Bispecific recombinant proteins and uses thereof

Abstract

The present invention discloses a bispecific recombinant protein comprising a first functional binding fragment, a second functional binding fragment, and an Fc region, wherein the first functional binding fragment targets a target antigen and comprises an antigen-binding fragment, wherein the C-terminus of the CL domain or the C-terminus of the CH1 domain of the antigen-binding fragment is linked directly or via a linker sequence to a second functional binding fragment having immunomodulatory and / or metabolic and / or endocrine regulatory functions. The present invention also discloses the use of the bispecific recombinant protein in the manufacture of a medicament for treating tumors, autoimmune diseases, infectious diseases, sepsis, graft-versus-host disease, metabolic disorders, and endocrine disorders. The recombinant protein enhances targeting of the second functional binding fragment of the bispecific recombinant protein and significantly reduces toxic side effects caused by targeting of non-target organs, tissues, and cells by non-target proteins containing the second functional binding fragment produced during the manufacturing process.

Description

Detailed Description of the Invention

[0001] This application claims priority to Chinese Patent Application No. 202010977716.2, filed on September 17, 2020, and Chinese Patent Application No. 202110933105.2, filed on August 12, 2021. This application cites the above Chinese patent applications in their entirety.

[0002] [Technical Field] The present invention is in the field of biomedicine and specifically relates to bispecific recombinant proteins and uses thereof.

[0003] [Background technology] The immune system is a physiological function of the body that distinguishes between "self" and "non-self" components, destroying and rejecting antigenic substances (such as bacteria) that invade the body, as well as damaged and tumor cells produced by the body itself, preventing the development and maintenance of tumor health. However, mutated cells can sometimes evade the body's immune surveillance through various mechanisms, rapidly proliferating within the body and forming tumors. The body also regulates its immune system through immune inhibition to avoid abnormal or persistent activation of the immune system, leading to immune inflammation, cytokine storms, and immune rejection of allografts, resulting in autoimmune diseases, sepsis, graft-versus-host disease (GVHD), and other organ damage.

[0004] Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restarting and maintaining the tumor immune cycle and restoring the body's normal anti-tumor immune response. Blocking these immune evasion mechanisms or "promoting" immune activation by regulating and controlling immune regulation and immune activation mechanisms are currently common tumor immunotherapy approaches, such as anti-PD-1 antibodies (nivolumab, pembrolizumab, etc.), anti-PD-L1 antibodies (durvalumab, atezolizumab, etc.), and anti-CD47 antibodies / fusion proteins (magrolimab, TTI-621, etc.).

[0005] Autoimmune diseases refer to diseases in which the body's own tissues are damaged by the body's immune response to autoantigens, and include organ-specific autoimmune diseases and systemic autoimmune diseases. Organ-specific autoimmune diseases mainly include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuropathy. Systemic autoimmune diseases generally include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, and ulcerative colitis.

[0006] Sepsis is an acute systemic infection caused by various pathogenic bacteria that invade the bloodstream, grow and proliferate in the blood, and produce toxins. Clinical symptoms generally include abrupt onset, chills, high fever, shortness of breath, tachycardia, rash, joint swelling and pain, hepatosplenomegaly, and mental and cognitive changes. Severe cases may develop acute organ dysfunction, and further progression may lead to septic shock, disseminated intravascular coagulation (DIC), and multiple organ failure. Sepsis is one of the most common causes of death in intensive care units. On the one hand, pathogenic or opportunistic pathogens rapidly proliferate in the bloodstream, and if the immune system is unable to eliminate them in time, they can cause acute systemic infection. On the other hand, toxins secreted by these pathogens, such as lipopolysaccharide (LPS), activate macrophages and release inflammatory factors such as TNF, IL-1β, and HMGB1, resulting in a systemic inflammatory response that further worsens sepsis and becomes life-threatening.

[0007] Graft-versus-host disease (GVHD) occurs when T lymphocytes in the allogeneic donor graft undergo a series of cytokine storms initiated by the recipient, resulting in a significantly enhanced immune response to recipient antigens and a cytotoxic attack on recipient target cells. The incidence of acute GVHD is 30% to 45%.

[0008] Numerous studies have shown that immune checkpoints or their ligands, cytokines, etc. play very important roles in the prevention and treatment of diseases.

[0009] Immune checkpoints refer to a series of molecules expressed on immune cells that can regulate the degree of immune activation. Immune checkpoints and their ligands play an important role in regulating autoimmunity. Abnormal expression and function of immune checkpoint molecules is one of the important causes of many diseases. For example, overexpression or excessive function of inhibitory immune checkpoint molecules inhibits immune function, weakening the body's immune system and making people more susceptible to diseases such as infections and tumors. Conversely, if the immune inhibitory function of inhibitory immune checkpoint molecules is too weak, the body's immune system becomes abnormally active. Tumor cells also express several substances that activate inhibitory immune checkpoints. When activated, these substances act like a "brake" to prevent antigens from being presented to T cells, blocking the antigen presentation process in the tumor immune system and inhibiting the immune function of T cells, allowing tumor cells to escape surveillance and survive. Common immune checkpoints and their ligands are, for example, PD-1 / PD-L1, LAG-3 / MHCII, CTLA-4 / B7-1 or B7-2, TIM-3 / Galectin-9, SIRPα / CD47, TIGIT / Nectin2 or Nectin3, and BTLA / HVEM.

[0010] PD-1 (programmed death receptor 1) is an important immune inhibitory molecule belonging to the immunoglobulin superfamily. The binding of PD-1 to PD-L1 triggers the programmed death of T cells, leading to immune evasion in tumor cells. Therefore, numerous clinical studies have demonstrated that blocking the immunomodulatory effects of the interaction between PD-1 and PD-L1 is crucial for anti-tumor, anti-infection, anti-autoimmune disease, and organ transplant survival.

[0011] Lymphocyte-activation gene 3 (LAG-3) belongs to the immunoglobulin superfamily and consists of three parts: the extracellular domain, the transmembrane domain, and the cytoplasmic domain. Inhibiting LAG-3 can restore the cytotoxicity of T cells and enhance their killing effect against tumors. At the same time, inhibiting LAG-3 can also reduce the function of T cells to inhibit immune responses.

[0012] CTLA-4 (cytotoxic T lymphocyte-associated antigen-4), also known as CD152, is a leukocyte differentiation antigen and a transmembrane receptor on T cells. It shares the B7 molecular ligand with CD28, and binds to CD80 and CD86 (also known as B7-1 and B7-2) with higher affinity and avidity than CD28, transmitting inhibitory signals to T cells and participating in the negative regulation of immune responses.

[0013] TIM-3, also known as HAVCR2, belongs to the TIM gene family. TIM-3 acts as a negative regulatory immune checkpoint and is present in various immune cell types, including T cells, regulatory T cells (Tregs), dendritic cells (DCs), B cells, macrophages, natural killer cells (NKs), and mast cells. TIM-3 is a type I membrane protein consisting of 281 amino acids. It consists of an extracellular domain, a single transmembrane domain, and a C-terminal cytoplasmic tail. TIM-3 and its ligand, Gal-9, inhibit tumor immunity by negatively regulating T cell immunity. Ligation of the TIM-3 IgV structural domain to Gal-9 can terminate Th1 immune responses. TIM-3 can induce immune tolerance and is associated with asthma, food allergies, and autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. TIM-3 also inhibits T cell immune responses and is involved in immune exhaustion, which leads to chronic viral infections.

[0014] CD47 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily that is expressed on the surface of almost all cells, including red blood cells. CD47 ligands include integrins, thrombospondin-1, and signal-regulatory receptor proteins (SIRPs). CD47 has various biological functions, including cell migration, T cell and dendritic cell activation, and axon development. Furthermore, CD47 inhibits macrophage phagocytosis by interacting with SIRPα. In this way, CD47 transmits a so-called "don't eat me" signal, protecting normal cells, such as red blood cells, B cells, and T cells, from phagocytosis by macrophages.

[0015] SIRP-alpha (SIRPα), a typical inhibitory immunoreceptor of the SIRP family, interacts with the ubiquitous transmembrane glycoprotein CD47 to transmit inhibitory signals. SIRPα fusion proteins can competitively block the binding of CD47 to SIRP-alpha on the surface of macrophages, alleviating the inhibitory effect of CD47 on tumor cells on macrophages, restoring the immune function of macrophages and exerting antitumor effects.

[0016] TIGIT (T cell immunoreceptor containing Ig and ITIM junction domains, also known as VS1G9, VSTM3, or WUCAM) is a member of the immunoglobulin poliovirus receptor family, a CD28 family-like receptor, and is expressed on a subset of T cells and natural killer (NK) cells. Its expression level is normally low, but upon activation of these cells, its protein level is upregulated. For example, in the tumor microenvironment, TIGIT is often highly expressed on tumor-infiltrating lymphocytes. TIGIT can bind to receptors CD155 (poliovirus receptor, PVR), CD112 (PVRL2, nectin-2), and CD113 (nectin-3) on immune cells, non-immune cells, and tumor cells, leading to T cell inactivation and inhibiting the cytotoxicity of T cells and natural killer cells.

[0017] BTLA is an important immune checkpoint molecule expressed on activated T and B lymphocytes. It shares a similar structure and intracellular signaling mechanism with other immune checkpoint molecules, such as PD-1 and CTLA-4 (a single immunoglobulin variable region (IgV) extracellular structural domain). Two ITIM structural domains in the cytoplasm activate the SHP-1 and SHP-2 kinases, inhibiting lymphocyte function. HVEM (Herpesvirus entry mediator), a member of the TNF receptor family, has been identified as a ligand for BTLA. It is widely expressed on human immune cells, including T cells, B cells, NK cells, myeloid cells, dendritic cells, and various tumor cells (including non-small cell lung cancer, melanoma, colorectal cancer, and lymphoma). Under normal physiological conditions, BTLA binds to its ligand, HVEM, and inhibits excessive lymphocyte activation in the body, preventing self-damage by the immune system. However, tumor cells, such as those from lung cancer, melanoma, colorectal cancer, and lymphoma, overexpress HVEM and bind to BTLA expressed by tumor-specific killer lymphocytes, thereby inhibiting the immune function of lymphocytes. High expression of HVEM in tumors is associated with poor prognosis.

[0018] Cytokines (CK) are small polypeptides or glycoproteins synthesized and secreted by various tissue cells (mainly immune cells). Cytokines can mediate cell-cell interactions and have various biological functions, such as regulating cell proliferation, differentiation and maturation, maintaining function, regulating immune responses, participating in inflammatory reactions, wound healing, and tumor progression. Depending on their structure and function, cytokines can be divided into the interleukin superfamily, interferon family, tumor necrosis factor superfamily, TGF-β superfamily, chemokine family, colony-stimulating factor family, and growth factor family.

[0019] Interleukins are cytokines produced by and act on a variety of cells. They were first produced by white blood cells and were named after the role they played among white blood cells, a name still used today. Their molecular structure and biological function have been fundamentally elucidated, and they play important regulatory roles. They are a type of cytokine with a unified name, and belong to the same group as blood growth factors. The two interact cooperatively to complete hematopoiesis and immunoregulatory functions. Interleukins play an important role in signal transduction, immune cell activation and regulation, T and B cell activation, proliferation and differentiation mediation, and inflammatory responses.

[0020] IL-2 is a multicellular cytokine (mainly produced by activated T cells) with pleiotropic effects, promoting the growth, proliferation, and differentiation of lymphocytes and playing an important role in the body's immune response and antiviral infection. IL-2 can activate T cells, promote cytokine production, stimulate NK cell proliferation, enhance NK cell killing activity, produce cytokines, induce the production of LAK cells, promote B cell proliferation and antibody secretion, and activate macrophages.

[0021] IL-10, also known as cytokine synthesis inhibitory factor (CSIF), possesses bidirectional immunomodulatory effects. IL-10 exerts immunosuppressive effects via antigen-presenting cells (APCs) and negative regulatory effects via T cells, negatively regulating immune responses in tumor environments. IL-10 also exerts stimulatory effects on T and B lymphocytes, and in tumor environments, stimulatory effects of IL-10. The bidirectional regulation of IL-10 has attracted considerable attention since its discovery. It not only influences the immune system but may also affect many pathophysiological processes, including angiogenesis, tumorigenesis, and infection, by regulating growth factors and cytokines. It can also play a role in peripheral tolerance by inducing regulatory T cells. IL-10 plays an important role in Crohn's disease, rheumatoid arthritis, psoriasis, HCV infection, and HIV infection. In nature, IL-10 forms a homodimer and binds to its receptor to form a complex, activating downstream signaling pathways and exerting its physiological effects. In 2000, Josephson et al. studied and designed IL-10 mutants that bind to the IL-10 receptor in the monomeric state and activate downstream signaling pathways (Josephson, K. Design and analysis of an engineered human interleukin-10 monomer. [J]. Journal of Biological Chemistry, 2000, 275(18):13552-7.).

[0022] Interferons are cytokines produced mainly by monocytes and lymphocytes. They are a group of multifunctional active proteins divided into three types: type I, type II, and type III. Type I interferons are mainly secreted by innate immune cells and mainly include IFN-α and IFN-β. Type II interferons are mainly secreted by activated T cells, such as IFN-γ, and type III interferons include several types of IFN-λ. Interferons have various biological activities, including broad-spectrum antiviral activity, cell proliferation and differentiation, and immune function regulation. Their specific functions are as follows: (1) Antiviral effect: Type I interferon is the main antiviral defense and regulatory factor in the immune system. During the early viral infection, type I interferon can control the growth and proliferation of the virus. On the one hand, it can directly activate immune cells, and on the other hand, it can indirectly inhibit the viral replication process.

[0023] (2) Antibacterial effects: Interferon can reduce the bacterial iron supply by downregulating transferrin receptors or directly inhibit intracellular bacteria by inducing the production of endogenous NO. It can also increase the bacterial lysis activity of lysosomes, the phagocytic bodies of monocyte-macrophages, and eliminate bacteria through the above pathways.

[0024] (3) Antiparasitic effect: Interferon can activate macrophages (Mφ), which express high levels of inducible nitric oxide synthase (iNOS) and catalyze the production of NO from L-arginine, which has inhibitory and killing effects on inoculated pathogens. It has also been reported that IFN-γ activates Mφ to produce NO, and its effect in promoting NO synthesis is dose-dependent, with higher doses being more significant. Daubener et al. (2001) demonstrated that stimulating human brain microvascular endothelial cells (HBMEC) with IFN-γ can induce resistance to toxoplasmosis. IFN-γ-stimulated HBMEC inhibits Toxoplasma growth and increases the expression of TNF-α, which is associated with IDO activity. Furthermore, the addition of excess tryptophan to HBMEC cultures completely inhibited IFN-γ-TNF-α-mediated anti-toxoplasmosis, indicating that IDO could mediate this protection, and it was reported that IFN-γ functions dependently on IDO expression.

[0025] (4) Involvement in immunoregulation: IFN-γ, also known as immunomodulatory interferon, is primarily involved in immunoregulation. Immunomodulatory interferons can be expressed by IgG Fc receptors, which are beneficial for antigen phagocytosis by macrophages, target cell killing by K and NK cells, and activation of T and B lymphocytes, thereby enhancing the body's immune response. IFN-γ can increase the expression of MHC class II molecules on the surface of macrophages, enhancing their antigen-presenting ability. Furthermore, by enhancing the expression of Fc receptors on the surface of macrophages, IFN-γ can promote the phagocytosis of immune complexes, antibody-coated pathogens, and tumor cells by macrophages. At the same time, it can also stimulate neutrophils, enhance their phagocytic activity, activate NK cells, and increase their cytotoxicity, thereby participating in immunoregulation.

[0026] (5) Antitumor effects: IFN-α and IFN-β have a wide range of antitumor effects, and IFN-γ has a multifaceted regulatory effect on the body's immune response. It can activate effector cells, increase the activity of natural killer cells, macrophages, and tumor-infiltrating lymphocytes, promote the circulation of monocytes, enhance the expression of antigens and antibodies on the surface of immune cells, stimulate the production of cytokines such as IL-2, tumor necrosis factor, and IFN-α, inhibit tumor cell division, and induce gene synthesis into complete antiviral proteins.

[0027] The tumor necrosis factor (TNF) superfamily is a cytokine expressed in serum that can kill cancer cells in mice. Major members include TNF-α, TNF-β, lymphotoxin-β, CD40L, FasL, CD30L, 4-1BBL, CD27L, and OX40L, TNF-related apoptosis-inducing ligand (TRAIL), receptor activator of light (RANKL), TNF-related apoptosis-inducing factor (TWEAK), proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), vascular endothelial cell growth inhibitor (VEGI), cytoplasmin A (EDA-A1, EDA-A2), and glucocorticoid-induced tumor necrosis factor receptor-related ligand (GITRL). Members of the TNF superfamily specifically recognize dozens of receptors, forming a ligand-receptor mechanism of action. TNF receptors (TNFRs) are transmembrane proteins primarily involved in physiological processes such as host defense, inflammation, apoptosis, autoimmunity, and immunity, as well as ectodermal and nervous system development and organogenesis. TNFs induce tumor cell necrosis (cell swelling, organelle destruction, and cell lysis) and apoptosis (cell shrinkage, aggregate formation, and DNA fragmentation), playing an important role in various immune and inflammatory processes.

[0028] Colony-stimulating factors (CSFs) are cytokines that stimulate the differentiation and maturation of immature bone marrow cells and stimulate colony formation in vitro. Different hematopoietic cell lines or cells at different differentiation stages form distinct cell colonies in semi-solid medium under different cytokine stimulation. These CSFs are named granulocyte CSF (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), multi-colony stimulating factor (also known as interleukin-3 [IL-3]), stem cell factor (SCF), and erythropoietin (EPO), respectively. They promote the proliferation and differentiation of hematopoietic stem cells at different developmental stages and are essential stimulators of hematopoiesis. Broadly speaking, all cytokines that stimulate hematopoiesis can be collectively referred to as CSFs. For example, leukemia inhibitory factor (LIF), which stimulates embryonic stem cells, and thrombopoietin, which stimulates platelets, both possess colony-stimulating activity. CSF also acts on various mature cells and has a multiphasic effect in promoting their functions.

[0029] Chemokines are small cytokines or signaling proteins secreted by cells. They are named chemokines because of their ability to direct chemokines to nearby responding cells. Common structural features of chemokine proteins are their small molecular weight (approximately 8-10 kDa) and the presence of four conserved cysteine ​​residues that ensure their tertiary structure. Chemokines are divided into four major subfamilies: CXC, CC, CX3C, and XC. All of these proteins exert their biological effects by interacting with transmembrane receptors (called chemokine receptors), which are G protein-coupled transmembrane receptors selectively expressed on the surface of target cells.

[0030] The primary function of chemokines is to regulate the migration (homing) of leukocytes to their respective locations during inflammation and homeostasis. Basal homing: These are basic homeostatic chemokines produced in the thymus and lymphoid tissues. The chemokines CCL19 and CCL21 (expressed on lymph node and lymphatic endothelial cells) and their receptor CCR7 (expressed on cells that home to these organs) are the best examples of the homeostatic function of homing. These ligands enable antigen-presenting cells (APCs) to migrate to lymph nodes during the adaptive immune response. Other homeostatic chemokine receptors include CCR9, CCR10, and CXCR5, which are important for tissue-specific leukocyte homing as part of cell addressing. CCR9 supports leukocyte migration to the intestine, CCR10 supports skin migration, and CXCR5 supports B cell migration to lymph node follicles. CXCL12 (SDF-1) produced in the bone marrow promotes the proliferation of B cell precursors in the bone marrow microenvironment. Inflammatory homing: Inflammatory chemokines are produced in high concentrations during infection or injury and determine the migration of inflammatory leukocytes to the site of injury. Typical inflammatory chemokines include CCL2, CCL3, and CCL5, CXCL1, CXCL2, and CXCL8.

[0031] Some chemokines are thought to be pro-inflammatory cytokines that direct cells of the immune system to enter sites of infection during an immune response, while others are thought to maintain the body's autoregulation and control cell migration during normal tissue maintenance or development.

[0032] Chemokines are classified according to the cell type on which they chemotactically affect:

[0033] Monocyte / macrophage chemokines: The major chemokines that attract monocytes / macrophages to sites of inflammation include CCL2, CCL3, CCL5, CCL7, CCL8, CCL13, CCL17, and CCL22.

[0034] T lymphocyte chemokines: The four important chemokines involved in the recruitment of T lymphocytes to inflammatory sites are CCL2, CCL1, CCL22, and CCL17. Furthermore, T cells induce the expression of CXCR3 after activation, and activated T cells are attracted to inflammatory sites and secrete the IFN-γ-inducing chemokines CXCL9, CXCL10, and CXCL11 at the inflammatory site.

[0035] Mast cell chemokines: Mast cells express multiple chemokine receptors on their surface, including CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4. The ligands for these receptors, CCL2 and CCL5, play an important role in the recruitment and activation of pulmonary mast cells. There is also evidence that CXCL8 may inhibit mast cells.

[0036] Eosinophil chemokines: Migration of eosinophils to various tissues is mediated by several chemokines of the CC family: CCL11, CCL24, CCL26, CCL5, CCL7, CCL13, and CCL3. The chemokines CCL11 (eotaxin) and CCL5 (rantes) act via the specific receptor CCR3 on the surface of eosinophils, and play an important role in the initial recruitment of eosinophils to the lesion.

[0037] Neutrophil chemokines: Primarily regulated by CXC chemokines. For example, CXCL8 (IL-8) is a neutrophil chemokine that activates neutrophil metabolism and degranulation.

[0038] The TGF-β superfamily is a group of cytokines that regulate cell proliferation and differentiation. In addition to TGF-β, this family includes activins, inhibitors, Mullerian inhibitor substance (MIS), and bone morphogenetic proteins (BMPs). TGF-β plays a role in inflammation, tissue repair, and embryonic development, and plays an important regulatory role in cell proliferation, differentiation, and immune function. Its specific functions are described below.

[0039] (1) Inhibition of the proliferation of immune-activated cells: (i) Inhibits colony formation of murine hematopoietic progenitor cells and LTBMC induced by IL-3, GM-CSF, and M-CSF, and reduces megakaryocyte responsiveness to IL-3T and CSF. (ii) Inhibits thymocyte proliferation induced by ConA or a combination of ConA, IL-2, and IL-6. (iii) Inhibits the proliferation of T cells stimulated by mitogens or alloantigens or the proliferation of IL-2-dependent T cells. (iv) Inhibits the proliferation of IL-2-dependent B cells after SAC stimulation.

[0040] (2) Modulation of cell phenotype: (i) Inhibition of IL-2-induced expression of T cell IL-2R, TfR, and TLiSA1 activation antigens without affecting CD3 expression, and (ii) Inhibition of IFN-γ-induced expression of MHC II antigens on melanoma cells.

[0041] (3) Inhibition of lymphocyte differentiation: (i) Inhibits IL-2 and BCDF-dependent IgM secretion from B cells and promotes the conversion of B cell-secreted Ig to IgA and IgE. (ii) Inhibits CTL, NK, and LAK functions in mixed lymphocyte cultures (MLCs), and this inhibitory effect can be reversed by TNF-α (mouse MIC) or IL-2 (human MLCs). (iii) Inhibits NK activity in PBMCs. (iv) Inhibits the activity of mouse thymic MHC-restricted killer cells synergistically induced by ConA, IL-2, and IL-6.

[0042] (4) Inhibiting cytokine production: For example, inhibiting the production of IFN-γ and TNF-α in PBMCs.

[0043] (5) Other regulatory effects: (i) promote the proliferation of fibroblasts, osteoblasts, and Schwann cells. TGF-β1 and TGF-β2 promote IL-6 production in human fibroblasts, possibly through the regulation of IL-6 gene transcription. (ii) inhibit the proliferation of epithelial cells, osteoclasts, and endothelial cells and the formation of adipocytes, cardiac muscle, and skeletal muscle. TGF-β can antagonize some biological functions of EGF. (iii) promote the expression of extracellular matrix (ECM) components such as collagen and fibronectin and inhibit their degradation, playing an important role in cell morphogenesis, proliferation, and differentiation, and is beneficial for embryonic development and cell repair. In vivo experiments in animals have shown that local injection of TGF-β promotes wound healing and typical granulation tissue formation. (iv) TGF-β is a chemokine for monocytes and fibroblasts but does not induce the production of colloid particles and oxidants. (v) inhibit the adhesion of lymphocytes and endothelial cells. (vi) promote the release of histamine from basophils.

[0044] (6) TGF-β1 and Proto-Oncogene Expression: TGF-β1 induces the expression of c-sis but inhibits the expression of c-myc. These inductions and inhibitions are related to the cell type and the different functions of TGF-β. For example, TGF-β induction of c-sis gene expression in fibroblasts is associated with the promotion of their proliferation in soft agar, while inhibition of epidermal keratinocyte proliferation is associated with the inhibition of c-myc gene expression. While TGF-β1, TGF-β2, and TGF-β3 share many similar biological effects, the inhibitory effect of TGF-β2 on the proliferation of vascular endothelial cells and hematopoietic progenitor cells is only 1% of that of TGF-β1 and TGF-β3, and significant differences may exist in some of their effects.

[0045] TGF-β has potential uses in treating wound healing, promoting cartilage and bone repair, and in treating autoimmune diseases and transplant rejection through immunosuppression.

[0046] Growth factors are cytokines secreted by various cells, which act on specific target cells to regulate cell division, matrix synthesis, and tissue differentiation. There are various types of growth factors, including platelet-like growth factors (platelet-derived growth factor, PDGF; osteosarcoma-derived growth factor, ODGF), epidermal growth factor (EGF), fibroblast growth factors (αFGF, βFGF), insulin-like growth factors (IGF-I, IGF-II), and nerve growth factor (NGF).

[0047] However, immunomodulation is often systemic, and immune activation is often accompanied by overactivation of the immune system, leading to immune-related toxic reactions such as those described below.

[0048] Skin: Symptoms include rash, papules, itching, dermatitis herpetiformis syndrome, etc. Endocrine: manifests as hypothyroidism, hyperthyroidism, primary adrenal insufficiency, hyperglycemia, etc. Liver: manifests mainly as elevated transaminases, Gastrointestinal: mainly manifested as diarrhea / colitis, Pulmonary: manifested as immune-mediated pneumonia, Other relatively rare side effects include neurotoxicity, hematotoxicity, nephrotoxicity, cardiac toxicity, and ocular toxicity.

[0049] Therapies targeting immune checkpoints or their ligands, cytokines or their receptors have already demonstrated therapeutic efficacy against diseases such as tumors, autoimmune diseases, sepsis, and graft-versus-host disease (GVHD). However, the drugs themselves have potential toxicities that limit their dosage and reduce their efficacy. Furthermore, immunosuppression is often accompanied by excessive weakening of the immune system, leading to bacterial, viral, and fungal infections, recurrent inflammation, and tumor development.

[0050] Therefore, single-target therapies such as various cytokines, monoclonal antibodies, and ligand / receptor Fc fusion proteins often fail to achieve optimal safety windows and therapeutic efficacy, leading to the concept of synergistic effects of two or more targets. In such cases, bispecific antibodies / fusion proteins (bispecific antibodies) or multispecific antibodies / fusion proteins (multispecific antibodies) that target two or more targets may offer novel therapeutic applications that are difficult to achieve with monoclonal antibodies. These include the design concept and clinical application of bispecific / multispecific antibodies / fusion proteins (e.g., blinatumomab) that target cell surface antigens (e.g., CD19, HER2) and immunomodulatory targets (e.g., CD3, PD-1, CD47) or carry immunomodulators (e.g., IL-2, 4-1BB, SIRPα, CD80).

[0051] Commonly seen symmetrical bispecific antibody molecules / bispecific fusion proteins have a certain target aggregation effect, allowing drug distribution to accumulate in target organs to a certain extent, making the drug development process, especially the purification process, relatively controllable. However, their structures still have certain limitations, especially the difference in drug concentration between target and non-target organs is not very significant. When non-target organs are used, such as subcutaneous or intravenous administration, the drug concentration in the blood still causes immunotoxicity and is a major issue limiting its safe dosage. Therefore, the affinity combination of the antigen-binding fragment / fusion protein targeting the target antigen and the antigen-binding fragment / fusion protein targeting the immune checkpoint / cytokine receptor must be strictly controlled to prevent the immunomodulatory targeting portion from already inducing immune-related toxic side effects if the target antigen-targeting portion of the bispecific molecule does not achieve the optimal concentration effect in target cells.

[0052] In addition to symmetric structures, bispecific antibodies / fusion proteins also use asymmetric structures. The difference in affinity between the left and right arms can further enhance the abundance of the drug on the surface of antigen-positive target cells and further reduce immune-related toxic side effects induced by targeting the immunomodulatory moiety. However, asymmetric structures have undesirable effects, such as increased difficulty in the pharmaceutical process, reduced yield, and increased manufacturing costs. Furthermore, monomeric or polymeric impurities in the immunomodulatory arm pose a significant risk for immune-related adverse events. For example, TTI-621 (a SIRPα-Fc fusion protein developed by Trillium Therapeutics Inc.) has been shown in published clinical studies to cause severe thrombocytopenia (grade III or higher) in over 20% of patients at very low doses (0.2 mg / kg). (TTI-621 binds to CD47, which is highly expressed on platelets, leading to their elimination by immune cells.) As shown in Figure 3 of CN108864290A, the double antibody structure potentially contains impurities of the right arm monomer / dimer structure (i.e., TTI-621 analogues) in the finished product during the manufacturing process. Typical tumor-targeting antibody drug dosages of 5mg / kg to 20mg / kg require extremely low impurity concentrations (1-4%) to reach the dosage levels at which TTI-621 causes serious toxic side effects. Therefore, this structure requires extremely strict control of the impurity content of the finished product, which significantly increases production costs.

[0053] Furthermore, some immune-modulating fusion proteins, such as interferon, generally have poor freeze-thaw stability due to their product characteristics. For example, the polymer content of recombinant human albumin interferon-α 2b fusion protein (rHSA-IFN-α 2b) gradually increases with repeated freezing and thawing, reaching 17.91% after four freeze-thaw cycles. Recombinant human albumin interferon-α 2b fusion protein is not suitable for freeze-thawing (Xia Yi et al., Study on the stability of recombinant human albumin interferon-α 2b fusion protein, Journal of Biology, 25(006):38-40). Therefore, there are many limitations in production, transportation, and use.

[0054] Therefore, there is an urgent need to invent a biantibody or bispecific structure that can target a target antigen and simultaneously exert an immunomodulatory effect. Such a structure not only achieves high concentration of target cells and exerts an immunomodulatory effect, but also does not simply rely on the immunomodulatory end to exert its immunomodulatory function in the absence of the target antigen even when it reaches a high concentration in the blood, thereby more accurately achieving immunomodulation of target organs, tissues, and cells, reducing the risk of immune-related toxic side effects, and improving product stability (e.g., freeze-thaw stability).

[0055] Summary of the Invention The first technical problem to be solved by the present invention is to provide a bispecific recombinant protein that has strong targeting ability to target cells containing the target antigen, and at the same time has a directional control effect of not binding or only weakly binding to non-target cells not containing the target antigen, thereby significantly improving the targeting of the second functional binding fragment of the recombinant protein to the target cells, directional control of the immune effect, and at the same time significantly reducing the toxic side effects caused by potentially risky impurities containing the second functional binding fragment generated during the production process that target organs, tissues, etc. containing non-target cells.

[0056] The second technical problem to be solved by the present invention is to provide a use of a bispecific recombinant protein with the above-mentioned direction-regulating effect in the production of medicines.

[0057] The third technical problem that the present invention aims to solve is to provide a bispecific recombinant protein that can improve product stability (e.g., freeze-thaw stability), thereby resolving the instability of some immunomodulatory fusion proteins (e.g., interferon) due to their inherent properties and the associated limitations on their production, transportation, and use.

[0058] In one embodiment, the present invention provides a bispecific recombinant protein, comprising a first functional binding fragment, a second functional binding fragment, and an Fc region, wherein the first functional binding fragment targeting a target antigen of the bispecific recombinant protein comprises an antigen-binding fragment, and wherein the C-terminus of the CL domain or the C-terminus of the CH1 domain of the antigen-binding fragment is linked directly or via a linker sequence to a second functional binding fragment having immunomodulatory and / or metabolic and / or endocrine regulatory functions.

[0059] Preferably, the antigen-binding fragment is linked to the N-terminus of the second functional binding fragment either directly or via a linker sequence, and the C-terminus of the second functional binding fragment is linked to the N-terminus of Fc either directly or via a linker sequence.

[0060] In one embodiment, the second functional binding fragment with immunomodulatory function targets an immune checkpoint, an immune checkpoint ligand, or a cytokine receptor. Optionally, the second functional binding fragment with immunomodulatory function targets PD-1 or its ligand, CD47 or its ligand, CD24 or its ligand, an interferon receptor (e.g., a type I or type II interferon receptor), or an interleukin receptor.

[0061] In one embodiment, the second functional binding fragment with metabolic regulatory function targets a metabolic regulator, a metabolic regulator receptor, or optionally, the second functional binding fragment with metabolic regulatory function targets an insulin receptor, a fibroblast growth factor receptor.

[0062] In one embodiment, the second functional binding fragment with endocrine regulatory function targets an endocrine regulator, an endocrine regulator receptor, or optionally, a hormone receptor.

[0063] In one embodiment, the variable region (V region) and constant region (C region) of the antigen-binding fragment are linked directly or via a linker sequence, or the antigen-binding fragment and Fc region are linked directly or via a linker sequence, or are linked using both of the above methods simultaneously.

[0064] In one embodiment, the linker sequence is (GGGGS)n (SEQ ID NO: 65) , (GGGS)n (SEQ ID NO: 66) , (GGS)n, (G)n, (GS)n, (EAAAK)n (SEQ ID NO: 67) or (XP)n, where n is a natural number. Preferably, n is a natural number from 0 to 5.

[0065] In one embodiment, the linker sequence is (GGGGS)n (SEQ ID NO: 65) where n=0, 1, 2, 3, 4, 5.

[0066] In one embodiment, the second functional binding fragment binds to a cytokine receptor or an immune checkpoint or an immune checkpoint ligand. Preferably, the second functional binding fragment is a cytokine or immune checkpoint ligand or immune checkpoint ligand binding protein, or a functional fragment or mutant thereof, and optionally a human SIRP family extracellular functional fragment, a human interferon family functional fragment, a tumor necrosis factor superfamily functional fragment, a TGF-β superfamily functional fragment, an interleukin system functional fragment, a chemokine family functional fragment, a colony-stimulating factor family functional fragment, a growth factor functional fragment, or a mutant thereof.

[0067] In one embodiment, the second functional binding fragment is the human SIRPα extracellular D1 domain or a mutant thereof. Preferably, the mutant is a low-affinity mutant, i.e., the binding affinity of the mutant to the human CD47 protein is not higher than the binding affinity of the wild-type protein to the human CD47 protein.

[0068] In one embodiment, the second functional binding fragment is human interferon gamma (IFN-γ) or human interferon alpha (IFN-α) or human interferon beta (IFN-β), a truncation thereof or a mutant thereof.

[0069] In one embodiment, the second functional binding fragment is an interleukin, a truncation thereof, or a mutant thereof.

[0070] In one embodiment, the interleukin is an immunomodulator or chemokine selected from the IL-1 family, IL-2 family, IL-3 family, IL-6 family, IL-8 family, IL-10 family, IL-12 family, and IL-17 family. Preferably, the second functional binding fragment is a fusion protein or a mutant thereof comprising a human IL-10 monomer mutant (amino acid sequence as set forth in SEQ ID NO: 52), human IL-12A or a mutant thereof (amino acid sequence as set forth in SEQ ID NO: 51), human IL-15 (amino acid sequence as set forth in SEQ ID NO: 53), and IL-15RαSUSHI (amino acid sequence as set forth in SEQ ID NO: 54), linked directly or via a linker sequence.

[0071] In one embodiment, the first functional binding fragment is selected from the group consisting of 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD74, CD79b, CD98, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CXCR4, DLL-4, EGFR, EGFRvIII, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GCC, The tumor targets one or more targets selected from GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Nectin-4, Notch2, Notch1, PD-L1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TIGIT, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138, TROP2, Siglec15, CD155, and AFP.

[0072] Preferably, when said second functional binding fragment comprises the human SIRPα extracellular D1 domain or a mutant thereof, the first functional binding fragment targets tumor cells or immune cells.

[0073] When the second functional binding fragment is human interferon alpha (IFN-α) or human interferon beta (IFN-β) or human interferon gamma (IFN-γ), a truncation or mutant thereof, the first functional binding fragment targets a tumor cell or an immune cell.

[0074] When the second functional binding fragment is an interleukin, a truncation or a mutant thereof, the first functional binding fragment targets a tumor cell or an immune cell.

[0075] In one embodiment, the bispecific recombinant protein is composed of an A chain and a B chain, and the A chain and the B chain are bound by intermolecular forces, or by covalent bonds such as interchain disulfide bonds, or by ionic bonds, or by a combination of two or three of the above-mentioned binding methods. The A chain is a protein sequentially composed of VH, CL / CH1, CH2, and CH3 domains, or a protein sequentially composed of VL, CL / CH1, CH2, and CH3 domains, where the domains are linked directly or via a linker sequence. The B chain is a protein sequentially composed of VL, CL / CH1, a second functional binding fragment, CH2, and CH3 domains, or a protein sequentially composed of VH, CL / CH1, a second functional binding fragment, CH2, and CH3 domains, where the domains are linked directly or via a linker sequence. For example, when "chain A" is VH-CH1-CH2-CH3, the "chain B" is VL-CL-second functional binding fragment-CH2-CH3; when "chain A" is VL-CL-CH2-CH3, the "chain B" is VH-CH1-second functional binding fragment-CH2-CH3; when "chain A" is VH-CL-CH2-CH3, the "chain B" is VL-CH1-second functional binding fragment-CH2-CH3; when "chain A" is VH-CL-CH2-CH3, the "chain B" is VL-CH1-second functional binding fragment-CH2-CH3; and when "chain A" is VL-CH1-CH2-CH3, the "chain B" is VH-CL-second functional binding fragment-CH2-CH3. The N-terminus of CH2 above further comprises a hinge region.

[0076] In one embodiment, the Fc region of the bispecific recombinant protein comprises a native Fc region sequence or a non-native Fc sequence, more preferably the Fc region is a human Fc region, and even more preferably, the Fc regions of the A and B chains are linked via knobs-into-holes.

[0077] In one embodiment, the Fc region is a human IgG Fc region, and preferably, the Fc region is a human IgG1 or IgG4 Fc region.

[0078] In one embodiment, the C-terminus of the CL domain or the C-terminus of the CH1 domain of the first functional binding fragment or the C-terminus of the second functional binding fragment is directly linked to the Fc region or is linked via a linker sequence.

[0079] In one embodiment, the first functional binding fragment is an antigen-binding fragment, optionally a human-mouse chimeric antigen-binding fragment, a human antigen-binding fragment, or a fully human antigen-binding fragment, and the first functional binding fragment is selected from the group consisting of 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD71, CD74, CD79b, CD80, CD83, CD86, CD98, CD206, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CCR5, CXCR4, DLL-4, EGFR, EGFRvIII, EGP-1, ENPP3, E and an antigen-binding fragment of a target antibody selected from any one or more of: phA3, ETBR, FGFR2, FN, FR-α, GCC, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Nectin-4, Notch2, Notch1, PD-L1, PD-L2, PD-1, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, TIGIT, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138, TROP2, Siglec15, CD155, and AFP, preferably, the first functional binding fragment is an antigen-binding fragment of a humanized or fully human antibody.

[0080] In one embodiment, when the first functional binding fragment targets CD20, EGFR, EGFRvIII, PD-L1, PD-L2, HER2, HER3, CD138, CD44, CD24, EpCAM, CLDN18.2, CD38, BCMA, MUC1, or TROP2, the second functional binding fragment comprises the SIRPα extracellular D1 domain or a mutant thereof. Preferably, the second functional binding fragment is as set forth in SEQ ID NO:50.

[0081] In one embodiment, when the first functional binding fragment targets Siglec15, PD-L1, PD-L2, CD71, CD80, CD86, CD206, or CCR5, the second functional binding fragment comprises a fusion protein formed between IFN-α, IFN-β, IFN-γ, or IL-10 monomer mutant, IL-12A, or IL-15 and IL-15RαSUSHI, or a truncation of the cytokine or a mutant that maintains the function of the cytokine. Preferably, the activity of the mutant for the corresponding cytokine receptor is no higher than the binding affinity of the wild-type cytokine to the corresponding cytokine receptor. More preferably, the cytokine contained in the second functional binding fragment is a fusion protein formed between SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53 and SEQ ID NO: 54.

[0082] In one embodiment, the first functional binding fragment is an antigen-binding fragment of the anti-CD20 antibody Ofatumumab, or the anti-EGFR antibody Panitumumab, or the anti-EpCAM antibody Catumaxomab, or the anti-CD24 antibody SWA11, and the second functional binding fragment is the human SIRPα extracellular D1 domain (as set forth in SEQ ID NO: 50).

[0083] In one embodiment, the first functional binding fragment comprises an antigen-binding fragment of the anti-alpha-fetoprotein (AFP) antibody tacatuzumab, and the second functional binding fragment is human IFN-α 2b or human IFN-β (SEQ ID NO: 55).

[0084] In one embodiment, the A and B chains of the bispecific recombinant protein are linked via IgG Fc, preferably via knobs-into-holes. For example, the knobs-into-holes are a protruding "knob" type formed by the T366W mutation and a recessed "hole" type formed by a single amino acid mutation (Y407V) or a recessed "hole" type formed by three amino acid mutations (T366S, L368A, and Y407V). Mutations are indicated from left to right as the original amino acid residue, the mutation site, and the replacement amino acid residue, respectively, according to the Kabat numbering scheme (Eu numbering scheme of Kabat et al. (1991)). For example, in T366W, T is the original amino acid residue, 366 is the mutation site, and W is the amino acid residue replacing T.

[0085] In some embodiments, the first functional binding fragment targets CD20, EpCAM, CD24, or EGFR, and the second functional binding fragment comprises the SIRPα extracellular D1 domain or a mutant thereof, and preferably the amino acid sequence of the second functional binding fragment is as set forth in SEQ ID NO:50, more preferably the amino acid sequence of the A chain is as set forth in SEQ ID NO:1, the amino acid sequence of the B chain is as set forth in SEQ ID NO:2 or 3, the amino acid sequence of the A chain is as set forth in SEQ ID NO:61, the amino acid sequence of the B chain is as set forth in SEQ ID NO:62, the amino acid sequence of the A chain is as set forth in SEQ ID NO:27, the amino acid sequence of the B chain is as set forth in SEQ ID NO:28, the amino acid sequence of the A chain is as set forth in SEQ ID NO:29, the amino acid sequence of the B chain is as set forth in SEQ ID NO:30, the amino acid sequence of the A chain is as set forth in SEQ ID NO:56, and the amino acid sequence of the B chain is as set forth in SEQ ID NO:57.

[0086] In some embodiments, the first functional binding fragment targets TIGIT, CD80, or PD-1, and the second functional binding fragment comprises IL-12A, an IL-10 monomer mutant, IL15, and an IL-15RαSUSHI complex, the amino acid sequences of which are as set forth in SEQ ID NOs: 51, 52, 53-54, respectively. More preferably, the amino acid sequence of the A chain is as set forth in SEQ ID NO: 35, the amino acid sequence of the B chain is as set forth in SEQ ID NO: 36, the amino acid sequence of the A chain is as set forth in SEQ ID NO: 37, the amino acid sequence of the B chain is as set forth in SEQ ID NO: 38, the amino acid sequence of the A chain is as set forth in SEQ ID NO: 39, and the amino acid sequence of the B chain is as set forth in SEQ ID NO: 40 or 41.

[0087] In some embodiments, the first functional binding fragment targets CD38 or AFP, and the second functional binding fragment comprises the SIRPα extracellular D1 domain or a mutant thereof, or IFN-β or a mutant thereof, and preferably the amino acid sequence of the second functional binding fragment is as set forth in SEQ ID NO: 50 or 55, more preferably the amino acid sequence of the A chain is as set forth in SEQ ID NO: 31, the amino acid sequence of the B chain is as set forth in SEQ ID NO: 32, the amino acid sequence of the A chain is as set forth in SEQ ID NO: 33, and the amino acid sequence of the B chain is as set forth in SEQ ID NO: 34, or the amino acid sequence of the A chain is as set forth in SEQ ID NO: 58, and the amino acid sequence of the B chain is set forth in SEQ ID NO: 60.

[0088] In another embodiment, the present invention provides nucleic acid molecules encoding said bispecific recombinant protein, wherein the nucleic acid molecule encoding said first functional binding fragment is on the same DNA strand as the nucleic acid encoding the second functional binding fragment, or wherein the nucleic acid molecule encoding said first functional binding fragment is on a different DNA strand than the nucleic acid encoding the second functional binding fragment.

[0089] Preferably, the nucleic acid molecule encoding the Fc region is on the same DNA strand as the nucleic acid encoding the first functional binding fragment or the second functional binding fragment, and the nucleic acid molecule encoding the Fc region is on a different DNA strand than the nucleic acid encoding the first functional binding fragment or the second functional binding fragment.

[0090] In another embodiment, the present invention provides an expression vector comprising the above-described nucleic acid molecule.

[0091] In another embodiment, the present invention provides a host cell transformed with the above expression vector.

[0092] In another embodiment, the present invention also provides a method for producing the bispecific recombinant protein, comprising using a host cell transformed with the expression vector described above, culturing the host cell under conditions suitable for expression, and obtaining the bispecific recombinant protein.

[0093] In another embodiment, the present invention provides the use of a bispecific recombinant protein in the manufacture of a medicament for treating a tumor, an autoimmune disease, an infectious disease, sepsis, graft-versus-host disease, a metabolic disorder, or an endocrine disorder.

[0094] In one embodiment, the tumor is a solid tumor or a hematological tumor. Preferably, the solid tumor is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell carcinoma, dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome. The hematological tumor is selected from myeloma, lymphoma, or leukemia. Preferably, the autoimmune disease is selected from any one of Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome. Preferably, the infectious disease is selected from any one of viral infections, bacterial infections, fungal infections, and other pathogenic infections.

[0095] In yet another embodiment, the present invention also provides a pharmaceutical or drug composition comprising the bispecific recombinant protein of the present invention and, optionally, an adjuvant, excipient, or pharmaceutically acceptable vector. The pharmaceutical composition may comprise a pharmaceutically acceptable vector. The composition may be in any form of drug formulation, including, but not limited to, an injection, a powder, a lyophilized powder, etc. The drug composition in the form of a drug formulation is prepared into a desired dosage form according to conventional formulation techniques, such as by fusing the pharmaceutically active ingredient, the bispecific recombinant protein or fusion protein of the present invention, with a drug vector and preparing the mixture according to conventional formulation techniques.

[0096] In one embodiment, the present invention also provides a pharmaceutical composition comprising an expression vector for a nucleic acid molecule encoding a bispecific recombinant protein of the present invention and any pharmaceutically acceptable vector.

[0097] In another embodiment, the present invention also provides a method for treating tumors, comprising administering to a patient or subject a therapeutically effective amount of a pharmaceutical composition according to the present invention, wherein the tumor expresses additional target molecules, such as 5T4, AGS-16, ALK1, ANG-2, B7-H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD20, CD22, CD24, EpCAM, CD30, CD32b, CD37, CD38, CD40, CD52, CD70, CD71, CD74, CD79b, CD80, CD83, CD86, CD98, CD206, CEA, and CE. ACAM5, CLDN18.2, CLDN6, CS1, CCR5, CXCR4, DLL-4, EGFR, EGFRvIII, EGP-1, ENPP3, EphA3, ETBR, FGFR2, FN, FR-α, GC C, GD2, GPC-3, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Nectin-4, Notch 2, Notch 1, PD-L1, PD-L2, PD-1, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, TIGIT, VEGFR, CD25, CD27L, DKK-1, CSF-1R, MSB0010718C, BCMA, CD138, TROP2, Siglec15, CD155, or AFP.

[0098] In yet another embodiment, the present invention also provides a method of in vivo gene therapy comprising the step of introducing into a patient or experimental subject a therapeutically effective amount of a nucleic acid molecule encoding a recombinant or fusion protein of the present invention, or a derivative thereof.

[0099] The term "recombinant protein" as used herein refers to a protein that is not naturally occurring but is artificially designed / constructed. The term "recombinant" in the "recombinant protein" of the present invention does not refer to the method of production, but is used only to indicate that the "recombinant protein" does not exist in nature. The recombinant protein of the present invention may be an expressed protein or an assembled protein.

[0100] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric protein of approximately 150,000 daltons consisting of two identical light chains (L) and two identical heavy chains (H) with identical structural characteristics. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region facing the first constant region of the heavy chain and the light chain variable region facing the heavy chain variable region. Certain amino acid residues form an interface between the light and heavy chain variable regions.

[0101] As used herein, the term "antigen-binding fragment" or "fab fragment" or "fab" refers to a fragment consisting of a light chain variable region (VL), a light chain constant region (CL), a heavy chain variable region (VH), and a heavy chain constant region 1 (CL1) domain, and capable of binding to an antigen. When referring to a fragment in which the variable region and the constant region in the antigen-binding fragment are directly linked or linked via a linker sequence (Linker), the constant region refers to the light chain constant region (CL) or the heavy chain constant region 1 (CH1).

[0102] As used herein, the term "first functional antigen" or "target antigen" refers to the antigen bound to the first functional binding fragment.

[0103] As used herein, the term "second functional antigen" refers to a protein bound to a second functional binding fragment.

[0104] As used herein, the term "Fc region" (fragment crystallizable, Fc) is composed of the IgG constant region CH2 and CH3 domains and the hinge region.

[0105] As used herein, the term "knobs-into-holes technology" or "punch and die" technology or "protrusion-into-cavity" technology or "button" technology refers to the use of genetic engineering techniques to introduce different mutations into the two CH3 domains of heavy chains to induce heterodimerization of the heavy chains, creating one knob in one heavy chain and a hole in the other heavy chain, which then preferentially interlock to form an asymmetric antibody (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996, 9(7):617-621). As known to those skilled in the art, multiple knobs and / or holes can be created in one heavy chain, and correspondingly, multiple holes and / or knobs can be created in the other heavy chain.

[0106] The term "mutant" as used herein refers to a functional protein or functional fragment having an amino acid sequence different from that of the wild-type functional protein, for example, a novel functional protein or functional fragment formed by inserting, deleting, or substituting one or more amino acids in the functional protein or functional fragment. A mutant of the second functional binding fragment in the bispecific recombinant protein described herein refers to a mutant having a lower binding affinity for the corresponding second functional antigen than the binding affinity of the first functional binding fragment of the bispecific recombinant protein to the first functional antigen.

[0107] As used herein, the term "substitution," when applied to amino acid residues, refers to the replacement of one or more naturally occurring or introduced amino acids with other amino acids in a peptide, polypeptide, or protein to form a novel peptide, polypeptide, or protein. Substitutions in a polypeptide or protein may result in an enhanced, decreased, or unchanged function of the polypeptide or protein. Substitutions may be "conservative substitutions," which, in relation to an amino acid sequence, refer to the replacement of one amino acid residue with another amino acid residue having a different side chain with similar physicochemical properties, or to the replacement of an amino acid that is not important for the activity of the polypeptide. For example, conservative substitutions can be made between nonpolar side chain amino acid residues (e.g., Met, Ala, Val, Leu, and Ile; Pro, Phe, and Trp); between uncharged polar side chain residues (e.g., Cys, Ser, Thr, Asn, Gly, and Gln); between acidic side chain residues (e.g., Asp and Glu); between basic side chain residues (e.g., His, Lys, and Arg); between β-branched side chain residues (e.g., Thr, Val, and Ile); between sulfur-containing side chain residues (e.g., Cys and Met); or between aromatic side chain residues (e.g., Trp, Tyr, His, and Phe). In some embodiments, substitutions, deletions, or additions can also be considered "conservative substitutions." The number of amino acids inserted or deleted can range from about 1 to 5. Conservative substitutions generally do not cause significant changes in the conformational structure of a protein and can maintain the biological activity of the protein.

[0108] As used herein, the term "double-positive expressing cell" or "target cell" or "target target cell" refers to a cell that can simultaneously interact with a first functional binding fragment and a second functional binding fragment.

[0109] As used herein, the term "second functional antigen monocytic cells" or "non-target cells" or "non-target cells" refers to cells that do not interact with the first functional binding fragment and only interact with the second functional binding fragment.

[0110] As used herein, the term "SIRPα" refers to signal regulatory protein α, also known as CD172a. Signal regulatory proteins (SIRPs) are transmembrane glycoproteins that include three family members: SIRPα (CD172a), SIRPβ (CD172b), and SIRPγ (CD172g). These three members have similar outer membrane edges but different inner membrane regions. The outer membrane edge contains three immunoglobulin (Ig)-like domains, the first of which belongs to the IgV region, and the second and third domains belong to the IgC region. The intramembrane region of SIRPα (CD172a) contains two inhibitory signaling domains that transmit inhibitory signals and inhibit corresponding cellular functions. Although the intracellular domains of SIRPβ (CD172b) and SIRPγ (CD172g) are short and lack signal transduction domains, SIRPβ (CD172b) can transmit activation signals via adaptor proteins (e.g., DAP12). SIRP proteins are primarily expressed in macrophages, dendritic cells (DCs), and neurons. This specification particularly refers to human SIRPα wild-type and its non-high-affinity CD47 mutant.

[0111] As used herein, the terms "D1, D2, D3" refer to the three extracellular Ig-like domains of SIRPα, which are, in order from the amino terminus of the protein, the D1 domain (Ig variable region-like domain, IgV region), the D2 domain (Ig constant region-like domain, IgC region), and the D3 domain (Ig constant region-like domain, IgC region) (Lee WY, et al. The Role of cis Dimerization of Signal Regulatory Protein α (SIRPα) in Binding to CD47. J Biol Chem, 2010, 285(49):37953-37963).

[0112] As used herein, the term "SIRPα-Fc fusion protein" refers to a fusion protein comprising an extracellular truncation of SIRPα, a linker sequence, and an Fc region. The linker sequence and / or Fc region contained in the sequence can be optionally substituted according to methods known to those skilled in the art or conventional linker sequences and / or Fc regions.

[0113] As used herein, the term "IFNα" or "IFN-α," i.e., "type α interferon-α" or "interferon α," includes all natural or recombinant type α interferons, which are members of the type I interferons, including 13 subtypes such as IFN-α1a, IFN-α1b, IFN-α2a, IFN-α2b, IFN-α4a, IFN-α4b, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17, and IFN-α21. In the present invention, the term "IFNα" also includes any substance that has the biological activity of IFNα, such as mutated or modified IFNα, such as a PEG derivative of IFNα (PEG-IFNα). In the present invention, the term "IFNα" is not limited to any particular source and can be obtained commercially or produced by conventional techniques known to those skilled in the art, including, but not limited to, biological source extraction and genetic engineering extraction, as described in detail in Pestka S. Arch Biochem Biophys. 1983 Feb 15;221(1):1-37. In some embodiments, the IFNα is selected from the group consisting of human, horse, cow, mouse, pig, rabbit, cat, dog, rat, goat, sheep, and non-human primate. Human α-type interferon is particularly preferred.

[0114] The terms "IFNα 2b" or "IFN-α 2b" or "IFNα 2b" or "interferon α 2b" or "interferon-α 2b" as used herein are subtypes of IFN-α and all refer to interferon-α 2b.

[0115] As used herein, the term "IFN-γ," i.e., gamma interferon (interferon-γ), is a water-soluble dimeric cytokine and the only member of the type II interferon family. It consists of a core of six α-helices and an extended fragment sequence in the C-terminal region, and always forms a homodimer of two antiparallel, interlocked units to exert its biological activity.

[0116] The term "IFN-β" or "IFNβ" as used herein, i.e., β-interferon (β-interferon, Interferon-β), is a type of human fibroblast interferon and is also a type of type I interferon. It is produced by many cells, such as fibroblasts, after being induced by viruses, nucleic acids, etc., and binds to the same interferon receptor as IFNα and has similar biological effects.

[0117] As used herein, the term "IFN-γ fusion protein" refers to a fusion protein comprising an IFN-γ truncation or mutant, a linker sequence, and an Fc region. The linker sequence and / or Fc region contained in the sequence can be optionally substituted according to methods known to those skilled in the art or conventional linker sequences and / or Fc regions.

[0118] As used herein, the terms "IL-10" or "IL10" refer to interleukin-10, and "IL-10M" or "IL10M" refer to an IL10 monomer mutant that activates downstream signaling pathways without forming homodimers (Josephson, K. Design and analysis of an engineered human interleukin-10 monomer. [J]. Journal of Biological Chemistry, 2000, 275(18):13552-7).

[0119] As used herein, the term "IL-12" or "IL12" refers to interleukin-12, a heterodimeric molecule composed of an alpha chain (p35 subunit, IL-12p35) and a beta chain (p40 subunit, IL-12p40), which are covalently linked by disulfide bonds to form a biologically active 74 kDa heterodimer. As used herein, the term "IL12A" or "IL-12A" refers to the alpha chain of IL-12.

[0120] As used herein, the term "IL-15" or "IL15" refers to interleukin 15, a cytokine important in the activity of NK cells, NKT cells, and CD8 memory T lymphocytes. It acts through a receptor consisting of three subunits, designated α, β, and γ, of which the α subunit is unique to the IL15 receptor. As used herein, the term "IL-15Rα SUSHI" or "IL15Rα SUSHI" refers to the SUSHI structural domain on the α chain of the IL-15 receptor and has its usual meaning in the art. The SUSHI structural domain influences the activation of downstream signaling pathways of IL15.

[0121] As used herein, the term "complex formed by IL-15 and IL15RαSUSHI" includes a fusion protein formed by IL15 and IL-15RαSUSHI, or a combination of proteins in which IL15 and IL-15RαSUSHI are bound by various physical or chemical methods, such as ionic bonds, covalent bonds, or van der Waals forces.

[0122] As used herein, the term "linker sequence" or "Linker" refers to a sequence that links different functional binding fragments (e.g., a first functional binding fragment and two functional binding fragments, a first functional binding fragment or a second functional binding fragment and an Fc region), or links the amino acid sequences of different structural domains within the same functional binding fragment.

[0123] As used herein, the terms "Ofa", "Ofatumumab", and "Anti-CD20 (Ofatumumab)" can be used interchangeably in the present invention and refer to the anti-CD20 antibody Ofatumumab.

[0124] As used herein, the terms "GC33" and "Codrituzumab" can be used interchangeably in the present invention and refer to the anti-GPC3 antibody Codrituzumab.

[0125] As used herein, the terms "atezolizumab" and "Atezolizumab" can be used interchangeably in the present invention and refer to the anti-PD-L1 antibody Atezolizumab.

[0126] As used herein, the term "host cell" generally refers to a single cell, cell line, or cell culture that may be a recipient of a plasmid or vector from a subject, or that contains a polynucleotide disclosed herein or expresses a protein heterodimer (e.g., a heterodimeric protein) of the present application. A host cell can include the progeny of a single host cell. Due to natural, accidental, or deliberate mutation, progeny may not necessarily be identical (in morphology or in full genomic DNA complement) to the original parent cell. A host cell can also include cells transfected in vitro with a vector disclosed herein. A host cell can be a bacterial cell (e.g., E. coli), yeast cell, or other eukaryotic cell, such as HEK293 cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, or myeloma cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a CHO cell.

[0127] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host that transfers an inserted nucleic acid molecule into and / or between host cells. The term can include vectors used primarily to insert DNA or RNA into cells, vectors used primarily to replicate DNA or RNA, and expression vectors used to transcribe and / or translate DNA or RNA. It also includes vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that may be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing an expression vector capable of producing a desired level of expression.

[0128] As used herein, the term "cell proliferation" or "proliferation" generally refers to a phenomenon in which cells change in number due to division. For example, cell proliferation can result in an increase in cell number. The term also includes cell proliferation in which cell morphology changes (e.g., an increase in size) consistent with a proliferation signal.

[0129] As used herein, the term "growth inhibition" or "inhibition of cell proliferation" generally refers to a decrease in the rate and / or proliferation of cancer cells. For example, this can include the death of cancer cells (e.g., by apoptosis). In some embodiments, the term can also refer to inhibiting the growth and / or proliferation of solid tumors and / or inducing a reduction in tumor size or elimination.

[0130] As used herein, the terms "treatment," "therapeutic method," and "cure" can be used interchangeably. The term "treatment" includes controlling the progression of a disease, illness, condition, and associated symptoms, preferably reducing the disease, illness, condition, or alleviating the effects of one or more symptoms of a disease, illness, or condition. The term includes curing a disease or completely eliminating symptoms. The term includes alleviating symptoms. The term also includes, but is not limited to, non-curative palliative treatments. The term "treating" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the invention to prevent, delay, reduce, or alleviate the progression of a disease, illness, condition, or the effects of one or more symptoms of a disease, illness, or condition.

[0131] As used herein, the term "administration" refers to the delivery of a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to an experimental subject. Administration may be systemic or local. Administration may be by an administration device such as a syringe. Methods of administration include, but are not limited to, implantation, nasal inhalation, spraying, injection, etc. Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration, etc.

[0132] Compared with the prior art, the present invention has the following beneficial effects: In the present invention, the C-terminus of the CL domain or the C-terminus of the CH1 domain of the antigen-binding fragment (fab segment) of a first functional binding fragment that targets a target antigen of a bispecific recombinant protein is linked directly or via a linker sequence to a second functional binding fragment that targets an immune checkpoint, immune checkpoint ligand, or cytokine receptor, and the C-terminus of the second functional binding fragment is linked directly or via a linker sequence to the N-terminus of the Fc region. This reduces impurities that produce homodimeric or homomultimeric structures of the second functional binding fragment that can target non-target cell immune checkpoints, immune checkpoint ligands, or cytokine receptors during the production of bispecific recombinant proteins, thereby unexpectedly reducing potential safety risks due to impurities (potentially risky impurities) in the product-related recombinant protein mixture in the fermentation product and improving the convenience of recombinant protein production.

[0133] In the present invention, the C-terminus of the CL domain or the C-terminus of the CH1 domain of the antigen-binding fragment (fab segment) of a first functional binding fragment targeting a target antigen of a bispecific recombinant protein is linked directly or via a linker sequence to a second functional binding fragment targeting an immune checkpoint, immune checkpoint ligand, or cytokine receptor, and the C-terminus of the second functional binding fragment is linked directly or via a linker sequence to the N-terminus of the Fc region. This unexpectedly balances and regulates the safety and efficacy of the two targets, and by mechanisms such as spatial conformational restriction, significantly reduces the binding of the bispecific recombinant protein to non-target cells or cytokine receptors (second functional antigens) that highly express single positive immune checkpoints, immune checkpoint ligands, or cytokine receptors, thereby enhancing the targeting of the bispecific recombinant protein and reducing immune-related toxic side effects such as cytokine storm that can occur when targeting non-target cells with common symmetric or asymmetric bispecific structures. This improves drug safety. Unexpectedly, while the first functional binding fragment of the bispecific recombinant protein remains effective or superior in target cells (double-positive expressing cells), the bispecific recombinant protein exhibits significantly reduced or no binding to second functional antigen-single-positive cells (non-target target cells) compared to the monomer containing the second functional binding fragment / homodimer / homomultimer. At the same time, however, the interaction between the bispecific recombinant protein and the second functional antigen on the double-positive expressing cells (i.e., target target cells) is unexpectedly not weaker or significantly stronger than that of the second functional binding fragment alone / homodimer / multimer. In other words, the bispecific recombinant protein technology of the present invention significantly reduces the toxic side effects caused by the binding of the second functional binding fragment to the second functional antigen on non-target cells, while at the same time strengthening the binding effect of the second functional binding fragment to the second functional antigen on target cells, thereby significantly enhancing the effect of the second functional binding fragment on target cells.For example, when a SIRPα extracellular truncation or non-high-affinity mutant is selected as the second functional binding fragment, the bispecific recombinant protein of the present invention significantly reduces the effect of its binding to CD47 single-positive non-target cells, such as erythrocytes or platelets, compared with a SIRPα-Fc fusion protein (e.g., TTI-621). However, at the same time, the bispecific recombinant protein of the present invention can significantly improve the interaction with double-positive expressing cells (target cells) compared with a SIRPα-Fc fusion protein (e.g., TTI-621), significantly improving the killing effect of immune cells (e.g., macrophages) against target cells, and at the same time, the competitive binding ability of the target cells is significantly stronger than that of the SIRPα-Fc fusion protein.

[0134] In addition, the bispecific recombinant protein of the present invention can also improve the freeze-thaw stability of some fusion proteins with immunomodulatory functions. For example, when the second functional binding fragment is IFN-α 2b or its mutant, after five freeze-thaw cycles, the purity is 95% or more, the appearance is transparent, and the freeze-thaw stability is significantly better than that of IFN-α 2b monomer or PEGylated IFN-α 2b.

[0135] The structure of the bispecific recombinant protein of the present invention is highly scalable and allows for simple screening design. The first functional binding fragment can be selected from a variety of antibody sequences, and the second functional binding fragment can be selected from cytokines, immune checkpoints, or immune checkpoint ligand proteins and truncations or mutants thereof, significantly reducing the time required for conventional antibody drug screening, improving drug screening efficiency, and reducing screening costs. Optionally, the second functional binding fragment can be selected from cytokines and / or immune checkpoint binding proteins and / or immune checkpoint ligand binding proteins or truncations thereof. Preferably, the second functional binding fragment can be selected from endogenous cytokines and / or immune checkpoint binding proteins and / or immune checkpoint ligand binding proteins or truncations thereof, thereby reducing potential immunogenicity.

[0136] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of the structure of the bispecific recombinant protein of the present invention.

[0137] FIG. 2 is an SDS-PAGE electrophoresis diagram of the bispecific recombinant protein after purification with Protein A in Example 2 of the present invention.

[0138] [Figure 3] Non-reducing SDS-PAGE electrophoresis diagram of the bispecific recombinant protein of Example 2 of the present invention after affinity capture.

[0139] FIG. 4 is a reducing SDS-PAGE electrophoresis diagram of the bispecific recombinant protein of Example 2 of the present invention after affinity capture.

[0140] FIG. 5 is a non-reducing SDS-PAGE electrophoresis diagram of the purified bispecific recombinant protein of Example 2 of the present invention.

[0141] FIG. 6 shows the binding curve of the bispecific recombinant protein of the present invention and CD20 single-positive cells (non-target cells, CHO-K1-hCD20) determined by flow cytometry in Example 3 of the present invention.

[0142] Figure 7A shows the binding curves of the bispecific recombinant protein of the present invention, whose second functional antigen is CD47, and the control sample to CD47 single-positive cells (non-target cells, HEK293 cells) determined by flow cytometry in Example 3 of the present invention.

[0143] [Figure 7B] Binding curves of the bispecific recombinant protein of the present invention, whose second functional antigen is CD47, and the control sample to CD47 single-positive cells (non-target cells, CHO-K1 cells) determined by flow cytometry in Example 3 of the present invention.

[0144] Figure 8 shows the binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD20 and the second functional antigen is CD47, and the control sample to CD20 / CD47 double-positive cells (target cells, Raji cells) determined by flow cytometry in Example 3 of the present invention.

[0145] [Figure 9A] Competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD20 and the second functional antigen is CD47, the corresponding potential risk impurity protein, and the control sample, in CD20 / CD47 double-positive cells (target cells, Raji cells) determined by flow cytometry in Example 3 of the present invention.

[0146] [Figure 9B] Competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is EpCAM and the second functional antigen is CD47, and the control sample, EpCAM / CD47 double-positive cells (target cells, CAPAN-2 cells), determined by flow cytometry in Example 3 of the present invention.

[0147] [Figure 9C] Competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD24 and the second functional antigen is CD47, and a control sample, determined by flow cytometry in Example 3 of the present invention, on CD24 / CD47 double-positive cells (target cells, MCF-7 cells).

[0148] [Figure 9D] Competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD38 and the second functional antigen is CD47, and a control sample, in CD38 / CD47 double-positive cells (target cells, Raji cells), as determined by flow cytometry in Example 3 of the present invention.

[0149] Figure 10 shows the binding activity curves of the bispecific recombinant protein of Example 4 of the present invention, in which the first functional antigen is GPC3 and the second functional binding protein is IFN-α 2b, and the control sample, in HepG2 liver cancer cells as target cells, as determined by flow cytometry.

[0150] Figure 11 shows a histogram of binding activity in the target cell line HuH-7 of the bispecific recombinant protein of Example 4 of the present invention, in which the first functional antigen is GPC3 and the second functional binding protein is IFN-α 2b, as well as a control sample, determined by flow cytometry.

[0151] FIG. 12 shows the ADCC activity curve of the bispecific recombinant protein in target cells HepG2 of Example 5 of the present invention determined by the LDH method.

[0152] [Figure 13] Figure 13 shows the inhibitory activity curves of bispecific recombinant proteins with different linker sequences against the proliferation of target cells HuH-7 in Example 6 of the present invention.

[0153] FIG. 14 shows the inhibitory activity curves of the bispecific recombinant protein having GPC3 antigen-targeting function of Example 6 of the present invention and the control sample on the proliferation of GPC3-positive target cells HuH-7.

[0154] Figure 15 shows the inhibitory activity curve of the bispecific recombinant protein of Example 7 of the present invention against the proliferation of PD-L1-positive target cells MDA-MB-231.

[0155] Figure 16 shows the inhibitory activity curve of the bispecific recombinant protein of Example 7 of the present invention on the proliferation of MDA-MB-231 cells after anti-PD-L1 antibody occlusion.

[0156] FIG. 17A shows the inhibitory activity curve of the bispecific recombinant protein of Example 8 of the present invention against the proliferation of CD38-positive target cells, Daudi.

[0157] [Figure 17B] The inhibitory activity curve of the bispecific recombinant protein of Example 8 of the present invention against the proliferation of CD38-negative non-target cells SK-BR3.

[0158] FIG. 18 shows the inhibitory activity curves of bispecific recombinant proteins containing different IFN-α 2b low-affinity mutants of Example 9 of the present invention against the proliferation of GPC3-positive target cells HuH-7.

[0159] FIG. 19 shows the inhibitory activity curves of bispecific recombinant proteins containing different IFN-α 2b low affinity mutants of Example 9 of the present invention against the proliferation of GPC3-negative target cells SW480.

[0160] FIG. 20 shows the inhibitory activity curves of bispecific recombinant proteins containing different IFN-α 2b low affinity mutants of Example 9 of the present invention against the proliferation of GPC3-negative target cells U266.

[0161] Figure 21 shows the inhibitory activity curve of potential risk impurities of the bispecific recombinant protein of Example 10 of the present invention on the proliferation of GPC3-negative non-target cells MDA-MB-231.

[0162] FIG. 22 shows the binding activity curve of the bispecific recombinant protein of Example 11 of the present invention to TIGIT-positive target cells H_IL12 Reporter 293 after anti-TIGIT antibody blockade.

[0163] FIG. 23 shows the detection results of the bispecific recombinant protein of Example 12 of the present invention on the activation level of P-STAT3 in THP1 cells.

[0164] Figure 24 shows the proliferative activity of the bispecific recombinant protein of Example 13 of the present invention on PD-1-positive hPBMCs after 48 hours of stimulation with OKT3.

[0165] Figure 25 shows the proliferation activity of the bispecific recombinant protein of Example 13 of the present invention against PD-1-negative non-target cells M-07e.

[0166] [Mode for Carrying Out the Invention] Example 1 Construction of an Expression Vector The bispecific recombinant proteins were directly synthesized by GENEWIZ after codon optimization according to the protein sequence, inserted into the pCDNA3.1 plasmid, and verified by sequencing. The different expression plasmids described above were mixed and transfected into expression cells in pairs to obtain bispecific recombinant proteins or control samples (see Table 1). Subsequent experimental materials were obtained by extracting from expression cells transfected with this series of plasmids.

[0167] [Table 1] JPEG0007814759000002.jpg232169JPEG0007814759000003.jpg232169JPEG0007814759000004.jpg233169JPEG0007814759000005.jpg72169

[0168] In Table 1 above, the first functional binding fragment of the bispecific recombinant protein is characterized by its target antigen, i.e., the first functional antigen. (H) refers to the structural domain consisting of the heavy chain VH and CH1, (L) refers to the structural domain consisting of the light chain VL and CL, D1 refers to the extracellular D1 domain of human SIRPα wild-type and its mutants, Fc refers to the wild-type Fc region, Fc1 refers to the Fc region with a hole or holes mutation, and Fc2 refers to the Fc region with a knob or knobs mutation. The sequence numbers corresponding to the sequence names are shown in Table 2. Here, the sequence of the signal peptide is shown in SEQ ID NO: 49. The amino acid sequence of codrituzumab is cited from Patent US7919086, with the amino acid sequence of the heavy chain as shown in SEQ ID NO: 19 and the amino acid sequence of the light chain as shown in SEQ ID NO: 20. The amino acid sequence of atezolizumab is taken from US Patent No. 20100203056, with the heavy chain amino acid sequence shown in SEQ ID NO: 21 and the light chain amino acid sequence shown in SEQ ID NO: 22. The heavy chain amino acid sequence of tiragolumab is shown in SEQ ID NO: 61 and the light chain amino acid sequence shown in SEQ ID NO: 62. The amino acid sequences of the heavy and light chain variable regions of palivizumab are taken from Patent No. WO199401715. Human IgG1 isotype control (B117901) was purchased from Biointron. Recombinantly expressed IFN-α 2b protein (Z03003) was purchased from Nanjing GenScript Biotech Co., Ltd. The IL10 used in IL10M-Fc is a monomeric mutant, and the corresponding sequence is described in the following literature (Josephson, K. Design and analysis of an engineered human interleukin-10 monomer. [J]. Journal of Biological Chemistry, 2000, 275(18):13552-7.). IL15-IL15RαSUSHI-Fc(C15Y) was purchased from Novoprotein.

[0169] [Table 2] JPEG0007814759000007.jpg223169JPEG0007814759000008.jpg231169JPEG0007814759000009.jpg67169

[0170] In Table 1, human IFN-α 2b (Genebank: AAP20099.1) was used to illustrate the design of the second functional binding fragment of IFN-α in the bispecific recombinant protein of the present invention. The IFN-α family contains 15 subtypes, each with similar structure and high sequence homology (80-99%). They bind to the same IFN receptor and all possess antiviral, growth inhibitory, antitumor, and immunomodulatory functions. Regarding the technical effects achieved by IFN-α 2b, other IFN-α subtypes can achieve the same technical effects (British Journal of Pharmacology (2013) 168 1048-1058), and this information will not be repeated here. IFN-β and IFN-α 2b share the same interferon receptor and are both type I interferons, possessing similar biological effects, and therefore can also achieve the same technical effects.

[0171] The structure of the bispecific recombinant protein of the present invention is as shown in Figure 1. The first functional binding fragment targeting a target antigen of the bispecific recombinant protein comprises an antigen-binding fragment, wherein the C-terminus of the CL domain or the C-terminus of the CH1 domain of the antigen-binding fragment is linked directly or via a linker sequence to a second functional binding fragment targeting an immune checkpoint, immune checkpoint ligand, or cytokine receptor. The variable region (V region) and constant region (C region) of the antigen-binding fragment of the first functional binding fragment are linked directly or via a linker sequence, or the antigen-binding fragment and Fc region are linked directly or via a linker sequence, or both methods are used simultaneously. The light chain VL or heavy chain VH of the first functional binding fragment is linked to the second functional binding fragment via knobs-into-holes. The light chain VL or heavy chain VH of the first functional binding fragment or the second functional binding fragment is linked to the Fc region directly or via a linker sequence. For example, in Figure 1, a and e represent that the C-terminus of the CL structural domain of the antigen-binding fragment of the first functional binding fragment is linked directly to the second functional binding fragment or via a linker sequence; b and f represent that the C-terminus of the CH1 structural domain of the antigen-binding fragment of the first functional binding fragment is linked directly to the second functional binding fragment or via a linker sequence; c, g, j, and k represent that the C-terminus of the CL structural domain of the antigen-binding fragment of the first functional binding fragment is linked directly to the second functional binding fragment or via a linker sequence, and the Fc region is linked via knobs-into-holes; and d, h, i, and l represent that the C-terminus of the CH1 structural domain of the antigen-binding fragment of the first functional binding fragment is linked directly to the second functional binding fragment or via a linker sequence, and the Fc region is linked via knobs-into-holes.

[0172] Example 2: Preparation of expression plasmid, cell transfection, and expression and purification of target protein 1. Expression Plasmid Preparation Glycerol bacteria containing the expression plasmid (1 mL of E. coli containing the expression plasmid was added to 0.5 mL of 60% sterile glycerol solution and mixed thoroughly) were inoculated into liquid LB medium at a ratio of 1:1000. After shaking at 37°C and 220 rpm for 16 hours, the bacteria were collected by centrifugation. The expression plasmid was extracted using an endotoxin-free plasmid kit (DP117, purchased from Tiangen Biotech (Beijing) Co., Ltd.) according to the standard procedure provided in the kit's instructions.

[0173] 2. Cell Transfection and Protein Expression The following method can be applied to bispecific recombinant proteins in which the second functional antigen is CD47, using LCB-001 as an example.

[0174] The resulting expression plasmid was filtered through a 0.22 μm filter, and then 3 mg of the plasmid (here, the ratio of the bispecific recombinant protein, its A-chain, and B-chain expression plasmids was 1:1 (molar ratio)) was aspirated and added to 50 mL of OptiMEM I Reduced Serum Medium (GIBCO) and mixed uniformly. 6 mg of the transfection reagent polyetherimide (PEI, purchased from Polysciences and dissolved in sterile ultrapure water at a concentration of 1 mg / mL) was aspirated and added to 50 mL of OptiMEM I Reduced Serum Medium and mixed uniformly. The resulting PEI solution was added to the OptiMEM I Reduced Serum Medium solution containing the plasmid and mixed uniformly. After leaving it at room temperature for 15 minutes, the mixture of plasmid and PEI was adjusted to a volume of 1 L and 3 × 10 6The suspension was slowly and uniformly added to a suspension of host cells (CHO-S) (Thermo Fisher) with a cell density of 1000 cells / mL, and cultured at 37°C in a 5% CO2 incubator. After 4 hours, a feed medium equivalent to 7% of the initial volume (the feed medium was prepared by dissolving 80 g of CD Efficient Feed C AGT (purchased from Gibco) and 75 g of 5x00483 (Kerry) per liter of water) was added. The culture temperature was lowered to 33°C, and the cells were harvested after 6 days of culture. The cell suspension was centrifuged at 10,000 g for 30 minutes at 10°C, and the supernatant obtained by centrifugation, i.e., the cell culture harvest solution, was used for target protein purification.

[0175] The method described below can be applied to bispecific recombinant proteins other than CD47 as the second functional antigen, using LCB-009 as an example.

[0176] The obtained expression plasmid was filtered through a 0.22 μm filter, and then 50 μg of the plasmid (where the mass ratio of A-chain and B-chain expression plasmid was 2:1 or 3:1) was aspirated and added to 2 mL of OptiPRO SFM Medium (GIBCO) and mixed uniformly. 160 μL of the transfection reagent ExpiFectamine CHO Reagent was aspirated and added to 2 mL of OptiPRO SFM Medium and mixed uniformly. The obtained transfection reagent mixed solution was added to the mixed solution containing the plasmid and mixed uniformly. The mixture of the plasmid and transfection reagent was adjusted to a volume of 50 mL and 6 × 10 6 The cells were slowly and uniformly added to a suspension of host cells (ExpiCHO-S, Thermo Fisher Scientific) with a cell density of 1000 viable cells / mL and cultured in an 8% CO2 incubator at 37°C. On day 1 (after 18-22 hours), 300 μL of ExpiCHO Enhancer and 8 mL of ExpiCHO Feed were added, and the culture temperature was cooled to 32°C. On day 5, a second 8 mL of ExpiCHO Feed was added, and the cells were harvested after 12 days of culture. The cell suspension was centrifuged at 8000 rpm for 15 minutes, and the supernatant obtained by centrifugation, i.e., the cell culture harvest solution, was used for target protein purification.

[0177] 3. Protein Purification 1) Sample capture (Protein A affinity capture) In the method below, LCB-001 is used as an example, but the method is applicable to all bispecific recombinant proteins of the present invention.

[0178] The LCB-001 cell culture harvest was centrifuged at 10,000 rpm for 30 minutes to remove cells and their fragments, and then loaded onto a Protein A affinity column (GE Healthcare). The target protein was recovered by elution. Protein purity was detected by SDS-PAGE.

[0179] The Protein A purification method is a conventional protein purification method well known to those skilled in the art. For specific test methods, please refer to the GE Healthcare Protein A Product Instructions and the GE Antibody Purification Manual.

[0180] 2) Sample purification The following method uses LCB-009 as an example to explain the purification steps of bispecific recombinant proteins, but is suitable for bispecific recombinant proteins that contain large amounts of aggregates after Protein A affinity capture.

[0181] The supernatant of the expression of the bispecific recombinant protein LCB-009 was purified by SULFATE 650F packing (TOSOH) to remove aggregates and other impurities in the sample, and the experimental process was as follows:

[0182] a) Equilibration: Equilibrate the chromatography column using an equilibration solution (50 mM NaAC-HAC, pH 5.5) until the UV detection line is stable; b) Sample loading: The sample is loaded by a sample pump, the retention time is 5 minutes, and the loading amount is ≦50 mg / mL; c) Re-equilibration: Wash the chromatography column with 5 column volumes using equilibration solution (50 mM NaAC-HAC, pH 5.5); d) Elution: The target protein was eluted using elution solution (50 mM NaAC-HAC, 250 mM, pH 5.5), and the purity of the protein was detected by SDS-PAGE.

[0183] The purification steps of the control samples in Table 1 were obtained with reference to the operation steps of 1) sample capture (protein A affinity capture).

[0184] The SDS-PAGE protein electrophoresis detection results of the purified bispecific recombinant protein, control samples, and potential risk impurities are shown in Figures 2 to 5.

[0185] The theoretical molecular weights of the four proteins, LCB-001, LCB-002, LCB-001-R, and LCB-002-R, are 111 kD, 114 kD, 123 kD, and 129 kD, respectively. As shown in Figure 2, the expression of the target proteins in each lane was normal, but LCB-001 and LCB-002 (lanes 1 and 2 in Figure 2) showed different degrees of left-arm dimers, right-arm dimers (LCB-001-R, LCB-002-R), and / or multimers.

[0186] The theoretical molecular weights of LCB-009, LCB-010, LCB-011, LCB-012, LCB-013, LCB-014, LCB-015, LCB-010-M1, LCB-010-M2, LCB-010-M3, LCB-010-M4, LCB-010-M5, LCB-011-M3, and LCB-011-M4 are all approximately 120 kD. The molecular weight of the control antibody Codrituzumab shown in Table 1 is 150 kD, the molecular weight of IFNα 2b-Fc is 88 kD, and the molecular weight of IFNα 2b alone is 19.2 kD. The results of non-reducing SDS-PAGE protein electrophoresis of the affinity capture sample (i.e., after purification with Protein A) are shown in Figure 3, and the results of reducing SDS-PAGE protein electrophoresis are shown in Figure 4. The electrophoresis results indicate that the antibody expressed by this construct contains a large amount of aggregates. The results of non-reducing SDS-PAGE protein electrophoresis after purification (i.e., after purification with SULFATE 650F) are shown in Figure 5, indicating that most of the aggregates can be removed by cation exchange chromatography.

[0187] The other bispecific recombinant proteins listed in Table 1 were subjected to affinity capture and purification, and the results of reducing and non-reducing SDS-PAGE protein electrophoresis of the samples were consistent with the theoretical molecular weights, so further explanation is omitted here.

[0188] Example 3: Detection of target affinity and target competitive binding activity of bispecific recombinant proteins in which the second functional antigen is CD47 1. Method for detecting CD47 and / or CD20 target affinity Detection of the affinity of bispecific recombinant proteins for target CD20 by flow cytometry: The binding affinity of the bispecific recombinant proteins to the target CD20 was measured by flow cytometry. The following method is applied to the detection of recombinant proteins whose first functional antigen is CD20, using LCB-001 or LCB-002 as an example.

[0189] CHO-K1-hCD20 (Chinese hamster ovary epithelial cells overexpressing hCD20) cells were cultured. Cells with good growth were collected, counted, centrifuged, and diluted to 3 × 10 with PBS + 2% FBS (purchased from Gibco). 6 The cells were resuspended at a concentration of 100 cells / mL. 100 μL of the cells were added to a 96-well U-shaped plate (product number: 3799, Corning) and incubated for at least 15 minutes. The plate was then centrifuged, the supernatant was aspirated, and eight dilutions of LCB-001, LCB-002, positive control ofatumumab, positive control rituximab, or negative control IgG (isotype) (starting at 100 nM, diluted 3-fold, for a total of eight concentrations) were added. The 96-well plate was incubated in a refrigerator at 4°C for 1 hour. After washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and incubated at 4°C for 1 hour. After washing and resuspension with PBS + 2% FBS, the fluorescence intensity was detected using a flow cytometer (BD).

[0190] Because CHO-K1 cells do not express the CD47 antigen, the binding affinities of recombinant proteins LCB-001 and LCB-002, positive control samples Ofatumumab and Rituximab, negative control IgG, and CD20 can be evaluated at the cellular level using CHO-K1-hCD20 cells.

[0191] The test results showed that, in addition to the negative control IgG being unable to bind to CHO-K1 cells, the recombinant proteins LCB-001, LCB-002, and the positive control samples Ofatumumab and Rituximab were all able to bind to CHO-K1 cells, and the binding affinity of LCB-001 and LCB-002 to CD20 was similar to that of the anti-CD20 antibodies Ofatumumab and Rituximab.

[0192] The above test data show that the recombinant protein of the present invention can specifically target tumor cell CD20 antigen at the cellular level, and its binding affinity to CD20 is not lower than that of a monoclonal antibody with the same target, CD20, and the recombinant protein of the present invention can target target cells with high affinity.

[0193] For example, as shown in Figure 6, the recombinant proteins LCB-001 and LCB-002, and the positive control samples Ofatumumab and Rituximab, all bound to CHO-K1-hCD20 cells. Specifically, the binding affinity of LCB-001 and LCB-002 to CD20 was similar to that of the anti-CD20 antibodies Ofatumumab and Rituximab.

[0194] Detection of the affinity of bispecific recombinant proteins for target CD47 by flow cytometry: The binding affinity of the bispecific recombinant proteins to the target CD47 was measured by flow cytometry. The following method is applied to the detection of recombinant proteins whose first functional antigen is CD47, using LCB-001, LCB-002, or LCB-017 as examples.

[0195] HEK293 (human embryonic kidney 293) cells (CD20- / CD47+, non-target cells) were cultured, and well-growing cells were collected, counted, centrifuged, and cultured in PBS + 2% FBS (Gibco) at 3 × 10 6The cells were resuspended at a concentration of 100 cells / mL in a 96-well U-shaped plate (product number: 3799, Corning) at 100 μL per well and left for at least 15 minutes. The plate was then centrifuged, the supernatant was aspirated, and seven dilutions of LCB-001, LCB-002, the anti-CD47 antibody Magrolimab, the anti-CD47 fusion protein TTI-621, or an IgG1 isotype (starting at 100 nM, diluted 4-fold for a total of seven concentrations) were added. The 96-well plate was then incubated in a refrigerator at 4°C for 1 hour. After washing with PBS + 2% FBS, goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added and the plate was incubated at 4°C for 1 hour. After washing and resuspension with PBS + 2% FBS, fluorescence was detected using a flow cytometer (BD).

[0196] Because HEK293 cells do not express the CD20 antigen, the binding affinities of the bispecific recombinant proteins LCB-001 and LCB-002, the positive control anti-CD47 antibody Magrolimab, the anti-CD47 fusion protein TTI-621, and the negative control IgG and CD47 can be evaluated at the cellular level using HEK293 cells.

[0197] The test results showed that, in addition to the negative control IgG being unable to bind to HEK293 cells, the anti-CD47 antibody Magrolimab, the anti-CD47 fusion protein TTI-621 (SIRPα D1-Fc7 fusion protein), and the recombinant proteins LCB-001 and LCB-002 were all able to bind to HEK293 cells, but the binding of the recombinant proteins LCB-001 and LCB-002 to HEK293 was significantly weaker than that of the anti-CD47 antibody Magrolimab and also significantly weaker than that of the anti-CD47 fusion protein TTI-621.

[0198] For example, as shown in Figure 7A, the anti-CD47 antibody Magrolimab, the anti-CD47 fusion protein TTI-621, and the recombinant proteins LCB-001 and LCB-002 all bound to HEK293 cells. Specifically, the affinity of the recombinant proteins LCB-001 and LCB-002 was significantly weaker than that of the anti-CD47 antibody Magrolimab and the anti-CD47 fusion protein TTI-621 (SIRPα D1-Fc fusion protein).

[0199] CD47-transfected CHO-K1 (Chinese hamster ovary cells) (CD38- / CD47+, non-target cells) were washed and digested, and well-growing cells were collected, counted, centrifuged, and diluted to 3 × 10 with DPBS + 2% FBS (Gibco). 6 The cells were resuspended at a concentration of 100 μL / well in a 96-well U-shaped plate (product number: 3799, Corning), left for at least 15 minutes, centrifuged, and the supernatant was aspirated. Seven dilutions of LCB-017, felzartamab, TTI-621, or SIRPα-D1m-Fc (starting at 200 nM, followed by 3-fold gradient dilutions, for a total of 11 concentrations) were added. The 96-well plate was incubated in a refrigerator at 4°C for 1 hour, washed with DPBS + 2% FBS, and then goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added. The plate was incubated at 4°C for 1 hour, washed with DPBS + 2% FBS, and resuspended. Fluorescence was detected using a flow cytometer (BD).

[0200] Because CD47-transfected CHO-K1 cells do not express the CD38 antigen, the binding affinities of the bispecific recombinant protein LCB-017, the negative control anti-CD38 antibody Felzartamab, the positive control anti-CD47 fusion protein TTI-621, and its high-affinity SIRPα D1m-Fc to CD47 can be evaluated at the cellular level using these CHO-K1 cells.

[0201] The test results showed that in addition to the inability of the negative control anti-CD38 antibody Felzartamab to bind to CHO-K1, the anti-CD47 fusion protein TTI-621 (SIRPα D1-Fc fusion protein), the high-affinity SIRPα D1m-Fc fusion protein, and the recombinant protein LCB-017 could all bind to HEK293 cells, but the binding of the recombinant protein LCB-017 to the non-target cell CHO-K1 was significantly weaker than that of the anti-CD47 fusion protein TTI-621.

[0202] For example, as shown in Figure 7B, the high-affinity SIRPα D1m-Fc fusion protein, the anti-CD47 fusion protein TTI-621, and the recombinant protein LCB-017 all bind to non-target CHO-K1 cells. Specifically, the affinity of the recombinant protein LCB-017 is significantly weaker than that of the anti-CD47 fusion protein TTI-621 (SIRPα D1-Fc fusion protein).

[0203] Other bispecific recombinant proteins of the present invention, in which the second functional antigen is CD47, were also observed to have low or no binding affinity to non-target cells.

[0204] The above test data demonstrate that the recombinant protein of the present invention can specifically target the CD47 antigen on tumor cells at the cellular level, and its binding affinity to CD47 is significantly weaker than that of the SIRPα D1-Fc fusion protein. Surprisingly, the bispecific recombinant protein of the present invention can also significantly reduce or avoid side effects, including non-tumor target cell killing, such as hemagglutination, anemia, and thrombocytopenia, caused by treatment with anti-CD47 antibodies or SIRPα D1-Fc fusion proteins.

[0205] Affinity of bispecific recombinant proteins for targets CD20 or CD47 by surface plasmon resonance analysis (referred to as "SPR analysis"): In the following method, LCB-002 is used as an example to detect a recombinant protein whose first functional antigen is CD20 and whose second functional antigen is CD47.

[0206] SPR analysis was performed using a Biacore T200 (GE Healthcare). Anti-human IgG (Fc) antibody (Human Antibody Capture Kit, GE Healthcare) was immobilized on a CM5 sensor chip. Human CD20-Fc or human CD47-Fc chimeric proteins (5 μg / mL) diluted in HBS-EP (GE Healthcare) were immobilized, and the amount of capture was measured. Subsequently, the complete recombinant protein LCB-002, the anti-CD20 antibody ofatumumab, and the anti-CD47 fusion protein TTI-621 were diluted to 50 nM in HBS-EP, and the binding amount of each to the human CD20-Fc chimeric protein or human CD47-Fc chimeric protein was measured. Next, HBS-EP+ buffer was added at a flow rate of 50 μL / min for 5 minutes, and the dissociation of the recombinant protein LCB-002, the anti-CD20 antibody Ofatumumab, the anti-CD47 fusion protein TTI-621, and the human CD20-Fc chimeric protein or human CD47-Fc chimeric protein was measured. Using a bivalent analyte model and Rmax analysis, the association rate constant (ka) and dissociation rate constant (kd) were calculated, and the association dissociation constant (KD) was calculated by dividing ka by kd. The Kd values ​​for the recombinant protein LCB-002, the anti-CD20 antibody Ofatumumab, and the anti-CD47 fusion protein TTI-621 with the human CD20-Fc chimeric protein or human CD47-Fc chimeric protein are shown in Table 3.

[0207] [Table 3]

[0208] Ofatumumab specifically targets the human CD20 protein and therefore does not bind to the human CD47-Fc chimeric protein, whereas the anti-CD47 fusion protein TTI-621 specifically targets the CD47 protein and therefore does not bind to the human CD20-Fc chimeric protein.

[0209] The test results showed that at the protein level, the recombinant protein LCB-002 could bind to human CD47 protein or human CD20 protein, respectively, and its affinity was not significantly different from that of the anti-CD20 antibody Ofatumumab or the anti-CD47 fusion protein TTI-621.

[0210] The above test data demonstrate that the recombinant protein of the present invention can specifically target tumor cell CD20 antigen and CD47 antigen at the protein level, and its binding affinity to CD47 or CD20 is comparable to that of the anti-CD20 antibody Ofatumumab or the anti-CD47 fusion protein TTI-621.

[0211] Detecting bispecific binding of targets CD20 and CD47 by flow cytometry: The binding affinity of the bispecific recombinant proteins to the targets CD20 and CD47 was measured by flow cytometry. The following method is applied to the detection of recombinant proteins whose first functional antigen is CD20 and whose second functional antigen is CD47, using LCB-001 or LCB-002 as an example.

[0212] Well-growing Raji cells (human B-cell lymphoma) (Cell Bank of Chinese Academy of Sciences, Shanghai) (CD20+ / CD47+, targeted cells) were collected, counted, centrifuged, and diluted to 3 × 10 in PBS + 2% FBS. 6The cells were resuspended at a concentration of 100 cells / mL. 100 μL / well of a 96-well U-shaped plate (product number: 3799, Corning) was added to eight dilutions of LCB-001, LCB-002, ofatumumab, rituximab, and IgG (starting at 100 nM, diluted three-fold, for a total of eight concentrations). The cells were incubated at 4°C for 1 hour, washed with PBS + 2% FBS, and then goat anti-human IgG Fc-FITC (F9512-2ML, Sigma) was added. The cells were incubated at 4°C for 1 hour, washed with PBS + 2% FBS, and resuspended. Fluorescence was detected using a flow cytometer (BD).

[0213] As shown in Figure 8, because CD20 and CD47 antigens are simultaneously expressed on the surface of Raji cells, both the anti-CD20 antibodies Ofatumumab and Rituximab can specifically bind to Raji cells. However, the recombinant proteins LCB-001 and LCB-002 can also bind to Raji cells, with binding avidity comparable to that of Ofatumumab and Rituximab.

[0214] 2. Detection of competitive binding activity to the target The following method is applied to bispecific recombinant proteins, such as LCB-001, LCB-005, LCB-006, and LCB-017, in which the second functional antigen is CD47.

[0215] Detection of competitive binding activity of LCB-001 by flow cytometry: The competitive binding activity of the bispecific recombinant proteins LCB-001 and LCB-002, which competitively bind to cell surface CD47, and the potential impurity control samples LCB-001-R and LCB-002-R with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was measured by flow cytometry. The following method uses LCB-001 or LCB-002 as an example and is applied to the detection of recombinant proteins whose first functional antigen is CD20 and whose second functional antigen is CD47.

[0216] Well-growing Raji cells (human B-cell lymphoma) (Cell Bank of Chinese Academy of Sciences, Shanghai) were collected, counted, centrifuged, and diluted to 3 × 10 cells in PBS + 2% FBS. 6 The cells were resuspended at a concentration of 100 cells / mL. 100 μL / well of the cells were added to a 96-well U-shaped plate (product number: 3799, Corning) and gradient diluted with LCB-001 (starting concentration: 200,000 ng / mL, 5-fold dilutions, total 8 concentrations). The final concentration of FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was 4 nM. LCB-001, SIRPα(CV1)-Fc-FITC, and Raji cells were co-cultured for 1 hour, centrifuged, the supernatant discarded, and the cells were resuspended in DPBS + 2% FBS. Detection was then performed using a flow cytometer.

[0217] The test results showed that the anti-CD47 antibody Magrolimab, the anti-CD47 fusion protein TTI-621, and the recombinant proteins LCB-001 and LCB-002 all competed to different degrees with FITC-labeled high-affinity SIRPα D1m-Fc for binding to the CD47 antigen on Raji cells, exerting competitive binding activity, while the potential impurities LCB-001-R and LCB-002-R could not compete with FITC-labeled high-affinity SIRPα D1m-Fc for binding to the CD47 antigen, and could not exert competitive binding activity.

[0218] For example, as shown in Figure 9A, the anti-CD47 antibody Magrolimab, the anti-CD47 fusion protein TTI-621, and the recombinant proteins LCB-001 and LCB-002 all competitively bound to the CD47 antigen with FITC-labeled high-affinity SIRPα D1m-Fc to varying degrees. The competitive binding activity of the recombinant protein LCB-002 was nontrivially significantly superior to that of the anti-CD47 fusion protein TTI-621, while the competitive binding activity of the recombinant protein LCB-001 was comparable to that of the anti-CD47 fusion protein TTI-621. These results demonstrate that a recombinant protein in which a first functional structural fragment and a second functional structural fragment are linked using an appropriate linker sequence can nontrivially and significantly improve the competitive binding ability of the second functional structural fragment with the second functional antigen. At the same time, the potentially risky impurity proteins LCB-001-R and LCB-002-R were unable to compete with FITC-labeled high-affinity SIRPα D1m-Fc to bind to the CD47 antigen. This result suggests that the potentially risky impurities that cannot be completely removed during the recombinant protein production process significantly reduce the competitive binding activity to the second functional antigen, and the binding of these potentially risky impurities to the second functional antigen on non-target cells is very weak, significantly reducing immune-related toxic side effects and providing excellent safety.

[0219] Detection of competitive binding activity of LCB-005 by flow cytometry: The competitive binding activity of the bispecific recombinant protein LCB-005, which competitively binds to cell surface CD47, with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was measured by flow cytometry. The following method uses LCB-005 as an example, and is applied to the detection of a recombinant protein whose first functional antigen is EpCAM and whose second functional antigen is CD47.

[0220] CAPAN-2 cells (human pancreatic cancer cells) (EpCAM+ / CD47+) (purchased from Nanjing Kebai), which showed good growth potential, were washed and digested, counted, centrifuged, and diluted to 3 × 106 The cells were resuspended at a concentration of 100 cells / mL. 100 μL / well of the cells were added to a 96-well U-shaped plate (product number: 3799, Corning) and gradient diluted with LCB-005 (starting concentration: 200 nM, 3-fold dilutions, total 8 concentration points). The final concentration of FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was 4 nM. LCB-006, SIRPα D1m-Fc, TTI-621, SIRPα(CV1)-Fc-FITC, and CAPAN-2 cells were co-cultured for 1 hour, centrifuged, the supernatant discarded, and the cells were resuspended in DPBS + 2% FBS and then analyzed by flow cytometry.

[0221] The test results showed that, compared with the anti-CD47 fusion protein TTI-621, which had no significant competitive binding activity with CAPAN-2 cells, the bispecific recombinant protein LCB-005 and high-affinity SIRPα D1m-Fc both competitively bound to the CD47 antigen of CAPAN-2 cells with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) to different degrees, exerting competitive binding activity. The competitive binding activity of the bispecific recombinant protein LCB-005 to CAPAN-2 was significantly superior to that of the anti-CD47 fusion protein TTI-621.

[0222] For example, as shown in Figure 9B, the bispecific recombinant protein LCB-005 and the high-affinity SIRPα D1m-Fc both competitively bind to the CD47 antigen with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) to varying degrees, demonstrating competitive binding activity, whereas the anti-CD47 fusion protein TTI-621 did not exhibit competitive binding activity. This indicates that the competitive binding activity of the bispecific recombinant protein LCB-005 is nontrivially significantly superior to that of the anti-CD47 fusion protein TTI-621. These results demonstrate that the bispecific recombinant protein of the present invention, in which a first functional structural fragment and a second functional structural fragment are linked using an appropriate linker sequence, can nontrivially and significantly improve the competitive binding ability of the second functional structural fragment with a second functional antigen.

[0223] Detection of competitive binding activity of LCB-006 by flow cytometry: The competitive binding activity of the bispecific recombinant protein LCB-006, which competitively binds to cell surface CD47, with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was measured by flow cytometry. The following method uses LCB-006 as an example to detect a recombinant protein whose first functional antigen is CD24 and whose second functional antigen is CD47.

[0224] MCF-7 cells (human breast cancer cells) (CD24+ / CD47+) (purchased from Nanjing Kebai), which are well-growing target cells, were washed and digested, counted, centrifuged, and diluted to 3 × 10 6 The cells were resuspended at a concentration of 100 cells / mL. 100 μL / well of the cells were added to a 96-well U-shaped plate (product number: 3799, Corning) and gradient diluted with LCB-006 (starting concentration: 200 nM, 30-fold dilutions, total 8 concentration points). The final concentration of FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was 4 nM. LCB-006, SIRPα D1m-Fc, TTI-621, SIRPα(CV1)-Fc-FITC, and MCF-7 cells were co-cultured for 1 hour, centrifuged, the supernatant discarded, and the cells were resuspended in DPBS + 2% FBS and then analyzed by flow cytometry.

[0225] The test results showed that, compared with the anti-CD47 fusion protein TTI-621, which had no significant competitive binding activity to MCF-7 cells, the bispecific recombinant protein LCB-006 and the high-affinity SIRPα D1m-Fc both competitively bound to the CD47 antigen of MCF-7 cells with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) to different degrees, exerting competitive binding activity. The competitive binding activity of the bispecific recombinant protein LCB-006 to MCF-7 cells was significantly superior to that of the anti-CD47 fusion protein TTI-621.

[0226] For example, as shown in Figure 9C, the bispecific recombinant protein LCB-006 and the high-affinity SIRPα D1m-Fc both competitively bind to the CD47 antigen with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) to varying degrees, demonstrating competitive binding activity, whereas the anti-CD47 fusion protein TTI-621 did not exhibit competitive binding activity. This indicates that the competitive binding activity of the bispecific recombinant protein LCB-006 is nontrivially significantly superior to that of the anti-CD47 fusion protein TTI-621. These results demonstrate that the bispecific recombinant protein of the present invention, in which a first functional structural fragment and a second functional structural fragment are linked using an appropriate linker sequence, can nontrivially and significantly improve the competitive binding ability of the second functional structural fragment with the second functional antigen.

[0227] Detection of competitive binding activity of LCB-017 by flow cytometry: The competitive binding activity of the bispecific recombinant protein LCB-017, which competitively binds to cell surface CD47, with FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was measured by flow cytometry. The following method uses LCB-017 as an example and is applied to the detection of a recombinant protein whose first functional antigen is CD38 and whose second functional antigen is CD47.

[0228] Well-growing Raji cells (human B-cell lymphoma) (Cell Bank of Chinese Academy of Sciences, Shanghai) (CD38+ / CD47+) were collected, counted, centrifuged, and diluted to 3 × 10 in DPBS + 2% FBS. 6The cells were resuspended at a concentration of 100 cells / mL. 100 μL / well of the cells were added to a 96-well U-shaped plate (product number: 3799, Corning) and gradient diluted with LCB-017 (starting concentration: 200 nM, 3-fold dilutions, total 7 concentration points). The final concentration of FITC-labeled high-affinity SIRPα D1m-Fc (SIRPα(CV1)-Fc-FITC) was 4 nM. LCB-017, SIRPα D1m-Fc, TTI-621, SIRPα(CV1)-Fc-FITC, and Raji cells were co-cultured for 1 hour, centrifuged, the supernatant discarded, and the cells were resuspended in DPBS + 2% FBS. Then, the cells were detected by flow cytometry.

[0229] The test results showed that the anti-CD47 fusion protein TTI-621, bispecific recombinant protein LCB-017, and high-affinity SIRPα D1m-Fc all competed with FITC-labeled high-affinity SIRPα D1m-Fc to the CD47 antigen of Raji cells to different degrees, and exhibited competitive binding activity. The competitive binding activity of the bispecific recombinant protein LCB-017 to MCF-7 was significantly superior to that of the anti-CD47 fusion protein TTI-621.

[0230] For example, as shown in Figure 9D, the anti-CD47 fusion protein TTI-621, bispecific recombinant protein LCB-017, and high-affinity SIRPα D1m-Fc all competitively bound to the CD47 antigen on Raji cells with FITC-labeled high-affinity SIRPα D1m-Fc to varying degrees, demonstrating competitive binding activity. The competitive binding activity of the bispecific recombinant protein LCB-017 (IC50 = 27.98) was non-trivially significantly superior to that of the anti-CD47 fusion protein TTI-621 (IC50 = 1067). These results demonstrate that the bispecific recombinant protein of the present invention, in which the first and second functional fragments are linked using an appropriate linker sequence, can significantly improve the competitive binding ability of the second functional fragment with the second functional antigen.

[0231] In summary, (Figures 9A-9D) show that the bispecific recombinant protein of the present invention, in which a first functional fragment and a second functional fragment are linked using an appropriate linker sequence, can nontrivially and significantly improve the competitive binding ability of the second functional fragment with the second functional antigen. Other bispecific recombinant proteins in which the second functional antigen is CD47 also showed results consistent with the above data in target antigen-positive target cells.

[0232] Example 4: Detection of binding activity of bispecific recombinant proteins targeting GPC3 to GPC3-highly expressing hepatic cancer cell lines (double-positive expressing cells, target cells) The binding affinity of GPC3-targeting bispecific recombinant proteins to the target GPC3-high-expressing hepatoma cell line was measured by flow cytometry. The following method is applied to the detection of bispecific recombinant proteins, LCB-009, LCB-010, and LCB-011, in which the first functional binding fragment binds to the GPC3 antigen and the second functional binding fragment binds to IFN-α 2b or its low-affinity mutant.

[0233] The test cells were HepG2 cells (Nanjing Kebai Biotechnology Co., Ltd.) and HuH-7 cells (Cell Bank of Chinese Academy of Sciences, Shanghai). Cells with good growth were collected, counted, centrifuged, and diluted to 1 × 10 in FACS buffer (PBS + 2% FBS). 6The cells were resuspended at a concentration of 100 cells / mL and added to a 96-well U-shaped plate (product number: 3799, Corning) at 100 μL / well. Seven dilutions of the bispecific recombinant protein or control protein (starting at 100 nM, diluted three-fold, for a total of 7 concentrations) were added and incubated at 4°C for 1 hour. After washing with FACS buffer, goat anti-human IgG (Alexa Fluor 488 goat anti-human IgG (H+L); Invitrogen) was added and incubated at 4°C for 1 hour. After washing and resuspension with FACS buffer, fluorescence was detected using a flow cytometer (Attune Nxt; Invitrogen).

[0234] The experimental results, as shown in Figures 10 and 11, showed that the bispecific recombinant proteins LCB-009, LCB-010, and LCB-011 all had a certain binding activity with the target cells HepG2 and HuH-7 cells, which co-express GPC3 and IFNα receptors, and the binding of the bispecific recombinant proteins to HepG2 and HuH-7 cells had a higher maximum mean fluorescence intensity than the anti-GPC3 monoclonal antibody (control sample Codrituzumab). At the same time, as shown in Figures 10 and 11, the length of the linker sequence had a weak effect on the binding activity of the bispecific recombinant proteins to the target cells, and a short linker sequence (for example, a linker sequence with a single GGGGS sequence) had a weak effect on the binding activity of the bispecific recombinant proteins to the target cells. (SEQ ID NO: 65) In this case, the binding activity (EC50) of the bispecific recombinant protein (e.g., LCB-009) to the target cells was relatively low.

[0235] Example 5: Detection of the activity of bispecific recombinant proteins targeting GPC3 against antibody-dependent cell-mediated cytotoxicity (ADCC) The ADCC activity of bispecific recombinant proteins against hepatoma cell lines highly expressing the target GPC3 was measured by the lactate dehydrogenase (LDH) assay. The following method is applied to detect the ADCC activity of bispecific recombinant proteins LCB-009, LCB-010, and LCB-011, in which the first functional binding fragment binds to the GPC3 antigen and the second functional binding fragment is IFN-α 2b or its low-affinity mutant.

[0236] The effector cells were human peripheral blood mononuclear cells (PBMCs), the target cells were HepG2 liver cancer cell line, which highly expresses GPC3, and the LDH detection kit was CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, G7892). The target and effector cells were plated at a ratio of 1:20, and the bispecific recombinant protein or control protein (starting from 150 nM, 5-fold gradient dilutions, total 8 concentrations) was added and co-cultured at 37°C for 4 hours. After 3.5 hours of incubation at 37°C, the control group was added with lysis reagent and the cells were observed under a microscope. After the cells were completely lysed, they were centrifuged at 10,000 rpm for 5 minutes. The supernatant was transferred to a 96-well black clear-bottom plate (Corning, 3904) and incubated with the reaction substrate at 37°C for 30 minutes. Stop solution was added and the plate was shaken in the dark for 3-5 minutes. The signal was detected using a microplate reader (SpectraMax M2). For detailed steps, please refer to the LDH detection kit instruction manual.

[0237] As shown in Figure 12, the bispecific recombinant proteins (e.g., LCB-009, LCB-010, LCB-011) retained ADCC activity, and the ADCC activity of the bispecific recombinant proteins was comparable to that of the anti-GPC3 monoclonal antibody (control sample Codrituzumab) (EC50 = 0.53).

[0238] Example 6: Detection of the growth inhibitory activity of bispecific recombinant proteins targeting GPC3 The growth inhibitory activity of bispecific recombinant proteins targeting GPC3 against different tumor cell lines was measured using the Cell Titer Glo kit (Promega, Cat. G7558). The following method is applied to the detection of bispecific recombinant proteins LCB-009, LCB-010, and LCB-011, in which the first functional binding fragment binds to the GPC3 antigen and the second functional binding fragment is IFN-α 2b or its low-affinity mutant.

[0239] GPC3-positive cell line HuH-7 or GPC3-negative tumor cell line U266 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) and GPC3-negative tumor cell line SW480 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were plated in a 96-well black clear-bottom plate (Corning, 3904). The bispecific recombinant protein or control protein (starting from 52 nM, 10-fold gradient dilution, a total of 6 points, or starting from 10 nM, 5-fold gradient dilution, a total of 8 points) was added and cultured in a carbon dioxide incubator at 37°C for 3 days. The signal was then detected using a Cell titer glo Multimode Plate Reader (PerkinElmer, Envision2105).

[0240] As a result, the growth inhibitory activity (IC50) of the bispecific recombinant proteins (e.g., LCB-009, LCB-010, LCB-011) against the GPC3-positive (GPC3+) target cell line HuH-7 was higher than that of the control IFN-α 2b (shown in Figure 13) and that of the anti-GPC3 monoclonal antibody (anti-GPC3 mAb, i.e., the control sample Codrituzumab, which did not exhibit any growth inhibitory effect against HuH-7). As shown in Figure 14, the growth inhibitory activity of the bispecific recombinant protein LCB-012, which lacks the GPC3-targeting function, and the IFN-α 2b Fc fusion protein (IFN-α 2b-Fc) on HuH-7 (GPC3-positive target cells) was significantly lower than that of the bispecific recombinant protein LCB-010, which has the GPC3-targeting function. This indicates that binding of the bispecific recombinant protein with the GPC3-targeting function to GPC3 on target cells significantly enhances the growth inhibitory activity of IFN-α 2b, while IFN-α 2b, a bispecific recombinant protein that does not have the effect of targeting target cells, has low growth inhibitory activity. This indicates that the bispecific recombinant protein of the present invention has a strong growth inhibitory effect only on target cells that have the target antigen, and has a weak effect on or does not bind to non-target cells that do not have the target antigen, demonstrating the high safety of the bispecific recombinant protein of the present invention.

[0241] Furthermore, as shown in the results in Table 4, the bispecific recombinant proteins with different linker sequences (e.g., LCB-009, LCB-010, and LCB-011) all had stronger growth inhibitory activity than IFN-α 2b against GPC3-positive (GPC3+) target cells (e.g., HuH-7). The growth inhibitory activity of the bispecific recombinant proteins with different linker sequences was slightly different, and the linker sequence was the same as that of the GGGGS (SEQ ID NO: 65)Bispecific recombinant proteins containing the bispecific recombinant proteins (i.e., bispecific recombinant proteins linked by a short linker sequence) (e.g., LCB-009) had relatively low activity (shown in Figure 13, Table 4), which is consistent with the binding activity results of recombinant proteins with different linker sequences as shown in Figure 11. However, in the GPC3-negative (GPC3-) non-target cell lines U266 and SW480, the growth inhibitory activity of bispecific recombinant proteins (e.g., LCB-009, LCB-010, LCB-011) was significantly lower than that of IFN-α 2b (i.e., the relative activity of the bispecific recombinant proteins was much lower than 1). This result indicated that the growth inhibitory activity of bispecific recombinant proteins was extremely low in cells that do not express GPC3 (i.e., non-target cells that do not express the target antigen) and that their safety was relatively high. At the same time, as shown in Table 4, the growth inhibitory activity of the bispecific recombinant protein against targeted cells was at least 700-fold higher than that of the bispecific recombinant protein against non-targeted cells.

[0242] [Table 4]

[0243] Example 7: Detection of the growth inhibitory activity of bispecific recombinant proteins targeting PD-L1 The growth inhibitory activity of bispecific recombinant proteins targeting PD-L1 against different tumor cell lines was measured using the cell titer glo kit (Promega, Cat: G7558). The following method applies to bispecific recombinant proteins LCB-013, LCB-014, and LCB-015, for example, in which the first functional binding fragment binds to the PD-L1 antigen and the second functional binding fragment is IFN-α 2b or its low-affinity mutant.

[0244] The PD-L1-positive cell line MDA-MB-231 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) was plated in a 96-well black clear-bottom plate (Corning, 3904), and the bispecific recombinant protein or control protein (starting at 52 nM, diluted 10-fold, for a total of 6 points) was added. The plate was then placed in a 37°C carbon dioxide incubator and cultured for 3 days. The signal was then detected using a Cell titer glo Multimode Plate Reader (PerkinElmer, Envision2105).

[0245] As shown in Figure 15, in the case of PD-L1-positive (PD-L1+) target cells, MDA-MB-231, the PD-L1-targeting bispecific recombinant proteins (LCB-013, LCB-014, LCB-015) all had growth inhibitory activity, and the activity was significantly higher than that of the control bispecific recombinant protein LCB-012 and IFN-α 2b, which do not have PD-L1 targeting function, indicating that targeting PD-L1 can significantly increase the growth inhibitory activity of IFN-α 2b. The binding of a bispecific recombinant protein with PD-L1 targeting function to PD-L1 in the target cell line MDA-MB-231 significantly enhanced the growth inhibitory activity of IFN-α 2b, whereas a bispecific recombinant protein without the effect of targeting target cells had low growth inhibitory activity with IFN-α 2b. This indicates that the bispecific recombinant protein of the present invention has a strong growth inhibitory effect only on target cells that have the target antigen, and has a weak effect on or does not bind to non-target cells that do not express the target antigen, demonstrating the high safety of the bispecific recombinant protein of the present invention.

[0246] PD-L1-positive target cells MDA-MB-231 were plated in a 96-well black clear-bottom plate (Corning, 3904) and incubated with 200 nM of the PD-L1 antibody atezolizumab or an isotype control at 37°C for 30 minutes. The bispecific recombinant protein or control protein (starting concentration 20 nM, 6-fold gradient dilution, total 6 points) was added and incubated in a 37°C carbon dioxide incubator for 3 days. Signal values ​​were then detected using a Cell titer glo Multimode Plate Reader (PerkinElmer, Envision2105).

[0247] As shown in Figure 16 , after adding atezolizumab to block the PD-L1 antigen-antibody binding site, the growth inhibitory activity of LCB-015 was significantly reduced. This result indicates that by blocking the binding function of the target antigen on the test cells, the growth inhibitory activity of the bispecific recombinant protein on the test cells is significantly reduced. From another perspective, the bispecific recombinant protein of the present invention has a weak effect on or does not bind to non-target cells that do not have the target antigen binding function, demonstrating the high safety of the bispecific recombinant protein of the present invention.

[0248] Example 8: Detection of the growth inhibitory activity of bispecific recombinant proteins targeting CD38 The growth inhibitory activity of bispecific recombinant proteins targeting CD38 against different tumor cell lines was measured using the Cell Titer Glo kit (Promega, Cat: G7558). The following method uses LCB-016 as an example to detect a bispecific recombinant protein whose first functional binding fragment binds to the CD38 antigen and whose second functional binding fragment is IFNα2b.

[0249] The CD38-positive cell line Daudi or the CD38-negative tumor cell line SK-BR-3 (purchased from Nanjing Kebai Biotechnology Co., Ltd.) was plated in a 96-well black clear-bottom plate (Corning, 3904), and the bispecific recombinant protein or control protein (starting at 2 nM, 5-fold gradient dilution, total 9 points) was added. The plate was placed in a carbon dioxide incubator at 37°C and cultured for 3 days. The signal was then detected using a Cell titer glo Multimode Plate Reader (PerkinElmer, Envision2105).

[0250] The results showed that the growth inhibitory activity (IC50) of the bispecific recombinant protein (e.g., LCB-016) against the CD38-positive (CD38+) target cell line Daudi was nontrivially stronger than that of the control IFN-α 2b (shown in Figure 17A). In comparison, the growth inhibitory activity (IC50) of the bispecific recombinant protein (e.g., LCB-016) against the CD38-negative (CD38-) non-target cell line SK-BR3 was nontrivially weaker than that of the control IFN-α 2b (shown in Figure 17B). That is, the bispecific recombinant protein (e.g., IFN-α 2b) as the second functional binding fragment increased the growth inhibitory activity of IFN-α 2b against target cells bearing the target antigen, while significantly reducing the growth inhibitory effect of IFN-α 2b against non-target cells not bearing the target antigen. The bispecific recombinant protein of the present invention has a strong growth inhibitory effect only on target cells that have the target antigen, and has a weak effect on or does not bind to non-target cells that do not have the target antigen, demonstrating the high safety of the bispecific recombinant protein of the present invention.

[0251] As shown in Figure 17A, in the case of the CD38-positive (CD38+) target cell Daudi, the CD38-targeting bispecific recombinant protein (LCB-016) had growth inhibitory activity, and the activity was significantly higher than that of IFN-α 2b without CD38 targeting function, indicating that targeting CD38 can significantly increase the growth inhibitory activity of IFN-α 2b. Binding of the CD38-targeting bispecific recombinant protein to PD-L1 on the target cell Daudi significantly enhanced the growth inhibitory activity of IFN-α 2b. As shown in Figure 17B, the growth inhibitory activity of IFN-α 2b, which lacks CD38-targeting function, on SK-BR3 (CD38-negative non-target cells) was significantly stronger than that of the bispecific recombinant protein LCB-016, which both have CD38-targeting function, indicating that the growth inhibitory activity of the bispecific recombinant protein on IFN-α 2b, a non-target cell line lacking the target antigen, was relatively low. The relative activity of LCB-016 on IFN-α 2b on the target cell line Daudi was at least 200-fold higher than its relative activity on the non-target cell line SK-BR3.

[0252] Example 9 Detection of growth inhibitory activity of bispecific recombinant proteins containing IFN-α 2b low affinity mutants Considering the difference between the human body's tolerance of IFN-α 2b and the effective dose of a typical antibody, and to better match the effects of the antigen-binding fragment targeting the target antigen and IFN-α 2b to achieve high efficacy and low toxicity, the present invention also designed a series of bispecific recombinant proteins containing low-affinity mutants of IFN-α 2b. In this example, using the mutation design based on LCB-010 as an example, bispecific recombinant proteins containing low-affinity mutants of IFN-α 2b were designed, and the growth inhibitory activity of bispecific recombinant proteins containing different low-affinity mutants of IFN-α 2b was examined. The experimental method was the same as in Example 7.

[0253] As shown in Figures 18 to 20, the results showed that the bispecific recombinant proteins carrying the IFN-α 2b low-affinity mutants exhibited reduced growth inhibitory activity against GPC3-positive target cells (HuH-7) or GPC3-negative non-target cells (U266, SW480), LCB-010-M2, LCB-010-M3, and LCB-010-M4, compared to LCB-010. For example, the growth inhibitory activity IC50 of LCB-010-M3 (A145G mutation) in HuH-7 (a GPC3-positive cell line, target cell) was comparable to that of IFN-α 2b (IC50 IFN-α 2b =0.1793, IC50 LCB-010-M3 = 0.179), whereas the relative growth inhibitory activity (IC50 IFN-α 2b / bispecific recombinant protein) of LCB-010-M3 in GPC3-negative non-target cells (U266) was weaker. For example, the relative growth inhibitory activity (IC50 IFN-α 2b / bispecific recombinant protein) of LCB-010-M3 in U266 was 0.000615, significantly weaker than IFN-α 2b and weaker than LCB-010. LCB-010-M4 (R149A mutation) also showed similar results.

[0254] The above results and Figures 19 to 21 show that the activity of the bispecific recombinant protein targeting GPC3 containing the IFN-α 2b low-affinity mutant in target antigen-positive cell lines is equivalent to that of IFN-α 2b, but the activity in non-target cell lines that do not express the target antigen is reduced, indicating that the bispecific recombinant protein containing the IFN-α 2b low-affinity mutant of the present invention has a higher safety profile.

[0255] In summary, the growth inhibitory activity of the bispecific recombinant protein of the present invention comprising a low affinity mutant of IFN-α 2b against target antigen-negative non-target cells is comparable to or significantly reduced compared to the growth inhibitory activity of the bispecific recombinant protein comprising wild-type IFN-α 2b against target antigen-negative non-target cells, and compared to the reduction in the growth inhibitory activity of the bispecific recombinant protein comprising a low affinity mutant of IFN-α 2b against target antigen-positive target cells.

[0256] Example 10: Detection of growth inhibitory activity of potential risk impurities of bispecific recombinant proteins on non-target cells To reduce the potential safety risk of impurities in the future manufacturing process of bispecific recombinant proteins, the present inventors took LCB-010 as an example to analyze the growth inhibition of its potential risk impurity (B-chain homodimer) on non-target cells.

[0257] During the purification process of LCB-010 (as described in Example 2), the B-chain homodimer of LCB-010 (Figure 3; the molecular weight of the homodimer is approximately 140 kD) was separated, and this potential risk impurity and IFN-α 2b-Fc (a potential risk impurity in the dual antibody structure shown in Figure 3A of CN108864290A, the right arm homodimer after D1 was replaced with IFN-α 2b) were detected to have growth inhibitory activity in the non-target cell line (GPC3-negative cell line) MDA-MB-231. As shown in Figure 21, at a concentration of 16.7 nM, the growth inhibition rate of IFN-α 2b against MDA-MB-231 (GPC3-negative cells, non-target cells) was 91.3%, the growth inhibition rate of IFN-α 2b-Fc against MDA-MB-231 (GPC3-negative cells, non-target cells) was 66.8% (a decrease of approximately 24.5% compared to IFN-α 2b), and the growth inhibition rate of LCB-010 B chain homodimer against MDA-MB-231 (GPC3-negative cells, non-target cells) was only 16.2% (a decrease of approximately 75.1% compared to IFN-α 2b and an increase of approximately 50% compared to IFN-α 2b-Fc). In summary, the potential risk impurities have very little effect on non-target cells, i.e., the potential safety risks or potential toxic side effects are very low.

[0258] Example 11 Detection of target affinity of bispecific recombinant proteins in which the second functional binding fragment is IL12 The binding affinity of the bispecific recombinant protein to the target TIGIT and IL-12 receptors was measured by flow cytometry. The following method is applied to the detection of a recombinant protein with TIGIT as the first functional antigen and IL-12A as the second functional binding fragment, using LCB-018 as an example.

[0259] H_IL12 Reporter 293 cells (TIGIT+, target cells) were plated in a 96-well white clear-bottom plate (Corning, 3903) and allowed to adhere to the plate overnight. The medium was then discarded, and 200 nM of the anti-TIGIT monoclonal antibody tiragolumab or isotype control antibody (isotype) was added. The cells were incubated at 37°C for 30 minutes. The medium was then discarded, and 200 nM of the bispecific recombinant protein LCB-018 and 2 μg / mL of IL12B were mixed in equal volumes and diluted 3-fold downward for a total of 10 points. 150 μL per well was then added to a 96-well plate and placed in a carbon dioxide incubator at 37°C for 6 hours. One-Glo was added, and the signal was detected using a Multimode Plate Reader (PerkinElmer, Envision 2105).

[0260] The results (shown in Figure 22) showed that the binding curve of the bispecific recombinant protein to the test cells was significantly shifted to the right when the anti-TIGIT monoclonal antibody Tiragolumab was added to block the binding, indicating that the bispecific recombinant protein of the present invention can non-trivially improve the binding ability of the bispecific recombinant protein to the target cells by using an appropriate linker sequence to link the first functional structural fragment (TIGIT targeting moiety) and the second functional structural fragment (IL12). Furthermore, the binding affinity of the bispecific recombinant protein of the present invention (e.g., LCB-018) to the target antigen-positive target cells (e.g., H_IL12 Reporter 293 cells) was approximately 500-fold weaker (EC50) than the binding affinity of the IL12A and IL12B complex to the same cells at the same concentration. LCB-018 = 30nM, EC50 IL12A / IL12B =0.06 nM), which also demonstrated that the targeting effect of the bispecific recombinant protein of the present invention significantly reduced the binding activity of IL12 to cells, thereby reducing the potential toxic side effects of IL12, especially the toxic side effects produced by the binding of IL12 to non-target cells.

[0261] Example 12: Detection of activation level of bispecific recombinant protein in which the second functional binding fragment is IL10M for P-STAT3 in THP1 cells The activation level of the bispecific recombinant protein against P-STAT3 in THP1 cells was measured by flow cytometry. The following method is applied to the detection of the recombinant protein LCB-019, whose first functional antigen is CD80 and whose second functional binding fragment is IL10M.

[0262] LCB-019, LCB-022 (a control bispecific recombinant protein that targets CD80 and is an ineffective control), Isotype, and IL10M-Fc fusion protein were diluted in basal medium (1640) to an equimolar concentration of 10 nM for use.

[0263] Considering that LPS (Sigma, L5418-2ML) can stimulate the expression of CD80 antigen on the surface of Thp1 cells (obtained from the Cell Bank of Chinese Academy of Sciences), this example used Thp1 cells after stimulation with LPS for 24 hours to simulate target CD80-positive double-positive cells (target cells).

[0264] Thp1 cells (obtained from the Cell Bank of Chinese Academy of Sciences) were cultured at 1 × 10 cells per well after stimulation with 1 μg / mL LPS (Sigma, L5418-2ML) for 24 h. 6The cells were resuspended in basal medium (1640) at a density of 100 μL. The resuspended cells after LPS stimulation and unstimulated Thp1 cells were plated in 96-well plates in a volume of 100 μL. The same volumes of diluted LCB-019, LCB-022 (control bispecific recombinant protein), isotype, and IL10M-Fc fusion protein were added and incubated at 37°C in a 5% CO2 incubator for 20 minutes. After incubation, the supernatant was removed by centrifugation, and the cells were fixed and permeabilized. The cells were then resuspended in 100 μL of FACS buffer (1x PBS + 2% FBS) containing 0.5 μL of LPE-P-STAT3 antibody (BD, 562072) and incubated at 4°C for 1 hour, protected from light. After washing twice with FACS buffer (1x PBS + 2% FBS), the cells were resuspended in 200 μL of FACS buffer (1x PBS + 2% FBS), and P-STAT3 levels were detected by FACS.

[0265] As shown in Figure 23, in Thp1 cells that highly express the target antigen CD80 after stimulation with LPS, the P-STAT3 level of the bispecific recombinant protein LCB-019, which has the effect of targeting the target antigen (CD80), was similar to that of the IL10M-Fc fusion protein and significantly higher than that of the control bispecific recombinant protein LCB-022, which does not have the function of targeting the target antigen. On the other hand, in Thp1 cells not stimulated with LPS, the P-STAT3 levels of LCB-019 and the control bispecific recombinant protein LCB-022 were similar and significantly weaker than that of the IL10M-Fc fusion protein.

[0266] These results indicate that when non-target cells expressing the target antigen weakly or not expressing it, the bispecific recombinant protein had a significantly weaker effect on the non-target cells than the IL10M-Fc fusion protein, demonstrating relatively high safety. However, when target cells expressing the target antigen highly, the bispecific recombinant protein targeting the target antigen showed STAT3 levels similar to those of the IL10M-Fc fusion protein, fully exerting and enhancing the effects and actions of IL10M in the bispecific recombinant protein. In comparison, the bispecific recombinant protein without target antigen targeting function had a weaker effect on these cells (non-target cells), demonstrating excellent safety.

[0267] Example 13 Detection of the proliferative activity of a bispecific recombinant protein in which the second functional binding fragment is IL15-IL15RαSUSHI The proliferation activity of bispecific recombinant proteins on PD-1-positive hPBMCs after 48 hours of OKT3 stimulation was measured by flow cytometry. The following method is applied to the detection of recombinant proteins LCB-020, LCB-021, LCB-023, and LCB-024, whose first functional antigen is PD-1 and whose second functional binding fragment is IL15-IL15RαSUSHI.

[0268] After resuscitation, hPBMC cells were placed in 6-well plates precoated with 100 ng / mL anti-CD3 antibody (OKT3, eBioscience, Cat. #16-0037-85) and cultured for 48 hours. Activated PBMCs were collected by centrifugation and washed once with PBS. The cells were then resuspended in medium and plated into 96-well plates at a density of 1.5E5 / 100 μL / well. 1.6 nM of the positive control C15Y and bispecific recombinant proteins LCB-020, LCB-021, LCB-023, and LCB-024 were added. The 96-well plates were placed in a 37°C carbon dioxide incubator and cultured for 96 hours. After the culture was completed, the cell membrane was stained with anti-CD4-APC (eBioscience, Cat. #17-0049-42) and anti-CD8-FITC (Invitrogen, Cat. #MHCD0801) antibodies for the first time. After staining, the cells were permeabilized by treatment with a fixative permeabilization reagent (eBioscience). TM Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, Cat. #00-5523-00). After permeabilization, cells were stained for 40 minutes using anti-Ki-67-PE (Biolegend, Cat. #350504) antibody. After staining, Ki-67 expression in CD4+ and CD8+ cell populations was analyzed using FACS.

[0269] As shown in Figure 24, all five samples showed a certain proliferation-promoting effect on hPBMCs with high PD-1 expression at a concentration of 1.6 nM. Among them, the positive control C15Y had the strongest activity. The proliferation-promoting effects of the bispecific recombinant proteins LCB-020 and LCB-021, which have the effect of targeting PD-1, and the control bispecific recombinant proteins RSV x IL-15R antibody (LCB-023, LCB-024), which have no effect of targeting PD-1, were weaker than that of the positive control C15Y. The proliferation-promoting effects of the bispecific recombinant proteins LCB-020 and LCB-021, which have the effect of targeting PD-1, were significantly stronger than that of the control bispecific recombinant proteins LCB-023 and LCB-024, which have no effect of targeting PD-1. At the same time, the proliferation-promoting effects of the control bispecific recombinant proteins LCB-023 and LCB-024, which have no effect of targeting PD-1, were relatively weaker. The results showed that, among bispecific recombinant proteins with target antigen-targeting function, IL15-IL15RαSUSHI significantly enhanced the proliferation-promoting effect on target cells, but this proliferation-promoting effect was weaker than that of the IL15-IL15RαSUSHI-Fc fusion protein (C15Y). The control bispecific recombinant proteins (LCB-023, LCB-024) showed a weak proliferation-promoting effect on PD-1-positive hPBMCs stimulated with OKT3 for 24 hours. This also demonstrated that the bispecific recombinant proteins of the present invention have a relatively weak effect on non-target cells that do not express the target antigen, and are relatively safe.

[0270] The proliferation activity of PD-1-targeting bispecific recombinant proteins against M-07e was measured using the Cell Titer glo kit (Promega, Cat. G7558). The following method is applied to the detection of recombinant proteins LCB-020, LCB-021, LCB-023, and LCB-024, in which the first functional antigen is PD-1 and the second functional binding fragment is IL15-IL15RαSUSHI.

[0271] Cytokine-dependent M-07e cells were harvested and washed once with PBS. Cells were diluted to a density of 2E4 / 50 μL in GM-CSF (R&D, Cat. #215-GM-050) growth factor-free medium, plated in a 96-well plate, and cultured for 4 hours for cytokine starvation. After 4 hours of starvation, gradient dilutions of C15Y (10 nM starting concentration, 3-fold dilution) and bispecific recombinant proteins LCB-020, LCB-021, LCB-023, and LCB-024 (333 nM starting concentration, 3-fold dilution) were added and co-cultured for 72 hours at 37°C in a carbon dioxide incubator. After 72 hours, viable cells were counted using Celltiter-glo (Promega, Cat. #G7573).

[0272] As shown in Figure 25, the positive control C15Y exerted a very strong proliferation-promoting effect (EC50 = 0.05267 nM) at an extremely low concentration, while the bispecific recombinant protein of the present invention exerted a proliferation-promoting effect only at a relatively high concentration (EC50 of approximately 21 to 37 nM), with no significant difference in EC50. Because the test cell M-07e does not express the target antigen corresponding to LCB-020, LCB-021, LCB-023, or LCB-024, M-07e can be considered a non-target cell. The bispecific recombinant protein of the present invention, in which the second functional binding fragment is IL15-IL15RαSUSHI, exerted a proliferation-promoting effect on non-target cells only at a relatively high concentration, with an EC50 that was more than 400-fold higher than that of the positive control.

[0273] From the above, the growth-promoting effect of the bispecific recombinant protein of the present invention on target antigen-positive target cells was significantly stronger than that of the bispecific recombinant protein without targeting function, but weaker than that of the positive control C15Y. Conversely, the growth-promoting effect of the recombinant protein of the present invention on target antigen-negative non-target cells was not significantly different, and the initial concentration of the growth-promoting effect was significantly higher than that of the positive control C15Y. Therefore, the bispecific recombinant protein with targeting function of the present invention can exert a growth-promoting effect on target cells at a relatively low concentration, but can exert a growth-promoting effect on non-target cells only at an extremely high concentration.

[0274] Example 14. Freeze-thaw stability studies of bispecific recombinant proteins Existing recombinant human albumin interferon-α2b fusion proteins have relatively poor freeze-thaw stability and are not suitable for repeated freeze-thaw cycles. For example, PEGylated long-acting interferon cannot be frozen or shaken, and has high requirements for transportation and storage conditions. To study the freeze-thaw stability of the bispecific recombinant protein of the present invention, a repeated freeze-thaw stability test was performed on the bispecific recombinant protein. The protein was placed in 20 mM NaAc (pH = 5) buffer and subjected to five freeze-thaw cycles at -40°C. Purity (size exclusion chromatography, SEC) and appearance analysis were performed on samples before and after freeze-thawing. The results are shown in Table 5. The LCB-011 mutants exhibited good freeze-thaw stability, with purities of over 95% and clear appearance after five freeze-thaw cycles. This indicates that the freeze-thaw stability of the IFN-α2b protein portion of the bispecific recombinant protein of the present invention is significantly superior to that of IFN-α2b alone or PEGylated IFN-α2b.

[0275] [Table 5]

[0276] Any examples or use of exemplary language (e.g., "such as") provided herein are intended to better describe the invention and do not limit the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0277] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety, just as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. It should be noted that the theories, mechanisms, evidence, or discoveries described herein are intended to further enhance the understanding of the invention, and are not intended in any way to limit the invention to such theories, mechanisms, evidence, or discoveries. While the invention has been shown and described in detail in the drawings and foregoing description, the invention should be considered illustrative and not limiting.

[0278] Although specific embodiments of the present invention have been described above, those skilled in the art will recognize that these are merely illustrative examples and may make various changes and modifications to these embodiments without departing from the principles and substance of the present invention. Therefore, the scope of protection of the present invention is limited by the appended claims. [Brief explanation of the drawings]

[0279] [Figure 1] FIG. 1 is a schematic diagram of the structure of the bispecific recombinant protein of the present invention. [Figure 2] FIG. 1 is an SDS-PAGE electrophoresis diagram of bispecific recombinant proteins after purification with Protein A in Example 2 of the present invention. [Figure 3] FIG. 1 shows a non-reducing SDS-PAGE electrophoresis diagram of the bispecific recombinant protein of Example 2 of the present invention after affinity capture. [Figure 4] FIG. 1 shows a reducing SDS-PAGE electrophoresis diagram of the bispecific recombinant protein of Example 2 of the present invention after affinity capture. [Figure 5] FIG. 1 shows a non-reducing SDS-PAGE electrophoresis diagram of the bispecific recombinant protein of Example 2 of the present invention after purification. [Figure 6] 1 shows the binding curve of the bispecific recombinant protein of the present invention to CD20 single-positive cells (non-target cells, CHO-K1-hCD20) determined by flow cytometry in Example 3 of the present invention. [Figure 7A] 1 shows the binding curves of the bispecific recombinant protein of the present invention, in which the second functional antigen is CD47, and the control sample to CD47 single-positive cells (non-target cells, HEK293 cells) determined by flow cytometry in Example 3 of the present invention. [Figure 7B] 1 shows the binding curves of the bispecific recombinant protein of the present invention, whose second functional antigen is CD47, and the control sample to CD47 single-positive cells (non-target cells, CHO-K1 cells) determined by flow cytometry in Example 3 of the present invention. [Figure 8] FIG. 1 shows the binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD20 and the second functional antigen is CD47, and the control sample to CD20 / CD47 double-positive cells (target cells, Raji cells) determined by flow cytometry in Example 3 of the present invention. [Figure 9A] FIG. 1 shows the competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD20 and the second functional antigen is CD47, the corresponding potential risk impurity protein, and the control sample CD20 / CD47 double-positive cells (target cells, Raji cells) determined by flow cytometry in Example 3 of the present invention. [Figure 9B] This figure shows the competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is EpCAM and the second functional antigen is CD47, as determined by flow cytometry in Example 3 of the present invention, and the control sample, EpCAM / CD47 double-positive cells (target cells, CAPAN-2 cells). [Figure 9C]FIG. 1 shows the competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD24 and the second functional antigen is CD47, and the control sample, in CD24 / CD47 double-positive cells (target cells, MCF-7 cells) determined by flow cytometry in Example 3 of the present invention. [Figure 9D] FIG. 1 shows the competitive binding curves of the bispecific recombinant protein of the present invention, in which the first functional antigen is CD38 and the second functional antigen is CD47, and the control sample, in CD38 / CD47 double-positive cells (target cells, Raji cells) determined by flow cytometry in Example 3 of the present invention. [Figure 10] Figure 1 shows the binding activity curves of the bispecific recombinant protein of Example 4 of the present invention, in which the first functional antigen is GPC3 and the second functional binding protein is IFN-α 2b, and the control sample, in HepG2 liver cancer cells as target cells, as determined by flow cytometry. [Figure 11] 1 shows a histogram of binding activity in the target cell HuH-7 of the bispecific recombinant protein of Example 4 of the present invention, in which the first functional antigen is GPC3 and the second functional binding protein is IFN-α 2b, and a control sample, determined by flow cytometry. [Figure 12] 1 shows the ADCC activity curves of the bispecific recombinant proteins in target cells HepG2 of Example 5 of the present invention determined by the LDH method. [Figure 13] 1 shows the inhibitory activity curves of bispecific recombinant proteins with different linker sequences against the proliferation of target cells HuH-7 in Example 6 of the present invention. [Figure 14] 1 shows the inhibitory activity curves of the bispecific recombinant protein having GPC3 antigen-targeting function of Example 6 of the present invention and a control sample on the proliferation of GPC3-positive target cells HuH-7. [Figure 15] 1 shows the inhibitory activity curve of the bispecific recombinant protein of Example 7 of the present invention against the proliferation of PD-L1 positive target cells MDA-MB-231. [Figure 16]Figure 10 shows the inhibitory activity curve of the bispecific recombinant protein of Example 7 of the present invention on the proliferation of MDA-MB-231 cells after anti-PD-L1 antibody occlusion. [Figure 17A] 1 shows the inhibitory activity curve of the bispecific recombinant protein of Example 8 of the present invention against the proliferation of CD38-positive target cells Daudi. [Figure 17B] 1 shows the inhibitory activity curve of the bispecific recombinant protein of Example 8 of the present invention against the proliferation of CD38-negative non-target cells SK-BR3. [Figure 18] 1 shows the inhibitory activity curves of bispecific recombinant proteins comprising different IFN-α 2b low affinity mutants of Example 9 of the present invention against the proliferation of GPC3-positive target cells HuH-7. [Figure 19] 1 shows the inhibitory activity curves of bispecific recombinant proteins comprising different IFN-α 2b low affinity mutants of Example 9 of the present invention against the proliferation of GPC3-negative target cells SW480. [Figure 20] 1 shows the inhibitory activity curves of bispecific recombinant proteins comprising different IFN-α 2b low affinity mutants of Example 9 of the present invention against the proliferation of GPC3-negative target cells U266. [Figure 21] 1 shows the inhibitory activity curve of potential risk impurities of the bispecific recombinant protein of Example 10 of the present invention against the proliferation of GPC3-negative non-target cell MDA-MB-231. [Figure 22] 1 shows the binding activity curve of the bispecific recombinant protein of Example 11 of the present invention to TIGIT-positive target cells H_IL12 Reporter 293 after anti-TIGIT antibody blockade. [Figure 23] 1 shows the detection results of the bispecific recombinant protein of Example 12 of the present invention on the activation level of P-STAT3 in THP1 cells. [Figure 24] 1 shows the results of detecting the proliferation activity of the bispecific recombinant protein of Example 13 of the present invention on PD-1-positive hPBMCs after 48 hours of stimulation with OKT3. [Figure 25]1 shows the results of detecting the proliferation activity of the bispecific recombinant protein of Example 13 of the present invention against PD-1-negative non-target cells M-07e.

Claims

1. A bispecific recombinant protein, comprising a first functional binding fragment, a second functional binding fragment, and an Fc region, wherein the first functional binding fragment, which targets a target antigen of the bispecific recombinant protein, comprises an antigen-binding fragment, and wherein the C-terminus of the antigen-binding fragment is linked directly or via a linker sequence to a second functional binding fragment having an immunomodulatory function and / or a metabolic and / or endocrine modulating function; the antigen-binding fragment is linked directly or via a linker sequence to the N-terminus of the second functional binding fragment, the C-terminus of the second functional binding fragment is linked directly or via a linker sequence to the N-terminus of Fc; and The bispecific recombinant protein is composed of an A chain and a B chain, and the A chain is bound to the B chain by an intermolecular force, a covalent bond, an ionic bond, or a combination of two or three of these bonds, and the structures of the A chain and the B chain are as follows: (i) the A chain comprises VH-CH1-CH2-CH3 and the B chain comprises VL-CL-second functional binding fragment-CH2-CH3; (ii) the A chain comprises VL-CL-CH2-CH3 and the B chain comprises VH-CH1-second functional binding fragment-CH2-CH3; (iii) the A chain comprises VH-CL-CH2-CH3 and the B chain comprises VL-CH1-second functional binding fragment-CH2-CH3; and (iv) the A chain comprises VL-CH1-CH2-CH3 and the B chain comprises VH-CL-second functional binding fragment-CH2-CH3; selected from the group consisting of the VH domain, VL domain, CH1 domain, and CL domain together form the first functional binding fragment, the Fc region comprising a CH2 domain and a CH3 domain, optionally, the CH2 domain further comprising a hinge region; and Domains (i) to (iv) are linked directly or via a linker; the second functional binding fragment having immunomodulatory function targets an immune checkpoint, an immune checkpoint ligand, or a cytokine receptor; or the second functional binding fragment having metabolic regulatory function targets a metabolic regulator or a metabolic regulator receptor, or A bispecific recombinant protein, wherein the second functional binding fragment having an endocrine regulatory function targets an endocrine regulatory factor or an endocrine regulatory factor receptor.

2. The second functional binding fragment having immunomodulatory function targets PD-1 or its ligand, CD47 or its ligand, CD24 or its ligand, an interferon receptor, or an interleukin receptor; or the second functional binding fragment with metabolic regulatory function targets the insulin receptor or the fibroblast growth factor receptor; or The bispecific recombinant protein of claim 1 , wherein the second functional binding fragment having an endocrine regulatory function targets a hormone receptor.

3. 2. The bispecific recombinant protein according to claim 1, wherein the linker sequence comprises (GGGGS)n (SEQ ID NO: 65), (GGGS)n (SEQ ID NO: 66), (GGS)n, (G)n, (GS)n, (EAAAK)n (SEQ ID NO: 67), or (XP)n, wherein n is a natural number from 0 to 5.

4. 2. The bispecific recombinant protein of claim 1, wherein the second functional binding fragment binds to a cytokine receptor, an immune checkpoint, or an immune checkpoint ligand; the second functional binding fragment is a cytokine, an immune checkpoint ligand, or an immune checkpoint ligand-binding protein, or a functional fragment thereof; and the second functional binding fragment is any one selected from a human SIRP family extracellular functional fragment, a human interferon family functional fragment, a tumor necrosis factor superfamily functional fragment, a TGF-β superfamily functional fragment, an interleukin system functional fragment, a chemokine family functional fragment, a colony-stimulating factor family functional fragment, and a growth factor functional fragment.

5. 5. The bispecific recombinant protein of claim 4, wherein the second functional binding fragment is a human SIRPα extracellular D1 domain; the second functional binding fragment is a type I or type II human interferon receptor; and / or the second functional binding fragment is an interleukin.

6. The interferon receptor is a human interferon gamma (IFN-γ) receptor or a human interferon beta (IFN-β) receptor; and / or the interleukin is any one selected from the IL-1 family, IL-2 family, IL-3 family, IL-6 family, IL-8 family, IL-10 family, IL-12 family, and IL-17 family. The bispecific recombinant protein of claim 5.

7. The first functional binding fragment includes CD20, GPC-3, PD-L1, CD38, EpCAM, CD24, TIGIT, PD-1, CD80, EGFR, AFP, 5T4, AGS-16, ALK1, ANG-2, B7. -H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD22, CD30, CD32b, CD37, CD40, CD52, CD70, CD71, CD74, CD79b, CD83, CD86, CD98, CD206, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CCR5, CXCR4, DLL-4, EGFRvIII, EGP-1, ENPP3, EphA3, ETBR, FGFR2, F N, FR-α, GCC, GD2, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Nectin-4, Notch 2, Notch 1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-IR, MSB0010718C, BCMA, CD138, TROP2, Siglec15, and CD155.

8. The bispecific recombinant protein of claim 7, wherein the second functional binding fragment comprises the human SIRPα extracellular D1 domain, human interferon beta or human interferon gamma, or an interleukin, and the first functional binding fragment targets tumor cells or immune cells.

9. A bispecific recombinant protein described in any one of claims 1 to 8, wherein the Fc region comprises a native Fc region sequence or a non-native Fc sequence.

10. The bispecific recombinant protein of claim 9, wherein the Fc region of the A chain is linked to the Fc region of the B chain by a knobs-into-holes connection, and / or the Fc region is the Fc region of human IgG.

11. The bispecific recombinant protein described in Claim 10, wherein the Fc region is the Fc region of human IgG1 or IgG4.

12. The first functional binding fragment includes CD20, GPC-3, PD-L1, CD38, EpCAM, CD24, TIGIT, PD-1, CD80, EGFR, AFP, 5T4, AGS-16, ALK1, ANG-2, B7. -H3, B7-H4, c-fms, c-Met, CA6, CD123, CD19, CD22, CD30, CD32b, CD37, CD40, CD52, CD70, CD71, CD74, CD79b, CD83, CD86, CD98, CD206, CEA, CEACAM5, CLDN18.2, CLDN6, CS1, CCR5, CXCR4, DLL-4, EGFRvIII, EGP-1, ENPP3, EphA3, ETBR, FGFR2, F N, FR-α, GCC, GD2, GPNMB, HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-3R, LIV-1, MSLN, MUC16, MUC1, NaPi2b, Nectin-4, Notch 2, Notch 1, PD-L2, PDGFR-α, PS, PSMA, SLTRK6, STEAP1, TEM1, VEGFR, CD25, CD27L, DKK-1, CSF-IR, MSB0010718C, BCMA, CD138, TROP2, Siglec15, and CD155, wherein the antigen-binding fragment is a human-mouse chimeric antigen-binding fragment, a humanized antigen-binding fragment, or a fully human antigen-binding fragment.

13. when the first functional binding fragment targets CD20, EGFR, EGFRvIII, PD-L1, PD-L2, HER2, HER3, CD138, CD44, CD24, EpCAM, CLDN18.2, CD38, BCMA, MUC1, or TROP2, the second functional binding fragment comprises the SIRPα extracellular D1 domain; 13. The bispecific recombinant protein of claim 12, wherein the first functional binding fragment targets TIGIT, Siglec15, PD-1, PD-L1, PD-L2, CD71, CD80, CD86, CD206, or CCR5, and the second functional binding fragment comprises a complex formed with IFN-β, IFN-γ, IL-10M, IL-12A, or IL-15 and IL15RαSUSHI.

14. the first functional binding fragment targets CD20, EpCAM, CD24, or EGFR, and the second functional binding fragment comprises a SIRPα extracellular D1 domain, the amino acid sequence of which is as set forth in SEQ ID NO:50; the first functional binding fragment targets TIGIT, CD80, or PD-1, and the second functional binding fragment comprises IL-12A, an IL-10 monomer mutant, or an IL15 and IL-15RαSUSHI complex, wherein the amino acid sequences of the IL-12A, IL-10 monomer mutant, IL15, and IL-15RαSUSHI are as set forth in SEQ ID NOs: 51, 52, 53, and 54, respectively; or 14. The bispecific recombinant protein of claim 13, wherein the first functional binding fragment targets CD38 or AFP, and the second functional binding fragment comprises the extracellular D1 domain of SIRPα or IFN-β, and the amino acid sequences of the extracellular D1 domain of SIRPα and the IFN-β are as set forth in SEQ ID NOs: 50 and 55, respectively.

15. The amino acid sequence of the A chain is as set forth in SEQ ID NO: 1, and the amino acid sequence of the B chain is as set forth in SEQ ID NO: 2 or 3; the amino acid sequence of the A chain is as set forth in SEQ ID NO:61, and the amino acid sequence of the B chain is as set forth in SEQ ID NO:62; the amino acid sequence of the A chain is as set forth in SEQ ID NO:27, and the amino acid sequence of the B chain is as set forth in SEQ ID NO:28; the amino acid sequence of the A chain is as set forth in SEQ ID NO:29 and the amino acid sequence of the B chain is as set forth in SEQ ID NO:30; the amino acid sequence of the A chain is as set forth in SEQ ID NO:56 and the amino acid sequence of the B chain is as set forth in SEQ ID NO:57; the amino acid sequence of the A chain is as set forth in SEQ ID NO:35 and the amino acid sequence of the B chain is as set forth in SEQ ID NO:36; the amino acid sequence of the A chain is as set forth in SEQ ID NO:37 and the amino acid sequence of the B chain is as set forth in SEQ ID NO:38; the amino acid sequence of the A chain is as set forth in SEQ ID NO: 39, and the amino acid sequence of the B chain is as set forth in SEQ ID NO: 40 or 41; the amino acid sequence of the A chain is as set forth in SEQ ID NO: 31, and the amino acid sequence of the B chain is as set forth in SEQ ID NO: 32; the amino acid sequence of the A chain is as set forth in SEQ ID NO:33 and the amino acid sequence of the B chain is as set forth in SEQ ID NO:34; or 15. The bispecific recombinant protein of claim 14, wherein the amino acid sequence of the A chain is as set forth in SEQ ID NO: 58 and the amino acid sequence of the B chain is as set forth in SEQ ID NO:

60.

16. 16. The nucleic acid molecule encoding a bispecific recombinant protein according to any one of claims 1 to 15, wherein the nucleic acid molecule encoding the first functional binding fragment and the nucleic acid encoding the second functional binding fragment are located on the same DNA strand, or the nucleic acid molecule encoding the first functional binding fragment and the nucleic acid encoding the second functional binding fragment are located on different DNA strands.

17. An expression vector comprising the nucleic acid molecule of claim 16.

18. A host cell transformed with the expression vector of claim 17.

19. A method for producing a bispecific recombinant protein according to any one of claims 1 to 15, comprising transforming a host cell according to claim 18 with the expression vector according to claim 17, and culturing the host cell under conditions suitable for expression to obtain the bispecific recombinant protein.

20. A medicament or pharmaceutical composition comprising a bispecific recombinant protein according to any one of claims 1 to 15.

21. 16. Use of a bispecific recombinant protein according to any one of claims 1 to 15 in the manufacture of a medicament for the treatment of tumors, autoimmune diseases, infectious diseases, sepsis, graft-versus-host disease, metabolic disorders, endocrine disorders.

22. The tumor is a solid tumor or a blood tumor, and the solid tumor is any one selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, esophageal cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell carcinoma, dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome, and the blood tumor is selected from myeloma, lymphoma, or leukemia, the autoimmune disease is any one selected from Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome; and / or 22. The use according to claim 21, wherein the infectious disease is any one selected from viral infections, bacterial infections, fungal infections and other pathogenic infections.

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