaceutical compositions described herein.

[0468] Subjects requiring treatment may be individuals who have, are at risk of having, or are suspected of having a disease associated with the PD-1-mediated signal transduction pathway. Such patients can be identified through routine medical examinations. For example, suitable subjects for treatment can be identified by examining whether such subjects carry PD-1, PD-L1, and / or PD-L2 positive cells. In one embodiment, subjects requiring treatment are patients who have, are suspected of having, or are at risk of having a disease, preferably a PD-1, PD-L1, and / or PD-L2 positive disease, and even more preferably a disease in which PD-1 and / or at least one PD-1 ligand is overexpressed. In such subjects, disruption of the PD-1 / PD-L1 and / or PD-1 / PD-L2 interactions can enhance the subject's immune response due to the administration of the bifunctional molecule or pharmaceutical composition according to the invention. In some embodiments, any of the humanized anti-PD-1 antibodies or pharmaceutical compositions described herein can be used to treat PD-1 positive cells.

[0469] cancer

[0470] It is known in the art that antibody blocking of PD-1 can enhance the immune response against cancer cells in patients. Therefore, in one aspect, the present invention provides a bifunctional molecule or pharmaceutical composition for use in treating a subject with cancer, comprising administering an effective amount of said bifunctional molecule or pharmaceutical composition to the individual, preferably to disrupt or inhibit PD1 / PD-L1 and / or PD1 / PD-L2 interactions.

[0471] In one implementation, the subject requiring treatment is a patient who has, is suspected of having, or is at risk of having the disease, preferably with PD-1 or PD-L1 positive cancer, and even more preferably with cancer in which PD-1 or PD-L1 is expressed or overexpressed. For example, patients suitable for treatment can be identified by examining whether such patients carry PD-L1 positive tumor cells. Alternatively, the subject suitable for treatment may be a subject with tumor-infiltrating T cells that express or overexpress PD-1.

[0472] In another embodiment, the subject is a patient who has, is suspected of having, or is at risk of cancer progression, preferably PD-L1 and / or PD-L2 positive cancer. In some embodiments, any of the bifunctional molecules or pharmaceutical compositions described herein can be used to treat PD-L1 and / or PD-L2 positive tumors. For example, identifying suitable human patients can be achieved by examining whether such patients carry PD-L1 and / or PD-L2 positive cancer cells.

[0473] In other aspects, a bifunctional molecule or pharmaceutical composition is provided for treating cancer, preferably PD-L1 and / or PD-L2 positive cancer, and even more preferably cancer in which PD-L1 and / or PD-L2 are overexpressed.

[0474] In a particular aspect, the bifunctional molecule or pharmaceutical composition according to the invention is used to treat cancer by activating exhausted T cells.

[0475] In another embodiment, the present invention provides the use of bifunctional molecules or pharmaceutical compositions as disclosed herein in the preparation of a medicament for treating cancer in a subject (e.g., inhibiting the growth of tumor cells (preferably PD-L1 or PD-L2 positive tumor cells)).

[0476] In one aspect of this disclosure, the cancer to be treated is associated with depleted T cells.

[0477] Preferably, “PD-L1 positive tumor cells” or “PD-L2 positive tumor cells” refers to a population of tumor cells in which PD-L1 or PD-L2 is expressed in at least 10% of tumor cells, preferably at least 20, 30, 40 or 50% of tumor cells, respectively.

[0478] Therefore, in one embodiment, the present invention provides a method for treating cancer (e.g., for inhibiting the growth of tumor cells in a subject), comprising administering to the subject a therapeutically effective amount of the bifunctional molecule or pharmaceutical composition according to the present invention. In particular, the present invention relates to treating a subject with a bifunctional molecule to inhibit the growth of cancer cells.

[0479] Any suitable cancer that can be treated using the bifunctional molecules provided herein can be a hematopoietic system cancer or a solid cancer. Such cancers include carcinomas, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, glioma, mesothelioma, melanoma, gastric cancer, urethral cancer, environmentally induced cancers, and any combination of said cancers. This invention is also applicable to the treatment of metastatic cancers, particularly metastatic cancers expressing PD-L1 (Iwai et al., (2005) Int. Immunol. 17:133-144). Furthermore, this invention includes refractory or recurrent malignancies.

[0480] In one particular aspect, cancer is a hematologic malignancy or solid tumor that highly expresses PD-1 and / or PD-L1. Such cancers can be selected from the following: lymphohematopoietic tumors, angioimmunoblastic T-cell lymphomas, myelodysplastic syndromes, and acute myeloid leukemia.

[0481] In one particular aspect, cancer is either virus-induced or associated with immune deficiency. Such cancers can be selected from the following: Kaposi's sarcoma (e.g., associated with Kaposi's sarcoma herpesvirus); squamous cell carcinoma of the cervix, anus, penis, and vulva, as well as oropharyngeal carcinoma (e.g., associated with human papillomavirus); B-cell non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmablastic lymphoma, primary central nervous system lymphoma, HHV-8 primary exudative lymphoma, classical Hodgkin's 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 hepatitis C viruses); Merkel cell carcinoma (e.g., associated with Merkel cell polyomavirus (MPV)); and cancers associated with human immunodeficiency virus infection (HIV).

[0482] Preferably, the cancer to be treated or prevented is selected from the following: metastatic or non-metastatic melanoma, malignant mesothelioma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, small cell lung cancer, metastatic Merkel cell carcinoma, gastric cancer or gastroesophageal cancer, and cervical cancer.

[0483] Preferred cancer treatments include those that typically respond to immunotherapy. Alternatively, preferred cancer treatments are those that do not respond to immunotherapy.

[0484] For example, and without wishing to be bound by theory, treatment with anticancer antibodies or anticancer immune conjugates or other current anticancer therapies that induce cancer cell death will enhance the PD-1-mediated immune response. Therefore, treatment for hyperproliferative diseases (e.g., cancerous tumors) may include combinations of bifunctional molecules as disclosed herein with anticancer therapies, simultaneously or sequentially, or any combination thereof, which may enhance the host's antitumor immune response. Preferably, the bifunctional molecules may be used in combination with other immunogenic agents, standard cancer therapies, or other antibodies as described below.

[0485] Infectious diseases

[0486] The bifunctional molecules, nucleic acids, nucleomes, vectors, host cells, or pharmaceutical compositions described in this invention can be used to treat patients who have been exposed to specific toxins or pathogens. Therefore, one aspect of this invention provides a method for treating an infectious disease in a subject, comprising administering to the subject a humanized anti-PD-1 antibody or an antigen-binding fragment thereof, or a pharmaceutical composition comprising thereof, preferably to treat the infectious disease in the subject.

[0487] Any suitable infection can be treated using the bifunctional molecules, nucleic acids, nucleomes, vectors, host cells, or pharmaceutical compositions provided in this article.

[0488] Examples of pathogenic viruses that cause infections treatable by the methods of the present invention include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, human papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and vector-borne encephalitis virus.

[0489] Specifically, the bifunctional molecular or pharmaceutical composition of the present invention is used to treat patients suffering from chronic viral infections caused by viruses selected from the following: retroviruses, circoviruses, porcine circoviruses, herpesviruses, varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), polyomavirus BK, polyomavirus, adeno-associated virus (AAV), herpes simplex virus type 1 (HSV-1), adenovirus, herpes simplex virus type 2 (HSV-2), Kaposi's sarcoma herpesvirus (KSHV), hepatitis B virus (HBV), GB virus C, papillomavirus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), hepatitis D virus (HDV), human T-cell leukemia virus type 1 (HTLV1), heterophile murine leukemia virus-associated virus (XMLV), rubella virus, rubella, parvovirus B19, measles virus, and Coxsackie virus.

[0490] Examples of pathogenic bacteria that cause infections treatable by the methods of this invention include Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis and Streptococcus, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulinum toxin, Anthrax, Plague, Leptospirosis and Lyme disease bacteria.

[0491] Some examples of pathogenic fungi that cause infections treatable by the method of the present invention include Candida (Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis, etc.), Cryptococcus neoformans, Aspergillus (Aspergillus fumigatus, Aspergillus niger, etc.), Mucorales (Mucor, Rhizopus, Rhizopus), Sporothrix schenkieri, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides imbricata, and Histoplasma capsulatum.

[0492] Some examples of pathogenic parasites that cause infections treatable by the method of the present invention include Entamoeba histolytica, Escherichia coli, Naegleria fowleri, Acanthamoeba, Giardia lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microsporum, Trypanosoma brevicornu, Trypanosoma krusei, Donovaliishmania, Toxoplasma gondii, and Sinusoids basidiospora.

[0493] In all of the above methods, the bifunctional molecule can be combined with other forms of immunotherapy such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2) or any therapy that enhances tumor antigen presentation.

[0494] combination therapy

[0495] In particular, the bifunctional molecule described in this invention can be combined with several other potential strategies to overcome immune evasion mechanisms of drugs in clinical development or already on the market (see Table 1, Antonia et al., Immuno-oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258–6268, 2014). This combination with the bifunctional molecule described in this invention can be particularly used for:

[0496] 1- Reverse the suppression of adaptive immunity (block the T cell checkpoint pathway);

[0497] 2-Activate adaptive immunity (using agonist molecules (especially antibodies) to promote T cell co-stimulatory receptor signal transduction);

[0498] 3- Improve the function of innate immune cells;

[0499] 4. Activate the immune system (enhance the effector function of immune cells), for example through vaccine-based strategies.

[0500] Therefore, this document also provides combination therapies for any disease associated with PD-1 signal transduction as described herein, using any one of the bifunctional molecules or pharmaceutical compositions containing them as described herein, and a suitable second agent. In one aspect, the bifunctional molecule and the second agent may be present in the pharmaceutical composition described above. Alternatively, as used herein, the terms "combination therapy" or "combined therapy" include the sequential administration of these agents (e.g., the bifunctional molecule as described herein and an additional or second suitable therapeutic agent), i.e., where each therapeutic agent is administered at different times, and where these therapeutic agents or at least two agents are administered substantially simultaneously. Sequential or substantially simultaneous administration of each agent can be achieved by any suitable route. The agents can be administered via the same route or via different routes. For example, the first agent (e.g., the bifunctional molecule) can be administered orally, and another therapeutic agent (e.g., an anticancer agent, an anti-infective agent, or an immunomodulatory agent) can be administered intravenously. Alternatively, the agents of the selected combination can be administered by intravenous injection, while other agents in the combination can be administered orally.

[0501] In another aspect, the present invention relates to a treatment method, particularly a combination product method, comprising as an active ingredient: a bifunctional molecule as defined above and an additional therapeutic agent, wherein said active ingredient is formulated for use alone, sequentially or in combination therapy, particularly in combination or sequentially.

[0502] As used herein, unless otherwise stated, the term "sequential" means characterized by a regular order or sequence. For example, if a dosing regimen involves the administration of a bifunctional molecule and an additional or second agent, a sequential dosing regimen may include the administration of the bifunctional molecule described herein before, simultaneously, substantially simultaneously, or after the administration of the second agent, but the two agents will be administered in a regular order or sequence. Unless otherwise stated, the term "separately" means to separate one from the other. Unless otherwise stated, the term "simultaneously" means to occur or be completed simultaneously, i.e., the agents described herein are administered simultaneously. The term "substantially simultaneously" means that the agents are administered within minutes of each other (e.g., within 15 minutes of each other) and is intended to include both combined and continuous administration, but if the administration is continuous, however, it is only spaced very short in time (e.g., the time required for a physician to administer two compounds separately).

[0503] It should be understood that any combination described herein can be used in any order to treat the conditions or diseases described herein. The combinations described herein can be selected based on a variety of factors, including but not limited to the utility of inhibiting or preventing the progression of the target disease, the utility of reducing the side effects of another agent in the combination, or the utility of reducing symptoms associated with the target disease. For example, the combination therapies described herein can reduce any side effects associated with each individual member of the combination.

[0504] The present invention also relates to a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the bifunctional molecule or pharmaceutical composition described herein and a therapeutically effective amount of an additional or second therapeutic agent.

[0505] When the bifunctional molecule or pharmaceutical composition described herein is used in combination with another therapeutic agent, a subtherapeutic dose of the bifunctional molecule, the composition, or the other or second pharmaceutical agent, or a subtherapeutic dose of both, may be used to treat a subject, preferably a subject who has or is at risk of developing a disease or condition related to PD-1-mediated cell signaling transduction.

[0506] In one aspect, the additional or second therapeutic agent may be selected from a non-exhaustive list including: alkylating agents, angiogenesis inhibitors, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, aurora kinase inhibitors, apoptosis promoters (e.g., Bcl-2 family inhibitors), death receptor pathway activators, Bcr-Abl kinase inhibitors, BiTE (bispecific T cell conjugate) antibodies, antibody-drug conjugates, biological response modulators, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, cell cycle inhibitors, cyclooxygenase-2 inhibitors, DVD, leukemia virus oncogene homolog (ErbB2) receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, hormone therapy, immunotherapies, apoptosis protein inhibitors (IA). Inhibitors of P), intercalating antibiotics, kinase inhibitors, kinin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, mitogen-activated extracellular signal-regulated kinase inhibitors, multivalent binding proteins, nonsteroidal anti-inflammatory drugs (NSAIDs), poly(ADP-ribose) polymerase (PARP) inhibitors, platinum-based chemotherapy agents, polo-like kinase (Plk) inhibitors, phosphoinositol-3 kinase (PI3K) inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs, receptor tyrosine kinase inhibitors, retinol / vitamin D alkaloids, small inhibitory RNA (siRNA), topoisomerase inhibitors, ubiquitin ligase inhibitors, hypomethylating agents, checkpoint inhibitors, peptide vaccines, etc., epitopes or neoepitaxes derived from tumor antigens, and one or more combinations of these agents.

[0507] For example, the additional therapeutic agents may be selected from the following: chemotherapy, radiotherapy, targeted therapy, anti-angiogenic agents, hypomethylating agents, cancer vaccines, epitopes or neoepitaxes derived from tumor antigens, bone marrow checkpoint inhibitors, other immunotherapies, and HDAC inhibitors.

[0508] In a preferred embodiment, the second therapeutic agent is selected from the following: chemotherapeutic agents, radiotherapy agents, immunotherapy agents, cell therapy agents (e.g., CAR-T cells), antibiotics, and probiotics. The immunotherapy agent may also be an antibody targeting a tumor antigen, particularly selected from the following: anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0509] In one embodiment, the present invention relates to combination therapies as defined above, wherein the second therapeutic agent is particularly selected from the following: therapeutic vaccines, immune checkpoint blockers or activators, particularly adaptive immune cells (T and B lymphocytes), and antibody-drug conjugates. Preferably, suitable agents for use in conjunction with humanized anti-hPD-1 antibodies or fragments thereof or pharmaceutical compositions according to the present invention include antibodies that bind to co-stimulatory receptors (e.g., OX40, CD40, ICOS, CD27, HVEM, or GITR), agents that induce immunogenic cell death (e.g., chemotherapeutic agents, radiotherapy agents, anti-angiogenic agents, or agents for targeted therapy), agents that inhibit checkpoint molecules (e.g., CTLA4, LAG3, TIM3, B7H3, B7H4, BTLA, or TIGIT), cancer vaccines, agents that regulate immunosuppressive enzymes (e.g., IDO1 or iNOS), agents that target Treg cells, agents for adoptive cell therapy, or agents that regulate myeloid cells.

[0510] In one embodiment, the present invention relates to a combination therapy as defined above, wherein the second therapeutic agent is an immune checkpoint blocker or activator selected from the following adaptive immune cells (T and B lymphocytes): anti-CTLA4, anti-CD2, anti-CD28, anti-CD40, anti-HVEM, anti-BTLA, anti-CD160, anti-TIGIT, anti-TIM-1 / 3, anti-LAG-3, anti-2B4 and anti-OX40, anti-CD40 agonist, CD40-L, TLR agonist, anti-ICOS, ICOS-L and B cell receptor agonist.

[0511] In one embodiment, the additional or second therapeutic agent is an antibody targeting a tumor antigen, particularly selected from the following: anti-Her2, anti-EGFR, anti-CD20, anti-CD19, and anti-CD52.

[0512] Specific examples of the second therapeutic agent are provided on pages 36-43 of WO 2018 / 053106, the disclosure of which is incorporated herein by reference.

[0513] Combination therapy also relies on the administration of bifunctional molecules in combination with surgery, chemotherapy (e.g., docetaxel or dacarbazine), radiation therapy, immunotherapy (e.g., antibodies targeting CD40, CTLA-4), gene targeting and modulation, and / or other agents (e.g., immunomodulators, angiogenesis inhibitors) and any combination thereof.

[0514] Reagent test kit

[0515] Any antibody or composition described herein may be included in the kits provided by this invention. This disclosure specifically provides kits for enhancing immune responses and / or treating diseases (e.g., cancer and / or infections) associated with PD-1 signaling transduction.

[0516] In the context of this invention, the term "kit" refers to two or more components (one of which corresponds to the bifunctional molecule, nucleic acid molecule, vector, or cell described in this invention) packaged in a container or other container. Therefore, a kit can be described as a set of products and / or devices sufficient to achieve a specific objective, which can be sold as a single unit.

[0517] Specifically, the kit according to the present invention may comprise:

[0518] - Bifunctional molecules as defined above

[0519] - Humanized anti-hPD1 antibodies or their antigen-binding fragments linked to immunotherapeutic agents

[0520] - A nucleic acid molecule or group of nucleic acid molecules encoding the bifunctional molecule.

[0521] - A vector containing the nucleic acid molecule or nucleic acid molecule set, and / or

[0522] - A cell containing the carrier or nucleic acid molecule or group of nucleic acid molecules.

[0523] Therefore, in suitable container means, the kit may contain the pharmaceutical composition and / or bifunctional molecule and / or host cell described in this invention, and / or carrier encoding the nucleic acid molecule and / or nucleic acid molecule described in this invention, and / or acid molecule or related reagent described in this invention. In some embodiments, means may be provided for obtaining samples from an individual and / or measuring samples. In some embodiments, the kit contains cells, buffers, cell culture media, carriers, primers, restriction enzymes, salts, etc. The kit may also contain components for containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0524] The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. In one embodiment, the present invention relates to a kit for a single-dose administration unit as defined above. The kit of the present invention may comprise a first container containing a dried / lyophilized bifunctional molecule and a second container containing an aqueous formulation. In some embodiments of the invention, kits comprising single-compartment and multi-compartment pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0525] The kit described in this invention is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed polyester film or plastic bottles), etc. Packaging for use with specific devices such as inhalers, intranasal administration devices (e.g., nebulizers), or infusion devices such as micropumps is also involved. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a needle-puncture stopper). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a needle-puncture stopper). At least one active agent in the composition is a bifunctional molecule described herein, comprising a humanized anti-hPD-1 antibody linked to an immunotherapeutic agent.

[0526] The compositions contained in the kit according to the invention can also be formulated into syringe-compatible compositions. In this case, the container component itself can be a syringe, pipette, and / or other such device from which the formulation can be applied to an infected area of ​​the body, and / or even applied to and / or mixed with other components of the kit. The components of the kit may alternatively be provided in the form of dry powder. When reagents and / or components are provided in dry powder form, soluble components can be reconstituted by adding a suitable solvent. It is envisioned that the solvent may also be provided in another container device and suitable for application.

[0527] In some embodiments, the kit further includes additional agents for treating cancer or infectious diseases, and these additional agents may be used in combination with the bifunctional molecule or other components of the kit described herein, or may be provided alone in the kit. In particular, the kit described herein may contain one or more additional therapeutic agents, such as those described in the "Combination Therapies" section above. The kit may be tailored to an individual's specific cancer and may include a corresponding second cancer therapy as described above for that individual.

[0528] Instructions for use relating to the bifunctional molecules or pharmaceutical compositions described herein typically include information regarding dosage, dosing regimen, route of administration for intended treatment, means of reconstructing the bifunctional molecule, and / or means of diluting the bifunctional molecule described herein. Instructions provided in kits described herein are typically written instructions on a label or packaging (e.g., paper pages included in the kit in the form of leaflets or instruction manuals). In some embodiments, the kit may contain instructions for use according to any of the methods described herein. The included instructions may include a description of administering a pharmaceutical composition comprising the bifunctional molecule to enhance an immune response and / or treat diseases as described herein. The kit may also contain a description of selecting individuals suitable for treatment based on identifying whether an individual has a disease related to PD-1 signaling transduction (e.g., those described herein). Example

[0529] The following figures and embodiments are intended to provide a complete disclosure and description of how to make and use the invention for those skilled in the art, and are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent all or only the experiments conducted. While the invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt particular circumstances, materials, composition, processes, or one or more process steps to the purpose, spirit, and scope of the invention. All such modifications are intended to be within the scope of the appended claims.

[0530] result

[0531] Example 1: Generation and binding characteristics of bifunctional anti-PD1 molecules and their interaction with PDL1 and ligand-specific interactions

[0532] Figure 1 The results show that the bifunctional molecule of the present invention, having a humanized anti-PD1 antibody fused to both the heavy and light chains, exhibits improved yield compared to a bifunctional molecule having a chimeric anti-PD1 antibody. For example... Figure 1 As shown, bifunctional molecules fused to type 1 (i.e., CD86, B (i.e., CD80, and C (i.e., SIRPa)) or type 2 (i.e., OX40L and B (i.e., 4-1BBL)) and cytokines (i.e., IL-7) on any heavy chain (A) or light chain (B) are produced better than bifunctional molecules of anti-PD1 antibodies in chimeric form. Figure 1 C shows that the yield of cytokines (i.e., IL-7) was also improved when fused with both the heavy and light chains. The molecule comprises four cytokines fused with the dimer antibody.

[0533] The yield of the bifunctional molecule with a humanized anti-PD-1 backbone was compared with that of two other anti-PD-1 backbones, Keytruda (pembrolizumab) and Opdivo (nivolumab). Figure 2 As shown in A and 2B, the humanized anti-PD-1 antibody described in this invention exhibits better yield of bifunctional molecules fused with either a cytokine (i.e., IL-7) or type I protein B (i.e., CD80) compared to other anti-PD-1 backbones. This improvement was observed using two different producer cell lines (CHO and HEK free-form) and two different production methods (shake flask vs. 12-well plate). To confirm this increase in other type I fusion proteins, the inventors generated bifunctional molecules fused with SIRPa using a pembrolizumab backbone. Figure 2As shown in Figure C, the humanized anti-PD-1 scaffold described in this invention achieved a higher yield in cell culture compared to the Keytruda scaffold. In fed-batch culture manufacturing (production from unoptimized fed-batch CHO cells in a bioreactor), the bifunctional molecule of humanized anti-PD-1 fused with CD80 type I protein showed good yield (1 g / L).

[0534] In parallel, the inventors constructed bifunctional molecules with various non-anti-PD-1 backbone antibodies fused to cytokines or type I or type II proteins on the heavy or light chain, and compared their yields with those of bifunctional molecules with humanized anti-PD-1 as described in this invention. Figure 3 The results show that all bifunctional molecules with the anti-PD-1 humanized backbone described in this invention have significantly higher yields compared to any other backbone, confirming the high prepareability of the humanized anti-PD-1 described in this invention.

[0535] In parallel, the inventors compared the binding of the bifunctional molecule to PD1 in chimeric or humanized anti-PD1 antibodies. (The following...) Figure 4 As shown in Table 1, immunotherapeutic agents of any type maintained good binding to the PD-1 antigen when fused to either the heavy or light chain of the antibody. The bifunctional molecule lost a slight binding efficacy compared to the control anti-PD-1 antibody alone, but it remained a strong PD-1 binder (EC50 < 10 ng / ml).

[0536] However, using a surface plasmon resonance assay (Biacore assay), the bifunctional molecule showed a similar affinity range for PD-1 to the control anti-PD-1 alone. Biacore is a standard assay for measuring protein affinity and is more sensitive and accurate than ELISA. For this assay, an anti-human Fc antibody on a sensor chip captures either the anti-PD-1 molecule alone or the bifunctional molecule. Different concentrations of recombinant PD-1 protein (6.25 to 100 nM) were then added to measure affinity. A KD of 3.46 nM was obtained for the anti-PD-1 molecule alone, 2.61 nM for the anti-PD-1 IL-7 molecule, and 3.83 nM for the anti-PD-1 SIRPa molecule.

[0537] Figure 5 and Figure 6 Comparison of those with heavy chains ( Figure 5 ) or light chain ( Figure 6The binding of bifunctional molecules of chimeric and humanized anti-PD1 antibodies fused to different immunotherapeutic agents (type I or II or cytokine proteins) was studied. The results, as well as Tables 2 and 3, show that the binding between chimeric and humanized versions of bifunctional molecules having immunotherapeutic agents fused to either the heavy or light chain of the antibody is comparable. The inventors also tested a bifunctional molecule having an anti-PD1 antibody fused to both the heavy and light chains, resulting in four proteins of the same type on a single antibody. The EC50 values ​​were comparable when comparing the data in Tables 1, 2, 3, and 4. However, as... Figure 1 As shown in C, the yield of bifunctional molecules with humanized anti-PD1 antibodies is still superior to that of bifunctional molecules with chimeric anti-PD1 antibodies.

[0538]

[0539] Table 1: From Figure 4 EC50 was determined by PD-1 binding ELISA.

[0540]

[0541]

[0542] Table 2: From Figure 5 EC50 is a bifunctional molecule containing a chimeric and humanized anti-PD-1 antibody fused with a heavy chain and an immunotherapeutic agent.

[0543]

[0544] Table 3: From Figure 6 EC50 is a bifunctional molecule containing a chimeric and humanized anti-PD-1 antibody with a light chain fused to an immunotherapeutic agent.

[0545] Anti-PD1 fuses with IL-7 on both heavy and light chains. EC50 (ng / mL) Inlay 2.11 Humanization 3.9

[0546] Table 4: From Figure 7 The EC50 is a bifunctional molecule comprising a chimeric and humanized anti-PD-1 antibody with both light and heavy chains fused to an immunotherapeutic agent. The cytokine fused to the anti-PD-1 antibody is IL-7.

[0547] Then, the antagonistic properties of each bifunctional molecule with anti-PD1 antibodies fused with different immunotherapeutic agents in PD1-PDL1 interaction were analyzed compared with those of individual anti-PD1 antibodies. Figure 8 Tables A and 5 present the results of the competitive ELISA assay. As previously mentioned, when the antibody is fused with the immunotherapeutic agent, the antagonistic properties of the bifunctional molecule containing the anti-PD1 antibody remain unchanged, while its binding to PD1 is slightly reduced (e.g., ...). Figure 4(As shown). The antagonistic properties of the bifunctional molecules anti-PD-1 VHIL7 or anti-PD-1 VHCD80 against PD1-PDL2 were also evaluated by ELISA. Figure 8 B). These two molecules effectively block PD-L2 from binding to PD-1.

[0548]

[0549] Table 5: From Figure 8 IC50 (ng / ml): Detection of competitive PD-1 / PD-L1 ELISA.

[0550] To test the binding of immunotherapeutic agents to their ligands, the inventors performed a bridging ELISA assay. Figure 9 Results for type I and type II proteins are shown, with histograms representing positive controls for the binding of individual immunotherapeutic agents to their ligands. Ligand binding is conserved when the immunotherapeutic agent is fused to either the heavy or light chain of an anti-PD1 antibody.

[0551] Example 2: In vitro characterization of T cell proliferation and activation by a bifunctional molecule containing an anti-PD1 antibody fused with an immunotherapeutic agent.

[0552] To measure the T cell activation and proliferation efficiency of the bifunctional anti-PD1 molecule, the inventors tested T cell proliferation after treatment with anti-PD1 antibody alone (pembrolizumab used as an anti-PD1 antibody control), or with an allotype control, or with an anti-PD1 antibody fused to the VH or VL chain. Results are as follows: Figure 10 As shown, any bifunctional molecule containing an anti-PD1 antibody induces T cell proliferation at least as well as, or even better than, anti-PD1 alone. Assays measuring T cell activation showed very good efficiency in T cell activation, such as through IFNg secretion. Figure 11 The demonstration showed that bifunctional molecules with anti-PD1 antibodies induced T cell activation at least as well as, or even better than, control anti-PD1 antibodies, indicating that the bifunctional molecule form can enhance the in vivo T cell proliferation and activation efficiency of anti-PD1 antibodies and may be a better form for targeting T cells into tumors.

[0553] Bifunctional molecules containing anti-PD1 antibodies fused with one, two, three, or four immunotherapeutic agents described in this invention exhibit good binding to PD1 and good inhibitory properties against the PD1-PDL1 interaction. They are produced better than bifunctional molecules with chimeric versions of the antibody. In vitro, they induce better proliferation and activation on human T cells, indicating that they are more effective in vivo than anti-PD1 antibodies alone, especially on tumor-bearing T cells.

[0554] Example 3: In vivo pharmacokinetics of Bicki anti-PD1 antibody

[0555] To analyze the pharmacokinetics of a bifunctional molecule containing humanized anti-PD-1, mice were intravenously injected with 5 mg / kg of the bifunctional molecule containing humanized anti-PD-1 fused to SIRPa on the heavy chain. Two different isoforms, IgG1 N298A and IgG4S228P, were compared. Plasma concentrations were assessed at multiple time points post-injection using ELISA. Figure 12 As shown, the bifunctional molecule with humanized anti-PD-1 constructed using IgG1 N298A exhibits better pharmacokinetic characteristics than the bifunctional molecule with humanized anti-PD-1 constructed using the IgG4 S228P isotype.

[0556] Materials and methods

[0557] Combined PD1 ELISA and bridging ELISA assays

[0558] For the ELISA assay of PD1 binding, recombinant hPD1 (Sino Biologicals, Beijing, China; reference number 10377-H08H) was immobilized on a plastic substrate at 0.5 μg / ml in carbonate buffer (pH 9.2), and purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference number 709-035-149) was added, and the assay was performed colorimetrically at 450 / 650 nM using a TMB substrate (3,3',5,5'-tetramethylbenzidine, BD Bioscience, San Jose, USA).

[0559] A similar method was used for the bridging ELISA assay. Recombinant hPD1 was immobilized, and purified bifunctional antibodies were added at sequence dilution. After incubation and washing, recombinant receptors of protein C, A, or B were added at a concentration of 1 μg / mL. Detection was performed using anti-receptor-specific mouse antibodies and peroxidase-labeled donkey anti-mouse IgG antibodies (reference number 715-036-151). Detection was performed using standard methods.

[0560] ELISA antagonists: Competition between PDL1 or PDL2 and humanized anti-PD1

[0561] A competitive ELISA assay was performed using a PD-1:PD-L1 inhibitor screening ELISA pair (AcroBiosystems; USA; reference number EP-101). In this assay, recombinant hPDL1 was immobilized on a plastic substrate at 2 μg / ml in PBS pH 7.4 buffer. Purified antibodies (at different concentrations) were mixed with 0.66 μg / ml of final (fixed concentration) biotinylated human PD1 (AcroBiosystems; USA; reference number EP-101) at 37°C for 2 hours to measure competitive binding. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring; USA; reference number SA-5004) was added to detect biotin-CD47Fc binding, which was revealed by standard methods. The same ELISA protocol was performed to assess PDL2 / PD1 antagonist activity by coating recombinant PD-L2 Fc protein (Sinobiological, #10292-H02H).

[0562] Assay for IFNγ secretion and T cell proliferation

[0563] Human T cells were purified from peripheral blood mononuclear cells using a contactless pan-T cell isolation kit (reference number 130-096-535, MACS Miltenyi Biotech; USA) and an Automacs Pro separator (Miltenyi). T cells (anti-CD3 clone OKT3 and anti-CD28 clone CD28.2, 3 μg / mL each) were incubated on CD3 / CD28-coated plates to stimulate cells and induce PD-1 expression. Twenty-four hours post-stimulation, in the presence of isotype control non-convergent anti-PD-1 or anti-PD-1 bifunctional antibody, T cells were collected, counted, and restimulated on anti-CD3 (clone OKT3, 2 μg / mL) + recombinant human PD-L1 (Sinobiological, reference number 10084-H02H, 5 μg / mL). On day 6, supernatant was collected to quantify IFNg secretion (human IFNg ELISA assay, BD Bioscience, USA, reference number 555142) and T cell proliferation was assessed by H3 thymidine incorporation.

[0564] Pharmacokinetics and Pharmacodynamics of Humanized Anti-PD1 Antibody in Mice

[0565] BalbcRJ females (6-9 weeks old) were injected intraorally with a single dose (34.4 nM / kg) of bifunctional humanized anti-PD-1 antibody. Plasma drug concentrations were determined by ELISA using diluted serum containing the anti-PD-1 antibody and immobilized anti-human light chain antibody (clone NaM76-5F3). Detection was performed using peroxidase-labeled donkey anti-human IgG (Jackson Immunoresearch; USA; reference number 709-035-149), with the addition of the donkey anti-human IgG and re-detection using standard methods.

[0566] Antibodies and bifunctional molecules

[0567] The following antibodies and bifunctional molecules have been used in different experiments disclosed herein: pembrolizumab (Keytrudra, Merck), nivolumab (Opdivo, Bristol-Myers Squibb), and the bifunctional molecules disclosed herein comprise anti-PD1 humanized antibodies or anti-PD1 chimeric antibodies, wherein the anti-PD1 humanized antibodies comprise a heavy chain as defined in SEQ ID NO:19, 22 or 24 and a light chain as defined in SEQ ID NO:28, and the anti-PD1 chimeric antibodies comprise a heavy chain as defined in SEQ ID NO:59 and a light chain as defined in SEQ ID NO:60.

Claims

1. A bifunctional molecule consisting of: (a) an antagonist humanized anti-human PD-1 antibody or antigen-binding fragment thereof comprising: (i) a heavy chain variable domain (VH) consisting of the amino acid sequence of SEQ ID NO: 19, 22, or 24, and (ii) a light chain variable domain (VL) consisting of the amino acid sequence of SEQ ID NO: 28, and (b) an immunotherapeutic agent selected from the group consisting of CD86, CD80, SIRPa, OX40L, 4-1BBL, and IL-7; wherein the C-terminal end of the heavy chain and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminal end of the immunotherapeutic agent as a fusion protein.

2. The bifunctional molecule of claim 1, wherein the C-terminal end of the heavy chain and / or light chain of the antibody or antigen-binding fragment thereof is covalently linked to the N-terminal end of the immunotherapeutic agent through a peptide linker as a fusion protein.

3. The bifunctional molecule of claim 1, 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 IgGl, IgG2, IgG3, or IgG4 heavy chain constant domain.

4. The bifunctional molecule of claim 1, 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 IgGl heavy chain constant domain having a substitution or combination of substitutions selected from the group consisting of: - T250Q and M428L; - M252Y, S254T, T256E, H433K, and N434F; - E233P, L234V, L235A, G236A, A327G, A330S, and P331S; - E333A; - S239D, A330L, and I332E; - P257I and Q311; - K326W and E333S; - S239D, I332E, and G236A; - N297A; - L234A and L235A; - N297A, M252Y, S254T, and T256E; - K322A and K444A; and - N297A, M252Y, S254T, T256E, L234A, and L235A.

5. The bifunctional molecule of claim 1, 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 and L235A; - S228P, M252Y, S254T, and T256E; and - K444A.

6. A vector comprising a nucleic acid or set of nucleic acid molecules encoding the bifunctional molecule of any one of claims 1-5.

7. A host cell comprising the vector of claim 6.

8. A method for producing the bifunctional molecule of any one of claims 1-5, comprising the steps of culturing the host cell of claim 7 and isolating the bifunctional molecule.

9. A pharmaceutical composition comprising the bifunctional molecule of any one of claims 1-5, the vector of claim 6, or the host cell of claim 7, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein it further comprises an additional therapeutic agent.

11. The pharmaceutical composition of claim 10, wherein the additional therapeutic agent is selected from the group consisting of: an alkylating agent, an angiogenesis inhibitor, an antimetabolite, an antimitotic agent, an antiproliferative agent, an antiviral agent, an apoptosis promoter, a death receptor pathway activator, a leukemia viral oncogene homolog receptor inhibitor, a growth factor inhibitor, a heat shock protein (HSP)-90 inhibitor, a histone deacetylase (HDAC) inhibitor, an agent used in hormone therapy, an immunologic, an intercalating antibiotic, a kinase inhibitor, a kinesin inhibitor, a Jak2 inhibitor, a mammalian target of rapamycin inhibitor, a microRNA, a multivalent binding protein, a non-steroidal anti-inflammatory drug (NSAID), a poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitor, a platinum-based chemotherapeutic agent, a proteasome inhibitor, a purine analog, a pyrimidine analog, a plant alkaloid, a small inhibitory ribonucleic acid (siRNA), a topoisomerase inhibitor, a ubiquitin ligase inhibitor, a checkpoint inhibitor, a peptide vaccine, an epitope peptide from a tumor antigen, and a combination of one or more of these agents.

12. The pharmaceutical composition of claim 10, wherein the additional therapeutic agent is selected from the group consisting of: a hypomethylating agent, a BiTE (bispecific T-cell engager) antibody, an antibody drug conjugate, an aurora kinase inhibitor, a Bcr-Abl kinase inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, a cyclin-dependent kinase inhibitor, a mitogen-activated extracellular signal-regulated kinase inhibitor, a polo-like kinase (Plk) inhibitor, a phosphoinositide-3 kinase (PI3K) inhibitor, a receptor tyrosine kinase inhibitor, an inhibitor of inhibitors of apoptosis proteins (IAPs), and a Bcl-2 family inhibitor.

13. Use of the pharmaceutical composition of claim 10 or 11 or the bifunctional molecule of any one of claims 1-5 in the manufacture of a medicament for the treatment of cancer, wherein the cancer is a solid tumor expressing PD-1 and / or PD-L1.

14. The use of claim 13, wherein the cancer is selected from the group consisting of: a cancer induced by a virus or associated with immunodeficiency, an anal cancer, a penile cancer, a vulvar squamous cell carcinoma, an oropharyngeal cancer; a metastatic or non-metastatic melanoma, a malignant mesothelioma, a non-small cell lung cancer, a renal cell carcinoma, a head and neck cancer, a urothelial cancer, a colorectal cancer, a hepatocellular carcinoma, a small cell lung cancer, a Merkel cell carcinoma, a gastric cancer, an esophageal cancer, and a cervical cancer.

15. The use according to claim 14, wherein - the cancer induced by a virus or associated with an immunodeficiency is Kaposi's sarcoma or a cancer associated with human immunodeficiency virus (HIV) infection.

16. The use according to any one of claims 13-15, wherein the pharmaceutical composition or bifunctional molecule is for use in combination with radiotherapy or an additional therapeutic agent selected from the group consisting of alkylating agents, angiogenesis inhibitors, antimetabolites, antimitotic agents, antiproliferative agents, antiviral agents, apoptosis promoters, death receptor pathway activators, leukemia viral oncogene homolog receptor inhibitors, growth factor inhibitors, heat shock protein (HSP)-90 inhibitors, histone deacetylase (HDAC) inhibitors, agents used in hormone therapy, immunologicals, intercalating antibiotics, kinase inhibitors, kinesin inhibitors, Jak2 inhibitors, mammalian target of rapamycin inhibitors, microRNAs, multivalent binding proteins, non-steroidal anti-inflammatory drugs (NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP) inhibitors, platinum-based chemotherapeutics, proteasome inhibitors, purine analogs, pyrimidine analogs, plant alkaloids, small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitors, ubiquitin ligase inhibitors, checkpoint inhibitors, peptide vaccines, epitope peptides from tumor antigens, and combinations of one or more of these agents.

17. The use according to claim 16, wherein the additional therapeutic agent is selected from the group consisting of hypomethylating agents, BiTE (bispecific T-cell engager) antibodies, antibody drug conjugates, aurora kinase inhibitors, Bcr-Abl kinase inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, cyclin-dependent kinase inhibitors, mitogen-activated extracellular signal-regulated kinase inhibitors, polo-like kinase (Plk) inhibitors, phosphoinositide-3 kinase (PI3K) inhibitors, receptor tyrosine kinase inhibitors, inhibitors of inhibitors of apoptosis proteins (IAPs), and Bcl-2 family inhibitors.

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