Bispecific antibodies that specifically bind to CD47 and PD-L1.

Abstract

The present invention relates to a bispecific antibody, which exhibits excellent antitumor effects and minimizes hemagglutination side effects by having high binding affinity to tumor cells expressing PD-L1 and / or CD47.

Description

[Technical Field]

[0001] This invention relates to a bispecific antibody. Specifically, this invention relates to a bispecific antibody that specifically binds to CD47 and PD-L1, its uses, and a method for producing it. [Background technology]

[0002] The body's immune function is regulated through co-stimulatory and co-inhibitory signals that occur simultaneously with antigen recognition. Cancer cells, however, evade immune attack by altering the tumor microenvironment or suppressing immune function. One such evasion strategy involves altering the immune barrier to suppress the function of immune cells. For example, cancer cells express specific cell surface proteins such as PD-L1 or CD47, which bind to immune cells such as T cells or macrophages, thereby suppressing their function.

[0003] With the elucidation of this mechanism, the development of immune barrier inhibitors that block the activity of immune barrier proteins, such as the aforementioned specific cell surface proteins, thereby activating immune cells to attack cancer cells, is actively underway. As a result, various anti-PD-L1 monoclonal antibodies such as Tecentriq (Atezolizumab), Imfinzi (Durvalumab), and Bavencio (Avelumab) have received FDA approval and are either commercially available or in clinical trials. In addition, many anti-CD47 monoclonal antibodies are being researched or are in clinical trials.

[0004] However, immunobarrier inhibitors have problems such as insufficient patient response rates, side effects, and low bioavailability, and the development of new immunobarrier inhibitors is still needed. [Overview of the project] [Problems that the invention aims to solve]

[0005] The purpose of this invention is to solve all of the aforementioned problems.

[0006] One objective of this invention is to provide a bispecific antibody that specifically binds to CD47 and PD-L1.

[0007] Another objective of this invention is to provide a bispecific antibody that specifically binds to CD47 and PD-L1 for cancer prevention or treatment.

[0008] The present invention also aims to provide applications for the production of cancer prophylactic or therapeutic agents using bispecific antibodies that specifically bind to CD47 and PD-L1.

[0009] Another objective of the present invention is to provide a pharmaceutical composition comprising bispecific antibodies that specifically bind to CD47 and PD-L1.

[0010] The present invention is further intended to provide a method for the prevention or treatment of cancer, comprising the step of administering a bispecific antibody that specifically binds to CD47 and PD-L1.

[0011] The present invention is further intended to provide polynucleotides, expression vectors, host cells, or methods for producing bispecific antibodies that specifically bind to CD47 and PD-L1.

[0012] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become clearer in the following description and will be achieved by the means and combinations described in the claims. [Means for solving the problem]

[0013] A typical configuration of the present invention for achieving the above objective is as follows:

[0014] According to one aspect of the present invention, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to PD-L1, wherein the first antigen-binding domain comprises a heavy chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 19, and CDR3 containing the amino acid sequence of SEQ ID NO: 21; and a light chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 23, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 27; and the second antigen-binding domain comprises a heavy chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 31, and CDR3 containing the amino acid sequence of SEQ ID NO: 33; and a light chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 35, CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0015] In a particular embodiment, the bispecific antibody comprises a first antigen-binding domain including the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 3, and a second antigen-binding domain including the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 7.

[0016] According to a particular embodiment, the bispecific antibody comprises a first antibody fragment (e.g., a Fab fragment) containing an Fc domain and an antigen-binding domain that specifically binds to CD47, and a second antibody fragment (e.g., an scFv fragment) containing an antigen-binding domain that specifically binds to PD-L1.

[0017] In a particular embodiment, in a bispecific antibody, the second antibody fragment (e.g., scFv fragment) is fused to the C-terminus of the Fc domain via a peptide linker.

[0018] According to a particular embodiment, the bispecific antibody comprises an Fc domain, two Fab fragments containing antigen-binding domains that specifically bind to CD47, and two scFv fragments containing antigen-binding domains that specifically bind to PD-L1.

[0019] According to a specific embodiment, the bispecific antibody comprises a single-chain variable fragment (scFv) comprising an antigen-binding domain that specifically binds to PD-L1.

[0020] According to a specific embodiment, the bispecific antibody comprises a Fab fragment comprising an antigen-binding domain that specifically binds to CD47.

[0021] According to a specific embodiment, the bispecific antibody comprises an Fc region derived from the heavy chain constant region (CH) of IgG1.

[0022] According to a specific embodiment, the Fc region of the bispecific antibody comprises heavy chain constant regions CH1, CH2 and CH3 derived from IgG1, wherein CH3 comprises the amino acid substitutions E239D and M241L with reference to SEQ ID NO: 11. Said Fc region can act on effector cells to exhibit an immunostimulatory effect, particularly an increased immunostimulatory effect.

[0023] According to a specific embodiment, the bispecific antibody comprises an antibody fragment comprising the antigen-binding domain that specifically binds to PD-L1, and said antibody fragment is fused to the C-terminus of said Fc domain via a peptide linker.

[0024] According to a specific embodiment, the single-chain variable fragment (scFV) of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 9.

[0025] According to a specific embodiment, the Fc region of the bispecific antibody comprises the amino acid sequence of SEQ ID NO: 11.

[0026] According to a specific embodiment, the bispecific antibody specifically binds to cancer cells that express PD-L1, CD47, or both.

[0027] According to another aspect of the present invention, the bispecific antibodies of the present invention are provided for use in the prevention or treatment of various cancers, including breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma.

[0028] According to yet another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of various cancers, including breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma, is provided, comprising the bispecific antibody of the present invention.

[0029] According to yet another aspect of the present invention, the bispecific antibody of the present invention may be used in combination with anticancer chemotherapy, radiotherapy and / or other immunoanticancer agents.

[0030] A further aspect of the present invention provides a method for the prevention or treatment of various cancers, including breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma, comprising the step of administering an effective amount of the bispecific antibody of the present invention to a subject in need of prevention or treatment of various cancers, including breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma.

[0031] According to yet another aspect of the present invention, a method is provided for inhibiting the growth of tumor cells in a subject, comprising the step of administering an effective amount of the bispecific antibody of the present invention to a subject having tumor cells.

[0032] According to yet another aspect of the present invention, a polynucleotide is provided that encodes the bispecific antibody of the present invention.

[0033] According to yet another aspect of the present invention, a host cell containing a polynucleotide that encodes the bispecific antibody of the present invention is provided.

[0034] According to yet another aspect of the present invention, a method for producing a bispecific antibody of the present invention is provided, comprising the steps of culturing host cells containing polynucleotides that encode the bispecific antibody under conditions suitable for the expression of the bispecific antibody, and recovering the bispecific antibody from the culture. [Effects of the Invention]

[0035] According to the present invention, bispecific antibodies that specifically bind to CD47 and / or PD-L1 can be effectively used for the prevention or treatment of a variety of cancers, including breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma. Specifically, the bispecific antibodies of the present invention exhibit superior antigen-binding ability, particularly much higher binding ability to the CD47 antigen, compared to anti-CD47 antibodies and / or anti-PD-L1 antibodies, while maintaining low binding ability to red blood cells (RBCs). As a result, the bispecific antibodies of the present invention exhibited excellent antitumor effects by having high binding affinity to tumor cells expressing CD47 and / or PD-L1, and were confirmed to exhibit low red blood cell (RBC) binding ability and minimized hemagglutination side effects. Furthermore, the bispecific antibodies of the present invention showed cancer-suppressing effects and NK cell and T cell activation effects in cells expressing PD-L1 and / or CD47 through ADCC (Antibody-Dependent Cellular Cytotoxicity) activity evaluation. Furthermore, the bispecific antibody of the present invention demonstrated a cancer growth inhibitory effect in the Xenograft model of breast cancer cell line using BALB / c nude mice lacking acquired immune cells, and in the Syngeneic model of the colorectal cancer CT26-hPD-L1 / hCD47 cell line using BALB / c-hPD-1 / hSIRPα mice. It also demonstrated an inhibitory effect on tumor re-induction through the generation of memory T cells. Therefore, the bispecific antibody of the present invention can be effectively used for the prevention, improvement, or treatment of various cancers, including breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma, through its cancer growth inhibitory effect and immune cell activation effect. [Brief explanation of the drawing]

[0036] [Figure 1] A schematic diagram of the bispecific antibody against CD47 and PD-L1 according to the present invention is shown.

[0037] [Figure 2]The results of 4% to 20% denaturation, reduction, and non-reduction, as well as SDS-PAGE analysis of the antibodies produced in Example 1.2, are shown. Molecular weight markers are expressed in kilodaltons.

[0038] [Figure 3] The analytical gel-filtration elution profile observed for the antibody prepared in Example 1.2 is shown (Column: Superdex 200 increase 10 / 300 GL; Absorption wavelength: 214 nm; Buffer: Phosphate-buffered saline (PBS); Temperature (°C): 19; Flow rate (ml / min): 0.75; SOP / WI used: WI PP 24.0; Lot number: 120520).

[0039] [Figure 4A-B] The results of ELISA analysis of single and dual ligand binding positivity for bispecific antibodies against CD47 and PD-L1, respectively, are shown.

[0040] [Figure 5] These show the binding affinity of bispecific antibodies to PD-L1-rich tumor cells and CD47-rich tumor cells, respectively.

[0041] [Figure 6A-D] These figures show the binding affinity of bispecific antibodies to MDA-MB-231 WT, PD-L1 knockout (KO), CD47 KO, and PD-L1 / CD47 dual KO cells, respectively.

[0042] [Figure 7] The results of RBC binding assays performed on bispecific antibodies are shown.

[0043] [Figure 8] The results of hemagglutination testing performed on bispecific antibodies are shown.

[0044] [Figure 9]The results of phagocytosis of bispecific antibodies against four cell types (MDA-MB-231, H1975, Raji, and SR786) are shown.

[0045] [Figure 10A-B] The results of the ADCC NFAT-Luc assay performed on bispecific antibodies in Raji cells and MDA-MB-231 cells are shown.

[0046] [Figure 11] The results of performing ADCC testing on NK cells using MDA-MB-231 cells against a bispecific antibody are shown.

[0047] [Figure 12] The results of MLR analysis performed to confirm the T-cell activation effect of the bispecific antibody are shown.

[0048] [Figure 13] The tumor inoculation sites of the mice mentioned in Examples 3.1 to 3.3 are shown.

[0049] [Figure 14] This is an efficacy study result confirming the suppression of cancer growth by a bispecific antibody in BALB / c nude mice transplanted with the human MDA-MB-231 cell line.

[0050] [Figure 15] This is an efficacy study that confirmed the suppression of cancer growth by a bispecific antibody in CT26-hPD-L1 / hCD47 tumor allogeneic transplanted BALB / c-hPD-1 / hSIRPα mice.

[0051] [Figure 16] The tumor volume curves in BALB / c-hPD-1 / hSIRPα mice treated with the CT26-hPD-L1 / hCD47 tumor allograft model, related to the re-induction study in Example 3.3, are shown. Data are presented mean ± SEM. [Modes for carrying out the invention]

[0052] The detailed description of the present invention, as described below, will be written with reference to specific drawings relating to certain embodiments in which the invention may be carried out, but the invention is not limited thereto and is limited only by the appended claims, along with all equivalents to those claimed thereto, where appropriately described. The technical and scientific terms used herein have the same meanings as those commonly used in the art to which the invention belongs, unless otherwise defined. The following definitions apply for the purposes of interpreting this specification, with singular terms including plural forms where appropriate, and vice versa.

[0053] definition

[0054] As used herein, the term "antigen-binding molecule" refers to a molecule that specifically binds to an antigen-determining factor. Examples of antigen-binding molecules include, but are not limited to, antibodies, antibody fragments, and scaffold antigen-binding proteins.

[0055] The term "antibody" is used in its broadest sense to include a wide variety of antibody structures, such as unrestricted monoclonal antibodies, polyclonal antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), antibody fragments with antigen-binding activity, and antibody fusions (e.g., fusions of antibodies with (poly)peptides or antibodies with compounds). In this specification, the prefix "anti-" means that the antibody is reactive with the antigen in question, when used in relation to an antigen. Antibodies reactive with a specific antigen may be produced by synthetic and / or recombinant methods, such as the selection of recombinant antibody libraries with phages or similar vectors, or by immunization of animals with the antigen or antigen-coding nucleic acids. A typical IgG antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain contains an invariant region and a variable region. The heavy chain variable region (HVR) and light chain variable region (LVR) each contain three sections referred to as "complementarity-determining regions" ("CDRs") or "hypervariable regions," which are primarily involved in binding to the antigen epitope. These are usually designated as CDR1, CDR2, and CDR3, sequentially numbered from the N-terminus. The more well-conserved regions within the variable regions outside the CDRs are referred to as "skeleton regions" ("FRs"). In this specification, antibodies may be, for example, animal antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0056] The term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially the same. That is, the individual antibodies in the population are identical to each other, or bind to the same epitope, except for possible mutant antibodies present in trace amounts (e.g., antibodies with spontaneously occurring mutations during monoclonal antibody production). In contrast to polyclonal antibody preparations, which contain different antibodies induced against typically different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is induced against a single determinant on an antigen.

[0057] The term "monospecific" refers to an antibody that has one or more binding sites, each binding site binding to the same epitope of the same antigen. The term "bispecific" means that an antigen-binding molecule can specifically bind to two or more distinct antigen-determining factors. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific to a different antigen-determining factor. In some embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigen-determining factors, for example, two antigen-determining factors expressed on two distinct cells. Furthermore, the bispecific antigen-binding molecules described in this application can form part of a multispecific antibody.

[0058] The term "antibody fragment" refers to a portion of an antibody or a polypeptide containing such fragment that has specific binding ability to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments and single-domain antibodies.

[0059] The term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are covalently linked to form a VH-VL heterodimer. The heavy chain (VH) and light chain (VL) are linked directly, by linking the N-terminus of VH to the C-terminus of VL, or by a peptide linker that links the C-terminus of VH to the N-terminus of VL.

[0060] The term "antigen-binding domain" refers to the portion of an antigen-binding molecule that specifically binds to an antigen-determining factor. The antigen-binding domain may be provided, for example, by one or more variable regions. Preferably, the antigen-binding domain may include an antibody light chain variable region and an antibody heavy chain variable region.

[0061] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, meaning an homodimer consisting of a portion of the hinge region, the CH2 and CH3 domains in each chain. The Fc region represents an intact antibody (e.g., a portion of IgG) produced by digestion with the enzyme papain. In one embodiment, the "Fc region" may be a natural sequence Fc region or a mutant Fc region. A "natural sequence Fc region" represents an amino acid sequence identical to the amino acid sequence of a naturally occurring and commonly found Fc region. In this invention, the Fc region may be an extended Fc region that includes a portion or all of the heavy chain invariant region 1 (CH1) and / or light chain invariant region 1 (CL1), excluding only the heavy chain and light chain variable regions of the immunoglobulin, as long as it has substantially equivalent or improved effects to the natural type. Alternatively, it may be a region from which a very long portion of the amino acid sequence corresponding to CH2 and / or CH3 has been removed. For example, in the present invention, the Fc region may be a combination of 1) CH1, CH2 and CH3, 2) CH1 and CH2, 3) CH1 and CH3, 4) CH2 and CH3, 5) one or more domains from CH1 to CH3 and an immunoglobulin hinge region (or part of a hinge region), or 6) a dimer of each domain of the heavy chain invariant region and the light chain invariant region. Furthermore, in the present invention, the Fc region includes not only the native amino acid sequence but also variants of this sequence. An amino acid sequence variant means that one or more amino acid residues in the native amino acid sequence have a different sequence due to fruiting, insertion, non-conservative or conservative substitution, or a combination thereof.

[0062] The term "CD47" refers to a protein known to be found on the surface of cancer cells. It is known to bind to SIRPα in macrophages and send a "Do-not-eat-me" signal, thereby blocking the phagocytic activity of macrophages.

[0063] The term "PD-L1" refers to a protein found on the surface of cancer cells. It binds to PD-1, a protein found on the surface of T cells, preventing T cells from attacking cancer cells.

[0064] The term "linker" can refer to a peptide linker having a length of 1 to 100 amino acids, specifically 2 to 50, and more specifically 5 to 30 amino acids. The linker may, but is not limited to, contain one or more amino acids selected from the group consisting of, for example, Gly, Asn, Ser, Thr, Ala, Asp, etc. As an example, the linker may be represented as (GGGGS)n, where n is the number of repeats of the (GGGGS) unit, which may be 1 to 10, specifically 1 to 5, taking into consideration the efficacy of the bispecific antibody. As yet another example, the linker may be a peptide fragment known to be able to link antibody fragment domains, and may have, for example, the amino acid sequence GGGGSGGGGSGGGGS.

[0065] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-spontaneously occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule of the present invention includes an antibody, or a continuous open reading frame encoding a fragment, derivative, mutain, or variant thereof.

[0066] The term "subject" is used interchangeably with "patient" and can refer to mammals requiring cancer prevention or treatment, such as primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), domestic animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In one embodiment of the present invention, the subject is a human.

[0067] The term “treatment” generally means obtaining a desired pharmacological and / or physiological effect. Such an effect is therapeutic in that it partially or completely cures the disease and / or side effects caused by such disease. Preferred therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, improvement of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or mitigation of the disease state, and recovery or improved prognosis. Preferably, “treatment” may mean a medical intervention for a disease or disorder that has already manifested.

[0068] The term "prevention" refers to preventive treatment, that is, obtaining an effect that prevents disease rather than treats it. "Prevention" means obtaining a desired preventive pharmacological and / or physiological effect in the sense of partially or completely preventing the disease or its symptoms.

[0069] The term "administration" means providing a substance (e.g., the bispecific antibody of the present invention) to a target body to achieve a prophylactic or therapeutic purpose.

[0070] The bispecific antibody of the present invention

[0071] The present invention provides a novel bispecific antibody comprising a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to PD-L1. The antibody can specifically bind to cancer cells expressing CD47, PD-L1, or both, and has particularly advantageous characteristics such as binding affinity, biological activity, immune cell activation, targeted efficacy, tumor reinduction suppression, and reduced side effects.

[0072] According to one aspect of the present invention, a bispecific antibody is provided comprising a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to PD-L1, wherein the first antigen-binding domain comprises a heavy chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 19, and CDR3 containing the amino acid sequence of SEQ ID NO: 21; and a light chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 23, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO: 27; and the second antigen-binding domain comprises a heavy chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 31, and CDR3 containing the amino acid sequence of SEQ ID NO: 33; and a light chain variable region including CDR1 containing the amino acid sequence of SEQ ID NO: 35, CDR2 containing the amino acid sequence of SEQ ID NO: 37, and CDR3 containing the amino acid sequence of SEQ ID NO: 39.

[0073] In some embodiments, the first antigen-binding domain that specifically binds to CD47 may include / include the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 3, or the second antigen-binding domain that specifically binds to PD-L1 may include the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 7. In particular, the second antigen-binding domain that specifically binds to PD-L1 may include the single-chain variable fragment (scFv) of SEQ ID NO: 9.

[0074] In some embodiments, the bispecific antibody of the present invention may include a first antibody fragment comprising an Fc region and an antigen-binding domain that specifically binds to the CD47, and a second antibody fragment comprising an antigen-binding domain that specifically binds to the PD-L1.

[0075] In some embodiments, the first antibody fragment may be a Fab fragment.

[0076] In some embodiments, the second antibody fragment may be a single-chain variable fragment (scFv).

[0077] In some embodiments, the Fc region can act on effector cells to exhibit an immunoactivating effect. Preferably, the Fc region may be derived from the heavy chain invariant region (CH) of IgG1. More preferably, the Fc region may include heavy chain invariant regions CH1, CH2, and CH3 derived from IgG1, or heavy chain invariant regions CH2 and CH3 derived from IgG1. Most preferably, the CH3 of the Fc region may include the amino acid substitutions E239D and M241L based on SEQ ID NO: 11.

[0078] In some embodiment, the Fc region may contain the amino acid sequence of SEQ ID NO: 11.

[0079] In some embodiments, the second antibody fragment can be fused to the C-terminus of the Fc region. More specifically, the second antibody fragment can be fused to the C-terminus of the Fc region through the heavy chain variable region (i.e., the N-terminus of the second antibody fragment) (see Figure 1). In this case, the second antibody fragment can be fused to the C-terminus of the Fc region through a linker. The linker may be a peptide linker and may have an amino acid sequence such as GGGGS, GGGGSGGGGSGGGGS, or GSGSGSGSGSGSGSGSGS.

[0080] In some embodiments, the second antibody fragment may include a variable region (e.g., a single-chain variable fragment scFv) in which a heavy-chain variable region and a light-chain variable region are linked via a linker. For example, the linker may be a peptide linker represented by (GGGGS)n, where n is the number of repeats of the (GGGGS) unit, and may be 1 to 10, specifically 1 to 5, taking into account the efficacy of the bispecific antibody. Preferably, the second antibody fragment may be an scFv fragment. More preferably, the scFv fragment may include the amino acid sequence of SEQ ID NO: 9.

[0081] In some embodiments, the bispecific antibody of the present invention may include two Fab fragments, each containing an Fc domain and an antigen-binding domain that specifically binds to CD47, and two scFv fragments, each containing an antigen-binding domain that specifically binds to PD-L1. In this case, the heavy chain variable domain of each scFv fragment may be fused to the C-terminus of the Fc domain (linked to the heavy chain of each Fab fragment) via a peptide linker.

[0082] In some embodiments, the bispecific antibody of the present invention comprises a full-length antibody that specifically binds to CD47 and includes two antibody heavy chains and two antibody light chains, and an antibody fragment (e.g., an scFv fragment) that includes a heavy chain variable region and a light chain variable region that specifically binds to PD-L1, wherein the heavy chain variable region of the antibody fragment may be fused directly to the C-terminus of the heavy chain of the full-length antibody or via a peptide linker.

[0083] In some embodiments, the bispecific antibodies of the present invention may include sequences having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the CDR sequence, heavy chain variable region sequence, and / or light chain variable region sequence.

[0084] In specific embodiments, amino acid sequence variants of the bispecific antibodies of the present invention are considered. For example, it is preferable to improve the antibody's binding affinity and / or other biological properties. Amino acid sequence variants of antibodies can be produced by introducing appropriate modifications into the nucleotide sequence encoding the molecule, or by peptide synthesis. Such modifications include, for example, the fruiting of residues from the amino acid sequence of the antibody, and / or the insertion of residues into such an amino acid sequence, and / or the substitution of residues within such an amino acid sequence. Any combination of various modifications, including fruiting, insertion, and substitution, can be performed to arrive at the final construct, but the final construct must possess the desired properties, such as antigen-binding properties. Sites of interest for substitutional mutagenesis include the heavy chain variable region (HVR) and the skeletal region (FR). Conservative substitutions are provided in Table 1 under the item “Preferred Substitutions,” and are further described below in relation to amino acid side chain groups (1) to (6). Amino acid substitutions can be introduced into the product screened for the molecule of interest and the desired activity, such as maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0085] [Table 1]

[0086] Amino acids can be grouped as follows based on the properties of their typical side chains:

[0087] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0088] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0089] (3) Acidic: Asp, Glu;

[0090] (4) Basicity: His, Lys, Arg;

[0091] (5) Residues that affect chain orientation: Gly, Pro;

[0092] (6) Aromatic: Trp, Tyr, Phe.

[0093] Non-conservative substitution involves replacing one member of one such category with another.

[0094] In this specification, the term “amino acid sequence variant” includes substantial variants in which amino acid substitutions are present in one or more hypervariable region residues of a parent antibody-binding molecule (e.g., a humanized or human antibody). Generally, generated variants selected for further study will have / have / substantially retain certain biological properties compared to the parent antibody-binding molecule, e.g., modifications, e.g., improvements (e.g., increased affinity, decreased immunogenicity), or substantially retain certain biological properties of the parent antigen-binding molecule. An exemplary substitution variant is an affinity-mature antibody, which can be conveniently generated, for example, by utilizing phage display-based affinity-mature techniques known in the art. Briefly, one or more HVR residues are mutated, and the variant antigen-binding molecule is displayed on a phage for screening of specific biological activity (e.g., binding affinity). In specific embodiments, substitutions, insertions, or fruitings may occur within one or more HVRs, provided that such modifications do not substantially reduce the ability of the antigen-binding molecule to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions, as provided herein) may be made in HVRs.

[0095] Amino acid sequence insertions can include not only the insertion of a single or multiple amino acid residues, but also amino-terminal and / or carboxyl-terminal fusions, which range in length from one residue to polypeptides containing more than one hundred residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the molecule can include fusions of polypeptides to the N-terminus or C-terminus to increase the serum half-life of the antibody. Other insertion variants of the molecule can include fusions of polypeptides to the N-terminus or C-terminus to facilitate crossing the blood-brain barrier (BBB).

[0096] Nucleic acids, vectors, host cells, and methods for manufacturing them

[0097] The bispecific antibodies of the present invention can be produced by any antibody production technique known in the industry.

[0098] According to another aspect of the present invention, an isolated nucleic acid (e.g., polynucleotide) is provided that encodes a bispecific antibody or fragment thereof as described in the present invention. Such a nucleic acid can encode an amino acid sequence containing the heavy chain variable region or heavy chain CDR region and / or an amino acid sequence containing the light chain variable region or light chain CDR region of the bispecific antibody.

[0099] In some embodiments, the bispecific antibodies of the present invention may be expressed by a single polynucleotide or by multiple polynucleotides that co-express, encoding the overall antigen-binding molecule. The polypeptide encoded by the multiple polynucleotides can be bound, for example, through disulfide bonds or other means that form a functional antigen-binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a polynucleotide separated from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide will bind to the light chain polypeptide for forming the immunoglobulin.

[0100] In one embodiment, the polynucleotide may include the sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 4. Furthermore, the polynucleotide sequence may also include the sequence of SEQ ID NO: 12.

[0101] In other embodiments, the polynucleotide may include the sequence of SEQ ID NO: 6 and the sequence of SEQ ID NO: 8. Furthermore, the polynucleotide sequence may include the sequence of SEQ ID NO: 10.

[0102] In further embodiments, the polynucleotide may be or contain the sequence of SEQ ID NO: 15 and / or the sequence of SEQ ID NO: 16.

[0103] In further embodiments, the polynucleotide may include a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or about 100% identity with the polynucleotide encoding the bispecific antibody of the present invention.

[0104] In another embodiment, the polynucleotide can encode the polypeptide contained in the bispecific antibody according to the present invention.

[0105] According to yet another aspect of the present invention, one or more vectors (e.g., expression vectors) containing the nucleic acid are provided. The term “vector” is a nucleic acid that can be used to introduce other nucleic acids linked to the nucleic acid into a cell. One type of vector is a “plasmid,” which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Yet another type of vector is a viral vector (e.g., non-replicating retroviruses, adenoviruses, and adeno-associated viruses), in which case additional DNA segments can be introduced into the viral genome. Certain vectors can replicate autonomously within the host cell into which they are introduced (e.g., bacterial vectors containing bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), upon introduction into a host cell, are integrated into the host cell's genome and replicate together with the host genome. An “expression vector” is a type of vector that can induce the expression of selected polynucleotides.

[0106] According to yet another aspect of the present invention, a host cell is provided comprising one or more polynucleotides that encode the bispecific antibody of the present invention. The host cell may be a prokaryotic or eukaryotic cell. For example, the host cell may be a Chinese hamster ovary (CHO) cell or lymphocyte. Antibodies can be produced in bacteria if glycation or the like is not required.

[0107] A further aspect of the present invention provides a method for producing a bispecific antibody of the present invention, comprising the steps of culturing host cells under conditions suitable for the expression of the bispecific antibody of the present invention, and recovering the bispecific antibody from the culture.

[0108] For the recombinant production of bispecific antibodies, for example, the polynucleotides encoding the bispecific antibodies described above are isolated and inserted into one or more vectors for additional cloning and / or expression within host cells. Such nucleic acids can be isolated or sequenced using conventional processes known in the art.

[0109] The method for culturing the transformed cells can be carried out using methods widely known in the industry. For example, bispecific antibodies can be produced in large quantities by culturing the transformants in a nutrient medium, and the medium and culture conditions can be appropriately selected to match those commonly used for host cells. During cultivation, conditions such as temperature, pH of the medium, and culture time can be appropriately adjusted to suit cell growth and large-scale protein production.

[0110] To recover the bispecific antibodies, any method known in the art for the purification of immunoglobulins, such as chromatography (ion exchange, affinity (e.g., protein A), size exclusion, etc.), centrifugation, differential solubility, or other standard techniques for protein purification may be used.

[0111] Prevention or treatment methods

[0112] According to yet another aspect of the present invention, the bispecific antibodies of the present invention may be used in preventive or therapeutic methods.

[0113] In some embodiments, the disease being treated is a proliferative disorder, which may be cancer in particular. Specifically, the bispecific antibodies of the present invention may be used to suppress cancer growth, survival, metastasis, and recurrence.

[0114] In some embodiments, a method is provided for inhibiting the growth of tumor cells in a subject, comprising the step of administering an effective amount of the bispecific antibody of the present invention to a subject having tumor cells. In some embodiments, a method is provided for the prevention or treatment of cancer, comprising the step of administering an effective amount of the bispecific antibody of the present invention to a subject requiring the prevention or treatment of cancer.

[0115] In some specific examples, the cancer may be a solid tumor or a hematological cancer.

[0116] In some embodiments, the cancer may be selected from, but is not limited to, the group consisting of ovarian cancer, colon cancer, colorectal cancer, breast cancer, lung cancer, myeloma, neuronocyte-induced CNS tumors, monocytic leukemia, B-cell-induced leukemia, T-cell-induced leukemia, B-cell-induced lymphoma, T-cell-induced lymphoma, and mast cell-induced tumors. Preferably, the cancer may be any one selected from the group consisting of ovarian cancer, lung cancer, B-cell-induced lymphoma, and T-cell-induced lymphoma.

[0117] The bispecific antibody according to the present invention can be administered in various ways depending on whether local or systemic treatment is desired and the area to be treated. The method of administering the bispecific antibody to the target body may vary depending on the purpose of administration, the site of the disease, the condition of the target body, etc. The route of administration may be parenteral, inhalation, local or local administration (e.g., intralesional administration). For example, parenteral administration may include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intraarterial, intramuscular, rectal, intravaginal, intra-articular, intraprostatic, intranasal, intraocular, intrabladder, intraspine, or intracardiac (e.g., intracerebroventricular) administration. Furthermore, when used in combination, the bispecific antibody of the present invention and the additional immunosuppressant can be administered via the same route or via different routes.

[0118] In the method described above, the effective dose of the bispecific antibody according to the present invention may vary depending on the age, sex, and weight of the individual (patient), and is generally about 0.01 mg to 100 mg or 5 mg to about 50 mg per kg of body weight, administered once to several times a day. However, the scope of the present invention is not limited thereto, as the dose may be increased or decreased depending on the route and duration of administration, the severity of the disease, sex, weight, age, etc.

[0119] In some embodiments, the bispecific antibodies of the present invention may be used or administered in combination with anticancer chemotherapy agents, radiotherapy and / or other immunoanticancer agents (e.g., anti-PD-1, anti-CTLA-4 antibodies, etc.). Such combination therapies include combination administration (administration in which two or more therapeutic formulations are contained in the same or separate compositions) and individual administration, in which case the administration of the bispecific antibodies may occur prior to, simultaneously with, and / or subsequently to the application / use of additional therapeutic agents / treatments.

[0120] Pharmaceutical compositions

[0121] According to yet another aspect of the present invention, a pharmaceutical composition comprising the bispecific antibody of the present invention is provided. The bispecific antibody may be included in the composition in a prophylactic or therapeutically effective amount.

[0122] In some embodiments, the pharmaceutical composition may be administered to a subject to suppress the growth, survival, metastasis, and recurrence of cancer. Therefore, the pharmaceutical composition may be administered to a subject for the prevention or treatment of cancer. In this case, the pharmaceutical composition may be used or administered in combination with anti-cancer chemotherapy agents, radiotherapy, and / or other immunosuppressant agents (e.g., anti-PD-1, anti-CTLA-4 antibodies). Such combination therapies include combination administration (administration in which two or more therapeutic formulations are contained in the same or separate compositions) and individual administration, in which case the administration of the bispecific antibody may occur prior to, simultaneously with, and / or subsequently to the application / use of the additional therapeutic agent / treatment.

[0123] In some specific examples, the cancer may be a solid tumor or a hematological cancer.

[0124] In some embodiments, the cancer is selected from, but is not limited to, the group consisting of ovarian cancer, colon cancer, colorectal cancer, breast cancer, lung cancer, myeloma, neuronocyte-induced CNS tumors, monocytic leukemia, B-cell-induced leukemia, T-cell-induced leukemia, B-cell-induced lymphoma, T-cell-induced lymphoma, and mast cell-induced tumors. Preferably, the cancer may be any one selected from the group consisting of ovarian cancer, lung cancer, B-cell-induced lymphoma, and T-cell-induced lymphoma.

[0125] To prepare the pharmaceutical composition of the present invention, the bispecific antibody of the present invention may be mixed with a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition may be prepared in the form of a lyophilized formulation or an aqueous solution. See, for example, the reference [Remington's Pharmaceutical Sciences and USPharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984)].

[0126] Acceptable carriers and / or excipients (including stabilizers) are non-toxic to the subject at the volume and concentration used and include: buffers (e.g., phosphates, citrates or other organic acids); antioxidants (e.g., ascorbic acid or methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately 10 residues or less) polypeptides; proteins (e.g., serum albumin This may include, but is not limited to, glycine, gelatin, or immunoglobulins; hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, e.g., glucose, mannose, or dextrin; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

[0127] The pharmaceutical composition of the present invention can be formulated in a suitable form known in the industry, depending on its route of administration.

[0128] In this specification, the terms “preventive or therapeutic effective dose” or “effective dose” mean the amount of the active ingredient of a composition effective in preventing or treating cancer in a subject, sufficient to prevent or treat cancer with a reasonable benefit / risk ratio applicable to the medical treatment, without causing side effects. The level of the effective dose may be determined by factors including the patient’s health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and elimination ratio, duration of treatment, drugs that are compounded or used concurrently, and other factors widely known in the medical field. At this time, it is important to administer the dose that yields the greatest effect with the minimum amount and with the minimum amount and without side effects, taking all of the above factors into consideration, which can be easily determined by a skilled technician.

[0129] Specifically, the effective amount of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the individual (patient). Generally, approximately 0.0001 mg to 100 mg, 0.001 to 50 mg, or 5 mg to approximately 50 mg per kg of body weight may be administered once to several times a day. However, the scope of the present invention is not limited to this, as the amount may be increased or decreased depending on the route and duration of administration, the severity of the disease, sex, weight, age, etc. [Examples]

[0130] The present invention will be described in further detail below with reference to the following embodiments. The following embodiments are provided to aid in understanding the present invention and are not intended to limit the scope in any way, nor should they be construed as such.

[0131] Example 1. Production of bispecific antibodies against CD47 and PD-L1

[0132] Example 1.1. Sequence used in the production of bispecific antibodies against CD47 and PD-L1 and the preparation of a vector containing the same.

[0133] To produce an antibody having the structure shown in Figure 1, we commissioned Fusion Antibodies plc. (North Island Belfast Material) to create vectors containing the nucleotide sequence of the heavy chain (SEQ ID NO: 15) and the nucleotide sequence of the light chain (SEQ ID NO: 16) of such an antibody.

[0134] Example 1.2. Production and purification of bispecific antibodies against CD47 and PD-L1

[0135] Transient transfection of the vector prepared in Example 1.1 was performed in a Chinese hamster ovary (CHO) cell line. After batch culture, the expressed antibodies were purified from the cell culture supernatant. Subsequently, quality control (QC) analysis was completed on the purified antibodies to confirm that they met the specified criteria shown in Table 2 below.

[0136] [Table 2]

[0137] The specific procedures for transfection, purification, and quality control analysis are as follows: The vector prepared in Example 1.1 was cloned into pETE V2 (Fusion antibodies), a mammalian primary expression plasmid. The antibodies were expressed using a CHO-based primary expression system, and the resulting antibody-containing cell culture supernatant was purified by centrifugation and filtration. The antibodies were purified from the cell culture supernatant by affinity chromatography (using state-of-the-art AKTA chromatography equipment). The purified antibodies were buffered in phosphate-buffered saline solution. The purity of the antibodies was confirmed to be >95%, as determined by reduction and denaturation of sodium dodecyl sulfate polyacrylamide gel (Figure 2). In Figure 2, each lane is as shown in Table 3 below.

[0138] [Table 3]

[0139] The antibody was analyzed by size exclusion chromatography (SEC) to obtain a chromatogram (Figure 3). The chromatogram showed a single major peak (total area exceeding 95%). The integration results for the chromatogram shown in Figure 3 are shown in Table 4 below (based on the retention volume of the control protein (data not presented)). The peak corresponding to the monomer fraction was >98%.

[0140] [Table 4]

[0141] Bacterial endotoxin levels were determined using the Endosafe®-PTS system and Endosafe®-PTS cartridge (Charles River Laboratories). Antibody concentrations were determined by measuring absorbance at 280 nm and calculating using the theoretical extinction coefficient A280, where 1.0 mg / ml = 1.63. Detailed information regarding the provided purified antibody product is summarized in Table 5.

[0142] [Table 5]

[0143] As can be seen from Table 5, the desired antibody was successfully expressed and purified to the extent that it met the specified criteria. The bispecific antibody that specifically binds to CD47 and PD-L1 produced in this manner is also referred to as "IMC-201".

[0144] The antibodies produced in this manner have the following heavy and light chain variable and CDR sequences. The Fc region has the amino acid sequence of SEQ ID NO: 11 (see sequence catalog).

[0145] [Table 6] TIFF0007914138000007.tif203170

[0146] Manufacturing Example 1. Production of anti-PD-L1 monoclonal antibody and anti-CD47 monoclonal antibody

[0147] The monoclonal antibodies produced in this manufacturing example refer to allore recombinant polypeptides that do not contain endogenous contaminants.

[0148] First, the variable region sequences (SEQ ID NOs. 1 and 2) of U.S. Patent No. 10,118,963 were used to produce anti-PD-L1 monoclonal antibodies. The light and heavy chain antibody sequences were completed by ligating the human IgG1 invariant region sequences, and the antibodies were produced using any expression system known in the industry. Specifically, the antibodies were produced by purifying the supernatant recovered from host cells via transient expression using a recombinant expression vector containing nucleic acids encoding the antibodies in a Chinese hamster ovary (CHO) cell line with Protein A.

[0149] Next, for the production of anti-CD47 monoclonal antibodies, the variable region sequences (SEQ ID NOs. 22 and 8) published in U.S. Patent No. 10,035,855 were used. Human IgG4 invariant region sequences were ligated to complete the light and heavy chain antibody sequences, and the antibodies were produced using any expression system known in the industry. Specifically, the antibodies were produced by purifying the supernatant recovered from host cells via transient expression using a recombinant expression vector containing nucleic acids encoding the antibodies in a Chinese hamster ovary (CHO) cell line with Protein A.

[0150] The anti-PD-L1 monoclonal antibody and anti-CD47 monoclonal antibody produced in this manner will be referred to as "IMC-001" and "IMC-002" below, respectively.

[0151] Example 2. Evaluation of antigen-binding ability and activity of IMC-201 (in vitro)

[0152] Example 2.1. Confirmation of ligand binding ability by ELISA

[0153] First, the binding affinity of IMC-201 to a single ligand (PD-L1) was measured by ELISA as follows: IMC-201 and IMC-001 were serially diluted (0.01 to 600 ng / ml, 3-fold dilution) and incubated with recombinant human PD-L1 pre-coated on plates at 75 ng / well. HRP-conjugated anti-human IgG Fcγ fragment-specific antibody (Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (#109-035-098), Jackson IR Lab) was used to detect IMC-201 and IMC-001.

[0154] Subsequently, the binding affinity of IMC-201 to a single ligand (CD47) was measured by ELISA. IMC-201 and IMC-002 were serially diluted (0.01 to 600 ng / mL, 3-fold dilution) and incubated with recombinant human CD47 pre-coated on plates at 50 ng / well. HRP-conjugated anti-human IgG Fcγ fragment-specific antibodies were used to detect IMC-201 and IMC-002.

[0155] Next, the dual ligand (PD-L1 and CD47) binding affinity of IMC-201 was measured by ELISA as follows: IMC-201, IMC-001, and IMC-002 were serially diluted (0.09 to 1800 ng / ml, 3-fold dilution) and incubated with recombinant human CD47-fc pre-coated on plates at 50 ng / well per 100 μL. After washing, they were incubated with recombinant human PD-L1-his at a concentration of 50 ng / well, and HRP-conjugated anti-his fragment-specific antibodies were used to detect IMC-201, IMC-001, and IMC-002.

[0156] As a result, the EC of IMC-001, a PD-L1 monoclonal antibody 50The value (ng / ml) was measured at 0.989, and IMC-201 was measured at 7.240 (left panel in Figure 4a). Additionally, the EC of IMC-002, a CD47 monoclonal antibody, was measured. 50 The value was measured at 2.769, while IMC-201 was measured at 4.091 (right-hand figure in Figure 4a). On the other hand, the measurement of the dual ligand simultaneous binding affinity of IMC-201 confirmed that only the IMC-201 biantibody specifically bound to both CD47 and PD-L1 simultaneously (EC 50 :17.380 ng / ml, Figure 4b). In conclusion, it was confirmed that IMC-201 exhibits similar binding affinity to the monoclonal antibodies IMC-001 and IMC-002.

[0157] Example 2.2. Confirmation of binding ability to tumor cells

[0158] The cell binding affinity of IMC-201 to tumor cells was measured as follows.

[0159] H1975 cell lines with high PD-L1 expression (ATCC #CRL-5908) and Jurkat cell lines with high CD47 expression (ATCC #TIB-152) were incubated with 3-fold diluted IMC-201, IMC-001, and IMC-002, respectively, at 4°C for 1 hour, and their binding strength was measured by flow cytometry (BD #LSRFortessa X-20).

[0160] As a result, contrary to the ELISA results, it was confirmed that IMC-201 had superior cell binding affinity compared to IMC-001 and IMC-002 (Figure 5).

[0161] Example 2.3. Confirmation of binding ability to knockout (KO) cell lines

[0162] The cell binding affinity of IMC-201 to knockout stable cell lines was measured as follows.

[0163] PD-L1 (Sigma) KO, CD47 (Sigma) KO, and PD-L1 and CD47 dual KO cells, obtained by knocking out cells using MDA-MB-231 (ATCC #HTB-26) and CRISPR-Cas9, were incubated with 3-fold dilutions of IMC-201, IMC-001, and IMC-002, respectively, at 4°C for 1 hour. Binding strength was measured by flow cytometry.

[0164] As a result, the binding ability of IMC-201 in CD47 KO cells was measured to be similar to that of the parent antibody IMC-001, but in PD-L1 KO cells, the binding ability of IMC-201 was measured to be significantly higher than that of the parent antibody IMC-002. In conclusion, as confirmed in Example 2.2 above, it was confirmed that the reason why IMC-201 has superior binding affinity to tumor cells compared to IMC-001 and IMC-002 is because IMC-201 has a much higher binding affinity to CD47 than monoclonal antibodies against CD47 (Figures 6a to 6d).

[0165] Example 2.4. RBC binding capacity test

[0166] The cell surface binding of IMC-201 to human erythrocytes (RBCs) was measured as follows. IMC-002 and Hu5F9 (manufactured according to the reference [Liu J, et al. Pre-clinical development of a humanized anti-CD47 antibody with anti-cancer therapeutic potential. PLoS One. 2015;10(9):e0137345]), both anti-CD47 antibodies, were used as comparative antibodies.

[0167] Human RBCs (5 × 10) obtained from healthy donors (n=3) 5Cells were incubated with 3-fold diluted IMC-201 or other comparative antibodies (IMC-002 and Hu5F9), and their binding strength was determined by measuring the geometric mean fluorescence intensity (MFI) of the anti-human IgG(H+L) secondary antibody using flow cytometry. hIgG1 (Sigma #I5029) and hIgG4 (Biolegend #403702) were used as isotype controls at a concentration of 300 μg / ml. Experiments were performed in duplicate for each donor, and the average MFI from three donors is plotted using error bars. EC 50 The values ​​were calculated using nonlinear regression in GraphPad Prism 5.

[0168] As a result, while the comparative antibody Hu5F9 showed high RBC binding affinity, IMC-201 showed almost no RBC binding affinity, which was no different from the isotype control group and the monoclonal antibody IMC-002. Therefore, it was confirmed that IMC-201 has high binding affinity to cancer cells (Figure 5) but low binding affinity to RBCs (Figure 7).

[0169] Example 2.5. Hemagglutination assay

[0170] Human erythrocytes from healthy donors (n=3) were diluted 1 / 10 with 1X DPBS and incubated overnight at 37°C with IMC-201 and other comparative antibodies (IMC-002 and Hu5F9) at the indicated concentrations.

[0171] As a result, while the comparative antibody Hu5F9 showed a high degree of hemagglutination, IMC-201 showed almost no hemagglutination. Similar results were obtained when compared with the control group (blank and isotype control group), and the results were also similar to those obtained with the monoclonal antibody IMC-002. Reflecting the low binding affinity to RBCs confirmed in Example 2.4, IMC-201 showed almost no hemagglutination (Figure 8).

[0172] Example 2.6. Confirmation of phagocytosis

[0173] Monocyte-derived macrophages (MDMs) were co-cultured with CFSE-labeled MDA-MB-231, H1975, Raji (ATCC#CCL-86), and SR786 (DSMZ#ACC369) cells in a 1:1 ratio for 2 hours in the presence of antibodies. For MDA-MB-231 cells, IMC-002 and IMC-201 were treated at concentrations of 5 and 10 μg / ml, respectively, while for H1975 cells, IMC-002 and IMC-201 were treated at concentrations of 0.37, 1.11, and 3.33 μg / ml. For Raji cells, IMC-002 and IMC-201 were administered at concentrations of 0.04, 0.12, and 0.37 μg / ml, respectively. For R786 cells, IMC-002 and IMC-201 were administered at concentrations of 0.01, 0.04, and 0.12 μg / ml.

[0174] The phagocytic activity of macrophages against tumor cells was evaluated by flow cytometry in the presence of a test antibody. Macrophages were defined as living single CD11b+ cells. Phagocytic activity % was defined as the percentage of CFSE+ macrophages.

[0175] As a result, it was confirmed that the phagocytic activity of IMC-201 was equivalent to or greater than that of IMC-002 in all four cell types, and the phagocytic activity of IMC-201 was particularly high in MDA-MB-231 cells and H1975 cells (Figure 9).

[0176] Example 2.7. Evaluation of ADCC activity in tumor cells

[0177] ADCC activity was evaluated using the ADCC assay kit (Promega_G7018). Target cells Raji were pre-coated on the day of the test, while target cells MDA-MB-231 were pre-coated on plates overnight. ADCC bioassay target cells were co-incubated at 4°C for 1 hour in the presence of antibodies (IMC-201, IMC-001, IMC-002, or Avelumab). During this time, the antibodies were used to treat Raji cells at a serial 4-fold dilution and MDA-MB-231 cells at a serial 7-fold dilution. After removing all antibodies not bound to the target cells by washing, the cells were co-incubated with effector cells in a 5% CO2 incubator for 6 hours. Luciferase was quantified using Bio-Glo reagent.

[0178] As a result, ADCC showed a surprising finding: only IMC-201 was effective in cells expressing CD47 (Raji) (Figure 10a). Furthermore, IMC-201 showed the highest efficacy in cells expressing both PD-L1 and CD47 (MDA-MB-231) (Figure 10b).

[0179] Example 2.8. Evaluation of ADCC activity in primary NK cells.

[0180] ADCC in primary NK cells was evaluated by CD107a degranulation assessment. Target cells, MDA-MB-231 cells, were pre-treated with antibodies (IMC-001, IMC-201) at concentrations of 0.1, 1, and 10 μg / ml for 30 minutes, and then co-cultured with CD56+ NK cells (effector cells) in a 1:1 ratio (target cells:effector cells) in a 37°C 5% CO2 incubator for 4 hours. CD107a was stained at the start of co-culture, and after 1 hour, the cells were treated with GolgiStop Protein Transport Inhibitor (containing monensin, purchased from BD Biosciences). Subsequently, NK cell markers were stained, and ADCC was evaluated in the percentage of CD107a in living NK cells (CD3-CD56+) by flow cytometry.

[0181] As a result, the NK cell activity induced by IMC-201 was comparable to that of the competing drug (data not presented), and it was confirmed to be significantly superior to IMC-001 at all concentrations (Figure 11). The increase in NK cell activity indicates that IMC-201 increases innate immunity activity.

[0182] Example 2.9. Confirmation of T cell activity using antibodies (MLR analysis)

[0183] Antibody-mediated T cell activity was confirmed through MLR (Mixed Lymphocyte Reaction) analysis using dendritic cells and T cells. Monocellular induced dendritic cells (moDCs) were differentiated from CD14+ monocytes, and CD4+ T cells were isolated and secured using a separation kit (stem cell #17952) with PBMCs (Peripheral blood mononuclear cells). Monocellular induced dendritic cells and CD4+ T cells were cultured together in a 1:10 ratio for 5 days in the presence of IMC-001 or IMC-201 (0.001~10 μg / mL, 10-fold dilution), and IFN-γ expression was confirmed in the culture medium of the secured cells by ELISA.

[0184] As a result, we confirmed that IFN-γ levels increased in a concentration-dependent manner with IMC-201 (Figure 12). IMC-201 showed similar efficacy to the parent antibody IMC-001, and MLR analysis suggests that increased IFN-γ expression may suppress cancer by increasing cytotoxicity against cancer cells in the tumor microenvironment.

[0185] Example 3. Confirmation of the effect of IMC-201 (in vivo)

[0186] Example 3.1. Cancer growth inhibitory effect in the Xenograft breast cancer cell line model.

[0187] For the in vivo efficacy study of IMC-201, the MDA-MB-231 breast cancer cell line was thawed and cultured, and these cells were injected into 40 BALB / c nude mice lacking acquired immune cells (obtained from Chemon) at 1×10 7 cells / 200 μl, and were subcutaneously inoculated into site 1 shown in Figure 13.

[0188] On the 17th day after cell inoculation, when the average tumor size reached 90.89 mm 3 , the mice were randomly assigned into 5 groups as shown in Table 7 below. Each group contained 8 mice. Administration of the test substance started on D0. The test substance was intraperitoneally administered once a week for 5 times in total.

[0189] Tumor volume and body weight were measured on D0, D4, D7, D11, D14, D18, D21, D25, D28, D32, D39, D46 and D53. Tumor volume is expressed in mm 3 using the following formula: TV = 0.5a×b 2 (where a and b are the longest diameter and the shortest diameter of the tumor, respectively).

[0190] Data are expressed as mean ± standard error of the mean (Mean±SEM). An Independent-Samples T test was performed for comparison between two groups. All data were analyzed with SPSS version 18.0. P<0.05 was considered statistically significant.

[0191] The efficacy study transplanted with breast cancer cell line was designed as follows:

[0192]

Table 7

[0193] In relation to the results obtained from the efficacy study, tumor growth inhibition based on tumor growth curves is shown in Figure 14.

[0194] The following points were confirmed from the results described above. At day 65, the endpoint of the efficacy study, there was no significant difference in tumor volume (TGItv=42.70%) in the IMC-001 (G2, 2.1 mpk, QW×5w) group compared to the isotype control group (G1, IgG1 kappa, 21.4 mpk, QW×5w). On the other hand, the IMC-002 (G3, 2.1 mpk, QW×5w) group showed a significant difference in tumor volume (TGItv=51.12%) compared to the control group (P<0.05). * Furthermore, the combined IMC-001 and IMC-002 treatment group (G4, 2.1 mpk + 2.1 mpk, QWX 5w) and the IMC-201 treatment group (G5, 3 mpk, QWX 5w) showed significant differences in TGI values ​​relative to tumor volume, at 67.14% and 92.78%, respectively (P<0.01). ** In particular, the IMC-201-treated group showed the most significant tumor growth inhibitory effect.

[0195] Example 3.2. Cancer growth inhibitory effect in the colorectal cancer cell line Xenograft model.

[0196] The colorectal cancer cell line used for the in vivo efficacy study of IMC-201 was CT26-hPD-L1 / hCD47, which was generated by knocking out the mouse PD-L1 and CD47 genes in CT26 WT cells (Kerafast, Cat No: ENH204) and inserting the constitutively expressed human PD-L1 and CD47 genes, and confirmed by flow cytometry.

[0197] CT26-hPD-L1 / hCD47 cells were thawed and subcultured, and these cells were fed 2 × 10⁶ cells to each of 48 BALB / c-hPD-1 / hSIRPα mice (purchased from GemPharmatech Co.,Ltd). 6 Cells were subcutaneously inoculated at site 3 shown in Figure 13 at a dose of 100 μL.

[0198] Seven days after cell inoculation, the average tumor size was 99.92 mm. 3Upon reaching [a certain point], the mice were randomly assigned to five groups as shown in Table 8 below. Each group contained eight mice. The day of grouping was indicated as Day 0 (D0). Administration of the test substance began on D0. The test substance was administered intraperitoneally twice a week (a total of four times). Tumor volume and body weight were measured on D0, D4, D7, D11, D14, D18, D21, D25, D28, D32, D39, D46, and D53. Tumor volume was measured in mm using the following formula. 3 Expressed as: TV = 0.5a × b 2 (Here, a and b are the longest and shortest diameters of the tumor, respectively.)

[0199] Data were expressed as mean ± standard error (Mean ± SEM). An independent-samples t-test was performed for comparison between the two groups. All data were analyzed using SPSS version 18.0. A p-value of <0.05 was considered statistically significant.

[0200] The efficacy study using transplanted colorectal cancer cell lines was designed as follows:

[0201] [Table 8]

[0202] Among the aforementioned test items, the isotype control group is IgG1 kappa (Crownbio).

[0203] In relation to the results of efficacy studies, the inhibition of tumor growth based on the tumor growth curve is shown in Figure 15.

[0204] The following points were confirmed from the results described above. At the end of the efficacy study, tumor volume (TGItv D28 = 27.08%) and tumor weight (TGItw = 21.11%) in G3 (IMC-002 administration group, 2.1 mpk, BIW × 2w) were not significantly different from the isotype control group G1 (IgG1 kappa, 21.4 mpk, BIW × 2w). In addition, tumor volume (TGItv D28 = 27.37%) and tumor weight (TGItw = 26.93%) in G4 (IMC-001 + IMC-002 combined treatment group, 2.1 mpk + 2.1 mpk, BIW × 2w) were not significantly different from G1. On the other hand, compared to G1, the IMC-001 treatment group (G2, 2.1 mpk, BIW × 2w) showed significant differences in tumor volume (TGItv D28 = 38.60%) and tumor weight (TGItw = 41.43%) (P < 0.05). * Furthermore, the tumor volume (TGItv D28 = 83.57%) and tumor weight (TGItw = 82.98%) of G5 (IMC-201, 3 mpk, BIW × 2w) showed statistically significant results (P < 0.001*). ** No abnormal weight changes were observed in any of the treatment groups, and significant suppression of cancer growth was observed with IMC-201 administration. A higher level of cancer growth suppression was confirmed compared to the combination treatment groups with the parent antibodies IMC-001 and IMC-002 compared to the monotherapy groups.

[0205] Example 3.3. Re-induction study

[0206] The re-induction study involved inducing CT26-hPD-L1 / hCD47 tumor cells into each mouse 53 days after the efficacy study grouping in Example 3.2, using 2 × 10⁶ cells. 6 The procedure was performed by subcutaneous inoculation of cells / 100 μL at site 4 shown in Figure 13. The endpoint of the re-induction study was estimated to be day 88. At the endpoint of the re-induction study, photographs of the mice and tumors were taken, and the weight of the tumors was measured. In relation to the results of the re-induction study, the mean tumor volume change over time for various groups is shown in Figure 16.

[0207] The following points were confirmed from the results described above: At day 88, the end of the study, the average tumor volume for G1 (G1:BALB / c-hPD-1 / hSIRPα mice) and G5 (3 mpk (BIW×2w) IMC-201 treated tumor-regressing mice) was 3233.74 mm², respectively. 3 and 0mm 3 The results were as follows: Compared to the control group (G1), the G5 group (3 mpk (BIW × 2w) IMC-201 treated tumor-regressing mice) showed a significant tumor-suppressing effect. Tumor formation occurred in mice that had not been administered IMC-201 antibody (G1) 53 days after the efficacy study group was established. On the other hand, in mice whose tumors were suppressed by IMC-201 antibody (G5), it was confirmed that no tumors were formed even after re-transplanting cancer cells to the opposite side of the already transplanted site, and complete remission (CR) was observed in 6 mice.

[0208] Through this, we confirmed that IMC-201(3 mpk) has an inhibitory effect on tumor re-induction by generating memory T cells in the same tumor re-induction study.

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to CD47 and a second antigen-binding domain that specifically binds to PD-L1, The first antigen-binding domain is A heavy chain variable region comprising CDR1 containing the amino acid sequence of SEQ ID NO: 17, CDR2 containing the amino acid sequence of SEQ ID NO: 19, and CDR3 containing the amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising CDR1 containing the amino acid sequence of SEQ ID NO: 23, CDR2 containing the amino acid sequence of SEQ ID NO: 25, and CDR3 containing the amino acid sequence of SEQ ID NO:

27. The aforementioned second antigen-binding domain is A bispecific antibody comprising a heavy chain variable region containing CDR1 with the amino acid sequence of SEQ ID NO: 29, CDR2 with the amino acid sequence of SEQ ID NO: 31, and CDR3 with the amino acid sequence of SEQ ID NO: 33; and a light chain variable region containing CDR1 with the amino acid sequence of SEQ ID NO: 35, CDR2 with the amino acid sequence of SEQ ID NO: 37, and CDR3 with the amino acid sequence of SEQ ID NO:

39.

2. The bispecific antibody according to claim 1, wherein the first antigen-binding domain includes the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO:

3.

3. The bispecific antibody according to claim 1, wherein the second antigen-binding domain comprises (i) the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 7, or (ii) the single-chain variable fragment (scFv) of SEQ ID NO:

9.

4. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a first antibody fragment having an Fc domain and an antigen-binding domain that specifically binds to CD47, and a second antibody fragment having an antigen-binding domain that specifically binds to PD-L1.

5. The bispecific antibody according to claim 4, wherein the first antibody fragment is a Fab fragment and the second antibody fragment is a single-chain variable fragment (scFv).

6. The bispecific antibody according to claim 4, wherein the second antibody fragment is fused to the C-terminus of the Fc domain via a peptide linker.

7. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises an Fc domain, two Fab fragments each containing an antigen-binding domain that specifically binds to CD47, and two scFv fragments each containing an antigen-binding domain that specifically binds to PD-L1.

8. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a single-chain variable fragment (scFv) containing an antigen-binding domain that specifically binds to PD-L1.

9. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises a Fab fragment having an antigen-binding domain that specifically binds to CD47.

10. The bispecific antibody according to claim 1, wherein the bispecific antibody includes an Fc region derived from the heavy chain invariant region (CH) of IgG1.

11. The bispecific antibody according to claim 10, wherein the Fc region includes heavy chain invariant regions CH1, CH2, and CH3 derived from IgG1, and CH3 includes amino acid substitutions E239D and M241L based on SEQ ID NO:

11.

12. The bispecific antibody according to claim 10, wherein the Fc region acts on effector cells to exhibit an immune-activating effect.

13. The bispecific antibody according to claim 10, wherein the bispecific antibody comprises an antibody fragment having an antigen-binding domain that specifically binds to PD-L1, and the antibody fragment is fused to the C-terminus of the Fc domain via a peptide linker.

14. The bispecific antibody according to claim 8, wherein the single-chain variable fragment (scFV) comprises the amino acid sequence of SEQ ID NO:

9.

15. The bispecific antibody according to claim 10, wherein the Fc region includes the amino acid sequence of SEQ ID NO:

11.

16. The bispecific antibody according to claim 1, wherein the bispecific antibody specifically binds to cancer cells that express PD-L1, CD47, or both.

17. A bispecific antibody according to claim 1, for use in the prevention or treatment of a cancer selected from the group consisting of breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma.

18. A pharmaceutical composition for the prevention or treatment of cancer, comprising a bispecific antibody as described in any one of claims 1 to 17.

19. The pharmaceutical composition according to claim 18, wherein the cancer is selected from the group consisting of ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma, neuronocyte-induced CNS tumor, monocytic leukemia, B-cell-induced leukemia, T-cell-induced leukemia, B-cell-induced lymphoma, T-cell-induced lymphoma, and mast cell-induced tumor.

20. The pharmaceutical composition according to claim 18, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, B-cell induced lymphoma, and T-cell induced lymphoma.

21. A pharmaceutical composition for use in inhibiting tumor cell growth, comprising a bispecific antibody as described in any one of claims 1 to 17.

22. The pharmaceutical composition according to claim 18, wherein the bispecific antibody is used or administered in combination with an anticancer chemotherapy agent, radiotherapy, and / or other immunoanticancer agents.

23. A method for preventing or treating cancer, comprising the step of administering an effective amount of a bispecific antibody described in any one of claims 1 to 17 to a subject (excluding humans) in need of cancer prevention or treatment.

24. A method for suppressing the growth of tumor cells in a subject, comprising the step of administering an effective amount of a bispecific antibody described in any one of claims 1 to 17 to a subject (excluding humans) having tumor cells.

25. A polynucleotide for encoding a bispecific antibody as described in any one of claims 1 to 17.

26. A host cell comprising the polynucleotide described in claim 25.

27. A method for producing a bispecific antibody as described in any one of claims 1 to 17, A method comprising the steps of culturing the host cells described in claim 27 under conditions suitable for the expression of the bispecific antibody, and recovering the bispecific antibody from the culture.

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