Bispecific single domain antibody to PD-l1 and CD47 and use thereof

Abstract

To provide an immune checkpoint inhibitor targeting immune checkpoint proteins PD-L1 and CD47.SOLUTION: Provided is a bispecific single domain antibody that bispecifically binds to PD-L1 and CD47, comprising a first VHH domain or an antigen-binding fragment thereof that specifically binds to PD-L1 and a second VHH domain or an antigen-binding fragment thereof that specifically binds to CD47, where the first VHH domain or the antigen-binding fragment thereof includes CDR1, CDR2, and CDR3 each having a specific amino acid sequence, and the second VHH domain or the antigen-binding fragment thereof includes CDR1, CDR2, and CDR3 each having a specific amino acid sequence.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present invention relates to bispecific single domain antibodies against immune checkpoint proteins PD-L1 and CD47, and uses thereof. [Background technology]

[0002] Recently, the therapeutic effectiveness of a newly developed immunotherapy using the human immune system has been proven, and conventional anti-cancer treatments using chemotherapeutic agents and targeted therapeutic agents are being replaced by immunotherapy.

[0003] In cancer patients, immune cells have acquired tolerance to cancer antigens. While they can recognize cancer cells, their function is suppressed and they are unable to effectively eliminate them. The core of immunotherapy is to awaken these immune cells and guide them into activated forms to destroy cancer cells. Examples of immunotherapy include cytokine treatments such as IFN-γ and IL-2, cancer vaccines using dendritic cells, cell therapy using T cells, and immune checkpoint inhibitors (ICIs) that block immune checkpoint proteins. These treatments are commonly referred to as immuno-oncology therapies. Among these, immune checkpoint inhibitors are the most competitively developed immuno-oncology therapies by international pharmaceutical companies.

[0004] Immune checkpoint proteins are cell membrane proteins that suppress the differentiation, proliferation, and activity of immune cells. Specifically, these proteins are typically expressed on activated T cells and suppress T cell proliferation, cytokine secretion, and cytotoxicity, thereby suppressing excessive T cell activity, hence the term "co-inhibitory molecule." T cells typically express the co-inhibitory receptors CTLA-4 and PD-1, which regulate T cell activity through binding with their respective ligands, B7.1 / 2 and PD-L1. On the other hand, PD-L1 is expressed on cancer cells and acts as an important molecular shield, protecting them from T cell immune attack by inactivating cancer-specific T cells and inducing apoptosis, potentially acting as an immune evasion mechanism for cancer. Furthermore, cancer patients with ectopic PD-L1 expression in their cancer cells have been reported to have a worse prognosis than those without PD-L1.

[0005] Immune checkpoint inhibitors are drugs that activate T cells and attack cancer cells by blocking the activity of immune checkpoint proteins involved in T cell inhibition. Antibodies that recognize CTLA-4, PD-1, and PD-L1 are typically used. The CTLA-4 inhibitor ipilimumab (Yervoy) was the first immune checkpoint inhibitor to receive FDA approval as a second-line treatment for metastatic melanoma in 2011. Subsequently, in 2014, the PD-1 blockers nivolumab (Opdivo) and pembrolizumab (Keytruda) were both approved by the FDA for the treatment of metastatic melanoma. Since then, the PD-L1 immune checkpoint inhibitors atezolizumab (Tecentriq) received FDA approval for bladder cancer in 2016, avelumab (Bavencio) for the treatment of metastatic Merkel cell carcinoma (a type of skin cancer), and durvalumab (Imfinzi) for bladder cancer in 2017. In 2018, the PD-1 inhibitor cemiplimab (Libtayo) received FDA approval for cutaneous squamous cell carcinoma. Currently, these agents are expanding their therapeutic indications and receiving FDA approval for the treatment of an increasing number of cancers. As of 2019, six PD-1 / PD-L1 inhibitors have been approved for a total of 18 cancer types. Furthermore, immune regulatory proteins such as B7-H4, ICOS, HVEM, PDL-2, and PVRIG are undergoing preclinical testing as novel targets. Most of these therapeutic agents target targets expressed on T cells.

[0006] To overcome this biased focus on T cells in target discovery, inhibitors targeting immune checkpoint proteins expressed in myeloid lineage cells such as macrophages and dendritic cells have been developed in recent years. Among these, CSF1R, CD47, and TLR7 have emerged as important targets.

[0007] PD-L1 (Programmed Death-Ligand 1), an immune checkpoint protein that inhibits T cell activity and enables tumor cells to evade immune attack, is primarily expressed on leukocytes and non-hematopoietic (Nongematopoietic) cells in lymphoid and non-lymphoid tissues, and is also expressed on the surface of various tumor cells, including those in colorectal cancer, pancreatic cancer, melanoma, and cervical cancer. Specifically, PD-L1 interacts with PD-1 (Programmed Death-1) expressed on the surface of activated T cells, thereby negatively regulating T cell immune responses by inhibiting TCR-mediated T cell activation, cytokine release, and T cell proliferation.

[0008] CD47 (Cluster of Differentiation 47), first identified in 1980 as a tumor antigen in human ovarian cancer, is expressed on many human tumor cells, including non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia, bladder cancer, and solid tumors.

[0009] CD47 is expressed on the cell surface and interacts with SIRP alpha (SIRPα), thrombospondin-1 (TSP1), and integrin proteins, and is involved in cell death, proliferation, and immune responses. In particular, CD47 expressed on tumor cells interacts with SIRP alpha (SIRPα) expressed on the surface of macrophages to send a "don't eat me" signal, thereby avoiding macrophage phagocytosis and inhibiting vascular proliferation and the role of effector T cells in the tumor environment, promoting the proliferation and growth of tumor cells.

[0010] Magrolimab, an antibody therapeutic targeting CD47 developed by the global pharmaceutical company Gilead, is currently undergoing clinical trials, and recently, multinational global pharmaceutical companies have been actively developing dual antibodies based on CD47 antibody therapeutics.

[0011] Therefore, the present inventors developed an immune checkpoint inhibitor that targets the immune checkpoint proteins PD-L1 and CD47, which led to the present application. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent Application Publication No. 2020-0181259 [Patent Document 2] International Publication No. 2011-143624 [Patent Document 3] U.S. Patent No. 8,008,449 [Patent Document 4] International Publication No. 94 / 04678 [Patent Document 5] US Patent Application Publication No. 2009 / 0307787 [Patent Document 6] U.S. Patent No. 8,754,287 [Patent Document 7] US Patent Application Publication No. 2015 / 0289489 [Patent Document 8] US Patent Application Publication No. 2010 / 0122358 [Patent Document 9] International Publication No. 2004 / 049794 [Patent Document 10] International Publication No. 99 / 37681 [Patent Document 11] International Publication No. 01 / 90190 [Patent Document 12] International Publication No. 03 / 025020 [Patent Document 13] International Publication No. 03 / 035694 [Patent Document 14] International Publication No. 00 / 43507 [Patent Document 15] International Publication No. 06 / 030220 [Patent Document 16] International Publication No. 06 / 003388 [Patent Document 17] U.S. Patent No. 4,816,567 [Patent Document 18] International Publication No. 1996 / 34103

Non-licensed literature

[0013]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

[0014] The present invention aims to provide bispecific single domain antibodies against immune checkpoint proteins PD-L1 and CD47, and uses thereof. [Means for solving the problem]

[0015] To achieve the objectives of the present invention, the present invention provides a bispecific antibody that dually binds to PD-L1 and CD47, comprising a first single domain antibody (first sdAb) or antigen-binding fragment thereof that specifically binds to PD-L1, and a second single domain antibody (second sdAb) or antigen-binding fragment thereof that specifically binds to CD47.

[0016] In one embodiment of the present invention, the first sdAb or antigen-binding fragment thereof may comprise a CDR1 consisting of the amino acid sequence represented by SEQ ID NO:2, a CDR2 consisting of the amino acid sequence represented by SEQ ID NO:3, and a CDR3 consisting of the amino acid sequence represented by SEQ ID NO:4.

[0017] The second sdAb or antigen-binding fragment thereof may also comprise a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:7, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:8, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:9.

[0018] Furthermore, the first sdAb or antigen-binding fragment thereof may comprise a VHH domain comprising FR1 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 13 or 17, FR2 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 14 or 18, FR3 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 15 or 19, and FR4 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 16 or 20; more specifically, it may comprise a VHH domain comprising (1) FR1 consisting of the amino acid sequence set forth in SEQ ID NO: 13, FR2 consisting of the amino acid sequence set forth in SEQ ID NO: 14, FR3 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO: 16, or (2) FR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, FR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, FR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO: 20; even more specifically, it may comprise a VHH domain comprising FR1 consisting of the amino acid sequence set forth in SEQ ID NO: 13, FR2 consisting of the amino acid sequence set forth in SEQ ID NO: 14, FR3 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO: 16. In some implementations, the first sdAb has the amino acid sequence set forth in SEQ ID NO:1.

[0019] The first sdAbs or antigen-binding fragments thereof may also be monovalent, bivalent, trivalent, tetravalent, or higher valent, and may be fused to one another via a peptide linker between the first sdAbs or antigen-binding fragments thereof, which in some embodiments has the amino acid sequence set forth in SEQ ID NO:21.

[0020] Furthermore, the second sdAb or antigen-binding fragment thereof may comprise a VHH domain comprising FR1 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 13 or 17, FR2 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 14 or 18, FR3 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 15 or 19, and FR4 consisting of the amino acid sequence set forth in either of SEQ ID NOs: 16 or 20; more specifically, it may comprise a VHH domain comprising (1) FR1 consisting of the amino acid sequence set forth in SEQ ID NO: 13, FR2 consisting of the amino acid sequence set forth in SEQ ID NO: 14, FR3 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO: 16, or (2) FR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, FR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, FR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO: 20; even more specifically, it may comprise a VHH domain comprising FR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, FR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, FR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO: 20. In some implementations, the second sdAb has the amino acid sequence set forth in SEQ ID NO:6.

[0021] The second sdAb or antigen-binding fragment thereof may be monovalent, bivalent, trivalent, tetravalent or of higher valency, and may be fused to each other via a peptide linker between the second sdAbs or antigen-binding fragments thereof.

[0022] In one aspect of the invention, the first sdAb or antigen-binding fragment thereof and / or the second sdAb or antigen-binding fragment thereof may be fused to each other via a peptide linker, which in some implementations has the amino acid sequence set forth in SEQ ID NO:11.

[0023] In one aspect of the invention, the first and / or second sdAb comprise at least one amino acid substitution, which may be a conservative substitution or a substitution of an amino acid with a non-genetically encoded amino acid or a synthetic amino acid.

[0024] In one aspect of the present invention, there is provided a heavy chain only antibody (HCAb) in which an Fc fragment is fused to said first sdAb or antigen binding fragment thereof, or a second sdAb or antigen binding fragment thereof.

[0025] In one embodiment of the present invention, the HCAb may be bispecific and multivalent (e.g., bivalent, trivalent, tetravalent, or higher) and Fc fragment-fused, comprising at least two copies of the first sdAb or antigen-binding fragment thereof and / or the second sdAb or antigen-binding fragment thereof. In some implementations, the HCAb may consist of the amino acid sequence set forth in SEQ ID NO: 12.

[0026] In one embodiment of the present invention, an sdAb may be fused to the Fc fragment via a peptide linker, and the Fc fragment may be human IgG1, IgG2, IgG3 or IgG4.

[0027] In one embodiment of the invention, the HCAb comprises at least one amino acid substitution, which may be a conservative substitution or a substitution of an amino acid with a non-genetically encoded amino acid or a synthetic amino acid.

[0028] In one embodiment of the present invention, an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug may be conjugated. Thus, the present invention provides an antibody conjugate comprising the bispecific antibody that bispecifically binds to PD-L1 and CD47, conjugated to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug.

[0029] The present invention also provides nucleic acid molecules encoding the bispecific antibodies that dually bind to PD-L1 and CD47.

[0030] Furthermore, the present invention provides an expression vector comprising the nucleic acid molecule.

[0031] The present invention also provides a host cell transformed with the expression vector.

[0032] Furthermore, the present invention provides (a) culturing the host cells under conditions that allow expression of the bispecific antibody; (b) recovering the expressed bispecific antibody; The present invention provides a method for producing a bispecific antibody that bispecifically binds to PD-L1 and CD47, comprising:

[0033] The present invention also provides pharmaceutical compositions for the prevention or treatment of cancer, which contain the bispecific antibody that bispecifically binds to PD-L1 and CD47 or the antibody conjugate as an active ingredient; methods for the prevention or treatment of cancer, which comprise the step of administering to an individual a pharmaceutically effective amount of the bispecific antibody that bispecifically binds to PD-L1 and CD47 or the antibody conjugate; and uses of the bispecific antibody that bispecifically binds to PD-L1 and CD47 or the antibody conjugate for the prevention or treatment of cancer.

[0034] In one form of the invention, the cancer may be selected from the group consisting of melanoma, lung cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, solitary myeloma, and aplastic anemia.

[0035] In one embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. [Effects of the Invention]

[0036] In the present invention, a single domain antibody that bispecifically binds to the immune checkpoint proteins PD-L1 and CD47 has been prepared, and its affinity for immune antigens and antitumor effect have been confirmed. Therefore, the bispecific single domain antibody can be useful as an immune checkpoint inhibitor in immunotherapy. [Brief explanation of the drawings]

[0037] [Figure 1A] Figure 2 shows the binding ability (EC50) of the CHO-K1_PD-L1 cell line (CHO-K1 cells induced to express PD-L1 antigen) prepared according to one example of the present invention to the PD-L1 antigen expressed on the cell surface when reacted with various concentrations of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1). [Figure 1B] Figure 1 shows the inhibitory effect (IC50) of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) on PD-L1 / PD-1 interaction after binding to a CHO-K1_PD-L1 cell line (CHO-K1 cells induced to express PD-L1 antigen) prepared according to one example of the present invention and PD-1 protein. [Figure 2A] Figure 2 shows the binding ability (EC50) of the CHO-K1_PD-L1 cell line (CHO-K1 cells induced to express PD-L1 antigen) prepared according to one example of the present invention to the PD-L1 antigen expressed on the cell surface when reacted with various concentrations of anti-PD-L1 bivalent HCAb (PP Nb-IgG4). [Figure 2B] Figure 1 shows the inhibitory effect (IC50) of anti-PD-L1 bivalent HCAb (PP Nb-IgG4) on PD-L1 / PD-1 interaction after binding to the CHO-K1_PD-L1 cell line (CHO-K1 cells induced to express PD-L1 antigen) prepared according to one example of the present invention and PD-1 protein. [Figure 3A]FIG. 1 shows the binding ability (EC50) of an Expi-CHO_CD47 cell line (Expi-CHO cells in which expression of the CD47 antigen has been induced) prepared according to one example of the present invention to various concentrations of anti-CD47 HCAb (CD47 Nb-IgG4) to the CD47 antigen expressed on the cell surface. [Figure 3B] This figure confirms the inhibitory potency (IC50) of anti-CD47 HCAb (CD47 Nb-IgG4) on the CD47 / SIRPalpha interaction after binding of the Expi-CHO_CD47 cell line (Expi-CHO cells induced to express CD47 antigen) prepared according to one embodiment of the present invention with SIRPalpha protein. [Figure 4A] Figure 1 shows the binding ability (EC50) of a CHO-K1_PD-L1 cell line (CHO-K1 cells induced to express PD-L1 antigen) prepared according to one example of the present invention to PD-L1 antigen expressed on the cell surface when reacted with various concentrations of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4). [Figure 4B] Figure 1 shows the binding ability (EC50) of the Expi-CHO_CD47 cell line (Expi-CHO cells induced to express CD47 antigen) prepared according to one example of the present invention to the CD47 antigen expressed on the cell surface when reacted with various concentrations of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4). [Figure 5A] Figure 1 shows the inhibitory effect (IC50) of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) on PD-L1 / PD-1 interaction after binding to a CHO-K1_PD-L1 cell line (CHO-K1 cells induced to express PD-L1 antigen) prepared according to one example of the present invention and PD-1 protein. [Figure 5B] Figure 1 shows the inhibitory effect (IC50) of anti-PD-L1 x CD47 HCAb (PPC Nb-IgG4) on CD47 / SIRPalpha interaction after binding of SIRPalpha protein to an Expi-CHO_CD47 cell line (Expi-CHO cells induced to express CD47 antigen) prepared according to one embodiment of the present invention. [Figure 6]Figure 1 shows the ability of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) prepared according to one example of the present invention to inhibit the PD-L1 / PD-1 interaction, using CHO-L1_PD-L1 cells (CHO-K1 cells induced to overexpress PD-L1 protein) and PD-1-expressing Jurkat cells. [Figure 7] Figure 1 shows the phagocytosis of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) prepared according to one embodiment of the present invention. [Figure 8] Figure 1 shows the human RBC binding ability of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) prepared in accordance with one embodiment of the present invention. [Figure 9] Figure 1 shows the hemagglutination activity of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) prepared according to one embodiment of the present invention. [Figure 10] Figure 1 shows the antitumor effect of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) prepared according to one example of the present invention, after intraperitoneal administration to C57BL / 6 mice in which tumor formation was induced with the B16F10_PD-L1 / CD47 cell line (a tumor cell line induced to express PD-L1 and CD47 antigens). [Figure 11] Figure 1 shows the antitumor effect of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) prepared according to one example of the present invention, after intravenous administration to C57BL / 6 mice in which tumor formation was induced with the B16F10_PD-L1 / CD47 cell line (a tumor cell line induced to express PD-L1 and CD47 antigens). DETAILED DESCRIPTION OF THE INVENTION

[0038] Hereinafter, the present invention will be described in detail by way of examples so that those skilled in the art can easily carry out the present invention. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art. Therefore, the examples of the present invention may be modified into various other forms, and the present invention is not limited to these examples.

[0039] As used herein, the term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes generally consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0040] The term "treatment" refers to any course of a disorder or disease disclosed herein, e.g., slowing, interrupting, ceasing, controlling, halting, alleviating, or ameliorating the symptoms or complications of the disease, or may even reverse its progression, but does not necessarily indicate the complete elimination of all disease or disorder symptoms.

[0041] The term "prevention" refers to a disease or disorder, e.g., prophylactic treatment of a disease or delaying the onset or progression of a disease or disorder.

[0042] The term "individual" or "subject" refers to a mammal, including, but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some implementations, the individual is a human.

[0043] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody" includes traditional four-chain antibodies, single-domain antibodies, and antigen-binding fragments thereof.

[0044] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five of these basic heterotetrameric units, along with an additional polypeptide called the J chain, and contain ten antigen-binding sites, whereas IgA antibodies contain two to five of these basic four-chain units that can polymerize to form multivalent assemblies with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, whereas the two H chains are linked to each other by at least one disulfide bond, depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. At the N-terminus, each H chain contains a variable domain (VH) followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its other end followed by a constant domain at its N-terminus. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. The combination of VH and VL together forms a single antigen-binding site. L chains in any vertebrate species are assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins are assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a designated heavy chain, α, δ, ε, γ, and μ. The γ and α classes are further divided into subclasses based on relatively few differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0045] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody that includes a heavy chain but lacks the light chain typically found in four-chain antibodies.

[0046] The terms "single domain antibody," "nanobody," or "sdAb" refer to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). An sdAb alone can bind to an antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered herein from camelid HCAbs, and their heavy chain variable domains are referred to as "VHHs" (variable domain of the heavy chain of a heavy chain antibody). A basic VHH has the following structure from N- to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0047] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy and light chain variable domains are sometimes referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site. Heavy-chain-only antibodies from Camelidae species have a single heavy-chain variable region, designated "VHH."

[0048] The term "variable" refers to the fact that certain fragments of the variable domains vary extensively within antibody sequences. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Instead, in both the heavy and light chain variable domains, it is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portions of the variable domains are called framework regions (FRs). Each naturally occurring heavy and light chain variable domain has four FR regions that form loops connected by the three CDRs and adopt a predominantly beta-sheet configuration, and in some cases form part of the beta-sheet structure. In each chain, the CDRs are held together in close proximity by the FR regions, and CDRs from other chains contribute to the formation of the antigen-binding site of antibodies (see Non-Patent Document 4). The constant domains are not directly involved in binding the antibody to the antigen but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity. The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. Constant domains include the CH1, CH2, and CH3 domains (collectively CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0049] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially complete form, as opposed to an antibody fragment. Specifically, full-length four-chain antibodies include those having a heavy chain and a light chain, including an Fc region. Full-length heavy-chain-only antibodies comprise a heavy chain variable domain (e.g., VHH) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Optionally, an intact antibody may have at least one effector function.

[0050] An "antibody fragment" or "antigen-binding fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody (scFv) molecules; single-domain antibodies (e.g., VHH), and multispecific antibodies formed from antibody fragments. An "Fv" is the minimum antibody fragment that contains an intact antigen recognition and binding site. The fragment consists of a dimer of one heavy-chain and one light-chain variable region domain in tight, non-covalent association. A "single-chain Fv," also abbreviated "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. "Diabodies" refer to small antibody fragments produced by constructing sFv fragments with a short linker (about 5-10 residues) between the VH and VL domains such that inter-chain, rather than intra-chain, pairing of the V domains is achieved, thereby resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0051] The term "humanized antibody" is used as a subset of "chimeric antibody."

[0052] "Humanized" forms of non-human (e.g., llama or camelid) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some implementations, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient CDRs (defined below) are replaced by residues from a CDR of a non-human species (donor antibody) having the desired specificity, affinity, and / or acceptor, e.g., mouse, rat, rabbit, camel, llama, alpaca, or non-human primate.

[0053] In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody performance, e.g., binding affinity.

[0054] The terms "hypervariable region," "HVR," or "HV," as used herein, refer to regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the highest degree of diversity among the three HVRs and is known to play a unique role in conferring microspecificity to antibodies. For example, see Non-Patent Documents 5 and 6.

[0055] The term "complementarity determining region" or "CDR" is used to refer to hypervariable regions as defined by the Kabat system. See Non-Patent Document 7. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used.

[0056] The term "framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0057] The term "specific" refers to the selective recognition of an antigen binding protein (e.g., an sdAb) for a particular epitope of an antigen.

[0058] Natural antibodies, for example, are monospecific. As used herein, the term "multispecific" indicates that an antigen-binding protein has polyepitopic specificity (i.e., can specifically bind to two, three, or more different epitopes on one biological molecule, or can specifically bind to epitopes on two, three, or more different biological molecules). As used herein, "bispecific" refers to an antigen-binding protein having two different antigen-binding specificities.

[0059] As used herein, the term "monospecific" refers to an antigen-binding protein having at least one respective binding site that binds the same epitope of the same antigen.

[0060] The term "valent" refers to the presence of a specific number of binding sites in an antigen-binding protein. For example, the terms "bivalent," "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two binding sites, three binding sites, four binding sites, five binding sites, and six binding sites in an antigen-binding protein.

[0061] "Antibody effector functions" refer to those biological activities attributable to the Fc region of an antibody (a native-sequence Fc region or an amino acid sequence variant Fc region) and vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind to their cognate antigen. "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins.

[0062] As used herein, the terms "Fc region" or "fragment crystallizable region" are used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Native-sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0063] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. Binding affinity is measured by the K d , K. off , K. on , or K a As used herein, the equilibrium dissociation constant "K D " or "K dThe term "dissociation constant" refers to the dissociation constant of a particular antibody-antigen interaction and describes the concentration of antigen required to occupy one-half of all antibody binding domains present in a solution of antibody molecules at equilibrium, and is expressed in units of M. D Measurement of the dissociation constant (K) assumes that all of the binding agent is in solution. D or K d ) is used as an index of the affinity of an antibody for an antigen. For example, a simple analysis can be performed by the Scatchard method, which uses antibodies marked with various marker preparations, or by using general pharmaceuticals and measurement kits according to the instructions and experimental procedures that come with the kit. The K that can be derived by these methods D The value is expressed in units of M (Mols).

[0064] "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity is achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNATAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be compared.

[0065] The present invention provides bispecific antibodies that bispecifically bind to PD-L1 and CD47, comprising a first single domain antibody (first sdAb) or antigen-binding fragment thereof (anti-PD-L1 sdAb) that specifically binds to PD-L1, and a second single domain antibody (second sdAb) or antigen-binding fragment thereof (anti-CD47 sdAb) that specifically binds to CD47 (hereinafter referred to as "anti-PD-L1xCD47 bsAb"), such as an anti-PD-L1xCD47 bsAb, specifically a bispecific sdAb comprising an anti-PD-L1 sdAb and an anti-CD47 sdAb fused together; an anti-PD-L1xCD47 heavy chain-only antibody (HCAb) (e.g., an anti-PD-L1xCD47 bsAb comprising a crystallizable fragment (Fc fragment) of human immunoglobulin G (IgG) fused to an anti-PD-L1 sdAb and / or an anti-CD47 sdAb); bsAb-Fc fusion protein), as well as its production and use.

[0066] Thus, the present invention provides bispecific antibodies that dually bind to PD-L1 and CD47, including anti-PD-L1xCD47 bsAb.

[0067] In the present invention, bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb, may be anti-PD-L1×CD47 bsAbs comprising a first single domain antibody (first sdAb) or an antigen-binding fragment thereof (anti-PD-L1 sdAb) that specifically binds to PD-L1 using a first antigen-binding portion, fused with a second single domain antibody (second sdAb) or an antigen-binding fragment thereof (anti-CD47 sdAb) that specifically binds to CD47 using a second antigen-binding portion.

[0068] In the present invention, the anti-PD-L1 sdAb comprises a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:2, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:4.

[0069] The anti-CD47 sdAb also comprises CDR1 consisting of the amino acid sequence shown in SEQ ID NO:7, CDR2 consisting of the amino acid sequence shown in SEQ ID NO:8, and CDR3 consisting of the amino acid sequence shown in SEQ ID NO:9.

[0070] The CDR sequences are shown in Tables 6 and 12.

[0071] In the present invention, the anti-PD-L1×CD47 bsAb may comprise any suitable sequence for the FR region. Specifically, the FR sequence may be any of the amino acid sequences shown in Tables 1 to 4 below.

[0072] [Table 1]

[0073] [Table 2]

[0074] [Table 3]

[0075] [Table 4]

[0076] More specifically, the anti-PD-L1 sdAb may comprise the following FR1, FR2, FR3 and FR4: FR1 consisting of the amino acid sequence represented by any one of SEQ ID NOs: 13 and 17; FR2 consisting of the amino acid sequence represented by any one of SEQ ID NOs: 14 and 18; FR3 consisting of the amino acid sequence represented by any one of SEQ ID NOs: 15 and 19; and FR4 consisting of the amino acid sequence represented by either SEQ ID NO: 16 or 20.

[0077] More specifically, the anti-PD-L1 sdAb may comprise the following FR1, FR2, FR3 and FR4: (1) FR1 consisting of the amino acid sequence represented by SEQ ID NO: 13; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 14; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 15; and FR4 consisting of the amino acid sequence represented by SEQ ID NO: 16 or (2) FR1 consisting of the amino acid sequence represented by SEQ ID NO: 17; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 18; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 19; and FR4 consisting of the amino acid sequence shown in SEQ ID NO:20.

[0078] More specifically, the anti-PD-L1 sdAb may comprise the following FR1, FR2, FR3 and FR4: FR1 consisting of the amino acid sequence represented by SEQ ID NO: 13; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 14; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 15; and FR4 consisting of the amino acid sequence shown in SEQ ID NO:16.

[0079] In the present invention, the anti-PD-L1 sdAb may comprise a VHH domain comprising the FR region.

[0080] Specifically, the anti-PD-L1 sdAb may comprise the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof having at least 80% sequence identity to the amino acid sequence (e.g., at least any of 80%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0081] The anti-CD47 sdAb may also comprise the following FR1, FR2, FR3 and FR4: FR1 consisting of the amino acid sequence represented by any one of SEQ ID NOs: 13 and 17; FR2 consisting of the amino acid sequence represented by any one of SEQ ID NOs: 14 and 18; FR3 consisting of the amino acid sequence represented by any one of SEQ ID NOs: 15 and 19; and FR4 consisting of the amino acid sequence represented by either SEQ ID NO: 16 or 20.

[0082] More specifically, the anti-CD47 sdAb may comprise the following FR1, FR2, FR3 and FR4: (1) FR1 consisting of the amino acid sequence represented by SEQ ID NO: 13; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 14; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 15; and FR4 consisting of the amino acid sequence represented by SEQ ID NO: 16 or (2) FR1 consisting of the amino acid sequence represented by SEQ ID NO: 17; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 18; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 19; and FR4 consisting of the amino acid sequence shown in SEQ ID NO:20.

[0083] More specifically, the anti-CD47 sdAb may comprise the following FR1, FR2, FR3 and FR4: FR1 consisting of the amino acid sequence represented by SEQ ID NO: 17; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 18; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 19; and FR4 consisting of the amino acid sequence shown in SEQ ID NO:20.

[0084] In the present invention, the anti-CD47 sdAb may comprise a VHH domain comprising the FR region.

[0085] Specifically, the anti-CD47 sdAb may comprise the amino acid sequence set forth in SEQ ID NO: 6, or a variant thereof having at least 80% (e.g., at least any of 80%, 58%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence.

[0086] In the present invention, the anti-PD-L1 sdAb binds to an epitope on PD-L1, and the anti-CD47 sdAb binds to an epitope on CD47.

[0087] Furthermore, the K values ​​of the binding of the anti-PD-L1×CD47 bsAb to PD-L1 and CD47 were D is 10 -6 M~10 -12 M, 10 -6 M~10 -11 M, 10 -6 M~10 -10 M, 10 -6 M~10 -9 M, or 10 -6 M~10 -8 It may also be M.

[0088] Furthermore, the EC 50 may be less than 500 nM in FACS analysis, specifically 0.1 nM to 500 nM, 0.1 nM to 400 nM, 0.1 nM to 300 nM, 0.1 nM to 0.00 nM, 0.1 to 50 nM, 0.1 to 10 nM, 1 nM to 500 nM, 1 nM to 400 nM, 1 nM to 300 nM, 1 nM to 200 nM, 1 nM to 100 nM, 1 nM to 50 nM, or 1 nM to 10 nM.

[0089] In the present invention, the anti-PD-L1×CD47 bsAb may have any suitable number of mantissas for each of the PD-L1 and CD47 epitopes. Specifically, the anti-PD-L1×CD47 bsAb may be bivalent, trivalent, tetravalent, pentavalent, hexavalent, or higher valent for each of PD-L1 and CD47. See, for example, Non-Patent Document 8.

[0090] Furthermore, the anti-PD-L1×CD47 bsAb may comprise an anti-PD-L1 sdAb and an anti-CD47 sdAb fused directly via a peptide bond or indirectly via a peptide linker. The length, degree of flexibility, and / or other properties of the peptide linker may have several effects on properties including, but not limited to, affinity, specificity, or binding capacity for at least one particular antigen or epitope. For example, a longer peptide linker may be selected to ensure that two adjacent domains do not sterically interfere with each other. In some implementations, the peptide linker contains flexible residues (e.g., glycine and serine) to allow the adjacent domains to move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker. The peptide linker may be of any suitable length. In some implementations, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length.

[0091] Furthermore, the peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence.

[0092] In some embodiments, the anti-PD-L1xCD47 bsAb may be bivalent to PD-L1, and the bivalent anti-PD-L1 sdAb has the amino acid sequence set forth in SEQ ID NO: 21. Alternatively, the anti-PD-L1xCD47 bsAb has the amino acid sequence set forth in SEQ ID NO: 11.

[0093] Single domain antibodies (sdAbs) of the present invention may include, but are not limited to, heavy chain variable domains from heavy chain-only antibodies (e.g., VHH (heavy chain variable domain of a heavy chain antibody) in Camelidae), light chains derived from traditional four-chain antibodies, binding molecules that naturally lack a single domain (e.g., VH or VL), humanized heavy chain-only antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and engineered domains and single domain scaffolds other than those derived from antibodies. sdAbs may be derived from any species, including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. They may also include naturally occurring sdAb molecules from species other than Camelidae.

[0094] Furthermore, sdAbs are derived from naturally occurring single-domain antigen-binding molecules known as heavy-chain antibodies lacking light chains. Such single-domain molecules are disclosed, for example, in Patent Document 4 and Non-Patent Document 5. Variable domains derived from heavy-chain molecules naturally lacking light chains are known herein as VHHs to distinguish them from conventional VHs of four-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced by Camelidae species, such as camel, llama, vicuña, dromedary, alpaca, and guanaco. Other species outside of Camelidae can naturally produce heavy-chain molecules lacking light chains, and such VHHs are within the scope of this specification.

[0095] sdAbs may also be recombinant, CDR-grafted, humanized, camelized, deimmunized and / or generated in vitro (e.g., selected by phage display). In some implementations, the amino acid sequence of the framework regions may be altered by "camelization" of specific amino acid residues within the framework regions. Camelization refers to the substitution or replacement of at least one amino acid residue in the amino acid sequence of a (naturally occurring) VH domain from a conventional four-chain antibody with at least one of the amino acid residues occurring at the corresponding position in the VHH domain of a heavy-chain antibody, and can be performed by methods known in the art.

[0096] Furthermore, the sdAb may be a human sdAb produced by transgenic mice or rats expressing a human heavy chain fragment (see, for example, Patent Documents 5 to 9).

[0097] Furthermore, naturally occurring VHH domains against a particular antigen or target may be obtained from a library (naive or immune) of Camelidae VHH sequences. Such methods may or may not involve screening such libraries using said antigen or target, or at least parts, fragments, antigenic determinants or epitopes thereof, using at least one screening technique known per se. Such libraries and techniques are disclosed, for example, in Patent Documents 10 to 13. Alternatively, improved synthetic or semi-synthetic libraries derived from (naive or immune) VHH libraries may be used, e.g., VHH libraries derived from (naive or immune) VHH libraries by techniques such as random mutagenesis and / or CDR shuffling, as described in Patent Document 14.

[0098] Alternatively, sdAbs may be generated from conventional four-chain antibodies (see, for example, Non-Patent Documents 9 and 10 and Patent Documents 15 and 16).

[0099] Furthermore, the sdAb of the present invention may be a chimeric antibody. Specific chimeric antibodies are disclosed, for example, in Patent Document 17 and Non-Patent Document 11. In some implementations, a chimeric antibody may comprise a non-human variable region (e.g., a variable region derived from a camelid species, such as a llama) and a human constant region. Alternatively, a chimeric antibody may be humanized. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises at least one variable domain in which the HVR, e.g., CDR, (or portions thereof) are derived from a non-human antibody and the FR (or portions thereof) are derived from a human antibody sequence. In some cases, a humanized antibody will also comprise at least a portion of a human constant region. In some implementations, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0100] In the present invention, the bispecific antibody that bispecifically binds to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb, may be an anti-PD-L1×CD47 HCAb or an antigen-binding fragment thereof.

[0101] Specifically, an anti-PD-L1xCD47 HCAb may comprise an anti-PD-L1xCD47 bsAb described herein fused to at least one CH2 and / or CH3 domain, e.g., an Fc fragment, and may further comprise an anti-PD-L1 sdAb and / or anti-CD47 sdAb described herein fused to at least one CH2 and / or CH3 domain, e.g., an Fc fragment.

[0102] The CH2 and / or CH3 domains are derived from an immunoglobulin. The immunoglobulin may be IgA, IgD, IgE, IgG, or IgM, and may specifically be IgG. In some implementations, the anti-CD47 HCAb may comprise an Fc fragment of an IgG, e.g., IgG1, IgG2, IgG3, or IgG4, and the Fc fragment may be a human Fc, e.g., a human IgG1 (hIgG1) Fc, hIgG2 Fc, hIgG3 Fc, or hIgG4 Fc.

[0103] The anti-PD-L1 x CD47 HCAbs may be monomeric or multimeric, and if multimeric, may be bispecific and multivalent (e.g., bivalent, trivalent, tetravalent or higher valency), comprising, for example, at least two copies of an anti-PD-L1 sdAb and an anti-CD47 sdAb described herein.

[0104] In the present invention, the CH2 and / or CH3 domains of the anti-PD-L1×CD47 bsAb, anti-PD-L1 sdAb, or anti-CD47 sdAb may be fused to the Fc fragment via a peptide linker. The length, degree of flexibility, and / or other properties of the peptide linker may have some influence on properties including, but not limited to, affinity, specificity, or binding capacity for at least one particular antigen or epitope. For example, a longer peptide linker may be selected to ensure that two adjacent domains do not sterically interfere with each other. In some implementations, the peptide linker contains flexible residues (e.g., glycine and serine) to allow the adjacent domains to move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker. The peptide linker may be of any suitable length. In some implementations, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length.

[0105] Furthermore, the peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, Patent Document 18. In some implementations, the peptide linker may be a hIgG1 hinge, a hIgG2 hinge, a hIgG3 hinge, a hIgG4 hinge, or a variant thereof.

[0106] In the present invention, the anti-PD-L1×CD47 HCAb may comprise the amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 80% (e.g., at least any of 80%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to said amino acid sequence.

[0107] In the present invention, bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb, may be bispecific Fab-like antibody fragments (bsFab) in which the C-terminus of the anti-PD-L1 sdAb and / or anti-CD47 sdAb is fused to the N-terminus of the CH1 and Cκ domains.

[0108] The CH1 and Cκ domains are derived from immunoglobulin. The immunoglobulin may be IgA, IgD, IgE, IgG, or IgM, and more specifically, IgG. The IgG may be IgG1, IgG2, IgG3, or IgG4, and may be human IgG1, IgG2, IgG3, or IgG4.

[0109] For the bsFab and its manufacturing technology according to the present invention, see Non-Patent Document 7.

[0110] In the present invention, bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb, include amino acid sequence variants. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the antibody amino acid sequence. Any combination of deletion, insertion, and substitution can be made to arrive at a final construct, provided that the final construct retains the desired characteristics, e.g., antigen binding. In some implementations, substitutions, insertions, or deletions may be made within at least one hypervariable region (HVR), so long as such changes do not substantially reduce the antibody's ability to bind antigen. For example, conservative changes that do not substantially reduce binding affinity may be made in HVRs. Such changes may be outside of HVR "hotspots" or CDRs.

[0111] The amino acid substitution may also be at least one (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitution. Furthermore, the at least one amino acid substitution may be a conservative substitution or a substitution with a non-genetically encoded amino acid or a synthetic amino acid. In some implementations, the amino acid substitution may be in the CDR region and may include at least one (e.g., any 1, 2, 3, or 4) amino acid substitution in CDR1, CDR2, and / or CDR3. In some implementations, the amino acid substitution may be in the FR region and may include at least one (e.g., any 1, 2, 3, 4, 5, or 6) amino acid substitution in FR1, FR2, FR3, and / or FR4.

[0112] Such amino acid sequence insertions also include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule may include the fusion to the N- or C-terminus of an antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody (e.g., in the case of ADEPT).

[0113] Furthermore, at least one amino acid modification may be introduced into the Fc region of bispecific antibodies that bispecifically bind to PD-L1 and CD47 (e.g., anti-PD-L1xCD47 HCAbs), including the anti-PD-L1xCD47 bsAbs provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc) that comprises an amino acid modification (e.g., a substitution) at at least one amino acid position.

[0114] In the present invention, bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb, may be linked to, fused to, bound (e.g., covalently or non-covalently) or otherwise associated with a diagnostic moiety or biocompatibility modifier, such as a peptide or polypeptide (e.g., a biotoxin, biomarker, purification tag, etc.), a protein, a polymer, a nucleic acid molecule, a small molecule, a mimetic, a synthetic drug, an inorganic molecule, an organic molecule, or a radioisotope.

[0115] Bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1xCD47 bsAb, may also be conjugated or associated with a diagnostic or detectable agent, marker, or reporter, which may be a biological molecule (e.g., a peptide or nucleotide), a small molecule, a fluorophore, or a radioisotope. Labeled modulators may be useful as part of a clinical trial procedure to monitor the development or progression of a PD-L1- and / or CD47-related disease, e.g., cancer, to determine the efficacy of a particular therapy involving the antibodies disclosed herein (i.e., theragnosis), or to determine future courses of treatment. Such markers or reporters are also useful for purifying the antibodies disclosed herein.

[0116] Furthermore, bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb, may be conjugated to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug. Thus, the present invention provides antibody conjugates comprising a bispecific antibody that bispecifically binds to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb of the present invention, conjugated to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug.

[0117] The present invention also provides nucleic acid molecules encoding the bispecific antibodies that dually bind to PD-L1 and CD47, including the anti-PD-L1xCD47 bsAb disclosed herein, expression vectors containing the nucleic acid molecules, and host cells transformed with the expression vectors.

[0118] Furthermore, the present invention provides a method for producing a bispecific antibody, comprising the steps of: (a) culturing host cells under conditions that allow expression of the bispecific antibody; and (b) recovering the expressed bispecific antibody.

[0119] In the present invention, DNA encoding bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb disclosed herein, can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains). Isolated and subcloned hybridoma cells (or phage- or yeast-derived colonies) serve as a preferred source of such DNA. In particular, the isolated DNA, which may be modified, may also be used to clone constant and variable region sequences for antibody preparation.

[0120] One exemplary method involves extraction of RNA from selected cells, conversion to cDNA, and amplification by PCR using antibody-specific primers. Suitable primers are well known in the relevant art and are readily available from many commercial sources, as exemplified herein. To express recombinant human or non-human antibodies isolated by screening of combinatorial libraries, DNA encoding the antibody is cloned into a recombinant expression vector and introduced into host cells, including mammalian cells, insect cells, plant cells, yeast, and bacteria. In some implementations, the modulator is introduced into and expressed by monkey COS cells, NSO cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce the desired construct.

[0121] In the present invention, the nucleic acid molecule is present in a vector, where appropriate, together with a promoter that controls the expression of the nucleic acid. The term vector is used in its most general sense and includes any intermediate vehicle for a nucleic acid that allows the nucleic acid to be introduced into, for example, a prokaryotic and / or eukaryotic cell and, where appropriate, integrated into the genome. Such vectors are preferably replicated and / or expressed intracellularly. Vectors may include plasmids, phagemids, bacteriophages, or viral genomes. The plasmid generally refers to an extrachromosomal genetic material construct, typically a circular DNA duplex, that can replicate independently of chromosomal DNA.

[0122] Those skilled in the relevant art can construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals using well-known methods, including, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0123] In the present invention, the term "host cell" or "recombinant host cell" refers to a cell into which an expression vector has been introduced. Recombinant host cell and host cell refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not be substantially identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such cells may contain the vector described above.

[0124] Additionally, substantial quantities of the antibodies disclosed herein may be produced using art-recognized molecular biology techniques and current protein expression methodologies. More particularly, nucleic acid molecules encoding such antibodies can be incorporated into well-known and commercially available protein production systems, including various types of host cells, to provide preclinical, clinical, or commercial quantities of the desired pharmaceutical product. In some implementations, nucleic acid molecules encoding the antibodies are engineered into vectors or expression vectors that provide efficient integration into the selected host cell and subsequent high expression levels of the antibody.

[0125] Preferably, nucleic acid molecules encoding the antibodies disclosed herein and vectors containing these nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells, although prokaryotic systems can also be used. Transfection can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules can be introduced into mammalian cells via viral vectors. Methods for transforming mammalian cells are well known in the art. Methods for transforming plant cells are also well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0126] Many different commercially available host-expression vector systems may be used to express the antibodies disclosed herein. Such host-expression systems not only represent vehicles in which a coding sequence of interest may be expressed and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the molecules of the invention in situ. Such systems include microorganisms, such as bacteria (e.g., E. coli, B. subtilis, Streptomyces), transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the modulator coding sequence; yeast (e.g., Saccharomyces, Pichia) transfected with recombinant yeast expression vectors containing the modulator coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the modulator coding sequence; recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco) containing the modulator coding sequence; and other host-expression systems. These include, but are not limited to, plant cell systems (e.g., Nicotiana, Arabidopsis, duckweed, maize, wheat, potato, etc.) infected with Triticum mosaic virus (TMV) or transfected with a recombinant plasmid expression vector (e.g., Ti plasmid) containing the modulator coding sequence; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) containing recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0127] When an antibody disclosed herein is produced by recombinant expression or any of the other techniques disclosed herein, it may be purified by any method known in the art for purification of immunoglobulins, or more generally, by any other standard technique for purification of proteins.

[0128] The present invention also provides pharmaceutical compositions for the prevention or treatment of cancer, which contain as an active ingredient a bispecific antibody that bispecifically binds to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb disclosed herein, or an antibody conjugate comprising such a bispecific antibody.

[0129] Furthermore, the present invention provides methods for preventing or treating cancer, comprising the step of administering to an individual a pharmaceutically effective amount of a bispecific antibody that bispecifically binds to PD-L1 and CD47, including the anti-PD-L1xCD47 bsAb disclosed herein, or a pharmaceutical composition comprising the bispecific antibody.

[0130] The present invention also provides uses of bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb disclosed herein, or antibody conjugates comprising said bispecific antibodies, for use in the prevention or treatment of cancer.

[0131] In the present invention, the cancer is a cancer that requires blocking the activity of immune checkpoint proteins to activate T cells, and may be selected from the group consisting of, for example, melanoma, lung cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, solitary myeloma, and aplastic anemia, but is not limited thereto.

[0132] In the present invention, the details of bispecific antibodies that bispecifically bind to PD-L1 and CD47, including the anti-PD-L1×CD47 bsAb disclosed herein, or antibody conjugates containing such bispecific antibodies, are the same as those described above. Therefore, the above details are to be referred to for specific details, and only the specific components of the pharmaceutical compositions and uses will be described below.

[0133] Pharmaceutical compositions of the present invention may comprise a bispecific antibody that bispecifically binds to PD-L1 and CD47, or an antibody conjugate comprising a bispecific antibody, including at least one (e.g. two or three) of the anti-PD-L1xCD47 bsAbs disclosed herein.

[0134] Cancer can be prevented or treated by administering the pharmaceutical composition of the present invention to an individual, particularly a cancer patient.

[0135] Pharmaceutical compositions of the present invention may also be formulated as desired using art-recognized techniques depending on the form of the antibody described herein, the intended mode of delivery, and numerous other variables. Furthermore, they may be formulated to contain suitable pharmaceutically acceptable carriers, including excipients and adjuvants, which are relatively inert substances well known in the art that facilitate administration or aid in processing the active compound into a pharmaceutically optimized formulation for delivery. A variety of pharmaceutically acceptable carriers, including, for example, vehicles, adjuvants, and diluents, are readily available from multiple commercial sources. Also available are a range of pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like. Specific, non-limiting, exemplary carriers include brine, buffered brine, dextrose, water, glycerol, ethanol, and combinations thereof.

[0136] The pharmaceutical compositions of the present invention may also be formulated for enteral, parenteral, or topical administration. In fact, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Excipients for parenteral and non-parenteral drug delivery, as well as formulations, are well known in the relevant art. Formulations suitable for parenteral administration include aqueous solutions of the active compound in water-soluble form, such as a water-soluble salt. Additionally, suspensions of the active compound in suitable oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. Optionally, the suspension may contain stabilizers. Furthermore, liposomes may be used to encapsulate the agent for delivery to cells.

[0137] Formulations suitable for enteral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

[0138] Generally, the antibodies disclosed herein are administered in vivo to a subject in need thereof by a variety of routes, including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intraspinal, or otherwise by implantation or inhalation. Appropriate formulations and routes of administration can be selected depending on the intended use and treatment.

[0139] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount for the treatment or prevention of cancer. The pharmaceutically effective amount refers to the amount of the antibody or pharmaceutical composition containing the antibody that elicits a biological or medical response in a subject as determined by a physician or other clinician. Furthermore, multiple doses of the antibody or pharmaceutical composition containing the antibody may be administered at a specific frequency to achieve a therapeutic amount having a preventive and / or therapeutic effect.

[0140] The pharmaceutically effective amount typically depends on the body weight, physical condition, extent of the condition being treated, and age of the subject being treated. Generally, the antibodies disclosed herein may be administered in amounts ranging from about 10 ng / kg body weight to about 100 mg / kg body weight, from about 50 μg / kg body weight to about 5 mg / kg body weight, from about 100 μg / kg body weight to about 10 mg / kg body weight, from about 100 μg / kg body weight to about 20 mg / kg body weight, or from 0.5 mg / kg body weight to about 20 mg / kg body weight per dose, but are not limited thereto. Furthermore, the antibodies may be administered at doses of at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, or at least about 10 mg / kg body weight, but are not limited thereto.

[0141] Furthermore, the pharmaceutical composition of the present invention may be administered in a dose of about 100 mg to about 10,000 mg, about 200 mg to about 9,000 mg, about 300 mg to about 8,000 mg, about 400 mg to 7,000 mg, or 500 mg to 5,000 mg, but is not limited thereto.

[0142] The pharmaceutical compositions of the present invention are typically administered to patients multiple times. Exemplary treatment regimens include administration once every two weeks, once a month, or once every three to six months. For example, a patient may receive the antibody (e.g., as an intravenous formulation) in cycles every four weeks, e.g., once every 28 days. The frequency of administration may be adjusted depending on the pharmacokinetic profile of the antibody in the patient. For example, the half-life of the antibody may require a two-week administration frequency. In some methods, at least two antibodies with different binding specificities may be administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.

[0143] Dosage and frequency depend on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency can vary depending on whether the treatment is prophylactic or therapeutic.

[0144] The duration of the treatment regimen will depend on the disease being treated, the age and condition of the patient, the stage and type of the patient's disease, how the patient responds to the treatment, etc. The clinician will closely monitor the effectiveness of the treatment and make any adjustments as needed. When agents are used in combination, two or more therapeutic agents can be administered simultaneously or sequentially in any order, i.e., an antibody disclosed herein can be administered prior to, conjointly with, or subsequent to the administration of a second therapeutic agent.

[0145] The present invention will be described in detail below with reference to examples and experimental examples.

[0146] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples. [Example]

[0147] Immunization and blood collection Human PD-L1 protein or human CD47 protein was mixed with an immunological adjuvant (GERBU) and immunized intramuscularly with one alpaca three times. After each of the three immunizations, 10 mL of blood was collected from each alpaca 14 days after the final immunization, and the immune response was analyzed by ELISA. To measure antibody production, the immunogen was dispensed into a 96-well microplate at a concentration of 1 μg / mL using coating buffer and coated overnight at 4°C. The 96-well microplate was washed three times with PBST and then blocked with 5% skim milk for 2 hours at room temperature to inhibit nonspecific binding. After washing three times with PBST, serum samples collected before immunization (day 0), 14 days (day 14), 28 days (day 28), and 42 days (day 42) after immunization were treated with stepwise dilutions. Thereafter, the 96-well microplate was washed five times with PBST, and then reacted with goat anti-Llama IgG HRP antibody at room temperature for 1 hour, and the presence or absence of antibody bound to the immunizing antigen was confirmed by TMB reaction. [Example]

[0148] Library creation and evaluation An immune library was constructed by amplifying genes encoding single domain antibodies that bind to the immunization antigen confirmed in Example 1. To construct the library, peripheral blood mononuclear cells (PBMCs) were isolated from blood using Ficoll. Gene fragments encoding single domain antibodies were amplified from total RNA extracted from the isolated PBMCs using specific primers and cloned into the pComb3x vector. The size of the constructed immune library was 5.4 x 10 8 It was. [Example]

[0149] Library amplification The immune library prepared in Example 2 was transformed into the XL1-blue strain. The transformed XL1-blue strain was added to 10 mL of 2xYT medium containing 2% glucose and 100 μg / mL ampicillin, and cultured at 37°C in a shaker. 600 The plate was cultured at 4°C for 1 hour at 4°C until the absorbance reached 0.5, and then 1 × 10 M13K07 phage (Invitrogen) was added. 11 The cells were incubated at 37°C for 30 minutes, followed by an additional 30 minutes at 200 rpm in a shaking incubator at 37°C. The culture was centrifuged at 4,000 rpm for 15 minutes at room temperature, and the supernatant was removed. The culture pellet was resuspended in 10 mL of 2xYT medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin and incubated overnight at 250 rpm in a shaking incubator at 30°C. The culture was then centrifuged at 4,000 rpm for 30 minutes at 4°C. The supernatant was precipitated using PEG precipitation and centrifuged at 12,000 rpm for 30 minutes at 4°C. The pellet was resuspended in PBS and centrifuged at 13,000 rpm for 5 minutes at 4°C. The supernatant was transferred to a new tube and stored at 4°C until use. [Example]

[0150] Bio-panning To select single domain antibodies specific to the immunizing antigen, the immunizing antigen was dispensed into a 96-well microplate at a concentration of 5 μg / mL using coating buffer and coated overnight at 4°C. The library used to select single domain antibodies (the library from Example 3) was dispensed into the 96-well microplate and incubated at room temperature for 30 minutes. The library was then transferred to new wells and incubated at room temperature for 30 minutes, and this process was repeated four times. This procedure was performed to reduce nonspecific binding of the library to the microplate wells. The library was transferred to a 1.7 mL tube and stored at 4°C until use. The microplate coated with the immunizing antigen was washed five times with PBST and then blocked with 5% Scheme's milk at room temperature for 2 hours. After washing five times with PBST, the library with reduced nonspecific binding was diluted with 5 x 10 mAb of binding solution (2.5% Scheme's milk, 0.5% Tween 20) to 5 x 10 mAb. 12 100 virions / well were dispensed and incubated at room temperature for 30 minutes. The plate was then washed 10 times with washing solution (PBS, 0.5% Tween 20) and then three times with PBST. Single domain antibodies that specifically bound to the immunizing antigen were selectively eluted by adding 5 μg of the immunizing antigen per well and incubating at room temperature for 30 minutes at 500 rpm. The eluted phages were infected into logarithmically growing XL-1 blue cells and then plated on 2xYT agar medium. The same conditions were repeated for the second round of panning. Single phage clones generated on agar medium were individually amplified and screened using FACS. [Example]

[0151] Phage screening To induce transient overexpression of the immunizing antigen in Expi-CHO cells, the gene encoding the immunizing antigen was inserted into the pCMV6-GFP vector to construct the pCMV6-immunizing antigen-GFP plasmid. Expi-CHO cells were washed with DPBS and then centrifuged at 1,200 rpm at room temperature for 3 minutes. The supernatant was removed, and the cells were resuspended in 2% skimmed milk and blocked at 4°C for 30 minutes. The cells were centrifuged at 1,200 rpm at room temperature for 3 minutes, the supernatant was removed, washed twice with DPBS, and plated at 3 × 10 cells in a 96-well microplate. 5 The cells were dispensed at 100 μL per well. Monoclonal phage was added to each well and incubated at 4°C for 1 hour, followed by washing twice with DPBS. An antibody (M13 major coat protein Alexa Flour 647 (Santacruz)) that specifically binds to the phage was dispensed onto the cells and incubated at 4°C for 30 minutes in the dark. The cells were washed twice with DPBS, resuspended in fresh DPBS, and subjected to FACS analysis using an Accuri C6 (BD) instrument. Clones screened using the FACS system were selected and sequenced. [Example]

[0152] Expression and purification of a single-domain antibody fused to a human IgG Fc domain <6-1> Expression and purification of monovalent single-domain antibodies fused with human IgG Fc domain The clones selected in Example 5 were cloned into the TGEX-Fc(IgG1) or TGEX-Fc(IgG4) expression vector. For the expression of single-domain antibodies fused with the human IgG Fc domain, Expi-CHO cells with a viability of 95-99% were counted and 7 x 10 6 The cells were added to 25 mL of Expi-CHO expression medium (Gibco) and cultured overnight at 125 rpm in a shaking incubator at 37°C with 8% CO2. Then, 80 μL of ExpiFectamine TM CHO Reagent (Gibco, 100033021) and 920 μL of OptiPROTM In a medium mixture, 20 μg of plasmid DNA encoding a single-domain antibody fused to a human IgG Fc domain and 1 mL of OptiPRO TM The medium mixture was added, and after 5 minutes of incubation at room temperature, it was added to the cultured cells. The cells were cultured at 125 rpm in a shaking incubator maintained at 8% CO for 20 hours. Then, 150 μL of ExpiFectamine, which enhances the expression of single-domain antibodies fused with the human IgG Fc domain, was added. TM The cells were cultured in a shaking incubator at 125 rpm at 32°C with 6 mL of CHO enhancer (Gibco) and 5% CO2 for 5 days. The cultured cells were centrifuged at 4,000 rpm for 30 minutes at 4°C, and the supernatant was filtered through a 0.2 μm syringe filter. The supernatant was then loaded onto a HiTrap protein G HP column (GE Healthcare), washed with PBS, and the single-domain antibody fused to the human IgG Fc domain was eluted from the column using IgG elution buffer (Thermo). The eluted sample was neutralized with 1 M Tris-HCl (pH 9.0) and stored at 4°C until use.

[0153] <6-2> Expression and purification of bivalent single-domain antibodies fused with human IgG4 Fc domains A bivalent single-domain antibody was constructed by linking the clones selected in Example 5 using two G2S linkers (GGSGGS). The nucleotides encoding the bivalent single-domain antibody were obtained by gene synthesis (Macrogen, Korea). The synthesized gene for expression and purification of the single-domain antibody fused with a human IgG4 Fc domain was cloned into a TGEX-Fc(IgG4) expression vector. The antibody was then expressed and purified using the same method as described in Example 6-1. [Example]

[0154] Expression and purification of a bispecific single-domain antibody fused to a human IgG4 Fc domain From the clones selected in Example 5, one single domain antibody specifically binding to PD-L1 and one to CD47 antigen were selected. The nucleotide sequences encoding each single domain antibody were linked using a peptide linker, resulting in two single domain antibodies specifically binding to PD-L1 and one single domain antibody specifically binding to CD47 (anti-PD-L1 sdAb x anti-PD-L1 sdAb x anti-CD47 sdAb). The nucleotide sequences encoding these trivalent bispecific single domain antibodies were obtained by gene synthesis (Macrogen, Korea). For expression and purification of bispecific single domain antibodies fused with a human IgG4 Fc domain, the synthesized genes were cloned into a TGEX-Fc(IgG4) expression vector and expressed and purified using the same method as described in Example 6-1. [Example]

[0155] Evaluation of the binding ability of mono- or bispecific single-domain antibodies fused with human IgG Fc domains to immunogens using FACS The binding ability of the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), anti-CD47 HCAb (CD47 Nb-IgG4), and anti-PD-L1×CD47 HCAb (PPC Nb-IgG4), purified in <Example 6> and <Example 7>, to the immunizing antigen was determined by FACS.

[0156] Specifically, the CHO-K1_PD-L1 cell line (CHO-K1 cells overexpressing the PD-L1 antigen) or Expi-CHO_CD47 cell line (Expi-CHO cells overexpressing the CD47 antigen) was washed with DPBS and then centrifuged at 1,200 rpm at room temperature for 3 minutes. The supernatant was removed, and the cells were resuspended in 2% skim milk and blocked at 4°C for 30 minutes. The cells were centrifuged at 1,200 rpm at room temperature for 3 minutes, the supernatant was removed, and then washed twice with DPBS. 3 x 10 cells were then added to each well. 5Cells were dispensed at 100 μL per well and then treated with various concentrations of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), anti-CD47 HCAb (CD47 Nb-IgG4), and anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4). An isotype control antibody was used as a negative control. Cells were incubated at 4°C for 1 hour and then washed twice with DPBS. Subsequently, cells were treated with an antibody specifically binding to the human Fc domain (Anti-human IgG Fc APC antibody (Biolegend)) and incubated for 30 minutes at 4°C in the dark. Cells were washed twice with DPBS and then resuspended in 100 μL of DPBS. FACS analysis was performed using an Accuri C6 (BD) instrument. [Example]

[0157] FACS-based evaluation of the inhibitory potential of mono- and bispecific single-domain antibodies fused with human IgG Fc domains against immune-antigen interactions The inhibitory effects of the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), anti-CD47 HCAb (CD47 Nb-IgG4), and anti-PD-L1×CD47 HCAb (PPC Nb-IgG4), purified in <Example 6> and <Example 7>, on the PD-1 / PD-L1 or CD47 / SIRPalpha interaction were evaluated.

[0158] Specifically, to evaluate the effects of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), or anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) on PD-1 / PD-L1 interaction, CHO-K1_PD-L1 cell line (CHO-K1 cells with constant expression of PD-L1 antigen) was plated at 2 × 10 cells per well in a 96-well microplate. 5Cells were then treated with 10μg / mL human PD-1-His protein. They were then treated with various concentrations of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), or anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4). An isotype control antibody was also used as a negative control. The cells were incubated at 4°C for 1 hour, washed three times with DPBS, and then treated with a His antigen-specific antibody (Goat anti-His APC) and incubated for 30 minutes at 4°C in the dark. The cells were washed three times with DPBS and then resuspended in 100 μL of DPBS. The amount of PD-1-His protein remaining in CHO-K1_PD-L1 cells (CHO-K1 cells with constant PD-L1 antigen expression) was determined using an Accuri C6 (BD) device to assess the inhibitory potential of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), or anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) on PD-1 / PD-L1 interaction.

[0159] Furthermore, to evaluate the effect of anti-CD47 sdAb (CD47 Nb-IgG4) or anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) on the CD47 / SIRPalpha interaction, Expi-CHO_CD47 cells (Expi-CHO cells with constant CD47 antigen expression) were plated at 2 × 10 per well in a 96-well microplate. 5Cells were aliquoted and treated with 10 μg / mL human SIRPalpha-His protein. They were then treated with various concentrations of anti-CD47 sdAb (CD47 Nb-IgG4) or anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4), and an isotype control antibody was used as a negative control. The amount of SIRPalpha-His protein remaining in Expi-CHO_CD47 cells (Expi-CHO cells with constant CD47 antigen expression) was then measured using an Accuri C6 (BD) instrument, as described above, to assess the inhibitory effect of anti-CD47 sdAb (CD47 Nb-IgG4) or anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) on the CD47 / SIRPalpha interaction. [Example]

[0160] Affinity evaluation of mono- and bispecific single-domain antibodies fused with human IgG Fc domains to immunogens Using an Octet RED 96e (ForteBio) device, the affinity (K) of the purified anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), anti-CD47 HCAb (CD47 Nb-IgG4), and anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) in Examples 6 and 7 to the immunogen protein was measured. d ) was measured.

[0161] Specifically, a biosensor chip (ForteBio) coated with anti-human Fc was saturate-coupled to 1.5 nM levels using 5 μg / mL anti-PD-L1 HCAb (PDL1 Nb#01-IgG1), anti-PD-L1 bivalent HCAb (PP Nb-IgG4), anti-CD47 HCAb (CD47 Nb-IgG4), and anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) in a 96-well microplate (Greiner). PD-L1 and CD47 antigens were serially diluted 2-fold in 1x kinetic buffer (ForteBio) from 10 to 400 nM and incubated at 30°C with stirring at 1,000 rpm. The binding and dissociation reactions were analyzed for 200 and 400 seconds, respectively. The resulting data were analyzed using a 1:1 interaction model (global fitting). [Example]

[0162] In vitro efficacy and safety evaluation of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) <11-1>In vitro efficacy evaluation against PD-L1 antigen The in vitro efficacy of the anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) purified in Example 7 was evaluated using CHO-K1_PD-L1 cells (CHO-K1 cells induced to overexpress PD-L1 antigen) and PD-1-expressing Jurkat cells.

[0163] Specifically, CHO-K1_PD-L1 cells (CHO-K1 cells induced to overexpress PD-L1 antigen) were cultured at 2 × 10 cells per well in a 96-well microplate. 4 The cells were then cultured in a 5% CO2 incubator for 16 hours. The medium was then removed, and the cells were treated with anti-PD-L1 x CD47 HCAb (PPC Nb-IgG4) at various concentrations for 1 hour. 5 x 10 5Jurkat cells were prepared at a concentration of 100μL and then treated with 0.5mg / mL PHA. This was then added to anti-PD-L1×CD47 HCAb (PPC Nb-IgG4)-treated CHO-K1_PD-L1 cells and incubated for 48 hours. IL-2 concentrations in the supernatants were then measured by ELISA to assess the in vitro efficacy of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4).

[0164] <11-2>In vitro efficacy and safety evaluation of CD47 antigen The in vitro efficacy of the anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) purified in <Example 7> was evaluated by measuring the degree of macrophage phagocytosis activation through CD47 antigen-specific binding, human RBC binding, and hemagglutination.

[0165] Specifically, 5 x 10 6 THP-1 cells were plated into 100 Pi dishes and cultured for 24 hours in an incubator maintained at 5% CO2. After incubation with 40 nM PMA for 24 hours, the medium was removed and stained with 1 μM deep red dye. After staining, the medium was replaced and the cells were rested for 48 hours. Trypsin-dissociated THP-1 cells were mixed with prey cells (Raji cells) stained with 3 μM CFSE at an 8:1 ratio and then co-cultured with various concentrations of anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) for 4 hours. Phagocytosis was then assessed using FACS.

[0166] To confirm the binding ability of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) to human RBCs (red blood cells), RBCs were washed seven times with DPBS and then diluted to 12% (v / v) with DPBS. 50 μL of 2× anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) was dispensed into a 96-well V-bottom plate, followed by the addition of 50 μL of 12% (v / v) RBCs and incubation at 4°C for 1 hour. The cells were washed with DPBS and then treated with a secondary antibody specifically recognizing human IgG4 (anti-human IgG4) and incubated at 4°C for 1 hour. The cells were then washed with DPBS and the extent of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) binding to RBCs was assessed via FACS.

[0167] To assess hemagglutination by anti-PD-L1×CD47 HCAb (PPC Nb-IgG4), RBCs were washed seven times with DPBS and then diluted to 6% (v / v) with DPBS. 50 μL of 2× anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) was dispensed into a 96-well U-bottom plate, followed by 50 μL of 6% (v / v) RBCs. After incubation at room temperature for 1 hour, hemagglutination by anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) was assessed by visual observation. [Example]

[0168] In vivo efficacy evaluation of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) The in vivo efficacy of the anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) purified in <Example 7> was evaluated by injecting a tumor cell line (B16F10 cells) induced to express human PD-L1 and human CD47 into C57BL / 6 mice, and assessing the anti-tumor effect of the anti-PD-L1×CD47 HCAb (PPC Nb-IgG4).

[0169] Specifically, 6- to 8-week-old C57BL / 6 female mice were inoculated with 8 × 10 B16F10_PD-L1 × CD47 cell lines (B16F10 cells induced to overexpress human PD-L1 and human CD47). 5 Cells were injected at a volume of 100 μL. Tumor formation was induced until the tumor size reached 3 × 3 mm (horizontal × vertical). Subsequently, 10 mpk of anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4) was intraperitoneally administered seven times at 2-day intervals, and tumor size was measured. After the final intraperitoneal administration, tumor size was measured at 2-day intervals for 1 week to evaluate the in vivo efficacy of anti-PD-L1 × CD47 HCAb (PPC Nb-IgG4).

[0170] To confirm the antitumor effect of intravenously administered anti-PD-L1×CD47 HCAb (PPC Nb-IgG4), the same mouse tumor model was used. Anti-PD-L1×CD47 HCAb (PPC Nb-IgG4) was administered intravenously seven times at 3-day intervals at doses of 5 and 10 mpk, and tumor size was then measured. Three days after the final intravenous administration, tumor size was measured to evaluate the in vivo efficacy of anti-PD-L1×CD47 HCAb (PPC Nb-IgG4).

[0171] <Experimental Example 1> Preparation of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and in vitro characterization <1-1> Preparation of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) Using human PD-L1 antigen as the immunizing antigen, a PD-L1 antigen-specific single domain antibody clone was selected and sequenced in the same manner as described in Example 5. The amino acid sequence of the selected anti-PD-L1 sdAb (PDL1 Nb#01) is shown in Tables 5 and 6 below.

[0172] Furthermore, using the selected anti-PD-L1 sdAb (PDL1 Nb#01), a PD-L1-specific monovalent single-domain antibody containing a human IgG1 Fc domain was expressed and purified in the same manner as described in Example <6-1>. The purified monovalent single-domain antibody was designated anti-PD-L1 HCAb (PDL1 Nb#01-IgG1). The amino acid sequence of anti-PD-L1 HCAb containing a human IgG1 Fc domain (PDL1 Nb#01-IgG1) is shown in Table 7 below.

[0173] Furthermore, using the selected anti-PD-L1 sdAb (PDL1 Nb#01) clone, a PD-L1-specific bivalent single domain antibody containing human IgG4 Fc was expressed and purified using the same method as described in Example <6-2>. The purified bivalent single domain antibody was designated anti-PD-L1 bivalent HCAb (PP Nb-IgG4). The amino acid sequence of the anti-PD-L1 bivalent sdAb (PP Nb), obtained by removing the human IgG4 Fc domain from the anti-PD-L1 bivalent HCAb (PP Nb-IgG4), is shown in Table 8 below, and the amino acid sequence of the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) containing the human IgG4 Fc domain is shown in Table 9 below.

[0174] [Table 5]

[0175] [Table 6]

[0176] [Table 7]

[0177] [Table 8]

[0178] [Table 9]

[0179] <1-2> Evaluation of the antigen binding ability of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4), and their ability to inhibit the PD-1 / PD-L1 interaction Similar to the methods described in <Example 8> and <Example 9>, the antigen-binding ability ( Figures 1A and 2A ) and inhibitory ability on PD-1 / PD-L1 interaction ( Figures 1B and 2B ) of the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Experimental Example 1> were assessed using FACS.

[0180] As a result, as shown in Figures 1A and 1B, the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) was confirmed to have an antigen-binding activity of 23.31 nM (EC50) in CHO-K1_PD-L1 cells (CHO-K1 cells that constantly express PD-L1 antigen), and an inhibitory activity against PD-1 / PD-L1 interaction of 4.60 nM (IC50).

[0181] Furthermore, as shown in Figures 2A and 2B, the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) exhibited an antigen-binding activity of 1.93 nM (EC50) in CHO-K1_PD-L1 cells (CHO-K1 cells that stably express PD-L1 antigen), and an inhibitory activity against PD-1 / PD-L1 interaction of 2.86 nM (IC50).

[0182] <1-3> Evaluation of the affinity of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) to the immunizing antigen The affinity of the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Experimental Example 1> for the PD-L1 antigen was evaluated using the same method as described in <Example 9>.

[0183] As a result, as shown in Table 10, the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) was confirmed to have an antigen affinity of 7.08 nM with the PD-L1 antigen, and the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) was confirmed to have an antigen affinity of 4.58 nM with the PD-L1 antigen.

[0184] [Table 10]

[0185] <Experimental Example 2> Preparation of anti-C47 HCAb (CD47 Nb-IgG4) and in vitro characterization <2-1> Preparation of anti-C47 HCAb (CD47 Nb-IgG4) Using human CD47 antigen as the immunizing antigen, single domain antibody clones specific to the CD47 antigen were selected and sequenced in the same manner as described in Example 5. The amino acid sequence of the selected anti-CD47 sdAb (CD47 Nb#01) is shown in Tables 11 and 12 below.

[0186] Furthermore, using the selected anti-CD47 sdAb (CD47_Nb_#01), a CD47-specific single-domain antibody containing a human IgG4 Fc domain was expressed and purified in the same manner as described in Example <6-1>. The purified single-domain antibody was designated anti-CD47 HCAb (CD47 Nb-IgG4). The amino acid sequence of anti-CD47 HCAb (CD47 Nb-IgG4) containing a human IgG4 Fc domain is shown in Table 13 below.

[0187] [Table 11]

[0188] [Table 12]

[0189] [Table 13]

[0190] <2-2> Evaluation of the antigen-binding ability of anti-CD47 HCAb (CD47 Nb-IgG4) and its inhibitory ability against CD47 / SIRPalpha interaction In the same manner as in <Example 8> and <Example 9>, the antigen-binding ability (Fig. 3A) and inhibitory ability on CD47 / SIRPalpha interaction (Fig. 3b) of the anti-CD47 HCAb (CD47 Nb-IgG4) purified in Experimental Example <2-1> were evaluated using FACS.

[0191] As a result, as shown in Figures 3A and 3B, anti-CD47 HCAb (CD47 Nb-IgG4) was confirmed to have an antigen-binding activity of 3.78 nM (EC50) in Expi-CHO_CD47 cells (Expi-CHO cells that constantly express CD47 antigen), and an inhibitory activity against the CD47 / SIRPalpha interaction of 7.28 nM (IC50).

[0192] <2-3> Evaluation of affinity between anti-CD47 HCAb (CD47 Nb-IgG4) and immunogen The affinity of the anti-CD47 HCAb (CD47Nb-IgG4) purified in <Experimental Example 2-1> to the CD47 antigen was evaluated in the same manner as described in <Example 10>.

[0193] As a result, as shown in Table 14, it was confirmed that anti-CD47 HCAb (CD47 Nb-IgG4) has an antigen affinity of 2.78 nM for the CD47 antigen.

[0194] [Table 14]

[0195] <Experimental Example 3> Preparation of anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) and in vitro characterization <3-1> Preparation of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) Using the clone selected in Experimental Example <1-1>, a single-domain antibody containing a human IgG4 Fc domain and bispecific for PD-L1 and CD47 antigens as immunizing antigens was prepared. Gene synthesis was performed using the nucleotide sequence encoding the anti-PD-L1 sdAb (PDL1 Nb#01) selected in Experimental Example <1-1> and the nucleotide sequence encoding the anti-CD47 sdAb (CD47 Nb#01) selected in Experimental Example <2-1> in the same manner as described in <Example 7>. The gene was then cloned into a TGEX-Fc(IgG4) expression vector, expressed, purified, and designated anti-PD-L1×CD47 HCAb (PPC Nb-IgG4).

[0196] The amino acid sequence of the PD-L1×CD47 trivalent sdAb, which is the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) minus the human IgG4 Fc domain, is shown in Table 15 below, and the amino acid sequence of the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) including the human IgG4 Fc domain is shown in Table 16 below.

[0197] [Table 15]

[0198] [Table 16]

[0199] <3-2> Evaluation of immunogen binding ability of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) Using a method similar to that described in <Example 8>, the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) purified in Experimental Example <3-1> was confirmed to have the binding ability to PD-L1 antigen and CD47 antigen using FACS.

[0200] As a result, as shown in Figures 4A and 4B, the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) exhibited an antigen-binding activity of 13.33 nM (EC50) in CHO-K1_PD-L1 cells (CHO-K1 cells with constant PD-L1 antigen expression) and 18.80 nM (EC50) in Expi-CHO_CD47 cells (Expi-CHO cells with constant CD47 antigen expression).

[0201] <3-3> Evaluation of the inhibitory effect of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) on PD-1 / PD-L1 and CD47 / SIRPalpha interactions The inhibitory ability of the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) purified in Experimental Example <3-1> against the interactions of PD-1 / PD-L1 and CD47 / SIRPalpha was assessed using FACS in the same manner as described in <Example 9>.

[0202] As a result, as shown in Figures 5A and 5B, the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) was confirmed to have an inhibitory effect on PD-1 / PD-L1 interaction of 9.15 nM (IC50), and an inhibitory effect on CD47 / SIRPalpha interaction of 22.44 nM (IC50).

[0203] <3-4> Evaluation of affinity between anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) and the immunogen The affinity of the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) purified in Experimental Example <3-1> for the PD-L1 antigen and CD47 antigen was evaluated using the same method as described in <Example 10>.

[0204] As a result, as shown in Table 17, the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) was confirmed to have excellent affinity for the PD-L1 antigen, 6.84 nM, and for the CD47 antigen, 3.62 nM.

[0205] [Table 17]

[0206] <Experimental Example 4> In vitro efficacy and safety evaluation of anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) The anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) purified in Experimental Example <3-1> was used to evaluate the efficacy and safety in vitro.

[0207] <4-1>In vitro efficacy evaluation of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) against PD-L1 antigen To confirm the in vitro efficacy of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) against the PD-L1 antigen, the interaction between CHO-K1_PD-L1 cells (CHO-K1 cells that stably express PD-L1) and PD-1-expressing Jurkat cells was inhibited by anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) in a manner similar to that described in Example <11-1>, and the concentration of IL-2 expressed by Jurkat cells was measured to evaluate the in vitro efficacy.

[0208] As a result, as shown in Figure 6, anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) interacted with PD-1-expressing Jurkat cells and PD-L1-expressing CHO-K1 cells, and inhibited the interaction between CHO-K1 cells and Jurkat cells with an IC50 of 1.45 nM.

[0209] <4-2>Evaluation of phagocytosis of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) against the CD47 antigen To confirm the in vitro efficacy of anti-PD-L1 × CD47 trivalent HCAb (PPC Nb-IgG4) against the CD47 antigen, THP-1 cells were transformed into macrophages using PMA in the same manner as described in Example <11-2>. The degree of phagocytosis by macrophages was then assessed via selective binding to prey cells (Raji cells) expressing the CD47 antigen.

[0210] As a result, as shown in Figure 7, the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) was confirmed to interact with THP-1 cells and prey cells (Raji cells) expressing the CD47 antigen, with a binding ability of 1.44 nM (EC50) to prey cells expressing CD47.

[0211] <4-3>Evaluation of human RBC binding ability of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) to the CD47 antigen Because human RBCs express CD47, antibodies that specifically bind to the CD47 antigen may cause side effects such as anemia through binding to human RBCs. Therefore, we investigated the binding ability of anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) to human RBCs and evaluated the safety of the antibody. Binding ability to human RBCs was assessed using FACS in a manner similar to that described in Example <11-2>.

[0212] As a result, as shown in Figure 8, the CD47 monoclonal antibody (Competitor) used as a positive control was confirmed to bind to human RBCs at concentrations of 2.93 nM or higher, whereas the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) did not bind to RBCs even at the maximum concentration used in the experiment, 3 μM.

[0213] <4-4>Hemagglutination assay of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) against the CD47 antigen Binding of human RBCs to CD47 antigen-specific antibodies induces hemagglutination. Therefore, the binding of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) to RBCs was assessed for hemagglutination using the same method as described in Example <11-2> to confirm the safety of the antibody.

[0214] As a result, as shown in Figure 9, the CD47 monoclonal antibody (Competitor) used as a positive control showed hemagglutination at concentrations of 11.72 nM or higher, whereas the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) did not show hemagglutination even at 3 μM, the maximum concentration used in the experiment.

[0215] <Experimental Example 5> Evaluation of the in vivo efficacy of anti-PD-L1 × CD47 trivalent HCAb (PPC Nb-IgG4) The in vivo efficacy of the anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) purified in Experimental Example <3-1> was evaluated using a mouse tumor model.

[0216] <5-1> Confirmation of the antitumor effect of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) administered intraperitoneally Using a method similar to that described in <Example 12>, anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) was injected into tumor cells (B16F10 cells) expressing PD-L1 and CD47 antigens, and the antitumor effect of intraperitoneal administration was observed in a mouse model in which tumors had formed.

[0217] As a result, as shown in Figure 10, the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) demonstrated an anti-tumor effect of approximately 49.4% compared to the negative control group (isotype group), and an anti-tumor effect of approximately 38.8% compared to the group in which each antibody constituting the anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) was co-treated (anti-PD-L1 bivalent HCAb (PP Nb-IgG4) + anti-CD47 HCAb (CD47 Nb-IgG4)).

[0218] <5-2> Confirmation of the antitumor effect of intravenous administration of anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) Using a method similar to that described in <Example 12>, anti-PD-L1×CD47 trivalent HCAb (PPC Nb-IgG4) was injected with tumor cells (B16F10 cells) expressing PD-L1 and CD47 antigens, and the antitumor effect of intravenous administration was observed in a mouse model in which tumors had formed.

[0219] As a result, as shown in Figure 11, it was confirmed that anti-PD-L1 x CD47 trivalent HCAb (PPC Nb-IgG4) exhibited an antitumor effect of up to approximately 74.4% compared to the negative control group (isotype group). [Industrial Applicability]

[0220] The single domain antibody according to the present invention exhibits excellent affinity for CD47, an immune checkpoint protein, and exhibits antitumor activity, and can therefore be usefully used as an immune checkpoint inhibitor in immunotherapy against cancer.

Claims

[Claim 1] A bispecific single domain antibody that bispecifically binds to PD-L1 and CD47, comprising a first VHH domain or antigen-binding fragment thereof that specifically binds to PD-L1, and a second VHH domain or antigen-binding fragment thereof that specifically binds to CD47, the first VHH domain or antigen-binding fragment thereof comprising: CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 2; CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 3; and It comprises a CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 4, the second VHH domain or antigen-binding fragment thereof comprises: CDR1 consisting of the amino acid sequence represented by SEQ ID NO:7; CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 8; and CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 9 1. A bispecific single domain antibody comprising:

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