PD-1 polypeptide variants

Abstract

To provide PD-1 polypeptide variants for cancer treatment.SOLUTION: Provided are PD-1 polypeptide variants including an extracellular domain that binds specifically to PD-L1, and a transmembrane domain or a fragment thereof. The disclosure also provides PD-1 Fc fusion proteins including an immunoglobulin Fc region, and a PD-1 polypeptide variant.SELECTED DRAWING: Figure 3

Description

Cross - reference to related applications

[0001] This application claims priority based on U.S. Patent No. 63 / 075,641 filed on September 8, 2020, which is hereby incorporated by reference in its entirety. Background of the Invention

[0002] Cancer is still one of the leading causes of death in the world. According to recent statistics, it has been reported that 13% of the world's population dies from cancer. According to the estimation of the International Agency for Research on Cancer (IARC), in 2012, there were 14.1 million new cancer cases and 8.2 million cancer - related deaths worldwide. By 2030, due to population growth, aging, exposure to risk factors such as smoking, unhealthy diet, and lack of exercise, the number of cancer incidences is expected to increase to 21.7 million cases globally and expand to 13 million cancer deaths. Furthermore, the pain and medical costs associated with cancer treatment cause a decline in the quality of life for both cancer patients and their families.

[0003] Programmed cell death protein 1 (PD - 1) is an immune checkpoint receptor that plays a role in regulating the immune response by down - regulating the immune system, suppressing the inflammatory activity of T cells, and promoting self - tolerance. Since PD - 1 is overexpressed in cancer, leading to an increase in T - cell exhaustion and a decrease in the anti - tumor response, blocking the PD - 1 / PD - L1 inhibitory pathway and enhancing T - cell activation has great potential for treating diseases such as cancer. Summary of the Invention

[0004] In the present application, there is provided a programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein the PD-1 polypeptide variant comprises: an extracellular domain that specifically binds to programmed cell death 1 ligand (PD-L1); and a transmembrane domain or a fragment thereof. In some embodiments, the PD-1 polypeptide variant comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the PD-1 polypeptide variant comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the PD-1 polypeptide variant comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0005] In some embodiments, the PD-1 polypeptide variant comprises SEQ ID NO: 4. In some embodiments, the PD-1 polypeptide variant comprises SEQ ID NO: 6. In some embodiments, the PD-1 polypeptide variant comprises SEQ ID NO: 8.

[0006] The transmembrane domain used in the present invention may be the wild-type transmembrane domain of PD-1 corresponding to amino acid residues 171 to 191 of SEQ ID NO: 11 or a fragment thereof. In some embodiments, the transmembrane domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues, including all ranges and sub-ranges bound by these values. For example, the transmembrane domain fragment can include at least 2 amino acid residues, at least 5 amino acid residues, or at least 10 amino acid residues. In some embodiments, the transmembrane domain has 2 amino acid residues corresponding to amino acid residues 171 to 172 of SEQ ID NO: 11. In some embodiments, the transmembrane domain has 3 amino acid residues corresponding to amino acid residues 171 to 173 of SEQ ID NO: 11. In some embodiments, the transmembrane domain has 4 amino acid residues corresponding to amino acid residues 171 to 174 of SEQ ID NO: 11. In some embodiments, the transmembrane domain has 5 amino acid residues corresponding to amino acid residues 171 to 175 of SEQ ID NO: 11.

[0007] Included in the definition of the transmembrane domain or a fragment thereof are variants of the wild-type transmembrane domain of PD-1 corresponding to amino acid residues 171 to 191 of SEQ ID NO: 11 that may be modified, added to, or substituted with 1, 2, 3, 4, and 5 amino acids, including integer values of 1 to 5 amino acids that are modified by addition, deletion, or substitution.

[0008] In the present application, there is provided a programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having at least 95% sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0009] In some embodiments, the polypeptide variant has an enhanced binding affinity for the PD-L1 molecule as compared to the wild-type PD-1 polypeptide. In some embodiments, the polypeptide variant has a binding affinity (K -8 ~1×10 -10 ) for the PD-L1 molecule of about 1×10 D M. In some embodiments, the polypeptide variant has a binding affinity (K -9 ) for the PD-L1 molecule of about 1.10×10 -9 M, about 1.037×10 -10 M, or about 7.14×10 D M.

[0010] Also provided herein is a PD-1Fc fusion protein comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the immunoglobulin Fc region comprises a sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16, or SEQ ID NO: 10 or SEQ ID NO: 16.

[0011] Also provided herein is a PD-1Fc fusion protein comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% sequence identity to residues 24-172 of SEQ ID NO: 11, and the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0012] Also provided in the present application is an Fc-fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and an scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody has an amino acid sequence having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker sequence.

[0013] Also provided in the present application is an Fc-fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and an scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% sequence identity to residues 24 to 172 of SEQ ID NO: 11, and the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139, and wherein the scFv against the anti-4-1BB antibody has an amino acid sequence having at least 95% sequence identity to an amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker sequence.

[0014] In some embodiments, the immunoglobulin Fc region comprises a sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16. In some embodiments, the scFv against the anti-4-1BB antibody is an amino acid sequence that is at least 70% identical to SEQ ID NO: 22 or SEQ ID NO: 23.

[0015] The BsAb antibody in the present invention has or does not have the effect of reduced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0016] The BsAb antibody in the present invention is shown to reduce or not reduce hepatotoxicity. In some embodiments, the fusion protein has enhanced binding affinity to the PD-L1 molecule as compared to the wild-type PD-1 polypeptide. In some embodiments, the fusion protein has a binding affinity (K -8 ~1×10 -10 M) for the PD-L1 molecule. In some embodiments, the fusion protein has a binding affinity (K D ) of about 1.10×10 -9 M, about 1.037×10 -9 M, or about 7.14×10 -10 M for the PD-L1 molecule. D )

[0017] Also provided herein is a nucleic acid comprising a sequence encoding a PD-1 polypeptide variant, wherein the polypeptide variant comprises a sequence having at least 95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. In some embodiments, the nucleic acid further comprises a sequence encoding an immunoglobulin Fc region, wherein the sequence encoding the immunoglobulin Fc region comprises SEQ ID NO: 9.

[0018] Also provided herein is an expression vector comprising any one of the nucleic acids provided herein. In some embodiments, the vector is a viral vector.

[0019] Also provided herein is a pharmaceutical composition comprising any one of the PD-1 polypeptide variants provided herein or any one of the PD-1 fusion proteins provided herein; and a pharmaceutically acceptable carrier.

[0020] Also provided herein is a method for treating a subject, comprising the step of treating a disease or condition by administering to the subject in need thereof any one of the pharmaceutical compositions provided herein. In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

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Mode for Carrying Out the Invention

[0022] The present disclosure describes a PD-1 polypeptide variant, which contains a mutation in the wild-type PD-1 protein that enhances the affinity for the PD-L1 molecule.

[0023] Definition: about : The term "about", as used herein with respect to a value, refers to a value similar in relation to the referenced value. Generally, one of ordinary skill in the art, being familiar with the context, will recognize the degree of related variation encompassed by "about" in that context. For example, in some embodiments, the term "about" may include a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0024] administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system to achieve delivery of a drug that is a composition or is included in a composition. One of ordinary skill in the art will be aware of the various routes that can be utilized for administration to a subject, such as a human, in an appropriate setting. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., intratracheal instillation), buccal, dermal (e.g., one or more or including any of topical to the dermis, intradermal, intercutaneous, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., within the liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., tracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may include only a single dose. In some embodiments, administration can be accompanied by the application of a fixed dose. In some embodiments, administration may include administration that is intermittent (e.g., multiple doses divided over time) and / or periodic (e.g., individual doses separated by a common period). In some embodiments, administration may include continuous dosing (e.g., perfusion) over at least a selected period of time.

[0025] affinity : As is known in the art, "affinity" is a measure of the strength with which a particular ligand binds to its partner. Affinity can be measured in a variety of ways. In some embodiments, affinity is measured by a quantitative assay. In some of said embodiments, the binding partner concentration can be fixed at an excess ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the binding partner concentration and / or the ligand concentration may be varied. In some of said embodiments, the affinity can be compared to a reference under equivalent conditions (e.g., concentration).

[0026] antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term includes any polypeptide or polypeptide complex that contains the structural elements of an immunoglobulin sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, the antibody agent can include one or more sequence elements such as humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for exploiting the structural and functional features of antibodies in alternative expressions. For example, in embodiments, the antibody agents used in accordance with the present invention can be intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies™, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and their isolated CDRs or sets thereof; single-chain Fvs; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies™); small modular immunopharmaceuticals ("SMIPs TM"); single-chain or tandem diabody (TandAb®); VHH; Anticalins®; Nanobodies®; minibody; BiTE®; ankyrin repeat protein or DARPINs®; Avimers®; DART; TCR-like antibody; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microprotein; Fynomers®; Centyrins®; and KALBITOR®), but is not limited to, formats selected from. In some embodiments, the antibody agent may lack covalent modifications (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, the antibody agent may include covalent modifications (e.g., attachment of a glycan, payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant groups [e.g., polyethylene glycol, etc.]). In many embodiments, the antibody agent is a polypeptide that includes or includes a polypeptide having one or more structural elements whose amino acid sequence is recognized by those skilled in the art as complementarity determining regions (CDRs); in some embodiments, the antibody agent is a polypeptide that includes or includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the CDRs included are substantially identical to the reference CDRs in that the sequences are identical or contain 1 to 5 amino acid substitutions compared to the reference CDRs. In some exemplary embodiments, the CDRs included are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs. In some embodiments, the CDRs included are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs.The CDRs included in some embodiments are substantially the same as the reference CDRs in that at least one amino acid within the included CDRs is deleted, added or substituted as compared to the reference CDRs, but otherwise the included CDRs have an amino acid sequence that is identical to that of the reference CDRs. In some embodiments, the included CDRs are substantially the same as the reference CDRs in that 1 to 5 amino acids within the included CDRs are deleted, added or substituted as compared to the reference CDRs, but otherwise the included CDRs have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially the same as the reference CDRs in that at least one amino acid within the included CDRs is substituted as compared to the reference CDRs, but otherwise the included CDRs have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially the same as the reference CDRs in that 1 to 5 amino acids within the included CDRs are deleted, added or substituted as compared to the reference CDRs, but otherwise the included CDRs have the same amino acid sequence as the reference CDRs. In some embodiments, the antibody agent is a polypeptide that includes or is a polypeptide having a structural element recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent includes or contains at least a portion of a chimeric antigen receptor (CAR).

[0027] antigen: As used herein, the term "antigen" refers to an agent that binds to an antibody agent. In some embodiments, the antigen may or may not bind to an antibody agent and induce a specific physiological response in an organism. Generally, an antigen may be any chemical entity, such as, for example, a small molecule, nucleic acid, polypeptide, carbohydrate, lipid, polymer (including biological polymers [e.g., nucleic acid and / or amino acid polymers] and polymers other than biological polymers [e.g., polymers other than nucleic acid or amino acid polymers]), and may include the same. In some embodiments, the antigen is a polypeptide or includes a polypeptide. In some embodiments, the antigen is a glycan or includes a glycan. One of ordinary skill in the art will generally understand that the antigen may be provided in an isolated or pure form, or alternatively, in a crude form (e.g., an extract of an antigen-containing source or other relatively crude preparation together with other materials such as a cell extract). In some particular embodiments, the antigen is present in the context of a cell (e.g., the antigen is expressed on the surface of the cell or expressed intracellularly). In some embodiments, the antigen is a recombinant antigen.

[0028] antigen-binding domain : As used herein, "antigen-binding domain" refers to an antibody agent or a portion thereof that specifically binds to a target moiety or entity. Typically, the interaction between the antigen-binding domain and its target is non-covalent. In some embodiments, the target moiety or entity may be of any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain may be a polypeptide (or a complex thereof) or include a polypeptide. In some embodiments, the antigen-binding domain is part of a fusion polypeptide. In some embodiments, the antigen-binding domain is part of a chimeric antigen receptor (CAR).

[0029] associated: As used herein, two events or entities are "associated" with each other if one presence, level, and / or form is correlated with another that is different. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or pathological condition if its presence, level, and / or form is correlated with the incidence and / or susceptibility of the disease, disorder, or pathological condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they directly or indirectly interact to be physically proximate to and / or maintain proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently bonded to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently bonded to each other, but rather are non-covalently bonded by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0030] binding : The term "binding" as used herein will generally be understood to refer to non-covalent bonds between or among two or more entities. "Direct" binding involves physical contact between entities or moieties; indirect binding involves physical interaction through physical contact with one or more intermediate entities. The binding between two or more entities can be evaluated from any of a variety of circumstances, typically including when the interacting entities or moieties are studied alone or in the context of a more complex system (e.g., where the carrier entity is covalently or otherwise associated with, and / or within a biological system or cell).

[0031] cancer: As used herein, the terms “cancer,” “malignant tumor,” “neoplasm,” “tumor,” and “carcinoma” refer to cells that exhibit a phenotype that is characterized by abnormal growth that is relatively uncontrolled and / or autonomous in this specification and that represents a loss of significant regulation of cell proliferation. In some embodiments, the tumor may be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure does not specifically identify particular cancers to which its teachings may particularly pertain. In some embodiments, the relevant cancer may be characterized by a solid tumor. In some embodiments, the relevant cancer may be characterized by a hematological tumor.

[0032] Generally, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinomas of solid tissues, squamous cell carcinoma of the mouth, pharynx, larynx, lung, liver cancer, genitourinary cancer, prostate cancer, cervical cancer, bladder cancer, endometrial cancer, renal cell cancer, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine cancers, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancers, benign lesions, papillary adenocarcinoma, and the like.

[0033] chemotherapeutic agent: As used herein, the term "chemotherapeutic agent" refers to one or more apoptosis promoters, cell growth inhibitors, and / or cytotoxic agents having the meaning understood in the art, including formulations recommended for use / not for use for treating one or more diseases, disorders, or conditions associated with unwanted cell growth. In many embodiments, the chemotherapeutic agent is useful for treating cancer. In some embodiments, the chemotherapeutic agent is one or more alkylating agents, one or more anthracyclines, one or more cytoskeleton disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansines, and their analogs), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or any one or more analogs (i.e., sharing related anti-proliferative activity), or may include the same. In some specific embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and one or more of their combinations, or may include the same. In some embodiments, the chemotherapeutic agent may be utilized in connection with an antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is found in an antibody-drug conjugate selected from the group consisting of hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumumab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, bolusertib mafodotin and robocertinib mertansine.

[0034] modified: Generally, the term "modified" refers to a state modified by human hands. For example, a polypeptide is considered "modified" when its polypeptide sequence is modified by human hands. For example, in some embodiments of the present invention, a modified polypeptide includes a sequence containing one or more amino acid mutations, deletions, and / or insertions introduced into a reference polypeptide sequence by human hands. In some embodiments, a modified polypeptide includes a polypeptide that has been fused (i.e., covalently bonded) to one or more additional polypeptides by human hands to form a fusion polypeptide that does not occur naturally in vivo. Similarly, a cell or organism is considered "modified" when its genetic information is modified (e.g., when new genetic material that did not previously exist is introduced by, for example, transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or when previously existing genetic material is modified or removed by, for example, substitution or deletion mutations, or mating protocols). It is a common practice and, as understood by those skilled in the art, derivatives and / or progeny of a modified polypeptide or cell are typically still referred to as "modified" even if the actual modification was made to a previous entity.

[0035] pharmaceutical composition : As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0036] pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material that is involved in carrying or transporting a useful compound in the present invention within or to a patient in order to perform an intended function. It has the meaning recognized in the art. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier should be compatible with the other components of the formulation containing the useful compound of the present invention and "acceptable" in the sense that it does not harm the patient. As used herein, "pharmaceutically acceptable carrier" also includes any and all coating agents, antibacterial and antifungal agents, absorption delaying agents, etc. that can be compatible with the activity of the useful compound in the present invention and are physiologically acceptable to the patient. The term "pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the useful compounds in the present invention. Other additional components that may be included in the pharmaceutical compositions used in the practice of the present invention are described, for example, in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference. "Pharmaceutically acceptable carrier" is generally useful for the preparation of pharmaceutical compositions that are compatible with the other components of the composition, not harmful to the recipient, and not biologically or otherwise undesirable. "Pharmaceutically acceptable carrier" includes one or more carriers. Embodiments include carriers for topical, ocular, parenteral, intravenous, intraperitoneal, intramuscular, sublingual, nasal, or oral administration. "Pharmaceutically acceptable carrier" further includes formulations for preparing aqueous dispersions and sterile powders or dispersions for injection.

[0037] pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" generally has the meaning recognized in the art and refers to derivatives of the compounds provided herein, where the parent compound is modified by converting an existing acidic or basic moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; etc. Pharmaceutically acceptable salts of the compounds provided herein include, for example, conventional non-toxic salts of the parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the compounds provided herein can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods.

[0038] Generally, such salts can be prepared by combining the free acid or base forms of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used. A list of suitable salts is described in the literature [Remington’s Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977)], which are hereby incorporated by reference in their entireties.

[0039] excipient : As used herein, the term "excipient" generally has the meaning recognized in the art and refers to physiologically compatible additives useful in the preparation of pharmaceutical compositions. Examples of pharmaceutically acceptable carriers and excipients are described, for example, in the literature [Remington Pharmaceutical Science, 16 th Ed].

[0040] polypeptide: As used herein, the term "polypeptide" generally has the meaning recognized in the art for polymers of at least three amino acids. One of ordinary skill in the art will understand that the term "polypeptide" includes not only polypeptides having the complete sequences recited herein, but also polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that possess at least one activity). Also, one of ordinary skill in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that generally maintains activity with other polypeptides of the same general class, shares an overall sequence identity of at least about 30-40%, often greater than about 50%, 60%, 70%, or 80% overall sequence identity, and further includes at least one region of higher identity that exceeds 90% or 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, and contains at least 3-4 and often 20 or more amino acids, is included within the term "polypeptide" as used herein. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the protein may include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody agent, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0041] recombinant: As used herein, "recombinant" refers to a polypeptide expressed using a recombinant expression vector transfected into a polypeptide host cell that has been designed, modified, prepared, expressed, created, manufactured, and / or isolated by recombinant means; a polypeptide isolated from a recombinant, combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or modified to express a gene component encoding a gene or genes, or one or more of its constituent(s), part(s), element(s), or domain(s) and / or to direct its expression; and / or a polypeptide produced, expressed, generated, or isolated by any other means involving splicing or ligating selected nucleic acid sequence elements to each other, or chemically synthesizing the selected sequence elements and / or generating a nucleic acid encoding a polypeptide or one or more of its component(s), part(s), element(s), or domain(s) and / or directing its expression. In some embodiments, one or more of such selected sequence elements exist in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements are generated from mutagenesis of known sequence elements from natural or synthetic sources, such as in the germline of an organism of interest (e.g., human, mouse, etc.).

[0042] specific binding: As used herein, the term "specific binding" refers to the ability to identify a binding partner that is possible in the environment where binding occurs. A binder that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is evaluated by detecting or determining the degree of association between the binder and its partner; in some embodiments, specific binding is evaluated by detecting or determining the degree of dissociation of the binder-partner complex. In some embodiments, specific binding is evaluated by detecting or determining the ability of the binder to compete with alternative interactions between its partner and another entity. In some embodiments, specific binding is evaluated by performing such detection or determination over a concentration range.

[0043] subject : As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments a prenatal human form). In some embodiments, the subject has a related disease, disorder, or medical condition. In some embodiments, the subject is susceptible to a disease, disorder, or medical condition. In some embodiments, the subject exhibits one or more symptoms or signs of a disease, disorder, or medical condition. In some embodiments, the subject does not exhibit any symptoms or signs of a disease, disorder, or medical condition. In some embodiments, the subject is a human having one or more characteristics characteristic of susceptibility or risk to a disease, disorder, or medical condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual on whom a diagnosis and / or treatment has been and / or is being performed.

[0044] therapeutic agent: As used herein, the term "therapeutic agent" generally refers to any agent that, when administered to an organism, produces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it has a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population can be defined by various criteria such as a particular age group, gender, genetic background, existing clinical condition, etc. In some embodiments, a therapeutic agent can be a substance used for alleviating, improving, reducing, suppressing, preventing, delaying onset, reducing severity, and / or decreasing incidence of one or more symptoms or signs of a disease, disorder, and / or medical condition. In some embodiments, a "therapeutic agent" is an agent that has been approved or requires approval by a government agency before being marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a prescription for administration to humans.

[0045] therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease, disorder, and / or condition in a population that has or is susceptible to having the disease, disorder, and / or condition when administered according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more of its characteristics, and / or delays onset. One of ordinary skill in the art will understand that the term "therapeutically effective amount" does not actually require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that blocks, stabilizes, attenuates, or reverses cancer-supportive processes arising from an individual when administered to an individual in need thereof in the therapeutic context of the present invention. In the context of cancer treatment, a "therapeutically effective amount" is an amount that prevents, stabilizes, inhibits, or reduces further progression of cancer in an individual when administered to an individual diagnosed with cancer. A particularly preferred "therapeutically effective amount" of the compositions described herein is (in a therapeutic treatment) useful for prolongation, such as reversing the progression of a malignancy such as pancreatic cancer or achieving remission of the malignancy. The therapeutically effective amount administered to an individual to treat that individual's cancer may be the same as or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. Similar to most cancer treatments, the treatment methods described herein should not be construed, limited, or restricted as "treatment" for cancer. Rather, the treatment method relates to the use of the described compositions for "treating" cancer, i.e., for bringing about a desirable or beneficial change to the health of an individual suffering from cancer.Such advantages are recognized by skilled medical providers in the field of oncology and include, but are not limited to, stabilization of the patient's condition, reduction in tumor size (tumor regression), improvement in life functions (e.g., improvement in the function of cancerous tissue or organs), further reduction or suppression of metastasis, reduction in opportunistic infections, increase in survival rate, reduction in pain, improvement in motor function, improvement in cognitive function, improvement in sense of energy (vitality, reduction in fatigue), improvement in sense of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Further (e.g., as a result of the treatments described herein), regression of a particular tumor in an individual can also be evaluated (e.g., as a result of treatment) by taking a sample of cancer cells from the site of the tumor, such as pancreatic adenocarcinoma, and testing the cancer cells for levels of metabolic and signaling markers and verifying whether the cancer cells regress to a less malignant phenotype at the molecular level in order to monitor the state of the cancer cells. For example, tumor regression induced by using the methods of the present invention is indicated by a decrease in any of the angiogenesis-promoting markers discussed above, an increase in the anti-angiogenesis markers described herein, normalization of metabolic pathways (i.e., a change to a state found in healthy individuals not suffering from cancer), discovery of an intracellular signaling pathway that shows abnormal activity in a solid that has received a cancer diagnosis, or an intercellular signaling pathway. One of ordinary skill in the art will understand that in some embodiments, a therapeutically effective amount can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount can be formulated and / or administered in multiple doses, for example, as part of a dosing schedule.

[0046] variant: As used herein, the term "variant" in the context of a molecule, such as a nucleic acid, protein, or small molecule, refers to a molecule that exhibits significant structural identity with a reference molecule in the presence, absence, or level of one or more chemical moieties, but is structurally different from the reference molecule. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is appropriately considered a "variant" of a reference molecule is determined based on the degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more of such characteristic structural elements, but differs from the reference molecule in at least one aspect. To give just a few examples, a polypeptide can have characteristic sequence elements composed of a plurality of amino acids that have specified positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function; a nucleic acid can have characteristic sequence elements composed of a plurality of nucleotide residues that have specified positions relative to others in linear or three-dimensional space. In some embodiments, a mutant polypeptide or nucleic acid can differ from a reference polypeptide or nucleic acid as a result of one or more differences in the amino acid or nucleotide sequence. In some embodiments, a variant polypeptide or nucleic acid exhibits an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid.

[0047] vectorAs used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid that has been ligated thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Further, certain vectors can direct the expression of a gene operably linked thereto. Such vectors are referred to herein as "expression vectors". Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the specifications of the manufacturer, or as commonly accomplished in the art, or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, see the reference [Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)], which is incorporated herein by reference for any purpose.

[0048] programmed cell death protein 1 (PD-1) Programmed cell death protein 1 (PD-1) is an inhibitory receptor expressed by all T cells during activation. PD-1 is expressed not only by conventional T cells, but also by regulatory T cells, B cells, natural killer (NK) cells, and some myeloid cell populations. PD-1 regulates the function of effector T cells during various physiological responses, including acute and chronic infections, cancer, autoimmunity, and immune homeostasis. Additionally, PD-1 often exhibits high and persistent expression levels during antigen encounter, which can occur in the context of chronic infections or cancer and can limit protective immunity.

[0049] In the tumor microenvironment, PD-1 and its ligand, programmed cell death ligand 1 (PD-L1), play important roles in tumor progression and survival by evading tumor neutralization and immune surveillance. Blocking the PD-1 and PD-L1 inhibitory pathways has been shown to enhance T cell activation and result in beneficial anti-tumor responses and long-term remissions in a subset of cancer patients. Therefore, the use of inhibitors that block the interaction between PD-L1 and PD-1 (e.g., against T cells) helps prevent PD-1 stimulation and thereby increase T cell function signaling and immune cell responses.

[0050] In some embodiments, specific mutations are introduced into wild-type PD-1 to generate PD-1 polypeptide variants, where the specific mutations induce preferential binding to PD-L1. In some embodiments, the PD-1 polypeptide variant is a soluble PD-1 protein. In some embodiments, the PD-1 polypeptide variant comprises an extracellular domain and a transmembrane domain or a fragment thereof. The transmembrane domain or a fragment thereof is described hereinabove.

[0051] In some embodiments, the PD-1 polypeptide variant comprises an amino acid sequence having 95% or greater (e.g., 96% or greater, 97% or greater, 98% or greater, or 99% or greater) sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, where the PD-1 polypeptide variant comprises an extracellular domain that specifically binds to PD-L1 and a transmembrane domain or fragment thereof. In some embodiments, the PD-1 polypeptide variant comprises the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

[0052] In some embodiments, the PD-1 polypeptide variant has an enhanced binding affinity for a PD-L1 molecule compared to a wild-type PD-1 polypeptide. In some embodiments, the polypeptide variant has an increased binding affinity for a PD-L1 molecule of about 1×10 -8 ~1×10 -10 M, preferably about 1 x 10 -9 ~1×10 -10 Binding affinity of M (K D In some embodiments, the polypeptide variant has an affinity for PD-L1 molecules of about 1.10×10 -9 M, approx. 1.037×10 -9 M, or approximately 7.14 x 10 -10 Binding affinity of M (K D ).

[0053] As used herein, "nucleic acid" is used to include any compound and / or substance, including polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA).

[0054] In some embodiments, the nucleic acid comprises a sequence encoding a PD-1 polypeptide variant, where the PD-1 polypeptide variant comprises at least one nucleotide sequence having 95% or greater (96% or greater, 97% or greater, 98% or greater, 99% or greater) sequence identity to SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

[0055] [Chem.] JPEG2025098135000003.jpg168161 JPEG2025098135000004.jpg124161

[0056] In all embodiments described herein, the PD-1 variant may contain additional residues at the C-terminus resulting from the choice of restriction endonucleases used for cloning. The additional residues resulting from the cloning choice are limited to 1, 2, 3, or 4 residues, and it is preferred that they be given a dipeptide addition, preferably an alanine - serine dipeptide. For example, when used as part of the PD-1 polypeptide variant itself or a fusion construct, in the case of euPD-1, the sequence of SEQ ID NO: 8 and the C-terminal AS dipeptide are included in the present invention. Such sequences are envisioned and included by the level of amino acid sequence identity defined for the PD-1 polypeptide variants of the present invention.

[0057] In some embodiments, the PD-1 polypeptide variant of the present invention includes a PD-1 variant comprising an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 - 172 of SEQ ID NO: 11, wherein said variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, vectors containing the nucleic acid, pharmaceutical compositions containing the PD-1 polypeptide variant of this embodiment, and methods of treating a disease or condition (e.g., cancer) of a subject in need of treatment by administering the pharmaceutical composition.

[0058] In a variant of this embodiment, the PD-1 polypeptide variant of the present invention includes a PD-1 variant comprising an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variants of this embodiment, vectors containing the nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variants of this embodiment, and methods of treating a disease or condition (e.g., cancer) of a subject in need of treatment by administering the pharmaceutical composition.

[0059] In some embodiments, the PD-1 polypeptide variant of the present invention includes a PD-1 variant comprising an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variants of this embodiment, vectors containing the nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variants of this embodiment, and methods of treating a disease or condition (e.g., cancer) of a subject in need of treatment by administering the pharmaceutical composition.

[0060] Preferred mutations include D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y, A125V, A132I, R139G.

[0061] Fc fusion protein and encoded nucleotide sequence As used herein, "Fc fusion protein" refers to a modified protein composed of the Fc domain of IgG linked to a polypeptide or protein of interest. Examples of Fc fusion polypeptides or proteins include, but are not limited to, single peptides, ligands that are activated upon binding to cell surface receptors, signaling molecules (e.g., cytokines), extracellular domains of receptors that are activated upon dimerization, and bait proteins used to identify binding partners for protein microarrays. In some embodiments, the Fc fusion protein acts as an antibody agent that specifically binds to a particular antigen via an antigen-binding domain. In some embodiments, the Fc fusion protein comprises a polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding.

[0062] Fc is a constant domain selected from IgG1 or variants thereof, IgG2 or variants thereof, IgG4 or variants thereof, and humanized IgG1 / 2 or variants thereof. Fc plays multiple roles in dimerization for the formation and maintenance of the Y-shaped structure of Ig, and in Fc-mediated effector functions and the extension of serum half-life. In some embodiments, monomeric Fc has two domains, the second constant domain (CH2) and the third constant domain (CH3). In some embodiments, the fusion protein comprises a linker that fuses the Fc domain with a particular peptide or protein together. In some embodiments, the immunoglobulin Fc region is linked to a particular peptide or protein by a peptide bond. In some embodiments, the immunoglobulin Fc region is linked to a particular peptide or protein by a peptide linker sequence. In some embodiments, the linker sequence comprises (G4S)2 (SEQ ID NO: 19), (G4S)3 linker (SEQ ID NO: 20), or 218 linker (SEQ ID NO: 21).

[0063] In some embodiments, a particular peptide or protein is linked to the carboxy-terminus of the immunoglobulin Fc region. In some embodiments, the Fc domain comprises a sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0064] In some embodiments, a PD-1 polypeptide variant is used to produce a PD-1 fusion protein. In some embodiments, the PD-1 fusion protein may comprise a PD-1 polypeptide variant and an immunoglobulin Fc region. In some embodiments, the PD-1Fc fusion protein comprises an immunoglobulin Fc region and a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0065] In some embodiments, the immunoglobulin Fc region comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0066] In some embodiments, the PD-1Fc fusion protein has enhanced binding affinity for the PD-L1 molecule as compared to the wild-type PD-1 polypeptide. In some embodiments, the PD-1Fc fusion protein has a binding affinity (K -8 ~1×10 -10 M, preferably about 1×10 -9 ~1×10 -10 M) for the PD-L1 molecule. In some embodiments, the PD-1Fc fusion protein has a binding affinity for the PD-L1 molecule of about 1.10×10 D ) for the PD-L1 molecule. In some embodiments, the PD-1Fc fusion protein has a binding affinity for the PD-L1 molecule of about 1.10×10 -9 M, about 1.037×10 -9M, or about 7.14×10 -10 The binding affinity (K D ) of M.

[0067] As used herein, "nucleic acid" is used to include any compound and / or substance that includes a polynucleotide. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA).

[0068] In some embodiments, the nucleic acid construct includes an immunoglobulin Fc region and a region encoding a PD-1 polypeptide variant. In some embodiments, the sequence encoding the Fc domain includes a nucleotide sequence having at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:9.

[0069]

Chemical formula

[0070] As described above, in all embodiments described herein, the PD-1 variant may include additional residues at the C-terminus that result from the choice of restriction endonuclease used for cloning. The additional residues resulting from cloning choices are preferably limited to 1, 2, 3, or 4 residues with dipeptide addition, preferably alanine-serine dipeptide. For example, when used as part of the PD-1 polypeptide variant itself or a fusion construct, in the case of euPD-1, the sequence of SEQ ID NO:8 and the C-terminal AS dipeptide are included in the present invention. Such sequences are contemplated and included by the level of amino acid sequence identity defined for the PD-1 polypeptide variants of the present invention. Thus, in the fusion constructs of this embodiment, the residues resulting from cloning may be located in front of the linker.

[0071] Also, a PD-1Fc fusion protein is included by the present invention, where multiple copies of a PD-1 polypeptide variant are each separated by a linker and present. The term "multiple copies of a PD-1 polypeptide variant" means that two, three, or four PD-1 polypeptide variants may be present, where the variants present may be the same or different. Also, the linkers separating each PD-1 polypeptide variant may be the same or different. Also, the linker between PD-1 polypeptide variants may be different from the linker between a PD-1 polypeptide variant and a polypeptide or protein of interest.

[0072] In some embodiments, the PD-1 polypeptide variant of the PD-1Fc fusion protein comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11, where the variant has a mutation at at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variant of this embodiment, vectors containing the nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variant of this embodiment, and methods of treating a disease or condition (e.g., cancer) of a subject in need of treatment by administering the pharmaceutical composition.

[0073] In a variant of this embodiment, the PD-1 polypeptide variant of the PD-1Fc fusion protein comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variants of this embodiment, vectors containing the nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variants of this embodiment, and methods of treating a disease or condition (e.g., cancer) of a subject in need of treatment by administering the pharmaceutical composition.

[0074] In some embodiments, the PD-1 polypeptide variant of the PD-1Fc fusion protein comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein the variant has mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139. The present invention includes nucleic acids encoding the PD-1 polypeptide variants of this embodiment, vectors containing the nucleic acids, pharmaceutical compositions containing the PD-1 polypeptide variants of this embodiment, and methods of treating a disease or condition (e.g., cancer) of a subject in need of treatment by administering the pharmaceutical composition.

[0075] Preferred mutations include D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y, A125V, A132I, R139G.

[0076] As described above, in some embodiments, the immunoglobulin Fc region comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0077] 4-1BB 4-1BB (sometimes referred to as CD137, TNFRSF9) is a receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily. 4-1BB is commonly expressed on activated T lymphocytes and is a costimulatory molecule involved in immune and autoimmune diseases (Kwon et al. PNAS 84:2896, 1987; Kwon et al. PNAS 86:1963, 1989; Son et al. Journal of Immunological Methods 286(1-2):187-201, 2004, each of which is incorporated herein by reference in its entirety). Human 4-1BB is a 255 amino acid protein that is expressed on the cell surface in monomeric (30 kDa) and dimeric (55 kDa) forms and trimerizes with 4-1BB ligand to transmit signals.

[0078] Furthermore, 4-1BB is constitutively expressed in many cells including Foxp3+ Tregs and dendritic cells (DCs), albeit at low levels. Activation by a number of agonists such as cytokines (e.g., IL-2, IL-4), polyclonal activators (e.g., Con A and PHA), cell surface molecules (e.g., anti-CD3, anti-CD28), and Ca2+-inducing and PKC-activating promoters (e.g., ionomycin, phorbol myristate acetate) further enhances the expression of 4-1BB.

[0079] Numerous studies on mouse and human T cells have shown that 4-1BB promotes improved cell proliferation, survival, and cytokine production. According to the research, some 4-IBB agonist monoclonal antibodies increase the expression of costimulatory molecules, significantly improve the cytotoxic T lymphocyte response, and bring about an anti-tumor effect in preventive and therapeutic settings. Also, tumor models of 4-1BB monotherapy and combination therapy have established a durable anti-tumor protective T cell memory response. 4-IBB agonists have also been shown to inhibit autoimmune reactions in various autoimmune models recognized in the art. Such dual activity of 4-1BB presents the possibility of providing anti-tumor activity while suppressing the autoimmune side effects associated with immunotherapy approaches.

[0080] In some embodiments of the present application, the fusion protein may include an anti-4-1BB antibody domain as an immunoglobulin Fc region. Specifically, the anti-4-1BB antibody domain can be produced using the 94kvt clone (International Publication WO2018-127787, the entire content of which is incorporated herein by reference) that holds the anti-4-1BB antibody domain (94kvt) as a single-chain Fv (scFv). Examples of scFvs suitable for anti-4-1BB antibodies include VH-218 linker-VL (HLC218) and VL-218 linker-VH (LHC218). The 94kvt VH sequence is shown in SEQ ID NO: 17, while the 94kvt VL sequence is shown in SEQ ID NO: 18, and the 218 linker is presented in SEQ ID NO: 21. Also, the scFv variant 94kvt HLC 218 is presented in SEQ ID NO: 22, and the scFv variant 94kvt LHC 218 is presented in SEQ ID NO: 23.

[0081] In some embodiments, the anti-4-1BB antibody domain (94kvt) as a single-chain Fv (scFv) comprises a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID NO: 23.

[0082] Accordingly, one embodiment of the present invention is an Fc fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant has an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and an scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to an amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker sequence.

[0083] Also provided is an Fc fusion BsAb (bispecific antibody) comprising an immunoglobulin Fc region; and a PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant has an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11; and an scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139, and wherein the scFv against the anti-4-1BB antibody has an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) sequence identity to an amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker.

[0084] In some embodiments, the immunoglobulin Fc region comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0085] In some embodiments, the scFv against the anti-4-1BB antibody has an amino acid sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID NO: 23.

[0086] Examples of linker sequences include (G4S)2 (SEQ ID NO: 19), (G4S)3 linker (SEQ ID NO: 20), or 218 linker (SEQ ID NO: 21).

[0087] The bispecific antibodies described herein have enhanced binding affinity for the PD-L1 molecule and specific binding affinity for 4-1BB as compared to the wild-type PD-1 polypeptide.

[0088] The BsAb antibodies of the present invention have a reduced antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) effect or have no such effect at all.

[0089] The BsAb antibodies of the present invention show reduced hepatotoxicity or no toxicity at all.

[0090] vector In some embodiments, the nucleic acid construct described above can be inserted into an expression vector or a viral vector by methods known in the art, and the nucleic acid molecule can be operably linked to an expression control sequence. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and virus-derived vectors (e.g., any adenovirus-derived vector (AV), cytomegalovirus-derived (CMV) vector, simian virus (SV40) vector, adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector). In some embodiments, the expression vector is a viral vector.

[0091] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to assist in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is widely known in the art.

[0092] In some embodiments, the nucleic acid molecule is inserted into a vector capable of expressing the PD-1 polypeptide variant or PD-1Fc fusion protein of the present disclosure when introduced into a suitable cell.

[0093] use in therapy In some embodiments, the PD-1 polypeptide variants, PD-1Fc fusion proteins, or nucleic acid constructs described herein can be used to treat a subject in need thereof. In some embodiments, the subject is diagnosed with a PD-L1-related disease. In some embodiments, the subject is diagnosed with a PD-L1-related cancer. In some embodiments, a pharmaceutical composition comprising a PD-1 polypeptide variant or a PD-1Fc fusion protein, and a pharmaceutically acceptable carrier can be administered to a subject diagnosed with a PD-L1-related disease. In some embodiments, the pharmaceutical composition can be administered with one or more additional anti-cancer therapies including, but not limited to, ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors.

[0094] The PD-1 / PD-L1 pathway represents an adaptive immune resistance mechanism used by tumor cells in response to endogenous immune anti-tumor activity. PD-L1 expressed on tumor cells binds to the PD-1 receptor of activated T cells and inhibits cytotoxic T cells. In some embodiments, the PD-1 polypeptide variant is used as a PD-L1 inhibitor for treating cancers including, but not limited to, non-small cell lung cancer, lung adenocarcinoma, gastric cancer, and breast cancer. Cancer may refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation form malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. A cancer or cancer tissue may contain a tumor.

[0095] Cancers suitable for treatment by the methods of the present disclosure can include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, cancers for treatment by the methods of the present disclosure can include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphomas), blastomas, sarcomas, and leukemias. In some embodiments, cancers can include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancers.

[0096] In some embodiments, the cancer may be a fetal tumor (Wilms tumor, hepatoblastoma, rhabdoid, neuroblastoma), germ cell tumor (yolk sac tumor, immature teratoma, and fetal carcinoma), carcinoma (hepatocellular carcinoma and squamous cell carcinoma of the lung), sarcoma (malignant rhabdoid tumor and RMS), or malignant melanoma.

[0097] The fusion proteins and bispecific antibodies of the present invention have the further advantage of having reduced or no antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) effects. Furthermore, the fusion proteins and bispecific antibodies of the present invention have reduced or no hepatotoxicity.

[0098] In the context of this specification, all publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety for all purposes as if fully set forth, unless otherwise indicated, and the entire contents thereof are to be regarded as part of the present disclosure.

[0099] Unless otherwise specified, all technical and scientific terms used in this specification shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, this specification, including definitions, shall prevail.

[0100] The foregoing description of the invention provides a method and process for making and using it so that one of ordinary skill in the art can make and use the same, and such possibilities are provided particularly with respect to the subject matter of the appended claims which form part of the original specification.

[0101] As used herein, phrases such as "selected from the group consisting of" and "selected from" include mixtures of specific substances.

[0102] Where numerical limits or ranges are expressly stated herein, endpoints are included. Also, all values and subranges within the numerical limitations or ranges are clearly included as if expressly written out.

[0103] The foregoing description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a particular use and its requirements. Various modifications to the preferred embodiments will be apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other embodiments and uses without departing from the spirit and scope of the invention. Accordingly, the invention is not intended to be limited to the embodiments presented, but rather should be accorded the widest scope consistent with the principles and features disclosed herein.

[0104] Without being limited to the following specific embodiments, the invention is illustrated by the following: (1) A programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, wherein the PD-1 polypeptide variant is: An extracellular domain that specifically binds to programmed cell death 1 ligand (PD-L1); and A transmembrane domain or a fragment thereof A programmed cell death 1 ligand (PD-L1) polypeptide variant comprising

[0105] (2) The PD-1 polypeptide variant according to (1), wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0106] (3) In (1) or (2), the PD-1 polypeptide variant, wherein the PD-1 polypeptide variant comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0107] (4) The PD-1 polypeptide variant according to any one of (1) to (3), wherein the PD-1 polypeptide variant comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0108] (5) The PD-1 polypeptide variant according to (1), wherein the PD-1 polypeptide variant comprises SEQ ID NO: 4.

[0109] (6) The PD-1 polypeptide variant according to (1), wherein the PD-1 polypeptide variant comprises SEQ ID NO: 6.

[0110] (7) The PD-1 polypeptide variant according to (1), wherein the PD-1 polypeptide variant comprises SEQ ID NO: 8.

[0111] (8) The PD-1 polypeptide variant according to any one of (1) to (7), wherein the transmembrane domain comprises at least 2 amino acid residues.

[0112] (9) The PD-1 polypeptide variant according to any one of (1) to (8), wherein the transmembrane domain comprises at least 5 amino acid residues.

[0113] (10) A PD-1 polypeptide variant as described in any one of (1) to (9), wherein the transmembrane domain herein contains at least 10 amino acid residues.

[0114] (11) A PD-1 polypeptide variant as described in any one of (1) to (10), wherein the polypeptide variant herein has an enhanced binding affinity for the PD-L1 molecule as compared to the wild-type PD-1 polypeptide.

[0115] (12) A PD-1 polypeptide variant as described in any one of (1) to (11), wherein the polypeptide variant herein has a binding affinity (K -8 ~1×10 -10 M) for the PD-L1 molecule. D )

[0116] (13) A PD-1 polypeptide variant as described in any one of (1) to (12), wherein the polypeptide variant herein has a binding affinity (K -9 M, about 1.037×10 -9 M, or about 7.14×10 -10 M) for the PD-L1 molecule. D )

[0117] (14) An immunoglobulin Fc region; and A PD-1 Fc fusion protein comprising a PD-1 polypeptide variant linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant herein comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0118] The PD-1Fc fusion protein described in (15)(14), wherein the immunoglobulin Fc region comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0119] The PD-1Fc fusion protein described in any one of (16)(14) to (15), wherein the fusion protein has an enhanced binding affinity for the PD-L1 molecule as compared to the wild-type PD-1 polypeptide.

[0120] (17) The PD-1Fc fusion protein described in any one of (14) to (16), wherein the fusion protein has a binding affinity (K -8 ~1×10 -10 M) for the PD-L1 molecule. D

[0121] (18) In any one of (14) to (17), the PD-1Fc fusion protein, wherein the fusion protein has a binding affinity (K -9 M, about 1.037×10 -9 M, or about 7.14×10 -10 M) for the PD-L1 molecule. D

[0122] (19) The PD-1Fc fusion protein described in any one of (14) to (18), wherein the PD-1 polypeptide variant is linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0123] (20) The PD-1Fc fusion protein described in any one of (14) to (19), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0124] A PD-1Fc fusion protein as described in any of (14)-(20), wherein one or more copies of a PD-1 polypeptide variant are present, and wherein said copies may be the same or different, and are linked by a peptide linker sequence.

[0125] A PD-1Fc fusion protein as described in (21), wherein two copies of a PD-1 polypeptide variant are present.

[0126] A PD-1Fc fusion protein as described in (21) or (22), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0127] A PD-1Fc fusion protein as described in any of (14)-(23), wherein the PD-1Fc fusion protein is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

[0128] A nucleic acid comprising a sequence encoding a PD-1 polypeptide variant, wherein the polypeptide variant comprises a sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0129] (26) As described in (25). Further comprising a sequence encoding an immunoglobulin Fc region, wherein the sequence encoding the immunoglobulin Fc region comprises SEQ ID NO: 9.

[0130] An expression vector comprising the nucleic acid as described in any of (25) or (26).

[0131] A vector as described in (27), wherein the vector is a viral vector.

[0132] (29) A PD-1 polypeptide variant as described in any one of (1) to (13) or a PD-1 fusion protein as described in any one of (14) to (24); and A pharmaceutically acceptable carrier.

[0133] (30) A method for treating a disease or condition in a subject in need of treatment, comprising administering the pharmaceutical composition of (29) to the subject and treating the disease or condition.

[0134] (31) The method according to (30), wherein the subject has cancer.

[0135] (32) The method according to (31), wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0136] (33) The method according to any one of (30) to (32), wherein when the pharmaceutical composition contains a PD-1 fusion protein as described in any one of (14) to (24), a decreased antibody-dependent cell cytotoxicity (ADCC) and / or complement-dependent cell cytotoxicity (CDC) effect is observed or no effect is observed.

[0137] (34) The method according to any one of (30) to (33), wherein when the pharmaceutical composition contains a PD-1 fusion protein as described in any one of (14) to (24), decreased hepatotoxicity is observed or no hepatotoxicity is observed.

[0138] A programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0139] (36) The PD-1 polypeptide variant according to (35), wherein said variant comprises a mutation at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139.

[0140] (37) The PD-1 polypeptide variant according to (35), wherein said variant comprises a mutation at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

[0141] (38) The PD-1 polypeptide variant according to any one of (35) to (37), wherein said mutation is D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y, A125V, A132I, or R139G.

[0142] (39) The PD-1 polypeptide variant according to any one of (35) to (38), wherein the PD-1 polypeptide variant comprises an amino acid sequence having a sequence identity of 97% or more with respect to residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139, a PD-1 polypeptide variant.

[0143] (40) The PD-1 polypeptide variant according to any one of (35) to (39), wherein the PD-1 polypeptide variant comprises an amino acid sequence having a sequence identity of 98% or more with respect to residues 24 to 172 of SEQ ID NO: 11, wherein said variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139, a PD-1 polypeptide variant.

[0144] (41) The PD-1 polypeptide variant according to any one of (35) to (40), wherein the polypeptide variant has an enhanced binding affinity for the PD-L1 molecule as compared to the wild-type PD-1 polypeptide, a PD-1 polypeptide variant.

[0145] (42) The PD-1 polypeptide variant according to any one of (35) to (41), wherein the polypeptide variant has a binding affinity (K -8 ~1×10 -10 ) for the PD-L1 molecule, a PD-1 polypeptide variant. D

[0146] (43) An immunoglobulin Fc region; and A PD-1Fc fusion protein comprising the PD-1 polypeptide variant according to any one of (35) to (42) linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence.

[0147] (44) The PD-1Fc fusion protein described in (43), wherein the immunoglobulin Fc region comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0148] (45) The PD-1Fc fusion protein described in (43) or (44), wherein the fusion protein has enhanced binding affinity for the PD-L1 molecule as compared to the wild-type PD-1 polypeptide.

[0149] (46) The fusion protein according to any one of (43) to (45), wherein the fusion protein has a binding affinity (K -8 ~1×10 -10 M) for the PD-L1 molecule of about 1×10 D .

[0150] (47) The PD-1Fc fusion protein according to any one of (43) to (46), wherein the PD-1 polypeptide variant is linked to the carboxy-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0151] (48) The PD-1Fc fusion protein according to any one of (43) to (47), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0152] (49) The PD-1Fc fusion protein according to any one of (43) to (48), wherein a plurality of copies of the PD-1 polypeptide variant are present, and the copies may be the same or different and are linked by a peptide linker sequence.

[0153] (50) The PD-1Fc fusion protein according to (49), wherein two copies of the PD-1 polypeptide variant are present.

[0154] A PD-1Fc fusion protein according to (51), (49) or (50), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0155] A nucleic acid comprising a sequence encoding a PD-1 polypeptide variant according to any one of (52), (35) to (51).

[0156] (53) As described in (52), A nucleic acid further comprising a sequence encoding an immunoglobulin Fc region.

[0157] An expression vector comprising the nucleic acid according to (52) or (53).

[0158] (55) A vector according to (54), wherein the vector is a viral vector.

[0159] (56) A PD-1 polypeptide variant according to any one of (35) to (42) or a PD-1 fusion protein according to any one of (43) to (51); and A pharmaceutically acceptable carrier A pharmaceutical composition comprising.

[0160] (57) A method for treating a disease or condition in a subject in need of treatment, Comprising administering the pharmaceutical composition of (56) to the subject to treat the disease or condition.

[0161] (58) The method according to (57), wherein the subject has cancer.

[0162] (59) The method according to (58), wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0163] A method in which, when the pharmaceutical composition according to any one of (57) to (59) contains the PD-1 fusion protein according to any one of (43) to (51), a decreased antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) effect is observed or not observed.

[0164] (61) A method in which, when the pharmaceutical composition according to any one of (57) to (60) contains the PD-1 fusion protein according to any one of (43) to (51), a decrease in hepatotoxicity is observed or hepatotoxicity is not observed.

[0165] (62) An immunoglobulin Fc region; A PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant has an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and A single-chain variable fragment (scFv) against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody has an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker sequence. A bispecific antibody comprising the above.

[0166] (63) The bispecific antibody according to (62), wherein the immunoglobulin Fc region has an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0167] A bispecific antibody according to (64), (62) or (63), wherein the PD-1 polypeptide variant is linked to the N-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0168] A bispecific antibody according to any one of (65)-(64), wherein the scFv against the anti-4-1BB antibody is linked to the C-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0169] A bispecific antibody according to any one of (66)-(62), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0170] A bispecific antibody according to any one of (67)-(62), wherein the scFv against the anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ ID NO: 23, or a sequence number that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID NO: 23.

[0171] A bispecific antibody according to any one of (68)-(62), wherein the bispecific antibody has enhanced binding affinity for the PD-L1 molecule compared to the wild-type PD-1 polypeptide and has specific binding affinity for 4-1BB.

[0172] A bispecific antibody according to any one of (69)-(62); and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0173] A method for treating a disease or condition in a subject in need of treatment, comprising: administering the pharmaceutical composition of (69) to the subject and treating the disease or condition.

[0174] The method according to (71)(70), wherein the subject has cancer.

[0175] The method according to (72)(71), wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0176] The method according to any one of (73)(70) to (72), wherein a decreased antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cellular cytotoxicity (CDC) effect is observed or no effect is observed.

[0177] The method according to any one of (74)(70) to (73), wherein decreased hepatotoxicity is observed or no hepatotoxicity is observed.

[0178] (75) Immunoglobulin Fc region; A PD-1 polypeptide variant linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the PD-1 polypeptide variant has an amino acid sequence having at least 95% (e.g., 96% or more, 97% or more, 98% or more, 99% or more) sequence identity to residues 24 to 172 of SEQ ID NO: 11, and wherein the variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139; and An scFv against an anti-4-1BB antibody linked to the N-terminus of an immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence comprising SEQ ID NO: 17 and SEQ ID NO: 18 linked by a peptide bond or a peptide linker sequence. A bispecific antibody comprising the same.

[0179] The bispecific antibody according to (76)(75), wherein the immunoglobulin Fc region comprises an amino acid sequence having at least 95% (e.g., at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO: 10 or SEQ ID NO: 16.

[0180] The bispecific antibody according to (77)(75) or (76), wherein the PD-1 polypeptide variant is linked to the N-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0181] The bispecific antibody according to any one of (78)(75) to (77), wherein the scFv against the anti-4-1BB antibody is linked to the C-terminus of the immunoglobulin Fc region by a peptide linker sequence.

[0182] The bispecific antibody according to any one of (79)(75) to (78), wherein the peptide linker sequence is selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0183] (80) The bispecific antibody according to any one of (75) to (79), wherein the scFv against the anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ ID NO: 23, or a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 22 or SEQ ID NO: 23.

[0184] (81) The bispecific antibody according to any one of (75) to (80), wherein the bispecific antibody has enhanced binding affinity for the PD-L1 molecule compared to the wild-type PD-1 polypeptide and has specific binding affinity for 4-1BB.

[0185] (82) The bispecific antibody according to any one of (75) to (81); and A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0186] (83) A method of treating a disease or condition in a subject in need of treatment, comprising: administering the pharmaceutical composition of (82) to the subject and treating the disease or condition.

[0187] (84) The method according to (83), wherein the subject has cancer.

[0188] (85) The method according to (84), wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, renal cell cancer, glioblastoma, and prostate cancer.

[0189] (86) The method according to any one of (83) to (85), wherein a decreased antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cellular cytotoxicity (CDC) effect is observed or not observed.

[0190] (87)(83) to (86), a method in which reduced hepatotoxicity is observed or no hepatotoxicity is observed.

Examples

[0191] The present disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0192] Example 1. Variants of PD-1 The PD-1Fc fusion protein containing the PD-1 polypeptide variant was generated using the PD1-G4S linker-Fc_pcDNA3.3 plasmid. The topology of the human PD-1 protein is shown in Table 1. The sequences of human PD-1 and the PD-1 polypeptide variant are shown in FIGS. 1 and 2. In addition, the mutations for each PD-1 polypeptide variant are listed in Table 2, where the amino acid residue numbers are related to the full-length human PD-1 wild-type sequence (SEQ ID NO: 11) shown in FIG. 1. The PD-1 polypeptide variant was designed to include the extracellular domain (24-170) and a part of the transmembrane domain (171-172) of the full-length human PD-1 wild-type sequence (SEQ ID NO: 11) shown in FIG. 1. Specifically, the PD-1Fc fusion protein was generated using the animal cell expression vector pcDNA3.3, where the restriction enzymes EcoRI and BamHI were inserted into the restriction enzyme sites. The human PD-1 signal peptide (sequence: Q15116, 1-23) was used as the signal peptide, and the 207aa-230aa IMGT allele IGHG1*03 and the 231aa-457aa IMGT allele IGHG2*01 were used for the Fc region. The DNA construct encoding the PD-1Fc fusion protein is shown in FIG. 3.

[0193]

Table 1

[0194]

Table 2

[0195] Example 2. Analysis and characterization of PD-1 polypeptide variants The PD-1 polypeptide variant was inserted into a plasmid and used to generate a PD-1Fc fusion protein using an Expi293 expression system (Invitrogen). The polypeptide variant was then purified using AktaPure (GE healthcare) and a FibroPrismA column (GE healthcare, catalog number 17-0618-01). The purified polypeptide variant was passed through a desalting column (GE healthcare, catalog number 17-1408-01), and the protein concentration was measured using a Multiskan GO (Thermo).

[0196] The results are shown in Table 3.

Table 3

[0197] Example 3. Analysis of PD-1 polypeptide variants by SDS-PAGE The PD-1 polypeptide variant was added to LDS sample buffer (Invitrogen, catalog number B0007), where the sample reducing agent (Invitrogen, catalog number B0004) was added to the reduction condition group and incubated at 70 °C for 10 minutes. The prepared sample was added to SDS running buffer (Bio-rad, catalog number 1610732), and the sample was run for 30 minutes using a Mini-Protein TGX Stain-Free Gel (Bio-rad, catalog number 456-8096). The results were analyzed using a Chemidoc (Bio-rad) (Figure 4).

[0198] Example 4. Analysis of affinity for PD-L1 molecule The affinity of the PD-1 polypeptide variant for the PD-L1 molecule was analyzed using surface plasmon resonance (SPR). After diluting the PD-1 polypeptide variant to a concentration of 2 μg / mL, it was immobilized on a CM5 chip (GE Healthcare, catalog number BR-1005-30). The PD-L1 molecule (Sino, catalog number 10084-H08H) was injected at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 nM for an association time of 150 seconds and a dissociation time of 240 seconds. Using a Biacore T200 (GE Healthcare), the affinity of each PD-1 polypeptide variant was measured and analyzed. The results show that the PD-1 polypeptide variant euPD-1 has the highest binding affinity for PD-L1 (Table 4).

[0199]

Table 4

[0200] Example 5. Size exclusion chromatography The PD-1 polypeptide variant was analyzed using HPLC (Agilent Technologies, 1260 infinity II LC system) and a size exclusion column (Tosoh, TSKgel G3000 SWXL, 7.8×300 mm, part number 0008541, column number 004E04320E). A gel filtration standard (BIO-RAD, catalog number 151-1901) was used as a control group (Figures 5a - 5e).

[0201] Example 6. Cell binding assay of PD-1 fusion protein The PD-L1 high-expression cell line MDA-MB-231 (human breast cancer cells) and the PD-L1 low-expression cell line MCF-7 (human breast cancer cells) were used. Figure 6 shows the expression levels of PD-L1 in each cell line. It was confirmed in Figure 6 that PD-L1 is highly expressed in MDA-MB-231 but not in MCF7.

[0202] 1.5×10 5Cells were incubated at 4°C for 20 minutes while being treated with antibodies. The treatment concentrations of euPD-1Fc, PD-1Fc, and Tecentriq (Genetech, atezolizumab, anti-PD-L1 antibody) were serially diluted 2-fold up to a total of 12 points at 877.19 nM. After washing once with FACS wash buffer (0.5% FBS + 0.1% NaN3 in DPBS), the anti-hFC-AF488 secondary antibody (Jackson ImmunoResearch) was treated at 1 μl / well for 20 minutes. After washing two more times, FACS analysis was performed.

[0203] As a result of FACS analysis, it was confirmed that there was almost no background signal due to the secondary antibody. As shown in Figure 7a, PD-1Fc did not adhere to PD-L1-positive cells in the experimental concentration range, whereas euPD-1Fc and Tecentriq adhered dose-dependently to PD-L1-positive cells. As a result of FACS analysis, as shown in Figure 7b, all three antibodies did not adhere to PD-L1-negative cells.

[0204] Example 7. Antigen binding assay of PD-1 fusion protein To confirm whether euPD-1Fc binds to the antigens PD-L1 or PD-L2, an antigen-binding assay was performed.

[0205] The assay method was carried out as shown in FIGS. 8a and 8c. Briefly, after coating a 96-well immunoplate with PD-L1 antigen or PD-L2 antigen at a concentration of 1 μg / mL at 4° C. overnight, 150 μl of 1× assay buffer (Biolegend) was treated for 1 hour to block non-specific binding. Each of euPD-1Fc, PD-1Fc, and tesentriq was treated with 100 uL and incubated for 2 hours. The treatment concentration of the antigen PD-L1-coated plate was serially diluted 3-fold at 30 μg / mL for a total of 15 points, and the treatment concentration on the antigen PD-L2-coated plate was serially diluted 3-fold up to 100 μg / mL for a total of 12 points. Anti-hFc-HRP (Biolegend) was treated with 100 μl at a concentration of 0.4 μg / mL. After incubation for 1 hour, color development was carried out with TMB, and the reaction was stopped with sulfuric acid after 3 minutes. Except for the steps after the TMB treatment, washing was carried out 3 times using a washing buffer, and all steps except the antigen coating step were carried out at room temperature. As a result of the experiment, as shown in FIG. 8b, the binding of euPD-1Fc and tesentriq to the antigen PD-L1 was confirmed in a dose-dependent manner. And in FIG. 8d, only PD-1Fc bound to the antigen PD-L2 in a dose-dependent manner, and euPD-1Fc and tesentriq did not bind.

[0206] Example 8. Blocking bioassay of PD-1 fusion protein To confirm the binding inhibitory effect of PD-1 and PD-L1 (Bicytogen), a blocking bioassay was carried out.

[0207] The bioassay product used (Promega, J1250) expresses luciferase under conditions where the binding of PD-1 and PD-L1 is inhibited. The bioassay and luciferase assay were carried out according to the protocol of Promega. Target cells were seeded at 4×10 4Cells were seeded at a density of [[number]] cells / 100 μL / well. After overnight incubation in a 37 °C CO2 incubator, the cells were treated with antibodies and effector cells. The antibodies used were euPD-1Fc, PD-1Fc, Tencentriq, and Keytruda (Merck, pembrolizumab, an anti-PD-1 antibody). The treatment concentrations were serially diluted 2.5-fold at 217.39 nM, for a total of 10 points. After incubation in a 37 °C CO2 incubator for 6 hours, luciferin was added and a luciferase assay was performed. As shown in Figure 9a, the results of the luciferase assay indicated that euPD-1Fc, Tencentriq, and Keytruda exhibited dose-dependent inhibitory effects. As shown in Figure 9b, euPD-1Fc showed an inhibitory effect that was 2.4- to 4.9-fold greater than that of PD-1Fc.

[0208] Example 9. In vivo efficacy study of PD-1 fusion protein To verify the efficacy of euPD-1Fc, an in vivo tumor growth inhibition test was performed. The experiment was conducted using female C57BL / 6 mice knocked-in with hPD-1.

[0209] As shown in Figure 10, MC38 cells expressing human PD-L1 were used as the tumor cells. [[number]] tumor cells per mouse were administered subcutaneously. One week after administration, the tumor size was measured, and the mean and standard deviation were similar (i.e., approximately 100 mm 6 ). 3 Please note that the number in [[number]] in the translation needs to be filled in according to the actual number in the original text which is not fully provided in the question. Also, the 6 and 3 tags seem to be incomplete in the original text and their full meaning is not clear for a perfect translation.) The administration groups were assigned accordingly. Three antibodies, euPD-1Fc, PD-1Fc, and tisentriq, were injected intravenously. As a negative control group, excipient DPBS (Gibco) was used. The dosage of the antibodies was set at 5 mg / kg and 2 mg / kg based on euPD-1Fc. For PD-1fc and tisentriq at 5 mg / kg, a concentration of 8.77 pM was used considering the molecular weight. The administration was performed at 100 μl and administered 5 times at 3-day intervals. The tumor size was also measured every 3 or 4 days, and the tumor size was measured until 2 days after the last administration. Also, blood was collected on the experimental end day to confirm toxicity, and the concentrations of ALT (alanine aminotransferase), AST (aspartate aminotransferase), BUN (blood urea nitrogen), and T-BIL (total bilirubin) were confirmed using a biochemical analyzer.

[0210] When observing the tumor size, as shown in Figures 11a and 11b, tumor growth was inhibited in both euPD-1Fc (8.77 μM) and tisentriq (8.77 μM) compared to the administration of the negative control group; PD-1Fc (8.77 μM) showed growth equivalent to that of the negative control group. Also, as shown in Figures 11c and 11b, euPD-1Fc reduced the tumor size by 33.9% compared to the administration of the negative control group in the low-concentration (2 mg / kg) administration group, and a 66.1% reduction in tumor size was observed in the high-grade (5 mg / kg) administration group, showing a dose-dependent inhibitory effect.

[0211] Furthermore, liver toxicity index analysis was performed and shown in Figure 12. In the case of liver toxicity index analysis, as shown in Figure 12, in the euPD-1Fc administration group, all four indicators were within the normal range (ALT: 17 - 77 U / L (Figures 12a and 12e), AST: 54 - 298 U / L (Figures 12b and 12f), BUN: 8 - 33 mg / dL (Figures 12c and 12g), T-BIL: 8 - 33 mg / dL (Figures 12d and 12h)), and no liver toxicity was confirmed within the experimental conditions.

[0212] Therefore, it was confirmed that the fusion proteins and bispecific antibodies containing euPD-1 and IgG1 variants did not show hepatotoxicity. Furthermore, the antibodies of the present invention lack ADCC and CDC effects.

[0213] Example 10. Design, manufacture, and characterization of PD-1 fusion protein euPD-1BsAb The Fc fusion BsAb using euPD-1 and anti-4-1BB antibody was designed as shown in Figure 13. To link euPD-1 and Fc, a (G4S)2 linker was used, and for the Fc region used in the BsAb, an Fc region modified with IgG1 (L234A, L235A, K322A, D356E, L358M) was used.

[0214] A BsAb in the form of linking two euPD-1s was also designed. To link the two euPD-1s, a (G4S)3 linker was used and it was designated as euPD-1.2.

[0215] On the C-terminal side, the anti-4-1BB antibody was bound to Fc in the form of scFv; at this time, it was linked with a (G4S)2 linker, and two types of scFv for the anti-4-IBB antibody, VH-218 linker-VL (HLC218) and VL-218 linker-VH (LHC218), were used.

[0216] The constructs in Table 5 or Figure 13 were prepared. Also, the sequences described above were provided.

[0217]

Table 5

[0218] Transient transfection was performed in Expi293F cells to generate euPD-1 BsAb (bispecific antibody); purification was performed using a Protein A column.

[0219] As a result of SDS-PAGE analysis, in the non-reduced state, euPD-1×94kvt HLC218 and euPD-1×94kvt LHC218 having one euPD-1 were observed to be approximately 160 kDa; in the reduced state, they were observed to be approximately 80 kDa. The euPD-1 double type, euPD-1.2×94kvt HLC218 and euPD-1.2×94kvt LHC218, were observed to be approximately 250 kDa; in the reduced state, they were observed to be approximately 125 kDa. As a result of size exclusion chromatography analysis, a single peak with a purity of 90% or more was observed (see Figure 15).

[0220] Surface plasmon resonance (SPR) analysis similar to that described in Example 4 was performed. Specifically, 25 μg / mL of anti-human Fc antibody was immobilized on a CM5 chip. 25 μg / mL of anti-human Fc antibody was immobilized on a CM5 chip. Then, four types of euPD-1BsAbs were flowed and captured at a flow rate of 10 μg / mL and 10 μl / min for 60 seconds, and K D analysis was performed using 100, 50, 25, 12.5, 3.25, 3.125, 0 nM of the PD-L1 antigen. Figure 16 shows the SPR results of the euPD-1BsAb constructs.

[0221] Example 11. Antigen binding assay using euPD-1BsAb construct An antigen binding assay was performed to confirm whether the euPD-1BsAbs bind to the antigen PD-L1.

[0222] The assay method was carried out as shown in Figure 17a. Briefly, after coating the PD-L1 antigen on a 96-well immunoplate at a concentration of 1 μg / mL at 4°C overnight, 150 μl of 1× assay buffer (Biolegend) was added and incubated for 1 hour to block non-specific binding. euPD-1BsAb was added at 100 L and incubated for 2 hours. The treatment concentration was serially diluted 10-fold up to a total of 3 points at 10 μg / mL. 100 μl of biotinylated 4-IBB at a concentration of 1 μg / mL was added and incubated for 1 hour. Avidin-HRP (Bioglend) was added at 100 μl at the concentration indicated in the protocol and incubated for 30 minutes. After developing color with TMB, the reaction was stopped with sulfuric acid after 3 minutes. In all steps except the steps after TMB treatment, washing was performed 3 times using wash buffer; all processes except the PD-L1 coating step were carried out at room temperature.

[0223] As a result of the experiment, as shown in Figure 17b, the binding of euPD-1BsAb to the antigen PD-L1 was confirmed to be dose-dependent.

[0224] Example 12. Bioassay of 4-1BB / PD-1 combination To confirm the binding inhibition effect between PD-1 and PD-L1 and the 4-1BB activation effect, a bioassay of the 4-1BB / PD-1 combination was performed.

[0225] In this experiment, the bioassay product (Promega, CS1978I10) is an assay system that expresses luciferase when 4-1BB is activated by 4-1BB antibody stimulation and at the same time the interaction between PD-1 / PD-L1 is inhibited. The bioassay and luciferase assay were performed according to the Promega protocol. MDA-MB-231 cells expressing PD-L1 were seeded in a 96-well white plate at 4×10 4Cells were seeded at 100 cells / 100 μL / well. After overnight incubation in a 37 °C CO2 incubator, the cells were treated with antibodies and PD1+4-1BB effector cells. As the antibodies, euPD-1×94kvt HLC218 and euPD-1×94kvt LHC218 were used. The treatment concentrations were serially diluted 4-fold from 60 ng / ml, for a total of 4 points. After culturing for 6 hours in a 37 °C CO2 incubator, luciferin was treated and a luciferase assay was performed.

[0226] As a result of the luciferase assay, as shown in Fig. 18, euPD-1×94kvt HLC218 and euPD-1×94kvt LHC218 activated 4-1BB and inhibited PD-1 / PD-L1 in a dose-dependent manner.

Claims

1. A programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having 95% or more sequence identity to SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, An extracellular domain that specifically binds to programmed cell death 1 ligand (PD-L1); and Transmembrane domain or fragment thereof A PD-1 polypeptide variant comprising:

2. 2. The PD-1 polypeptide variant of claim 1, comprising the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:

8.

3. 2. The PD-1 polypeptide variant of claim 1, wherein the transmembrane domain comprises at least two amino acid residues.

4. The PD-1 polypeptide variant of claim 1, which has enhanced binding affinity to a PD-L1 molecule compared to a wild-type PD-1 polypeptide.

5. Approximately 1 × 10 for PD-L1 molecules －８ ~1×10 －１０ The binding affinity of M (K Ｄ 5. The PD-1 polypeptide variant of claim 4, having the following structure:

6. 1. A programmed cell death 1 (PD-1) polypeptide variant comprising an amino acid sequence having 95% or greater sequence identity to residues 24-172 of SEQ ID NO:11, wherein the PD-1 polypeptide variant has a mutation in at least one residue selected from the group consisting of D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

7. 7. The PD-1 polypeptide variant of claim 6, comprising mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, Q99, L122, A125, A132, and R139.

8. 7. The PD-1 polypeptide variant of claim 6, comprising mutations at D26, P34, V43, T45, T59, V64, L65, N66, Y68, M70, N74, K78, C93, Q99, R114, L122, A125, A132, and R139.

9. 7. The PD-1 polypeptide variant of claim 6, wherein the mutation is D26E, P34A, V43L, T45A, T59A, V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93H, Q99R, R114Q, L122Y, A125V, A132I, or R139G.

10. An immunoglobulin Fc region; and The PD-1 polypeptide variant of claim 1, linked to the carboxy terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence. A PD-1Fc fusion protein comprising:

11. The PD-1Fc fusion protein of claim 10, wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10 or SEQ ID NO:

16.

12. 11. The PD-1Fc fusion protein of claim 10, wherein there are two copies of said PD-1 polypeptide variant, wherein said copies are linked by a peptide linker, which may be the same or different.

13. The PD-1Fc fusion protein of claim 10, which has enhanced binding affinity to a PD-L1 molecule compared to a wild-type PD-1 polypeptide.

14. Approximately 1 × 10 for PD-L1 molecules －８ ~1×10 －１０ The binding affinity of M (K Ｄ 14. The PD-1Fc fusion protein of claim 13, comprising:

15. An immunoglobulin Fc region; and The PD-1 polypeptide variant of claim 6, linked to the carboxy terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence. A PD-1Fc fusion protein comprising:

16. 16. The PD-1Fc fusion protein of claim 15, wherein there are two copies of said PD-1 polypeptide variant, said copies being the same or different and linked by a peptide linker.

17. A nucleic acid comprising a sequence encoding a PD-1 polypeptide variant, wherein the polypeptide variant comprises a sequence having 95% or greater sequence identity to SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:

7.

18. 18. The nucleic acid of claim 17, further comprising a sequence encoding an immunoglobulin Fc region, wherein the sequence encoding the immunoglobulin Fc region comprises SEQ ID NO:

9.

19. An expression vector comprising the nucleic acid of claim 17.

20. The vector of claim 19 which is a viral vector.

21. 10. The PD-1 polypeptide variant of claim 1, or a fusion protein comprising an immunoglobulin Fc region and the PD-1 polypeptide variant linked to the carboxy terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and Pharmaceutically acceptable carriers 13. A pharmaceutical composition comprising:

22. 1. A method of treating a disease or condition in a subject in need of treatment, comprising:

22. A method comprising administering to a subject the pharmaceutical composition of claim 21, thereby treating a disease or condition.

23. 23. The method of claim 22, wherein the subject has cancer.

24. 24. The method of claim 23, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

25. 23. The method of claim 22, wherein a reduced or no effect of antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) is observed when the pharmaceutical composition comprises the fusion protein.

26. 23. The method of claim 22, wherein reduced or no hepatotoxicity is observed when the pharmaceutical composition comprises the fusion protein.

27. 7. The PD-1 polypeptide variant of claim 6, or a fusion protein comprising an immunoglobulin Fc region and the PD-1 polypeptide variant linked to the carboxy terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and Pharmaceutically acceptable carriers 13. A pharmaceutical composition comprising:

28. 1. A method of treating a disease or condition in a subject in need of treatment, comprising:

30. A method comprising administering to a subject the pharmaceutical composition of claim 27, thereby treating a disease or condition.

29. 30. The method of claim 28, wherein the subject has cancer.

30. 30. The method of claim 29, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

31. 29. The method of claim 28, wherein a reduced or no antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) effect is observed when the pharmaceutical composition comprises the fusion protein.

32. 29. The method of claim 28, wherein reduced or no hepatotoxicity is observed when the pharmaceutical composition comprises the fusion protein.

33. Immunoglobulin Fc region; 10. The PD-1 polypeptide variant of claim 1, which is linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and An scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody comprises an amino acid sequence having 95% or more sequence identity to an amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker sequence. A bispecific antibody comprising:

34. 34. The bispecific antibody of claim 33, wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10 or SEQ ID NO:

16.

35. 34. The bispecific antibody of claim 33, wherein the PD-1 polypeptide, the scFv, or both are linked to an immunoglobulin Fc region by a peptide linker selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:

21.

36. The bispecific antibody of claim 33, wherein the scFv against the anti-4-1BB antibody comprises a sequence that is at least 90% identical to SEQ ID NO:22 or SEQ ID NO:

23.

37. The bispecific antibody of claim 33, wherein the scFv against the anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ ID NO:

23.

38. 34. The bispecific antibody of claim 33, which has enhanced binding affinity for a PD-L1 molecule as compared to a wild-type PD-1 polypeptide and has specific binding affinity for 4-1BB.

39. The bispecific antibody of claim 33; and Pharmaceutically acceptable carriers 13. A pharmaceutical composition comprising:

40. 1. A method of treating a disease or condition in a subject in need of treatment, comprising:

40. A method comprising the step of administering to a subject the pharmaceutical composition of claim 39, thereby treating the disease or condition.

41. 41. The method of claim 40, wherein the subject has cancer.

42. 42. The method of claim 41, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

43. 41. The method of claim 40, wherein a reduced or no effect of antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) is observed when the pharmaceutical composition comprises the fusion protein.

44. 41. The method of claim 40, wherein reduced or no hepatotoxicity is observed when the pharmaceutical composition comprises the fusion protein.

45. Immunoglobulin Fc region; 7. The PD-1 polypeptide variant of claim 6, which is linked to the N-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence; and An scFv against an anti-4-1BB antibody linked to the C-terminus of the immunoglobulin Fc region by a peptide bond or a peptide linker sequence, wherein the scFv against the anti-4-1BB antibody comprises an amino acid sequence having 95% or more sequence identity to an amino acid sequence comprising SEQ ID NOs: 17 and 18 linked by a peptide bond or a peptide linker sequence. A bispecific antibody comprising:

46. 46. ​​The bispecific antibody of claim 45, wherein the immunoglobulin Fc region comprises an amino acid sequence having 95% or greater sequence identity to SEQ ID NO: 10 or SEQ ID NO:

16.

47. 46. ​​The bispecific antibody of claim 45, wherein the PD-1 polypeptide, the scFv, or both are linked to an immunoglobulin Fc region by a peptide linker selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:

21.

48. The bispecific antibody of claim 45, wherein the scFv against the anti-4-1BB antibody comprises a sequence that is at least 90% identical to SEQ ID NO:22 or SEQ ID NO:

23.

49. The bispecific antibody of claim 45, wherein the scFv against the anti-4-1BB antibody comprises SEQ ID NO: 22 or SEQ ID NO:

23.

50. 46. ​​The bispecific antibody of claim 45, which has enhanced binding affinity for a PD-L1 molecule as compared to a wild-type PD-1 polypeptide and has specific binding affinity for 4-1BB.

51. 46. ​​The bispecific antibody of claim 45; and Pharmaceutically acceptable carriers 13. A pharmaceutical composition comprising:

52. 1. A method of treating a disease or condition in a subject in need of treatment, comprising:

52. A method comprising administering to a subject the pharmaceutical composition of claim 51, thereby treating a disease or condition.

53. 53. The method of claim 52, wherein the subject has cancer.

54. 54. The method of claim 53, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

55. 55. The method of claim 54, wherein a reduced or no effect of antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) is observed when the pharmaceutical composition comprises the fusion protein.

56. 55. The method of claim 54, wherein reduced or no hepatotoxicity is observed when the pharmaceutical composition comprises the fusion protein.

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