Single domain antibodies against PD-L1 and their uses

Abstract

The present invention relates to a single domain antibody against PD-L1 and its use. More specifically, a single domain antibody that specifically binds to PD-L1, an immune checkpoint protein, has been prepared, and its affinity to immune antigens and antitumor effect have been confirmed, so that the single domain antibody can be usefully used as an immune checkpoint inhibitor in immunotherapy.

Description

[Technical Field]

[0001] The present invention relates to a single domain antibody against PD-L1, an immune checkpoint protein, 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] Therefore, the present inventors developed an immune checkpoint inhibitor that targets an immune checkpoint protein, which led to the present application. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 8,008,449 [Patent Document 2] International Publication No. 94 / 04678 [Patent Document 3] US Patent Application Publication No. 2009 / 0307787 [Patent Document 4] U.S. Patent No. 8,754,287 [Patent Document 5] US Patent Application Publication No. 2015 / 0289489 [Patent Document 6] US Patent Application Publication No. 2010 / 0122358 [Patent Document 7] International Publication No. 2004 / 049794 [Patent Document 8] International Publication No. 99 / 37681 [Patent Document 9] International Publication No. 01 / 90190 [Patent Document 10] International Publication No. 03 / 025020 [Patent Document 11] International Publication No. 03 / 035694 [Patent Document 12] International Publication No. 00 / 43507 [Patent Document 13] International Publication No. 06 / 030220 [Patent Document 14] International Publication No. 06 / 003388 [Patent Document 15] U.S. Patent No. 4,816,567 [Patent Document 16] International Publication No. 1996 / 34103 [Patent Document 17] International Publication No. 93 / 08829 [Patent Document 18] U.S. Patent No. 5,731,168 [Patent Document 19] International Publication No. 2009 / 089004 [Patent Document 20] U.S. Patent No. 4,676,980 [Patent Document 21] US Patent Application Publication No. 2011 / 0028695 [Non-patent literature]

[0010] [Non-Patent Document 1] Pol Specenier (2016): Ipilimumab in melanoma, Expert Review of Anticancer Therapy. [Non-patent document 2] DB Johnson, C Peng et al.Nivolumab in melanoma:latest evidence and clinical potential.Ther Adv Med Oncol 2015, Vol.7(2)97-106 [Non-licensed document 3] Liu J, Wang L, Zhao F, Tseng S, Narayanan C, Shura L, et al. (2015) Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential.PLoS ONE 10(9):e0137345.

Non-licensed Document 4

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0011] The present invention aims to provide a single domain antibody against PD-L1, an immune checkpoint protein, and uses thereof. [Means for solving the problem]

[0012] To achieve the objectives of the present invention, the present invention provides an antibody or antigen-binding fragment thereof, including a single domain antibody (sdAb) that specifically binds to PD-L1, comprising: The present invention provides an antibody or antigen-binding fragment thereof, wherein the sdAb comprises 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.

[0013] In one aspect of the invention, the sdAb may comprise a VHH domain having FR1 consisting of the amino acid sequence set forth in SEQ ID NO:6, FR2 consisting of the amino acid sequence set forth in SEQ ID NO:7, FR3 consisting of the amino acid sequence set forth in SEQ ID NO:8, and FR4 consisting of the amino acid sequence set forth in SEQ ID NO:9, and in some embodiments has the amino acid sequence set forth in SEQ ID NO:1.

[0014] In one aspect of the invention, the sdAb 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.

[0015] In one embodiment of the invention, the sdAbs may be monomeric or multimeric, and if multimeric, may be fused via an inter-sdAb peptide linker, which in some implementations has the amino acid sequence set forth in SEQ ID NO: 10.

[0016] In one aspect of the present invention, the sdAb provides a heavy chain-only antibody (HCAb) fused to an Fc fragment. In some implementations, the HCAb may consist of the amino acid sequence set forth in either SEQ ID NO: 5 or 11.

[0017] In one embodiment of the invention, the HCAb may be monomeric or multimeric.

[0018] 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.

[0019] 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.

[0020] In one aspect of the invention, there is provided an antibody comprising: (a) a first antigen-binding portion comprising the sdAb; and (b) a second antigen-binding portion that specifically binds to a second epitope.

[0021] In one aspect of the invention, the antibody may be bispecific or multispecific.

[0022] In one embodiment of the present invention, the second antigen-binding portion may be fused to the first antigen-binding portion via a peptide linker, and the second antigen-binding portion may be a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), scFv-scFv, minibody, diabody or second sdAb.

[0023] 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 attached. Thus, the present invention provides an antibody conjugate comprising an antibody or antigen-binding fragment thereof attached to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug.

[0024] The present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0025] Furthermore, the present invention provides an expression vector comprising the nucleic acid molecule.

[0026] The present invention also provides a host cell transformed with the expression vector.

[0027] Furthermore, the present invention provides (a) culturing the host cells under conditions that allow expression of the antibody; (b) recovering the expressed antibody or antigen-binding fragment thereof; The present invention provides a method for producing an antibody or antigen-binding fragment thereof, comprising:

[0028] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which contains the antibody or its antigen-binding fragment, or the antibody complex as an active ingredient; a method for preventing or treating cancer, which comprises the step of administering a pharmaceutically effective amount of the antibody or its antigen-binding fragment, or the antibody complex to an individual; and uses of the antibody or its antigen-binding fragment, or the antibody complex for use in the prevention or treatment of cancer.

[0029] 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.

[0030] In one embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0031] The present invention also provides (i) contacting the antibody or antigen-binding fragment thereof with a sample; (ii) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and PD-L1 or determining the amount of the complex; The present invention provides a method for detecting PD-L1 or determining the amount of PD-L1 in a sample, comprising: [Effects of the Invention]

[0032] In the present invention, single domain antibodies that specifically bind to PD-L1, an immune checkpoint protein, have been prepared, and their affinity for immune antigens and antitumor effects have been confirmed. Therefore, these single domain antibodies can be useful as immune checkpoint inhibitors in immunotherapy. [Brief explanation of the drawings]

[0033] [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 3]Figure 1 shows the ability of anti-PD-L1 bivalent HCAb (PP Nb-IgG4) prepared according to one example of the present invention to inhibit PD-L1 / PD-1 interaction, using CHO-K1_PD-L1 cells (CHO-K1 cells induced to overexpress PD-L1 protein) and PD-1-expressing Jurkat cells. [Figure 4] Figure 1 shows the antitumor effect of anti-PD-L1 bivalent HCAb (PP 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 cell line (tumor cells induced to express PD-L1 antigen). DETAILED DESCRIPTION OF THE INVENTION

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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."

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The term "humanized antibody" is used as a subset of "chimeric antibody."

[0049] "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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The term "framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0054] The term "specific" refers to the selective recognition of an antigen binding protein (e.g., an sdAb) for a particular epitope of an antigen.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] "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.

[0059] 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.

[0060] "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).

[0061] "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.

[0062] The present invention relates to antibodies or antigen-binding fragments thereof, including single domain antibodies (sdAbs) that specifically bind to PD-L1 (hereinafter referred to as "anti-PD-L1 sdAbs"), such as anti-PD-L1 sdAbs, anti-PD-L1 heavy chain-only antibodies (HCAbs) (such as anti-PD-L1 sdAb-Fc fusion proteins in which an anti-PD-L1 sdAb is fused to the crystallizable fragment (Fc fragment) of human immunoglobulin G (IgG)), or other sdAbs, or multispecific antigen-binding proteins in which an anti-PD-L1 sdAb is fused to a full length four-chain antibody or antigen-binding fragment thereof (e.g. Fab or scFv), and their preparation and uses.

[0063] Thus, the present invention provides antibodies or antigen-binding fragments thereof, including anti-PD-L1 sdAbs.

[0064] In the present invention, the antibody or antigen-binding fragment thereof comprising the anti-PD-L1 sdAb may be an anti-PD-L1 sdAb or antigen-binding fragment thereof.

[0065] 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.

[0066] The CDR sequences are shown in Table 6.

[0067] In the present invention, the anti-PD-L1 sdAb 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.

[0068] [Table 1]

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] 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: 6; FR2 consisting of the amino acid sequence represented by SEQ ID NO: 7; FR3 consisting of the amino acid sequence represented by SEQ ID NO: 8; and FR4 consisting of the amino acid sequence shown in SEQ ID NO:9.

[0073] In the present invention, the anti-PD-L1 sdAb may comprise a VHH domain comprising the FR region.

[0074] 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%).

[0075] In the present invention, the anti-PD-L1 sdAb may be monomeric or multimeric. If multimeric, it may be multispecific and multivalent (e.g., bispecific and bivalent), for example, containing an anti-PD-L1 sdAb different from the anti-PD-L1 sdAbs described herein, or it may be monospecific and multivalent (e.g., bivalent), containing at least two copies of the same anti-PD-L1 sdAb. Furthermore, sdAbs may be fused together via a peptide linker. In some implementations, the peptide linker contains flexible residues (e.g., glycine and serine) to allow 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 embodiments, 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. In some embodiments, the anti-PD-L1 sdAb may comprise the amino acid sequence set forth in SEQ ID NO: 10, 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.

[0076] In the present invention, the anti-PD-L1 sdAb binds to an epitope on PD-L1.

[0077] Furthermore, the K 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.

[0078] 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.

[0079] 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.

[0080] 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 2 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.

[0081] 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.

[0082] 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 3 to 7).

[0083] 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 8 to 11. 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 12.

[0084] Alternatively, sdAbs may be generated from conventional four-chain antibodies (see, for example, Non-Patent Documents 8 and 9 and Patent Documents 13 and 14).

[0085] Furthermore, the sdAB of the present invention may be a chimeric antibody. Specific chimeric antibodies are disclosed, for example, in Patent Document 15 and Non-Patent Document 10. 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. A humanized antibody will also optionally 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.

[0086] In the present invention, the antibody or antigen-binding fragment thereof comprising the anti-PD-L1 sdAb may be an anti-PD-L1 HCAb or antigen-binding fragment thereof.

[0087] Specifically, the anti-PD-L1 HCAb comprises an anti-PD-L1 sdAb described herein fused to at least one CH2 and / or CH3 domain, e.g., an Fc fragment.

[0088] The CH2 and / or CH3 domains are derived from an immunoglobulin. The immunoglobulin may be IgA, IgD, IgE, IgG, or IgM, specifically IgG. In some implementations, the anti-PD-L1 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., human IgG1 (hIgG1) Fc, hIgG2 Fc, hIgG3 Fc, or hIgG4 Fc.

[0089] The anti-PD-L1 HCAbs may be monomeric or multimeric, and if multimeric, may be multispecific and multivalent (e.g., bispecific and bivalent), e.g., containing an anti-PD-L1 sdAb that is different from the anti-PD-L1 sdAbs described herein, or may be monospecific and multivalent (e.g., bivalent), containing at least two copies of the same anti-PD-L1 sdAb.

[0090] In the present invention, the anti-PD-L1 sdAb and the CH2 and / or CH3 domain, specifically the Fc fragment, may be fused 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. 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 16. In some implementations, the peptide linker may be a hIgG1 hinge, a hIgG2 hinge, a hIgG3 hinge, a hIgG4 hinge, or a variant thereof.

[0092] In the present invention, the anti-PD-L1 HCAb may comprise the amino acid sequence set forth in either SEQ ID NO: 5 or 11, or a variant thereof having at least 80% sequence identity to said amino acid sequence (e.g., at least any of 80%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%).

[0093] In the present invention, the antibody or antigen-binding fragment thereof comprising the anti-PD-L1 sdAb may also be a multispecific antigen-binding protein (MABP) in which the anti-PD-L1 sdAb is fused to another sdAb, or a full-length four-chain antibody or its antigen-binding fragment (e.g., a bispecific antigen-binding protein (BABP) fused to an anti-PD-L1 sdAb (hereinafter referred to as anti-PD-L1 BABP)), or an antigen-binding fragment thereof.

[0094] The anti-PD-L1 BABP comprises (a) a first antigen-binding portion that comprises an anti-PD-L1 sdAb described herein, and (b) a second antigen-binding portion that specifically binds to a second epitope.

[0095] The second epitope may be an antigen other than PD-L1 or a second epitope in PD-L1.

[0096] The second antigen-binding portion may be a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), scFv-scFv, minibody, diabody, or second sdAb. The second antigen-binding portion may also comprise a heavy chain having a VH and a light chain having a VL. In some implementations, the first antigen-binding portion may be fused to the second antigen-binding portion at the N-terminus of the heavy chain, the N-terminus of the light chain, the N-terminus of the Fc region, the C-terminus of the heavy chain, or the C-terminus of the light chain. In some implementations, the second antigen-binding portion may comprise a Fab or scFv. In some implementations, the first antigen-binding portion may be fused to the second antigen-binding portion at the C-terminus of the Fab or scFv. In some implementations, the second antigen-binding portion may comprise a full-length four-chain antibody. In some implementations, the first antigen-binding portion may be fused to the second antigen-binding portion via a peptide linker. In some implementations, the second antigen-binding portion can include an Fc region, for example, an IgG1 Fc, an IgG2 Fc, an IgG3 Fc, or an IgG4 Fc.

[0097] The anti-PD-L1 MABP comprises at least two antigen-binding moieties that specifically bind at least two different epitopes. Portions of the at least two antigen-binding moieties may be identical, so long as the MABP has binding sites for two different epitopes. Furthermore, the anti-PD-L1 MABP may comprise any of 1, 2, 3, 4, 5, 6, 7, 8 or more different antigen-binding moieties, each of which comprises an anti-PD-L1 sdAb described herein.

[0098] Additionally, an anti-PD-L1 MABP may have any suitable number of mantissas and any suitable number of specificities for a first epitope and / or a second epitope on PD-L1. In some implementations, an anti-PD-L1 MABP may be bivalent, trivalent, tetravalent, pentavalent, hexavalent, or higher valency with respect to PD-L1. In some implementations, an MABP may be trispecific or tetraspecific.

[0099] Techniques for producing multispecific antibodies according to the present invention include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (e.g., Non-Patent Documents 11, 12, and 17), and "knobs-in-holes" engineering (e.g., Patent Document 18). Multispecific antibodies may also be produced by manipulating electrostatic steering effects to produce antibody Fc-heterodimeric molecules (Patent Document 19); cross-linking at least two antibodies or fragments (e.g., Patent Document 20 and Non-Patent Document 13); using leucine zippers to produce bispecific antibodies (e.g., Non-Patent Document 14); using "diabody" technology to produce bispecific antibody fragments (e.g., Non-Patent Document 15); and using single-chain Fv (sFv) dimers (e.g., Non-Patent Document 16); as well as preparing trispecific antibodies, as described, for example, in Non-Patent Document 17; and creating polypeptides comprising tandem single-domain antibodies (e.g., Patent Document 21 and Non-Patent Document 18).

[0100] In the present invention, antibodies or antigen-binding fragments thereof comprising the anti-PD-L1 sdAb 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 amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at a final product, provided that the final product 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.

[0101] 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.

[0102] 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).

[0103] Additionally, at least one amino acid modification may be introduced into the Fc region of an antibody or antigen-binding fragment thereof (e.g., an anti-PD-L1 HCAb, or an anti-PD-L1 MABP), including an anti-PD-L1 sdAb, 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) comprising an amino acid modification (e.g., a substitution) at at least one amino acid position.

[0104] In the present invention, the antibodies or antigen-binding fragments thereof, including the anti-PD-L1 sdAbs, may be linked, fused, 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.

[0105] Antibodies or antigen-binding fragments thereof, including the anti-PD-L1 sdAbs, 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-associated disease, such as cancer, to determine the efficacy of a particular therapy (i.e., theragnosis) involving the antibodies disclosed herein, or to determine future courses of treatment. Such markers or reporters are also useful for purifying the antibodies disclosed herein.

[0106] Furthermore, the anti-PD-L1 sdAb-containing antibody or antigen-binding fragment thereof 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 an antibody conjugate comprising an anti-PD-L1 sdAb-containing antibody or antigen-binding fragment thereof of the present invention conjugated to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent, or drug.

[0107] The present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, including the anti-PD-L1 sdAbs disclosed herein, expression vectors containing the nucleic acid molecules, and host cells transformed with the expression vectors.

[0108] Additionally, the present invention provides a method for producing an antibody or antigen-binding fragment thereof, comprising the steps of: (a) culturing a host cell under conditions that allow expression of the antibody; and (b) recovering the expressed antibody or antigen-binding fragment thereof.

[0109] In the present invention, DNA encoding antibodies or antigen-binding fragments thereof, including the anti-PD-L1 sdAbs disclosed herein, may 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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).

[0117] 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.

[0118] The present invention also provides pharmaceutical compositions for the prevention or treatment of cancer, which contain as an active ingredient an antibody or antigen-binding fragment thereof comprising the anti-PD-L1 sdAb disclosed herein, or an antibody conjugate comprising said antibody or antigen-binding fragment.

[0119] Furthermore, the present invention provides methods for preventing or treating cancer, comprising the step of administering to an individual a pharmaceutical composition comprising a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof comprising an anti-PD-L1 sdAb disclosed herein, or an antibody conjugate comprising said antibody or antigen-binding fragment thereof.

[0120] The present invention also provides the use of an antibody or antigen-binding fragment thereof comprising an anti-PD-L1 sdAb disclosed herein, or an antibody conjugate comprising said antibody or antigen-binding fragment thereof, for use in the prevention or treatment of cancer.

[0121] 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.

[0122] In the present invention, the details regarding antibodies or antigen-binding fragments thereof comprising the anti-PD-L1 sdAb, and antibody conjugates comprising the antibodies or antigen-binding fragments thereof, are the same as those described above, so the details described above are hereby incorporated by reference. Only the specific components of the pharmaceutical compositions and uses will be described below.

[0123] Pharmaceutical compositions of the invention may comprise an antibody or antigen-binding fragment thereof comprising at least one (e.g. two or three) anti-PD-L1 sdAb as described herein, or said antibody or antigen-binding fragment thereof.

[0124] Cancer can be prevented or treated by administering the pharmaceutical composition of the present invention to an individual, particularly a cancer patient.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] The present invention also provides (i) contacting the sample with an antibody or antigen-binding fragment thereof, including an anti-PD-L1 sdAb disclosed herein; (ii) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and PD-L1 or determining the amount of the complex; The present invention provides a method for detecting PD-L1 or determining the amount of PD-L1 in a sample, comprising:

[0136] In the present invention, the details regarding antibodies or antigen-binding fragments thereof comprising the anti-PD-L1 sdAb, and antibody complexes comprising the antibodies or antigen-binding fragments thereof, are the same as those described above, and therefore the details are hereby incorporated by reference.

[0137] In the present invention, the sample may be a cell sample, ie, a sample containing cells such as cancer cells.

[0138] The antibodies or antigen-binding fragments of the present invention can be used to detect PD-L1 or PD-L1-expressing cells or to determine the amount of PD-L1 or PD-L1-expressing cells. PD-L1 or PD-L1-expressing cells are detected or the amount of PD-L1 or PD-L1-expressing cells is determined by detecting or determining the amount of complex between PD-L1 and the antibodies or antigen-binding fragments of the present invention. The formation of a complex indicates the presence of PD-L1 or PD-L1-expressing cells. Such detection or determination of amount may be carried out by a number of methods, including, but not limited to, immunodetection using the antibodies or antigen-binding fragments of the present invention. Methods using antibodies to detect peptides or proteins are well known and include ELISA, competitive binding assays, and similar methods. Generally, such assays utilize an antibody or antibody fragment that specifically binds to a target peptide or protein that is directly or indirectly linked to a label that provides for detection, such as an indicator enzyme, radiolabel, fluorophore, or paramagnetic particle. The methods of the invention allow quantitative and / or qualitative evaluations, eg absolute and / or relative evaluations, of PD-L1 levels or levels of PD-L1 expressing cells.

[0139] The present invention will be described in detail below with reference to examples and experimental examples.

[0140] 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]

[0141] Immunization and blood collection Human PD-L1 protein was mixed with an immunological adjuvant (GERBU) as the immunizing antigen and immunized three times via intramuscular injection into one alpaca. After 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 immunizing antigen 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]

[0142] Library creation and evaluation A 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]

[0143] 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]

[0144] 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 PBST and then mixed with a binding solution (2.5% Scheme's milk, 0.5% Tween 20) to form a 5 x 10 mAb solution. 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]

[0145] 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]

[0146] 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 IgG1 Fc domain The clones selected in Example 5 were cloned into a TGEX-Fc (IgG1) expression vector. For the expression of a single-domain antibody fused with a human IgG1 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 OptiPRO TMIn a medium mixture, 20 μg of plasmid DNA encoding a single-domain antibody fused to a human IgG1 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. After that, 150 μL of ExpiFectamine, which enhances the expression of single-domain antibodies fused with the human IgG1 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 human IgG1 Fc domain-fused single-domain antibody 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.

[0147] <6-2> Expression and purification of PD-L1 antigen-specific bivalent single-domain antibodies fused to 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]

[0148] Evaluation of the binding ability of a single domain antibody fused with a human IgG Fc domain to an immunogen using FACS The binding ability of the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Example 6> to the immunizing antigen was confirmed by FACS.

[0149] Specifically, the CHO-K1_PD-L1 cell line (CHO-K1 cells overexpressing PD-L1 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% 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, and the cells were then washed twice with DPBS. 3x10 cells were then added to each well. 5 Cells were dispensed at 100μL / well and then treated with various concentrations of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4), with an isotype control antibody used as a negative control. The cells were incubated at 4°C for 1 hour and then washed twice with DPBS. Subsequently, the cells were treated with an antibody that specifically binds to the human Fc domain (Anti-human IgG Fc APC antibody (Biolegend)) and incubated for 30 minutes at 4°C in the dark. The cells were washed twice with DPBS and then resuspended in 100μL of DPBS. FACS analysis was performed using an Accuri C6 (BD) instrument. [Example]

[0150] FACS-based evaluation of the inhibitory potential of 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) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Example 6> on PD-1 / PD-L1 interaction were evaluated.

[0151] Specifically, to evaluate the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) on the PD-1 / PD-L1 interaction, CHO-K1_PD-L1 cell line (CHO-K1 cells that consistently express PD-L1 antigen) was plated at 2 × 10 cells per well in a 96-well microplate. 5 Cells were aliquoted and treated with 10 μg / mL human PD-1-His protein. They were then treated with various concentrations of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4), as well as an isotype control antibody 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 PE). The cells were incubated in the dark for 30 minutes at 4°C. 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 expression of PD-L1 antigen) was determined using an Accuri C6 (BD) device to assess the inhibitory effects of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) on PD-1 / PD-L1 interaction. [Example]

[0152] Affinity evaluation of single-domain antibodies fused with human IgG Fc domains to immunogens The affinity (Kd) of the anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in Example 6 for the immunogen protein was measured using an Octet RED 96e (ForteBio) instrument.

[0153] Specifically, a biosensor chip (ForteBio) coated with anti-human Fc was saturated at the 1.5 nM level with 5 μg / mL anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) in a 96-well microplate (Greiner). PD-L1 antigen was 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 of the samples were analyzed for 200 and 400 seconds, respectively. The resulting data were analyzed using a 1:1 interaction model (global fitting). [Example]

[0154] In vitro efficacy evaluation of anti-PD-L1 bivalent HCAb (PP Nb-IgG4) The in vitro efficacy of the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in Example 6 was evaluated using CHO-K1_PD-L1 cells (CHO-K1 cells induced to overexpress PD-L1 antigen) and PD-1-expressing Jurkat cells.

[0155] 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 bivalent HCAb (PP Nb-IgG4) at various concentrations for 1 hour. 5 x 10 5 Jurkat cells were prepared at a concentration of 100µL and treated with 0.5mg / mL PHA. These were then added to CHO-K1_PD-L1 cells treated with anti-PD-L1 bivalent HCAb (PP Nb-IgG4) 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 bivalent HCAb (PP Nb-IgG4). [Example]

[0156] In vivo efficacy evaluation of anti-PD-L1 bivalent HCAb (PP Nb-IgG4) The in vivo efficacy of the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Example 6> was evaluated by injecting a tumor cell line (B16F10 cells) induced to express human PD-L1 into C57BL / 6 mice, and evaluating the anti-tumor effect of the anti-PD-L1 bivalent HCAb (PP Nb-IgG4).

[0157] Specifically, 6- to 8-week-old C57BL / 6 female mice were inoculated with 8 × 10 B16F10_PD-L1 cell lines (B16F10 cells induced to overexpress PD-L1). 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 bivalent HCAb (PP 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 bivalent HCAb (PP Nb-IgG4).

[0158] <Experimental Example 1> Preparation of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) Using human PD-L1 antigen as the immunizing antigen, single domain antibody clones specific for PD-L1 protein were selected and sequenced using the same method as described in Example 5. The amino acid sequences of the selected anti-PD-L1 sdAb (PDL1 Nb#01) are shown in Tables 5 and 6 below.

[0159] Furthermore, using the selected anti-PD-L1 sdAb (PD-L1 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.

[0160] 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 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.

[0161] [Table 5]

[0162] [Table 6]

[0163] [Table 7]

[0164] [Table 8]

[0165] [Table 9]

[0166] Experimental Example 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 PD-1 / PD-L1 interaction Similar to the methods described in <Example 7> and <Example 8>, 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.

[0167] 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).

[0168] 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).

[0169] <Experimental Example 3> Evaluation of the affinity of anti-PD-L1 HCAb (PDL1 Nb#01-IgG1) and anti-PD-L1 bivalent HCAb (PP Nb-IgG4) with 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>.

[0170] 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.

[0171] [Table 10]

[0172] <Experimental Example 4> In vitro efficacy evaluation of anti-PD-L1 bivalent HCAb (PP Nb-IgG4) The in vitro efficacy was evaluated using the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Experimental Example 1>.

[0173] Specifically, in a similar manner to that described in Example 10, 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 bivalent HCAb (PP Nb-IgG4), and the concentration of IL-2 expressed by Jurkat cells was measured to assess in vitro efficacy.

[0174] As shown in Figure 3, the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) interfered with the interaction between PD-1-expressing Jurkat cells and PD-L1-expressing CHO-K1 cells, demonstrating an inhibitory effect of 0.80 nM (IC50) on the interaction between CHO-K1 cells and Jurkat cells.

[0175] Experimental Example 5: Evaluation of the in vivo efficacy of anti-PD-L1 bivalent HCAb (PP Nb-IgG4) The in vivo efficacy of the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) purified in <Experimental Example 1> was evaluated using a mouse tumor model.

[0176] Specifically, using a method similar to that described in Example 11, anti-PD-L1 bivalent HCAb (PP Nb-IgG4) was injected with tumor cells (B16F10 cells) expressing the PD-L1 antigen, and the antitumor effect of intraperitoneal administration was observed in a mouse model in which tumors had formed.

[0177] As a result, as shown in Figure 4, it was confirmed that the anti-PD-L1 bivalent HCAb (PP Nb-IgG4) exhibited an antitumor effect of approximately 39.2% compared to the negative control group (isotype group). [Industrial Applicability]

[0178] The single domain antibody of the present invention exhibits excellent affinity and antitumor effect for PD-L1, an immune checkpoint protein, and can therefore be usefully used as an immune checkpoint inhibitor in immune anticancer therapy.

Claims

1. A single domain antibody (sdAb) or antigen-binding fragment thereof that specifically binds to PD-L1, The sdAb or an antigen-binding fragment thereof, wherein the sdAb consists of the amino acid sequence represented by SEQ ID NO:

10.

2. 2. The sdAb or antigen-binding fragment thereof of claim 1, wherein the sdAb is a heavy chain-only antibody (HCAb) fused to an Fc fragment.

3. 3. The sdAb or antigen-binding fragment thereof of claim 2, wherein the HCAb is monomeric or multimeric.

4. 3. The sdAb or antigen-binding fragment thereof of claim 2, wherein the Fc fragment is human IgG1, IgG2, IgG3 or IgG4.

5. 3. The sdAb or antigen-binding fragment thereof of claim 2, wherein the sdAb is fused to an Fc fragment via a peptide linker.

6. The sdAb or antigen-binding fragment thereof according to claim 2, wherein the HCAb consists of the amino acid sequence represented by SEQ ID NO:

11.

7. 2. The sdAb or antigen-binding fragment thereof of claim 1, comprising: (a) a first antigen-binding portion comprising the sdAb; and (b) a second antigen-binding portion that specifically binds to a second epitope.

8. 8. The sdAb or antigen-binding fragment thereof of claim 7, wherein the second antigen-binding portion is a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv), scFv-scFv, minibody, diabody or second sdAb.

9. 8. The sdAb or antigen-binding fragment thereof of claim 7, wherein the first antigen-binding portion and the second antigen-binding portion are fused to each other via a peptide linker.

10. 10. The sdAb or antigen-binding fragment thereof of claim 1 conjugated to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent or drug.

11. 10. An antibody conjugate comprising the sdAb or antigen-binding fragment thereof of claim 1 conjugated to an immunomodulatory agent, cytokine, cytotoxic agent, chemotherapeutic agent, diagnostic agent, antiviral agent, antimicrobial agent or drug.

12. A nucleic acid molecule encoding the sdAb or antigen-binding fragment thereof of claim 1.

13. An expression vector comprising the nucleic acid molecule described in claim 12.

14. A host cell transformed with the expression vector described in claim 13.

15. (a) culturing the host cell of claim 14 under conditions that allow expression of the sdAb; (b) recovering the expressed sdAb or antigen-binding fragment thereof; 2. A method for producing an sdAb or antigen-binding fragment thereof, comprising:

16. A pharmaceutical composition for preventing or treating cancer, comprising the sdAb or antigen-binding fragment thereof according to claim 1, or the antibody complex according to claim 11, as an active ingredient.

17. 17. The pharmaceutical composition of claim 16, wherein the cancer is 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.

18. 17. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

19. (i) contacting the sdAb or antigen-binding fragment thereof of claim 1 with a sample; (ii) detecting complex formation or determining the amount of complex between the sdAb or antigen-binding fragment thereof and PD-L1; A method for detecting PD-L1 or determining the amount of PD-L1 in a sample, comprising:

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