Patent Application
20240270841
The disclosure relates to an anti-LILRB1 antibody and uses thereof. More specifically, an anti-LILRB1 antibody or an antigen-binding fragment thereof, and a use thereof for cancer therapy are provided.
The present application includes a Sequence Listing filed in electronic format. The Sequence Listing is entitled “3570-819_ST25.txt” created on Dec. 21, 2022 and is 292,811 bytes in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.
Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1; also known as ILT2, CD85j, or LIR-1) is an inhibitory receptor, which is expressed in cells such as B cells, T cells, NK cells, dendritic cells, macrophages, and other immune cells. LILRB1 participates in a signal transduction mechanism of inhibiting activities of immune cells by binding classical and non-classical MHC class I.
Meanwhile, it has been reported that various cancer cells overexpress MHC class I such as HLA-G for immune evasion. It has been expected that blocking the binding of LILRB1 to MHC Class I allows recovery of the inhibited activities of immune cells, thereby exhibiting anti-cancer effects.
Therefore, it is required to develop novel agent binding to LILRB1 and blocking the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I.
This disclosure provides antibodies, which bind to LILRB1, act on LILRB1-expressing immune cells, regulate activities of the immune cells, and exhibit anti-cancer effects, and uses thereof for cancer therapies.
An embodiment provides an anti-LILRB1 antibody, which binds to LILRB1, or an antigen-binding fragment thereof. The anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to block the binding of LILRB1 to MHC Class I and/or blocking the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to inhibit immune evasion of cancer cells. Furthermore, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect. The anti-cancer effect may be against a cancer cell expressing or overexpressing MHC Class I on its cell surface.
Another embodiment provides a pharmaceutical composition for treatment and/or prevention of a cancer, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a pharmaceutical composition for inhibition of binding of LILRB1 to MHC Class I and/or blocking the interaction between LILRB1 and MHC Class I, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a pharmaceutical composition for inhibiting immune evasion of cancer cell, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
An embodiment provides an anti-LILRB1 antibody, which binds to LILRB1, or an antigen-binding fragment thereof. The anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to block the binding of LILRB1 to MHC Class I and/or blocking the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity to inhibit immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect.
The anti-LILRB1 antibody or an antigen-binding fragment thereof may comprise the following complementarity determining regions (CDRs):
In a specific embodiment, combinations of 6 CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that can be comprised in the anti-LILRB1 antibody or an antigen-binding fragment thereof provided in this disclosure are illustrated in Table 1:
In an embodiment, the anti-LILRB1 antibody or an antigen-binding fragment thereof may comprise:
More specifically, the anti-LILRB1 antibody or an antigen-binding fragment thereof may comprise:
In a specific embodiment, combinations of a light chain variable region and a heavy chain variable region that can be comprised in the anti-LILRB1 antibody or an antigen-binding fragment thereof provided in this disclosure are illustrated in Table 2:
In this disclosure, the expression “an antibody or an antigen-binding fragment (for example, CDR, variable region, or heavy chain /light chain) comprising, consists of, or represented by a certain amino acid sequence” may refer to an antigen-binding fragment that consists essentially of (1) the certain amino acid sequence or (2) an amino acid sequence wherein an insignificant mutation (for example, substitution, deletion, and/or addition of an amino acid residue(s); leading to no impact on the activity of the antibody) is introduced in the amino acid sequence (1).
The anti-LILRB1 antibody or an antigen-binding fragment thereof provided in this disclosure may have a binding affinity (KD) to LILRB1 (for example, human LILRB1) of 10mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM, or 0.15 mM or less, for example, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM, 0.001 nM to 5 mM, 0.005 nM to 5 mM, 0.01 nM to 5 mM, 0.05 nM to 5 mM, 0.1 nM to 5 mM, 0.5 nM to 5 mM, 1 nM to 5 mM, 0.001 nM to 1 mM, 0.005 nM to 1 mM, 0.01 nM to 1 mM, 0.05 nM to 1 mM, 0.1 nM to 1 mM, 0.5 nM to 1 mM, 1 nM to 1 mM, 0.001 nM to 0.5 mM, 0.005 nM to 0.5 mM, 0.01 nM to 0.5 mM, 0.05 nM to 0.5 mM, 0.1 nM to 0.5 mM, 0.5 nM to 0.5 mM, 1 nM to 0.5 mM, 0.001 nM to 0.2 mM, 0.005 nM to 0.2 mM, 0.01 nM to 0.2 mM, 0.05 nM to 0.2 mM, 0.1 nM to 0.2 mM, 0.5 nM to 0.2 mM, 1 nM to 0.2 mM, 0.001 nM to 0.15 mM, 0.005 nM to 0.15 mM, 0.01 nM to 0.15 mM, 0.05 nM to 0.15 mM, 0.1 nM to 0.15 mM, 0.5 nM to 0.15 mM, or 1 nM to 0.15 mM, when measured by surface plasmon resonance (SPR).
Another embodiment provides a pharmaceutical composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient. For example, the pharmaceutical composition may be a pharmaceutical composition for treating and/or preventing a cancer. The pharmaceutical composition may have an activity to inhibit the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I. The cancer may be a cancer associated with the interaction between LILRB1 and MHC Class I. In an embodiment, the pharmaceutical composition may have an activity to inhibit immune evasion of a cancer cell. The cancer cell may be a cell expressing or overexpressing MHC Class I on cell surface.
Another embodiment provides a composition for blocking the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a composition for inhibiting immune evasion of a cancer cell, the composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a method of treating and/or preventing a cancer, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of treating and/or preventing the cancer.
Another embodiment provides a method of blocking the binding of LILRB1 to MHC Class I and/or a method of blocking the interaction between LILRB1 and MHC Class I, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of inhibiting the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I.
Another embodiment provides a method of inhibiting immune evasion of a cancer cell, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal including human) in need of inhibiting immune evasion of the cancer cell.
The methods provided in this disclosure may further comprise a step of identifying the subject in need of treating and/or preventing the cancer, inhibiting the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I, and/or inhibiting immune evasion of the cancer cell, prior to the step of administering.
Another embodiment provides a nucleic acid molecule (polynucleotide) encoding at least one polypeptide selected from the group consisting of CDR (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, a combination of CDR-L1, CDR-L2, and CDR-L3, or a combination of CDR-H1, CDR-H2, and CDR-H3), a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3; a light chain comprising the light chain variable region, and a heavy chain comprising the heavy chain variable region, of the anti-LILRB1 antibody described above.
Another embodiment provides a recombinant vector comprising the nucleic acid molecule. In an embodiment, the recombinant vector may comprise a nucleic acid molecule encoding the light chain variable region or light chain, and a nucleic acid molecule encoding the heavy chain variable region or heavy chain, respectively (e.g., in two separate vectors) or all together (e.g., in one vector). The recombinant vector may be used as an expression vector.
Another embodiment provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.
Another embodiment provides a method of preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, comprising expressing the nucleic acid molecule in a cell. The step of expressing the nucleic acid molecule may comprise culturing the recombinant cell.
As described herein, the antigen-binding fragment of an anti-LILRB1 antibody may refer to a fragment which is derived from an anti-LILRB1 antibody and retain antigen (LILRB1) binding affinity of the antibody. In an embodiment, the antigen-binding fragment may be an polypeptide comprising the 6 CDRs of an anti-LILRB1 antibody as described above, and, for example, may be scFv, scFv-Fc, scFv-Ck(kappa constant region), scFv-Cλ(lambda constant region), (scFv)2, Fab, Fab′, or a F(ab′)2, but not be limited thereto. In an embodiment, the antigen-binding fragment may be scFv, a fusion polypeptide (scFv-Fc) wherein scFv is fused with a Fc region of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.), or a fusion polypeptide (scFv-Ck or scFv-CA) wherein scFv is fused with a constant region (e.g., kappa or lambda) of a light chain.
The anti-LILRB1 antibody or an antigen-binding fragment thereof may have a regulatory activity, for example, an antagonistic or agonistic activity, on LILRB1 protein. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity of blocking the binding of LILRB1 to MHC Class I and/or the interaction between LILRB1 and MHC Class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an activity of inhibiting immune evasion of a cancer cell. Furthermore, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect.
A protein LILRB1, which is an antigen of an anti-LILRB1 antibody or an antigen-binding fragment thereof provided in this disclosure, may be derived from mammal. For example, LILRB1 as an antigen may be a human LILRB1 (e.g., GenBank accession numbers AAH15731.1 (SEQ ID NO: 348), NP_001265328.2, NP_001265327.2, NP_001075108.2, NP_001075107.2, NP_001075106.2, NP_006660.4, NM_001081637.2, NM_001081638.3, NM_001081639.3, NM_001278398.2, NM_001278399.2, etc.), but not be limited thereto.
MHC Class I may be one of classes of major histocompatibility complex (MHC) molecules. In an embodiment, the MHC Class I may be a human MHC Class I and may be at least one selected from the group consisting of HLA(human leukocyte antigen)-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, but not be limited thereto.
As described herein, the term “antibody” may refer to a protein that specifically binds to a specific antigen, and may be a protein produced by stimulation of an antigen in the immune system, or a protein produced by chemical synthesis or recombinant production, with no specific limitation. The antibody may be non-naturally occurring, for example, produced by recombinant or synthetic production. The antibody may be an animal antibody (e.g., a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal or polyclonal antibody.
In the anti-LILRB1 antibody or an antigen-binding fragment thereof provided herein, the portion, except for the heavy-chain CDR and light-chain CDR portions or the heavy-chain variable and light-chain variable regions as defined above, may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like), and, for example, derived from the framework portions, and/or light-chain constant region and/or heavy-chain constant region. In an embodiment, the anti-LILRB1 antibody provided in this disclosure may be an antibody in a form of human IgG, for example, IgG1, IgG2, IgG3, or IgG4, but not be limited thereto.
An intact antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, in which each light chain is linked to a corresponding heavy chain via a disulfide bond. The constant region of an antibody is divided into a heavy-chain constant region and a light-chain constant region. The heavy-chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) or alpha2 (α2) as its subclass. The light chain constant region is of either a kappa (κ) or lambda (λ) type.
As used herein, the term “heavy chain” may be intended to encompass a full-length heavy chains and fragments thereof, wherein the full-length heavy chain may comprise a variable region VH including amino acid sequences sufficient to provide specificity to antigens, three constant regions CH1, CH2, and CH3, and a hinge. The term “light chain” may be intended to encompass full-length light chains and fragments thereof, wherein the full-length light chain may comprises a variable region VL including amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.
The term “complementarity determining region (CDR)” may refer to a portion that confers antigen-binding specificity in a variable region of an antibody, and may refer to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contacting residues that play an important role in the binding of an antibody to its antigen or an epitope of the antigen. As used herein, the terms “specifically binding” and “specifically recognizing” may have the same general meaning as known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological reaction.
In this disclosure, unless differently stated, the term “antibody” may encompass not only an intact antibody but also an antigen-binding fragment of the antibody possessing an antigen-binding capability.
The term “antigen-binding fragment” used herein may refer to a polypeptide in any type, which comprises a portion (e.g., 6 CDRs as described herein) capable of binding to an antigen, and, for example, may be scFv, (scFv)2, scFv-Fc, Fab, Fab′, or F(ab′)2, but is not limited thereto. In addition, as described above, the antigen-binding fragment may be scFv, a fusion polypeptide wherein scFv is fused with a Fc region of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) or a constant region (e.g., kappa or lambda).
Among the antigen-binding fragments, Fab includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region CH1.
Fab′ is different from Fab in that Fab′ comprises a hinge region having at least one cysteine residue at the C-terminal of CH1.
F(ab′)2 antibody is formed thro/h disulfide bridging of the cysteine residues in the hinge region of Fab′.
Fv is a minimal antibody fragment composed of only a heavy chain variable region and a light chain variable region. Recombination techniques of generating an Fv fragment are widely known in the art.
Two-chain Fv comprises a heavy chain variable region and a light chain variable region which are linked to each other by a non-covalent bond. Single-chain Fv generally comprises a heavy-chain variable region and a light-chain variable region which are linked to each other by a covalent bond via a peptide linker or directly linked at the C-terminals to have a dimer structure like two-chain Fv.
The antigen-binding fragments may be obtained using protease (for example, Fab may be obtained by restrictively cleaving a whole antibody with papain, and an F(ab′)2 fragment may be obtained by cleaving with pepsin), or may be prepared by using a genetic recombination technique.
The term “hinge region” may refer to a region between CH1 and CH2 domains within heavy chain of an antibody, which functions to provide flexibility for the antigen-binding site in the antibody.
The anti-LILRB1 antibody may be a monoclonal or polyclonal antibody and, for example, a monoclonal antibody. A monoclonal antibody can be prepared using a method widely known in the art, for example, using a phage display technique. Alternatively, the anti-LILRB1 antibody may be constructed in the form of a mouse-derived monoclonal antibody by a conventional method.
Meanwhile, individual monoclonal antibodies can be screened using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format, based on the binding potential against LILRB1. Inhibitory activities can be verified through functional analysis such as competitive ELISA for verifying the molecular interaction of binding assemblies or functional analysis such as a cell-based assay. Then, with regard to monoclonal antibody members selected on the basis of their strong inhibitory activities, their affinities (Kd values) to LILRB1 may be each verified.
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, in addition to the active ingredient (the anti-LILRB1 antibody or an antigen-binding fragment thereof). The pharmaceutically acceptable carrier may be anyone selected from those commonly used for the formulation of antibodies. For example, the pharmaceutically acceptable carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition may further comprise one or more selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, preservative, and the like, which can be commonly used for manufacturing pharmaceutical composition.
The pharmaceutical composition, or the antibody or an antigen-binding fragment thereof may be administered orally or parenterally in a pharmaceutically effective amount. The parenteral administration may be intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, intranasal administration, intrapulmonary administration, rectal administration or intralesional local administration. Since proteins or peptides are digested when administered orally, the active ingredient in the compositions for oral administration may be coated or formulated to prevent digestion in stomach. In addition, the antibody or the compositions may be administered using an optional device that enables the active ingredient to be delivered to target cells (e.g., cancer cells).
The anti-LILRB1 antibody or an antigen-binding fragment thereof may be comprised in the pharmaceutical composition or administered to a subject in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” may refer to an amount of an active ingredient (the antibody or fragment thereof) at which the active ingredient can exert desired effects (e.g., anti-cancer effect). The pharmaceutically effective amount may be prescribed in a variety of ways, depending on various factors, such as age, body weight, gender, pathologic conditions, diets, excretion speed, and/or reaction sensitivity of a subject, formulation types, administration time, administration interval, administration route, administration manner, and the like. For example, anti-LILRB1 antibody or an antigen-binding fragment thereof may be administered at the amount of 0.005 ug/kg to 1000 mg/kg, 0.005 ug/kg to 500 mg/kg, 0.005 ug/kg to 250 mg/kg, 0.005 ug/kg to 100 mg/kg, 0.005 ug/kg to 75 mg/kg, 0.005 ug/kg to 50 mg/kg, 0.01 ug/kg to 1000 mg/kg, 0.01 ug/kg to 500 mg/kg, 0.01 ug/kg to 250 mg/kg, 0.01 ug/kg to 100 mg/kg, 0.01 ug/kg to 75 mg/kg, 0.01 ug/kg to 50 mg/kg, 0.05 ug/kg to 1000 mg/kg, 0.05 ug/kg to 500 mg/kg, 0.05 ug/kg to 250 mg/kg, 0.05 ug/kg to 100 mg/kg, 0.05 ug/kg to 75 mg/kg, or 0.05 ug/kg to 50 mg/kg per day, but not be limited thereto. The daily dosage may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container.
The pharmaceutical compositions may be formulated into a form of a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further comprise a dispersing or a stabilizing agent for the formulation.
The subject, to whom the antibody, pharmaceutical composition, or method provided in this disclosure is applied, may be selected from mammals including a mammal including primates such as humans and monkeys, rodents such as rats and mice, and the like.
The cancer may be a solid cancer or blood cancer. The cancer may be, but not limited to, one or more selected from the group consisting of lung cancer (e.g., squamous cell carcinoma of the lung, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung), peritoneal carcinoma, skin cancer, squamous cell carcinoma, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, leukemia (e.g., chronic or acute leukemia), lymphocytic lymphoma, hepatoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, renal cell carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, bone osteosarcoma, and the like. The cancer may be a primary cancer or a metastatic cancer. The cancer may be a cancer characterized by expression or overexpression of MHC Class I on a surface of cancer cell, and, for example, may be colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma, bone osteosarcoma, renal cell carcinoma, or gastric cancer. The overexpression of MHC Class I may refer to an overexpression compared to that of a normal cell or a cancer cell which is non-responsive or resistant to the immunotherapy, for example, T-cell (e.g., cytotoxic T-cell) mediated immunotherapy.
As used herein, the term “treatment of cancer” may refer to all anti-cancer actions that prevent, alleviate or ameliorate the symptoms of cancer, or partially or completely remove a cancer, such as, cancer cell death, inhibition of cancer cell proliferation, inhibition of cancer metastasis, and the like.
The anti-LILRB1 antibody or an antigen-binding fragment thereof provided in this disclosure may be co-administered with another drug, for example, at least one selected from the group consisting of conventionally used agents for immunotherapy, anti-cancer agents, cytotoxic agents, and the like. Accordingly, an embodiment provides a pharmaceutical composition of combined administration for treating and/or preventing a cancer, comprising (1) an anti-LILRB1 antibody or an antigen-binding fragment thereof, and (2) at least one selected from the group consisting of agents for immunotherapy, anti-cancer agents, cytotoxic agents, and the like. Another embodiment provides a method of treating and/or preventing a cancer, comprising administering (1) an anti-LILRB1 antibody or an antigen-binding fragment thereof, and (2) at least one selected from the group consisting of agents for immunotherapy, anti-cancer agents, cytotoxic agents, and the like, to a subject in need of treating and/or preventing the cancer. The agents for immunotherapy, anti-cancer agents, and cytotoxic agents may include any drugs which are conventionally used for cancer therapy, and/or have cytotoxic activity, and for example, they may be at least one selected from the group consisting of proteins such as antibodies, nucleic acid molecules such as siRNA, and/or small molecular chemicals such as paclitaxel, docetaxel, and the like, but not limited thereto.
Another embodiment provides a polypeptide molecule comprising a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), a combination thereof; or heavy chain variable region, light chain variable region, or a combination thereof, of the anti-LILRB1 antibody as described above. The polypeptide molecule may be used in preparing an antibody as a precursor of antibody, or comprised in a protein scaffold having an antibody-like structure (e.g., peptibody), a bispecific antibody, or a multispecific antibody, as a component thereof. In another embodiment, the polypeptide molecule may be used as a target (antigen) recognition domain or a secreted antibody, in cell therapeutics for target therapy, such as CAR-T. In another embodiment, the polypeptide molecule may be used for constructing anti-LILRB1 antibody-secreting cells as cell therapeutics.
Another embodiment provides a nucleic acid molecule encoding a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a heavy chain variable region, or a heavy chain, of the anti-LILRB1 antibody.
Another embodiment provides a nucleic acid molecule encoding a light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), a light chain variable region, or a light chain, of the anti-LILRB1 antibody.
Another embodiment provides a recombinant vector comprising a nucleic acid molecule encoding a heavy chain variable region or a heavy chain of the anti-LILRB1 antibody, and a light chain variable region or a light chain of the anti-LILRB1 antibody, respectively in two separate vectors or all together in one vector.
Another embodiment provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.
The term “vector” refers to a means for expressing a target gene in a host cell, as exemplified by a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, a lentivirus vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be constructed from or by manipulating a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (for example, λgt4λB, λ-Charon, λΔz1, M13, etc.), or a virus vector (for example, SV40, etc.), which is commonly used in the art.
In the recombinant vector, the nucleic acid molecule may be operatively linked to a promoter. The term “operatively linked” is intended to pertain to a functional linkage between a nucleotide sequence of interest and an expression regulatory sequence (for example, a promoter sequence). When being “operatively linked”, the regulatory element can control the transcription and/or translation of a polynucleotide of interest.
The recombinant vector may be constructed typically as a cloning vector or an expression vector. For recombinant expression vectors, a vector generally available in the relevant art for expressing a foreign protein in plant, animal, or microbial cells may be employed. Various methods well known in the art may be used for the construction of recombinant vectors.
For use in hosts, such as prokaryotic or eukaryotic cells, the recombinant vector may be constructed accordingly. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., a pLλ promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter, etc.), a ribosomal binding site for initiating translation, and transcriptional/translational termination sequences. On the other hand, an expression vector for use in a eukaryotic host includes an origin of replication operable in a eukaryotic cell, such as an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and a BBV origin of replication, but is not limited thereto. In addition, the expression vector typically includes a promoter derived from genomes of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, adenovirus late promoter, vaccinia virus 7.5 K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence.
The recombinant cell may be prepared by introducing the recombinant vector into a suitable host cell. As long as it allows the sequential cloning and expression of the recombinant vector in a stable manner, any host cell known in the art may be employed in the present disclosure. Examples of the prokaryotic host cell available for the present disclosure may be selected from E. coli such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus spp. such as Bacillus subtilis and Bacillus thuringiensis, and enterobacteriaceae strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that may be used for transformation may selected from, but are not limited to, Saccharomyces cerevisiae, insect cells, and animal cells, such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK, etc.
The nucleic acid molecule or a recombinant vector carrying the same may be introduced (transfected) into a host cell using a method well known in the relevant art. For example, this transfection may be carried out using a CaCl2 or electroporation method when the host cell is prokaryotic. For eukaryotic host cells, the genetic introduction may be achieved using, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle bombardment.
To select a transformed host cell, advantage may be taken of a phenotype associated with a selection marker according to methods well known in the art. For example, when the selection marker is a gene conferring resistance to a certain antibiotic, the host cells may be grown in the presence of the antibiotic in a medium to select a transformant of interest.
Another embodiment provides a method of preparing the anti-LILRB1 antibody or an antigen-binding fragment thereof, comprising expressing the nucleic acid molecule or a recombinant vector in a host cell. The step of expressing may be conducted by culturing the recombinant cell comprising the nucleic acid molecule (for example, in a recombinant vector) under a condition allowing the expression of the nucleic acid molecule. The method may further comprise isolating and/or purifying the antibody or its fragment from the cell culture, after the step of expressing or culturing.
The anti-LILRB1 antibody or an antigen-binding fragment thereof provided in this disclosure can have high anti-cancer effect by inhibiting the immune evasion mechanism of cancer cells, allowing that the immune cells can exhibit their anti-cancer effect.
Hereafter, the present invention will be described in detail by examples.
The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.
In order to select antibodies that specifically recognize human LILRB1, a phage display screening was performed using a library composed of human scFv antibodies. As an antigen, human LILRB1-His (Cat. No. 8989-T2) and human LILRB1-Fc (Cat. No. 2017-T2) (RnD systems) were used respectively. Each antigen was conjugated with biotin by EZ-Link Sulfo-NHS-Biotin kit (ThermoFisher Scientific) for use.
The phage display screening was performed using total 4-types of LILRB1 antigens (LILRB1-His, LILRB1-Fc, LILRB1-His-Biotin, and LILRB1-Fc-Biotin) through solid-phase screening and solution-phase screening. Additional screenings were performed by gradually decreasing the concentration of the used antigen, competitively eluting with control antibodies against LILRB1, conducting negative selection to Fc when LILRB1-Fc is used as an antigen, etc. The selected products were confirmed for their binding to the antigen through polyclonal phage ELISA.
Genes encoding the scFvs, which were verified to bind the antigen in Example 1.1, were amplified by PCR to prepare expression vectors. For each selection, a certain number of transformants were transferred to a 96 well culture plate for screening. Antibodies in a scFv form were expressed using Autoinduction media (Studier, F. W. (2005) Protein Expression and Purification 41, 207-34) and then analyzed for their binding to the antigen by performing DELFIA immune assay (PerkinElmer). In addition, after allowing a certain amount of each scFv antibody to be captured on the surface, DELFIA for the antigen was performed to determine the ranking for antigen-antibody binding affinity.
Among the clones which were confirmed to bind to the antigen in Example 1.2, a total of 376 clones were selected, and the DNA sequences of genes encoding the selected scFvs were analyzed by a general DNA sequencing to remove duplicate clones. In addition, a total of 93 clones were selected based on the ranking of the antigen-antibody binding affinity determined in Example 1.2. Genes encoding a heavy chain variable region(VH) and a light chain variable region(VL) were respectively amplified by PCR from each of the genes encoding the selected scFvs, and inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of vectors comprising CMV promoter or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and kappa or lambda light chain constant region can be used), wherein the expression vector was designed for encoding a human IgG4 antibody(IgG4 Fc: SEQ ID NO: 341, Kappa constant region: SEQ ID NO: 342, Lambda constant region: SEQ ID NO: 343). The DNA sequence of the expression vector was confirmed by sequencing.
The vectors constructed in Example 1.3 were purified using Plasmid Plus Maxi kit (Qiagen). The purified vectors were used for expressing antibodies in ExpiCHO-S™ cells or Expi293™ cells.
In particular, the vectors constructed in Example 1.3 were transfected into ExpiCHO-S™ cells(Gibco) (1.5×108 cells/Culture Volume 25 mL) by adding 80 μL of ExpiFectamine™ CHO reagent (Thermo Fisher). One day post-transfection, 150 μL of ExpiCHO™ Enhancer (Thermo Fisher) and 4 mL of ExpiCHO™ Feed (Thermo Fisher) were added to the culture. On day 5, 4 mL of ExpiCHO™ Feed was added to the culture. The transfected cells were cultured under the conditions of 32° C. and 5% CO2 for 7-11 days in total.
In addition, the vectors constructed in Example 1.3 were transfected into Expi293F™ cells (Gibco) (3×108 cells/Culture Volume 100 mL) by adding 320 μL of ExpiFectamine™ 293 Reagent (Gibco) according to manufacturer's protocol. One day post-transfection, ExpiFectamine™ 293 Enhancer 1 (Thermo Fisher), ExpiFectamine™ Enhancer 2 (Thermo Fisher), and glucose were added in the amount of 0.6 mL per Culture Volume 100 mL, 6 mL per Culture Volume 100 mL, and 3.6 g per 1 liter, respectively. The transfected cells were cultured under the conditions of 36.5° C. and 5% CO2 for 5 days in total. The cultured cells of two types were respectively centrifuged at 4000 rpm at 4° C. for 20 minutes, and then, filtrated using 0.22 um bottle-top filter system (Corning). The culture supernatant was harvested and purified using AKTA Pure L (GE healthcare). The culture supernatant was loaded into AKTA Pure L equipped with Hitrap MabSelectSure 1 mL column (GE healthcare) at the flow rate of 1 mL/min,, and the column was washed with 20 column volumes (CV) of 1× PBS. Then, elutionbuffer (0.1 M sodium citrate pH 3.4 buffer) was loaded to the column, to elute a protein of interest. The eluate was concentrated using Amicon Ultra Filter Device (MWCO 10K, Merck), centrifuged and subjected to buffer exchange with 1×PBS buffer.
The purified antibody samples were diluted with 1× PBS, to make the final concentration about 1 mg/mL. Ten (10) μL of Reducing Loading Buffer (3×) or Non-reducing Loading Buffer (3×) and 20 μL of the purified antibody sample were mixed and left in 95° C. heating bath for 2 minutes, and then, brought out and cooled. The sample was injected into SDS-PAGE Gradient Gel (4-20% or 4-12%) equipped on an electrophoresis device at the amount of 10 μg per well and developed on the gel. In order to analyze molecular weight of the sample, Precision Plus Protein™ Dual Color Standards (BIO-RAD) was injected to another separate well. The gel was stained with Coomassie staining solution and destained to obtain gel images.
Among 93 antibodies, gel electrophoresis images for antibodies A10, B3, E3, G1, G9 and H2 were representatively shown in
The binding affinities of the 93 antibodies, which were selected in Example 1.3, to the antigen, LILRB1, were measured using Biacore T200 (GE healthcare). An anti-human IgG (Fc) antibody (GE healthcare, Cat. No. BR-1008-39, final concentration of 25 μg/mL) was flowed at the flow rate of 5 μL/min for 360 seconds to be immobilized at 5000-7000 RU on Series S Sensor Chip CM5 (GE healthcare, Cat. No. BR-1005-30) using Amine Coupling Kit (GE healthcare, Cat. No. BR-1000-508). The antigen, human LILRB1 protein (LILRB1-His, RnD systems Cat. No. 8989-T2) was injected thereto in 4˜9 different concentrations from 3.13 nM to 1600 nM at the flow rate of 30 μL/min to determine ka and kd values as shown in Table 3 and calculate KD value therefrom.
Among the 93 antibodies, 20 antibodies showing excellent binding affinities (KD values) were selected and summarized in Table 3. Among them, SPR sensorgrams for antibody B3 showing the LILRB1 binding affinity (KD) of about 99.8 nM and for antibody E3 showing the LILRB1 binding affinity (KD) of about 101.2 nM are shown in
In the 20 antibodies which are analyzed for antigen binding affinity in Example 1.5, amino acid sequences of the CDRs defined according to Kabat numbering, light chain variable region, heavy chain variable region, light chain, and heavy chain, and nucleic acid sequence encoding the light chain variable region and the heavy chain variable region were analyzed by general amino acid sequencing and DNA sequencing methods and summarized in Tables 4-23:
In order to test whether or not 93 antibodies selected in Example 1.4 bind LILRB1 expressed on surface of immune cells, natural killer cell (NK cell) surface binding assay was performed. A human NK cell, KHYG-1 cell (JCRB) was cultured in RPMI 1640 medium (Gibco) supplemented with 10%(w/v) of FBS (Gibco) and 100 U/mL of interleukin-2 (Novartis). KHYG-1 cells were added to a U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 5×104 cells/well. Each of the selected antibodies was added to the well to the final concentration of 50 μg/mL per well and incubated at 4° C. for 1 hour.
In order to see the level of LILRB1-specific binding of the selected antibodies, a human IgG4 isotype control antibody (Biolegend) was treated in the same manner. After washing with FACS buffer, the cells were treated with an anti-human Fc-biotin antibody (life technologies) and incubated at 4° C. for 1 hour. After washing with FACS buffer, the cells were treated with streptavidin PE (BD Pharmigen) and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were resuspended and subjected to analysis using iQue screener (Sartorius).
Among the obtained results, the results for antibodies A10, E3, E4, F12, G1, G9, G11, H2 and H11 are representatively compared with that of human IgG4 isotype (control), which are shown in Table 24. The flow cytometry diagrams for A10, E3 and human IgG4 isotype (control) are shown in
As shown in Table 24 and
In order to test whether or not the antibodies selected in Example 1.5 exert an inhibitory effect on binding of LILRB1 to its ligand, HLA-G, the degree of blocking by the selected antibodies was analyzed.
For this purpose, JEG-3 cells (ATCC cat #HTB-36), which show high expression level of HLA-G, were used. JEG-3 cells were cultured in MEM medium (Gibco) supplemented with 10%(v/v) of FBS (Gibco) and 1%(v/v) of pen-strep (Gibco). The JEG-3 cells were added to U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 5×104 cells/well. The well plate was washed with 1× PBS buffer. Each of the antibodies selected in Example 1.5 (A10, E3, F12, G1, G9, H2 and H11) and LILRB1-Fc (RnD systems) were mixed in FACS buffer (1× PBS+1% BSA+1 mM EDTA) to the final concentrations of 10 μg/mL and 5 μg/mL, respectively. The cells were treated with 100 uμL of the mixture solution per well and incubated on ice for 2 hours. An anti-LILRB1 antibody (clone HP-F1, Abcam) as a positive control and an anti-lysozyme IgG4 antibody (clone D1.3) as a negative control were treated in the same manner. After washing with FACS buffer twice, the cells were treated with PE-anti-hulgG-Fc antibody (Biolegend, 10 μg/mL) and incubated on ice for one hour. After washing with FACS buffer twice, the cells were resuspended in 100 μL of the same buffer and subjected to analysis using iQue screener (Sartorius).
The obtained results are shown in
In order to test whether or not the selected antibodies increase the degree of cancer cell lysis by NK cells, the cell death rate of HLA-G-overexpressing HEK293 cell by NK cell KHYG-1 was analyzed. KHYG-1 cells (JCRB) were addeded to 96-well tissue culture plate (BD Falcon) at the amount of 2×104 cells/well (4×104 cells/mL, total volume 50 μL). The cells were treated with each antibody (Table 25) to the final concentration of 20 μg/mL per well, and left at 37° C. for one hour.
As a negative control, a human IgG4 isotype control antibody (Biolegend) was treated in the same manner.
HLA-G-overexpressing HEK293 cells (which were prepared by transduction of HEK293 cells (American Typo Culture Collection) with lentivirus constructed for expressing HLA-G) were stained with IncuCyte CytoLight Rapid Red Reagent (Sartorius) according to the manufacturer's protocol. After one hour, the HLA-G-overexpressing HEK293 cells were added to the plate at the amount of 1×104 cells/well (2×104 cells/mL, total volume 50 μL). The plate was placed in IncuCyte S3(Sartorius) equipped in an incubator under the condition of 37° C. and 5% CO2, and images thereof were taken for 72 hours. Red area confluence indicating the density of live HLA-G-overexpressing HEK293 cells was measured, and cell viability was calculated. The obtained cell viabilities are shown in Table 25 (wherein the cell viabilities are shown as a relative value to that of control antibody (cell viability of IgG4 isotype-treated well=1)):
As shown in Table 25, all the tested antibodies including A10, B9, D3, E1, E3, F12, G1, G6, G9, G11, H2 and H11 increase cell death of HLA-G-overexpressing HEK293 cells by KHYG-1, compared to that of human IgG4 isotype control antibody.
Among the antibodies selected in Example 1.5, two antibodies (E3 and B3) were tested for their in vivo anti-cancer efficacies. For this purpose, it was tested whether or not administration of the two antibodies reduces tumor size where the tumor was generated by engrafting human colorectal carcinoma cells (Bioware Brite Cell Line HCT116 Red-Fluc colorectal carcinoma cells (PerkinElmer)) and THP-1 derived macrophages to the mice. As a negative control, human colon cancer xenograft mice prepared as above were treated with a human IgG1 isotype control antibody (BioXcell, Cat. No. BP0297). Hereinafter, the processes are described in detail:
The THP-1 derived macrophages used above were prepared by differentiating THP-1 cells (ATCC) with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma), 20 ng/ml of interferon gamma (Peprotech) and 10 pg/ml of lipopolysaccharide (LPS, Sigma).
5-week old female CIEA NOG mice [NOG immunodeficient mouse] (Central Institute for Experimental Animals, Japan) were subcutaneously injected with a mixture of 3×106 cells of HCT116 Red-Fluc colorectal carcinoma cells, 3×106 cells of THP-1 derived macrophages and each of two test antibodies (E3 or B3 antibody; 20 μg per mouse). From the 4th day after tumor grafting, the antibody was administered to the mouse model at the dosage of 5 mg/kg by intraperitoneal injection twice a week. Then, the size (mm3) of the grafted tumor was measured and shown in
The nucleic acid sequence encoding the full-length heavy chain (SEQ ID NO: 302) of antibody E3, which was confirmed to have particularly significant effect in Example 3, was amplified by PCR. The nucleic acid sequence encoding the region from Ser1 to Leu110 of the light chain variable region (VL) (SEQ ID NO: 221) of antibody E3 was amplified by PCR and ligated to a nucleic acid sequence encoding the lambda constant region (Lambda CL.1, SEQ ID NO: 344) to amplify the nucleic acid sequence encoding lambda light chain by PCR. The amplified sequences were inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of vectors comprising CMV promoter or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and lambda light chain constant region, can be used), wherein the expression vector was designed for encoding a human IgG4 antibody. The DNA sequence of the expression vector was confirmed by sequencing.
An antibody (E3.1) was prepared using the constructed expression vector referring to Example 1.4, and the sequence of the antibody was analyzed referring to Example 1.6 and summarized in Table 26:
Example 5: Generation of Human LILR-overexpressing cell lines The nucleic acid sequences encoding the full-length human LILR family proteins (Table 27) were amplified by PCR, and each of the amplified sequences was inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of vectors comprising CMV promoter or CMV/CHO beta-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and lambda light chain constant region, can be used). The DNA sequence of the expression vector was confirmed by sequencing. The constructed vector was transfected into CHO cells, to generate 11 stable cell lines overexpressing each LILR protein on its surface.
In order to determine EC50 values of the antibodies prepared in Examples 1 and 4 for binding to human LILRB1-overexpressing cell lines, a cell surface binding assay was performed. Representing the prepared antibodies, EC50 values of E3.1 and H11 antibodies were measured. The CHO cells prepared in Example 5, which overexpress LILRB1 on surface, were added to U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 1×105 cells/well. Threefold serial dilutions of E3.1 and H11 antibodies were prepared starting from the final concentrations of 600 ug/mL and 27 ug/mL, respectively. The cells were treated with each of the diluted antibodies and incubated at 4° C. for 60 minutes. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (Invitrogen) and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin (BD Pharmigen) labeled with PE fluorescence and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were resuspended and subjected to analysis using iQue screener (Sartorius). EC50 values were calculated using nonlinear regression formula of GraphPad Prism software, and the obtained results are shown in Table 28:
In order to confirm whether or not the selected antibodies bind to human LILR family proteins other than LILRB1, a cell surface binding assay was performed. The CHO cells (prepared in Example 5) expressing each of various LILR family proteins on surface were added to U-bottom 96-well tissue culture plate (BD Falcon) at the amount of 1×105 cells/well. The cells in each well were treated with the selected antibody in the final concentration of 20 ug/mL and incubated at 4° C. for 60 minutes. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (Invitrogen) and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin (BD Pharmigen) labeled with PE or FITC fluorescence and incubated at 4° C. for 30 minutes. After washing with FACS buffer, the cells were resuspended and subjected to analysis using iQue screener (Sartorius). The cells treated with each LILR protein specific antibody (Table 27) were used as a positive control, and the cells treated with human IgG4 isotype control antibody (Biolegend) were used as a negative control.
The results obtained for antibody E3.1 are shown in
In order to confirm whether the E3.1 and H11 antibodies increase the level of cytotoxicity of NK cells, an enzyme-linked immune absorbent spot (ELISPOT) assay was performed. The level of cytotoxicity was determined by the release of cytotoxic granules, granzyme B and perforin, in NK cells.
5×103 cells of LILRB1-expressing KHYG-1 cell lines (JCRB) and 5×103 cells of HLA-G-overexpressing K562 cells (which were prepared by transduction of K562 cells (American Type Culture Collection) with lentivirus constructed for expressing HLA-G) were co-cultured in U-bottom 96-well tissue culture plate. Each antibody(E3.1, H11 or human IgG4 isotype control antibody) was added thereto with final concentration of 50 ug/mL per each well and left incubated at 37° C. for 30 minutes. The co-cultured cells were transferred onto 96 well plates (Immunospot, Cat. HGZBPFN-2M) (PVDF membrane) for ELISPOT, which were coated with anti-perforin antibody and anti-granzyme B antibody, respectively, and further incubated at 37° C. for 8 hours. The PVDF membranes were washed with a washing solution (0.05% tween 20 in PBS), then treated with anti-granzyme B-HRP and anti-perforin-biotin antibodies. Then, detection processes were performed according to the manufacturer's protocol. The PVDF membranes were dried at room temperature for 24 hours, and the number of spots for granzyme B and perforin were counted by ELISPOT analyzer (Immunospot).
The results are shown in
In order to see if antibodies provided by the examples show higher efficacy compared to pre-existing antibodies, a chimeric GHI/75 antibody comprising a variable region of GHI/75 antibody (Biolegend, cat #333721), which is a mouse-derived anti-human LILRB1 antibody, and a constant region of human antibody was prepared.
More specifically, the amino acid sequence of the GHI/75 antibody was analyzed through peptide mapping, and a vector, in which the nucleic acid sequence corresponding to the variable region (VH and VL domain) of a human IgG4 antibody was replaced by the nucleic acid sequence corresponding to the variable region (VH and VL domain) of mouse GHI/75 antibody, was prepared. In the vector, the region corresponding to upper hinge of human IgG4 was substituted with the nucleic acid sequence corresponding to the amino acid sequence (EPKSCDKTHT; SEQ ID NO: 359) of human IgG1 upper hinge. The vector was expressed as described in Example 1.4, and the obtained antibody was purified and used as a comparative antibody in examples below.
In order to confirm whether the antibodies prepared in Examples 1 and 4 inhibit signaling by LILRB1, a luciferase reporter assay was performed. Among the antibodies prepared in Examples 1 and 4, the assay was performed representatively for E3.1 and H11 antibodies, and the chimeric GHI/75 antibody prepared in Example 9 was used for comparison. Jurkat cells expressing LILRB1 and interleukin 2 (IL-2) promoter luciferase (which were prepared by inserting IL-2 promoter luciferase vector (Promega) into Jurkat cell line (American Type Culture Collection) followed by transduction with lentivirus constructed for expressing LILRB1) and HLA-G-overexpressing K562 cells were used. Ninety six-well plates were coated with anti-CD3 antibody (Biolegend) by incubating with the antibody overnight at 4° C. On the next day, Jurkat cells expressing LILRB1 and IL-2 promoter luciferase were added to U-bottom 96-well plates at the amount of 1×105 cells/well, and the plates were treated with each antibody (E3.1, H11, chimeric GHI/75 or human IgG4 isotype (control)) to the final concentration of 20 ug/mL and incubated at 37° C. for one hour. HLA-G-overexpressing K562 cells (1×105 cells/well) were added to the plates and incubated at 37° C. for 30 minutes. The obtained suspension was transferred to the plate coated with anti-CD3 antibody, and anti-CD28 antibody (Biolegend) was added thereto to the final concentration of 10 ug/mL. The plate was incubated at 37° C. for 6 hours. Steady-Glo® solution (Promega) was added to each well, and the luminescence intensity was recorded using a luminometer (Envision, PerkinElmer).
The results are shown in
For analysis of anti-cancer effects of selected antibodies, referring to Example 3, a mixture of 3×106 cells of HCT116 Red-Fluc colorectal carcinoma cells, 3×106 cells of THP-1 derived macrophages and an antibody (20 μg/mouse) was subcutaneously injected to 5-week old female CIEA NOG mice (NOG immunodeficient mouse; Central Institute for Experimental Animals, Japan) The antibody used was E3.1 or H11 antibody, and human IgG4 isotype was used as a control antibody for comparison. From the 4th day after grafting tumor cells, the antibody was administered to the mouse model at the dosage of 5 mg/kg by intraperitoneal injection twice a week, and the tumor volume was measured and shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0173414 | Dec 2019 | KR | national |
| 10-2020-0061907 | May 2020 | KR | national |
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/KR2020/018931 filed on Dec. 22, 2020, which claims the benefits of KR 10-2019-0173414 filed on Dec. 23, 2019 and KR 10-2020-0061907 filed on May 22, 2020 with the Korean Intellectual Property Office, the entire disclosures of which are herein incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/018931 | 12/22/2020 | WO |
