CN118165119A - Recombinant proteins comprising IL-15/IL-15Rα complexes and uses thereof - Google Patents

Recombinant proteins comprising IL-15/IL-15Rα complexes and uses thereof Download PDF

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CN118165119A
CN118165119A CN202211578437.4A CN202211578437A CN118165119A CN 118165119 A CN118165119 A CN 118165119A CN 202211578437 A CN202211578437 A CN 202211578437A CN 118165119 A CN118165119 A CN 118165119A
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seq
fragment
antibody
tigit
amino acid
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胡俊杰
刘志刚
刘子彬
张雪萍
周晓巍
郝小勃
刘玉兰
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Beijing Wisdomab Biotechnology Co ltd
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Beijing Wisdomab Biotechnology Co ltd
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Abstract

The present application provides recombinant proteins comprising a T cell immunoglobulin and an ITIM domain protein (TIGIT) binding moiety and a complex of IL-15 and IL-15 ra. The present application provides single domain antibodies that bind to human TIGIT. The application also provides a nucleic acid molecule encoding the recombinant protein or the antibody, a pharmaceutical composition comprising the recombinant protein or the antibody, and the use of the recombinant protein, the antibody or the pharmaceutical composition in the preparation of a medicament for tumor treatment.

Description

Recombinant proteins comprising IL-15/IL-15Rα complexes and uses thereof
Technical Field
The present application relates generally to the fields of genetic engineering and biological medicine; in particular, the application relates to recombinant proteins comprising TIGIT binding moieties and IL-15/IL-15 ra complexes, single domain antibodies that bind human TIGIT and their uses.
Background
IL-15 is a pleiotropic cytokine with the function of activating T cells, B cells and NK cells and mediating proliferation and survival of these cells. IL-15 exists in two forms, one is a soluble monomeric form of IL-15 and the other is IL-15 binds to its proprietary receptor IL-15Rα to form IL-15/IL-15Rα complex 1.
IL-15 and IL-15Rα are commonly co-expressed in cells and are presented as complexes on the cell surface, with high expression in myeloid cells, especially dendritic cells, monocytes, macrophages, etc. 2. IL-15 signaling requires two additional receptor subunits, IL-2Rβ and yc, which are highly expressed in NK cells and CD8+ T cells 3.
IL-15 produces an anti-tumor effect by promoting proliferation and activation of CD8+ T cells and NK cells. In various experimental animal tumor models (e.g., B16F10 melanoma and CT26 colon cancer models), treatment with IL-15/IL-15 ra can promote tumor regression, reduce tumor metastasis, and increase survival 4.
TIGIT (CD 226), all known as T cell immunoglobulins and ITIM domain proteins belonging to the immunoglobulin superfamily (IgSF), is expressed in a variety of lymphocytes, including activated cd8+ and cd4+ T cells, NK cells, regulatory T cells (tregs) and follicular helper T cells 5, and is a co-inhibitory receptor. CD155 is a ligand of TIGIT, CD155 is hardly expressed or weakly expressed in normal human tissues, but is usually overexpressed 6 in human malignancies. TIGIT is widely expressed in TIL, especially depleted cd8+ T cells, NK cells, CD155 inhibits T cell and NK cell function by interacting with TIGIT, blocking TIGIT/CD155 signaling can inhibit tumor growth, TIGIT is a potential immunotherapeutic target 7 for tumor treatment.
IL-15 can promote proliferation and activation of CD8+ T cells and NK cells, and TIGIT antibodies block the binding of TIGIT and CD155 to release the inhibition of T cell and NK cell functions. The TIGIT antibody and the IL-15 combined can enhance the activity of NK cells and reduce metastasis 8 of melanoma tumors in mice, and the combination of the IL-15 and the TIGIT antibody is expected to become a novel immunotherapy.
Summary of The Invention
In a first aspect, the application provides recombinant proteins comprising a T cell immunoglobulin and ITIM domain protein (TIGIT) binding moiety and a complex of IL-15 and IL-15Rα.
In some embodiments of the first aspect, the TIGIT binding moiety is in the form of a single domain antibody.
In some embodiments of the first aspect, the TIGIT binding moiety comprises 1 or 2 TIGIT single domain antibodies.
In some embodiments of the first aspect, the TIGIT binding moiety comprises HCDR1 as shown in SEQ ID No. 10, HCDR2 as shown in SEQ ID No. 11, and HCDR3 as shown in SEQ ID No. 12; wherein the amino acid sequence of HCDR is defined according to Kabat.
In some embodiments of the first aspect, the TIGIT binding moiety comprises the amino acid sequence as set forth in SEQ ID NO: 9. 13, 14 or 16.
In some embodiments of the first aspect, the recombinant protein comprises:
SEQ ID NO: 13. 7 and 8;
SEQ ID NO: 14. 7 and 8; or alternatively
SEQ ID NO: 16. 7 and 8.
In some embodiments of the first aspect, the TIGIT binding moiety and the complex of IL-15 and IL-15 ra are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the amino acid position of the antibody constant region is determined according to EU numbering.
In some embodiments of the first aspect, the TIGIT binding moiety and the complex of IL-15 and IL-15 ra are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 234, 235 and 331 of the first and second Fc fragments are F, E and S, respectively; wherein the amino acid position of the antibody constant region is determined according to EU numbering.
In some embodiments of the first aspect, one of the first Fc fragment and the second Fc fragment is linked to the TIGIT binding moiety and the other of the first Fc fragment and the second Fc fragment is linked to one of IL-15 and IL-15 ra.
In a second aspect, the present application provides a single domain antibody that binds to human T cell immunoglobulin and ITIM domain protein (TIGIT), comprising
As set forth in SEQ ID NO: the HCDR1 shown in figure 10,
As set forth in SEQ ID NO:11, and
As set forth in SEQ ID NO:12, HCDR3;
wherein the amino acid sequence of HCDR is defined according to Kabat.
In a third aspect, the application provides a nucleic acid molecule encoding the recombinant protein of the first aspect or the single domain antibody of the second aspect that binds TIGIT.
In a fourth aspect, the application provides a pharmaceutical composition comprising a recombinant protein according to the first aspect or a single domain antibody according to the second aspect, and a pharmaceutically acceptable excipient, diluent or carrier.
In a fifth aspect, the application provides the use of a recombinant protein according to the first aspect, a single domain antibody according to the second aspect or a pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for the prevention or treatment of a tumour.
Brief Description of Drawings
FIG. 1 shows the ability of N9C9-Fc and its humanized molecules N9C9-h1-Fc and N9C9-h2-Fc to bind to the cell surface hTIGIT.
FIG. 2 shows the results of activation activity of hIL-15/IL-15Rα complex.
FIG. 3 shows the results of the binding capacity of recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2xN9C9-h1+hIL-15/IL-15 Rα to CHO-hTIGIT.
FIG. 4 shows the ability of recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2xN9C9-h1+hIL-15/IL-15 Rα to promote NK cell proliferation.
FIG. 5 shows the tumor inhibition by recombinant protein N9C9-h1+hIL-15/IL-15Rα.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 shows the amino acid sequence of the full-length human (homo sapiens) TIGIT (hTIGIT).
SEQ ID NO. 2 shows the amino acid sequence of the extracellular region (hTIGIT-ECD) of human (homo sapiens) TIGIT.
SEQ ID NO. 3 shows the amino acid sequence of the extracellular region of the recombinant antigen IL-2Rβ (IL-2 Rβ -ECD).
SEQ ID NO. 4 shows the amino acid sequence of the His tag.
SEQ ID NO. 5 shows the amino acid sequence of the Fc segment (mFc) of the mouse (mus musculus) antibody IgG2 a.
SEQ ID NO. 6 shows the amino acid sequence of the Fc segment (IgG 1-Fc) of the human (homo sapiens) antibody IgG 1.
SEQ ID NO. 7 shows the amino acid sequence of human (homosapiens) IL-15 (hIL-15).
SEQ ID NO. 8 shows the amino acid sequence of human (homo sapiens) IL-15Rα (hIL-15 Rα).
SEQ ID NO. 9 shows the amino acid sequence of clone N9C9 binding recombinant protein hTIGIT, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 10-12, respectively.
SEQ ID NO. 13 shows the amino acid sequence of the humanized molecule N9C9-h 1.
SEQ ID NO. 14 shows the amino acid sequence of the humanized molecule N9C9-h 2.
SEQ ID NO. 15 shows the amino acid sequence of the GS flexible peptide.
SEQ ID NO. 16 shows the amino acid sequence of 2 x N9C9-h 1.
SEQ ID NO. 17 shows the amino acid sequence of the Fc-segment mutant mIgG2a-K of the murine antibody IgG2 a.
SEQ ID NO. 18 shows the amino acid sequence of the Fc-segment mutant mIgG2a-H of the murine antibody IgG2 a.
SEQ ID NO. 19 shows the amino acid sequence of the Fc-segment mutant mIgG1-K of the murine antibody IgG 1.
SEQ ID NO. 20 shows the amino acid sequence of the Fc-segment mutant mIgG1-H of the murine antibody IgG 1.
SEQ ID NO. 21 shows the amino acid sequence of a mutant IgG1m3-K of the Fc segment of human antibody IgG 1.
SEQ ID NO. 22 shows the amino acid sequence of a mutant IgG1-K of the Fc segment of human antibody IgG 1.
SEQ ID NO. 23 shows the amino acid sequence of a mutant IgG1m3-H of the Fc segment of human antibody IgG 1.
SEQ ID NO. 24 shows the amino acid sequence of a mutant IgG1-H of the Fc segment of human antibody IgG 1.
SEQ ID NO. 25 shows the amino acid sequence of N9C9-h1-IgG1m 3-FcK.
SEQ ID NO. 26 shows the amino acid sequence of 2X N9C9-h1-IgG1m 3-FcK.
SEQ ID NO. 27 shows the amino acid sequence of N9C9-h1-IgG 1-FcK.
SEQ ID NO. 28 shows the amino acid sequence of 2X N9C9-h1-IgG 1-FcK.
SEQ ID NO. 29 shows the amino acid sequence of hIL-15Rα -IgG1m 3-FcH.
SEQ ID NO. 30 shows the amino acid sequence of hIL-15Rα -IgG 1-FcH.
SEQ ID NO. 31 shows the nucleotide sequence of primer PCal-CH 2R.
SEQ ID NO. 32 shows the amino acid sequence of the heavy chain variable region of the DP47 antibody.
SEQ ID NO. 33 shows the amino acid sequence of the light chain variable region of the DP47 antibody.
Detailed Description
The inventor prepares the complex of IL-15 and IL-15 Ralpha and the TIGIT binding part into recombinant protein through genetic engineering means, carries the complex of IL-15 and IL-15 Ralpha to lymphocyte through the TIGIT binding part combined with lymphocyte surface, thus mediating the proliferation and survival of the cells, promoting the regression of tumor, reducing the metastasis of tumor and achieving the purpose of treating tumor. In various aspects of the application, novel recombinant proteins comprising a complex of IL-15 and IL-15 ra with a TIGIT binding moiety, single domain antibodies that bind to human TIGIT, nucleic acid molecules encoding the recombinant proteins or the single domain antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods of making and purifying the recombinant proteins or the single domain antibodies, and medical and biological uses of the recombinant proteins or the single domain antibodies are provided. According to the sequence of the recombinant protein or the single domain antibody combined with the human TIGIT, the recombinant protein or the single domain antibody combined with the human TIGIT can be constructed and used as a medicament for clinically preventing or treating tumors.
The practice of the present application employs, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.
Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.
Definition of the definition
The term "recombinant protein" as used herein refers to a protein obtained using recombinant DNA or recombinant RNA technology, the route of which can be divided into in vitro and in vivo methods. Both methods employ gene recombination technology to obtain recombinant vectors linked with gene fragments that can be translated into the protein of interest, which are then transferred into host cells that can express the protein of interest to express the particular recombinant protein molecule.
The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full length) antibodies, but also antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv), variants thereof, fusion proteins comprising an antibody moiety, single domain antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.
Typically, an intact or full length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second and third constant regions (CH 1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The full length antibody may be any kind of antibody, such as IgD, igE, igG, igA or IgM (or subclasses thereof as described above), but the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant region of the heavy chain. Typically, immunoglobulins have five main classes IgA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
The term "single domain antibody" as used herein refers to a heavy chain single variable domain antibody that naturally lacks a light chain, such antibody comprising a heavy chain variable region (VHH) and conventional CH2 and CH3 regions (e.g., one or two sets (heavy chain variable region (VHH) and conventional CH2 and CH3 regions)). The VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity to the antigen, the smallest unit known to bind the antigen of interest. Single domain antibodies are also known as nanobodies (Nb).
The term "complex" as used herein refers to an aggregate of two or more molecules bound together with a certain physiological, chemical function or apparent physicochemical property. Are common in the biological arts, such as immune complexes, receptor complexes, polysome complexes, and the like.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of antibodies that is substantially homogeneous, i.e., the individual antibodies that make up the population are identical except for the naturally occurring mutations that may be present in a small number of individuals. The monoclonal antibodies described herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
The term "binding moiety" or "binding fragment" as used herein is used interchangeably to refer to a portion or region of an intact antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3.
It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence on the affinity and specificity of an antibody. There are two common ways of defining CDR sequences for VH or VL, namely Kabat definition and Chothia definition. (see, e.g., Kabat,"Sequences of Proteins of Immunological Interest",National Institutes of Health,Bethesda,Md.(1991);A1-Lazikani et al.,J.Mol.Biol.273:927-948(1997); and Martin et al, proc. Natl. Acad. Sci. USA86:9268-9272 (1989)). For a given antibody variable region sequence, the CDR region sequences in VH and VL sequences may be determined according to the Kabat definition or Chothia definition. In an embodiment of the application, the CDR sequences are defined using Kabat.
For a given antibody variable region sequence, the CDR region sequences in the variable region sequence can be analyzed in a number of ways, for example, as determined using on-line software Abysis (http:// www.abysis.org /).
For a typical antibody, examples of antigen binding fragments include, but are not limited to, (1) Fab fragments, which can be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) A F (ab ') 2 fragment, which may be a bivalent fragment having two Fab' fragments linked by a disulfide bridge of the hinge region (i.e., a dimer of Fab); (3) Fv fragments having VL and VH domains of a single arm of an antibody; (4) A single chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; (5) (scFv) 2, which may comprise two VH domains connected by a peptide linker and two VL domains combined with the two VH domains via a disulfide bridge; and (6) a single domain antibody format.
The terms "Fc fragment," "Fc domain," and "Fc portion" as used herein are used interchangeably to refer to a portion of an antibody heavy chain constant region, including hinge region (range), CH2 fragment and CH3 fragment of the heavy chain constant region, and are determined with reference to EU numbering of a human IgG1 antibody.
The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
The term "tumor" as used herein refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors may be benign, premalignant or malignant.
The term "malignancy" as used herein refers to or describes a physiological condition of a mammal that is typically characterized by unregulated cell growth. Exemplary malignancies include: carcinomas, solid tumors, melanoma sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignancy include: melanoma, colon cancer, renal cell carcinoma, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous carcinoma, bladder cancer, breast cancer, cervical cancer, liver cancer (hepatic carcinoma), stomach cancer including gastrointestinal cancer, prostate cancer, pancreatic cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer, liver cancer (LIVER CANCER), urinary tract cancer, hepatoma, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, squamous cell carcinoma (e.g., squamous cell carcinoma), vulval cancer, thyroid cancer, anal cancer, penile cancer, multiple myeloma and B-cell lymphoma, brain cancer and head and neck cancer, and related metastases.
The term "hematological neoplasm" as used herein refers to the fact that the site of origin of abnormal cells is in most cases bone marrow, which is also where blood cells are produced, due to uncontrolled growth and proliferation of such abnormal cells. Exemplary hematological tumors include various types of leukemia, multiple myeloma, and malignant lymphoma. More specific examples of hematological neoplasms include: acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), acute lymphoblastic leukemia, acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), hairy Cell Leukemia (HCL), T cell prolymphocytic leukemia, large granule lymphoblastic leukemia, juvenile granule-monocytic leukemia, B cell prolymphocytic leukemia, burkitt's leukemia and adult T cell leukemia, non-Hodgkin lymphoma, B cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytoid lymphoma, primary macroglobulinemia Splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intranodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, burkitt's lymphoma, B-cell chronic lymphocytic lymphoma, classical hodgkin's lymphoma, nodular lymphomas are primary hodgkin's lymphoma, adult T-cell lymphoma, extranodal nasal NK/T-cell lymphoma, intestinal T-cell lymphoma, hepatosplenic T-cell lymphoma, maternal NK-cell lymphoma, mycosis fungoides, sjogren's syndrome, primary cutaneous CD30 positive T-cell lymphoproliferative disease, primary skin anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblast T-cell lymphoma, non-peripheral T-cell lymphoma, and anaplastic large cell lymphoma.
The term "solid tumor" as used herein refers to a tangible tumor that can be reached through clinical examinations such as radiographs, CT scans, B-mode or palpation. Clinically diagnosed solid tumors are classified into malignant and benign. Malignant solid tumors include: hodgkin lymphoma in children: lymphocyte is main type, nodulizing type, mixed cell type and lymphocyte reducing type; non-hodgkin lymphoma in children: pre-lymphoblastic lymphoma, small, non-split cell lymphoma (burkitt/non-burkitt lymphoma), diffuse large B cell lymphoma, anaplastic large cell lymphoma, and the like; renal tumor in children: wilms' tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primary neuroectoblastoma, etc.; childhood neuroblastoma: neuroblastoma, gangliocytoma, gangliocytoneuroma; children extracranial germ cytoma: mature teratomas, immature teratomas, endoplasmic sinus tumors (yolk sac tumors), seminomas, asexual cytomas, chorionic epithelial cancers, embryonal cancers, etc.; osteosarcoma and chondrosarcoma; rhabdomyosarcoma in children: embryo type, acinus type, polymorphic type, etc.; soft tissue sarcoma: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, acinar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, and fibroproliferative microcytoma; you Wenshi family sarcomas: ewing's sarcoma, primary neuroectoblastoma; liver tumor in children: hepatoblastomas (embryonic, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoma, mesothelioma, and the like. Benign solid tumors include: lymphangioma, hemangioma, ungulate lingual cyst, etc.
In a first aspect, the application provides recombinant proteins comprising a T cell immunoglobulin and ITIM domain protein (TIGIT) binding moiety and a complex of IL-15 and IL-15Rα.
In some embodiments of the first aspect, the TIGIT binding moiety is in the form of a single domain antibody.
In some specific embodiments of the first aspect, the TIGIT binding moiety comprises 1 or 2 TIGIT single domain antibodies.
In some embodiments of the first aspect, when the TIGIT binding moiety comprises 2 TIGIT single domain antibodies, the TIGIT single domain antibodies are linked by a linker, the linker being a linker comprising a GS flexible peptide, e.g. (GGGGS) n, wherein n is an integer of ≡1. In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 15).
In some embodiments of the first aspect, the TIGIT binding moiety comprises HCDR1 as shown in SEQ ID No. 10, HCDR2 as shown in SEQ ID No. 11, and HCDR3 as shown in SEQ ID No. 12; wherein the amino acid sequence of HCDR is defined according to Kabat.
In some embodiments of the first aspect, wherein the TIGIT binding moiety comprises the amino acid sequence as set forth in SEQ ID NO: 9. 13, 14 or 16.
In some embodiments of the first aspect, the TIGIT binding moiety has an amino acid sequence that hybridizes to SEQ ID NO: 9. 13, 14 or 16 differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the TIGIT binding moiety has an amino acid sequence that hybridizes to SEQ ID NO: 9. 13, 14 or 16 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology.
In some embodiments of the first aspect, SEQ ID NO: 9. 13, 14 or 16 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar functionality of the TIGIT binding moiety.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO: 9. 13, 14 or 16, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence, the resulting amino acid sequence still retaining similar functionality of the TIGIT binding moiety.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO: 9. 13, 14 or 16, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added or deleted in a region other than the C-terminal or N-terminal of the amino acid sequence as long as the altered amino acid sequence substantially retains similar function of the TIGIT binding moiety.
In some embodiments of the first aspect, wherein IL-15 comprises an amino acid sequence as set forth in SEQ ID NO: 7.
In some embodiments of the first aspect, the amino acid sequence of IL-15 hybridizes to the amino acid sequence of SEQ ID NO:7 differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the amino acid sequence of IL-15 hybridizes to the amino acid sequence of SEQ ID NO:7 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology.
In some embodiments of the first aspect, SEQ ID NO: the C-terminal or N-terminal region of the amino acid sequence shown in fig. 7 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar IL-15 function.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO:7 to the C-terminal or N-terminal region of the amino acid sequence shown in FIG. 7,1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added, the resulting amino acid sequence still retains similar IL-15 function.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO:7 or a region other than the C-terminal or N-terminal of the amino acid sequence shown in fig. 7, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added or deleted, provided that the altered amino acid sequence substantially retains similar IL-15 function.
In some embodiments of the first aspect, wherein IL-15 ra comprises the amino acid sequence as set forth in SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.
In some embodiments of the first aspect, the amino acid sequence of IL-15 ra hybridizes to SEQ id no:8 differ by about 1,2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the first aspect, the amino acid sequence of IL-15 ra hybridizes to SEQ id no:8 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology.
In some embodiments of the first aspect, SEQ ID NO:8 may also be truncated by about 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar IL-15 ra function.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO:8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence shown in fig. 8, the resulting amino acid sequence still retains similar IL-15 ra function.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO:8 or a region other than the C-terminal or N-terminal of the amino acid sequence shown in fig. 8, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, provided that the altered amino acid sequence substantially retains similar IL-15 ra function.
In some embodiments of the first aspect, the recombinant protein comprises SEQ ID NO: 13. 7 and 8.
In some embodiments of the first aspect, the recombinant protein comprises SEQ ID NO: 14. 7 and 8.
In some embodiments of the first aspect, the recombinant protein comprises SEQ ID NO: 16. 7 and 8.
In some embodiments of the first aspect, the TIGIT binding moiety and the complex of IL-15 and IL-15 ra are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the amino acid position of the antibody constant region is determined according to EU numbering.
In some embodiments of the first aspect, the TIGIT binding moiety and the complex of IL-15 and IL-15 ra are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; wherein the amino acid position of the antibody constant region is determined according to EU numbering.
In some embodiments of the first aspect, the TIGIT binding moiety and the complex of IL-15 and IL-15 ra are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein the amino acids at positions 234, 235 and 331 of the first and second Fc fragments are F, E and S, respectively; wherein the amino acid position of the antibody constant region is determined according to EU numbering.
In some embodiments of the first aspect, one of the first Fc fragment and the second Fc fragment is linked to the TIGIT binding moiety and the other of the first Fc fragment and the second Fc fragment is linked to one of IL-15 and IL-15 ra.
In some embodiments of the first aspect, the TIGIT binding moiety (e.g., at the C-terminus) is linked to the first Fc fragment (e.g., having an amino acid sequence as set forth in SEQ ID NOs 17, 19, 21, or 22).
In some embodiments of the first aspect, the fusion protein formed by the TIGIT binding moiety (e.g., at the C-terminus) linked to the first Fc fragment (e.g., having the amino acid sequence set forth in SEQ ID NOs 17, 19, 21, or 22) comprises the sequence set forth in SEQ ID NOs: 25. 26, 27 or 28, or a sequence represented by SEQ ID NO: 25. 26, 27 or 28.
In some embodiments of the first aspect, IL-15Rα (e.g., at the C-terminus) is linked to the second Fc fragment (e.g., having an amino acid sequence as set forth in SEQ ID NO:18, 20, 23 or 24).
In some embodiments of the first aspect, the fusion protein formed by ligating IL-15Rα (e.g., at the C-terminus) to the second Fc fragment (e.g., having the amino acid sequence set forth in SEQ ID NO:18, 20, 23 or 24) comprises the sequence set forth in SEQ ID NO:29 or 30, or a sequence represented by SEQ ID NO:29 or 30.
In some embodiments of the first aspect, the recombinant protein is formed from a three-part assembly, wherein the first part is a fusion protein comprising one of the first and second Fc fragments and the TIGIT binding moiety, the second part is a fusion protein comprising the other of the first and second Fc fragments and one of IL-15 and IL15 ra, and the third part is the other of IL-15 and IL15 ra.
In some embodiments of the first aspect, the TIGIT binding moiety (e.g., at the C-terminus) is linked to the first Fc fragment (e.g., having an amino acid sequence as set forth in SEQ ID NOs 17, 19, 21, or 22) to form a first moiety.
In some embodiments of the first aspect, the first portion comprises SEQ ID NO: 25. 26, 27 or 28, or a sequence represented by SEQ ID NO: 25. 26, 27 or 28.
In some embodiments of the first aspect, IL15Rα (e.g., at the C-terminus) is linked to the second Fc fragment (e.g., having an amino acid sequence as set forth in SEQ ID NO:18, 20, 23 or 24) to form a second moiety.
In some embodiments of the first aspect, the second portion comprises SEQ ID NO:29 or 30, or a sequence represented by SEQ ID NO:29 or 30.
In some embodiments of the first aspect, IL-15 is a third moiety.
In some embodiments of the first aspect, the TIGIT binding moiety (e.g., at the C-terminus) is linked to the first Fc fragment (e.g., having an amino acid sequence as set forth in SEQ ID NOs 17, 19, 21, or 22) to form a first moiety; IL15Rα (e.g., at the C-terminus) is linked to the second Fc fragment (e.g., having an amino acid sequence as set forth in SEQ ID NO:18, 20, 23 or 24) to form a second moiety; and IL-15 as the third moiety.
In a second aspect, the present application provides a single domain antibody that binds to human T cell immunoglobulin and ITIM domain protein (TIGIT), comprising
As set forth in SEQ ID NO: the HCDR1 shown in figure 10,
As set forth in SEQ ID NO:11, and
As set forth in SEQ ID NO:12, HCDR3;
wherein the amino acid sequence of HCDR is defined according to Kabat.
In some embodiments of the second aspect, the single domain antibody that binds to human TIGIT comprises a sequence as set forth in SEQ ID NO: 9. 13, 14 or 16, and a polypeptide comprising the amino acid sequence shown in seq id no
In some embodiments of the second aspect, the amino acid sequence of the single domain antibody that binds human TIGIT differs from the amino acid sequence set forth in SEQ ID No. 9, 13, 14, or 16 by about 1,2, 3,4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or additions.
In some embodiments of the second aspect, the amino acid sequence of the single domain antibody that binds human TIGIT has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID No. 9, 13, 14 or 16.
In some embodiments of the second aspect, SEQ ID NO: 9. 13, 14 or 16 may also be truncated by about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still retaining similar functionality of the TIGIT-binding single domain antibody.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO: 9. 13, 14 or 16, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence, the resulting amino acid sequence still retains similar function of the TIGIT-binding single domain antibody.
In some embodiments of the first aspect, the sequence set forth in SEQ ID NO: 9. 13, 14 or 16, 1,2,3, 4, 5,6, 7, 8, 9, 10, 11,12,13, 14, 15, 20, 25 or more amino acids are added or deleted in a region other than the C-terminal or N-terminal of the amino acid sequence as long as the altered amino acid sequence substantially retains similar function of the TIGIT-binding single domain antibody.
In a third aspect, the application provides a nucleic acid molecule encoding the recombinant protein of the first aspect or the single domain antibody of the second aspect.
In some embodiments of the third aspect, the nucleic acid molecule may include a DNA molecule and an RNA molecule. The nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.
In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.
In a fourth aspect, the application provides a pharmaceutical composition comprising a recombinant protein according to the first aspect or a single domain antibody according to the second aspect, and a pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments of the fourth aspect, the pharmaceutical composition is for preventing or treating a tumor.
In some embodiments of the fourth aspect, the tumor is selected from the group consisting of: melanoma, colon carcinoma, renal cell carcinoma, non-small cell lung carcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, bladder carcinoma, acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma.
In some embodiments of the fourth aspect, the pharmaceutical composition may further comprise one or more of lubricants such as talc, magnesium stearate and mineral oil; a wetting agent; an emulsifying agent; a suspending agent; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
In some embodiments of the fourth aspect, the pharmaceutical compositions of the present application may be formulated in the form of tablets, pills, powders, troches, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules and the like.
In some embodiments of the fourth aspect, the pharmaceutical compositions of the present application may be delivered using any physiologically acceptable mode of administration including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
In some embodiments of the fourth aspect, the pharmaceutical composition for therapeutic use may be formulated for storage in the form of a lyophilized formulation or an aqueous solution by mixing an agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or the like.
In a fifth aspect, the application provides the use of a recombinant protein according to the first aspect, a single domain antibody according to the second aspect or a pharmaceutical composition according to the fourth aspect in the manufacture of a medicament for the prevention or treatment of a tumour.
In some embodiments of the fifth aspect, the tumor is selected from the group consisting of: melanoma, colon carcinoma, renal cell carcinoma, non-small cell lung carcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, bladder carcinoma, acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma.
In a sixth aspect, the application provides a method of preventing or treating a tumor comprising administering to a subject in need thereof a recombinant protein according to the first aspect, a single domain antibody according to the second aspect, or a pharmaceutical composition according to the fourth aspect.
In some embodiments of the sixth aspect, the tumor is selected from the group consisting of: melanoma, colon carcinoma, renal cell carcinoma, non-small cell lung carcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, bladder carcinoma, acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma.
The application also provides a vector comprising a nucleic acid molecule encoding the recombinant protein of the first aspect, or the single domain antibody of the second aspect, and a host cell comprising the nucleic acid molecule or vector. In other aspects, the application also provides methods of producing a recombinant protein according to the first aspect or a single domain antibody according to the second aspect. In some embodiments, the method of producing a recombinant protein according to the first aspect or an antibody according to the second aspect comprises culturing the host cell so that the nucleic acid molecule is expressed. In some embodiments, the method of producing a recombinant protein of the first aspect, or a single domain antibody of the second aspect, further comprises recovering the recombinant protein or the single domain antibody from the host cell culture medium.
It should be understood that the foregoing detailed description is only for the purpose of making apparent to those skilled in the art the contents of the application, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will occur to those skilled in the art.
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the application.
Examples
EXAMPLE 1 preparation of recombinant proteins
1.1 Preparation of recombinant antigens
The recombinant antigen human TIGIT extracellular domain (hTIGIT-ECD, SEQ ID NO: 2) was used in the preparation of anti-TIGIT monoclonal antibodies, and the recombinant antigen IL-2Rβ extracellular domain (IL-2 Rβ -ECD, SEQ ID NO: 3) was used in the identification of IL-15/IL-15Rα complex. These proteins have post-translational modifications (e.g., glycosylation or disulfide bonds, etc.), and thus the use of mammalian cell expression systems is more advantageous in maintaining the structure and function of the recombinant protein. In addition, to facilitate purification of recombinant proteins and identification of single domain antibody functions, his tag (His, SEQ ID NO: 4) or Fc segment of human antibody IgG1 (IgG 1-Fc, SEQ ID NO: 6) or Fc segment of murine antibody IgG2a (mFc, SEQ ID NO: 5) was added to the C-terminus of these recombinant proteins.
The genes (including His tag or Fc and mFc coding genes) of the various recombinant proteins are designed and synthesized according to the amino acid sequences of the various target recombinant proteins in the Uniprot database. The synthesized recombinant protein genes are cloned into proper eukaryotic expression vectors (such as pcDNA3.1 of the company of invitrogen) by using conventional molecular biology technology, and then the prepared recombinant protein expression plasmids are transfected into HEK293 cells (such as HEK293F of the company of invitrogen) by using liposomes (such as 293fectin of the company of invitrogen) or other cationic transfection reagents (such as PEI and the like), and are cultured for 3-4 days under serum-free suspension culture conditions. The culture supernatant is then harvested by centrifugation or the like.
The recombinant protein expressed by His tag fusion is purified in one step by using a metal chelating affinity chromatography column (such as HISTRAP FF of GE company, etc.). The Fc and mFc fusion expressed recombinant proteins were purified in one step using a ProteinA/G affinity column (e.g., mabSelect SURE, GE, etc.). The recombinant protein preservation buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., hitrap desaulting from GE). If necessary, the sample may be sterilized by filtration and then stored in aliquots at-20 ℃.
1.2 Preparation of recombinant antibodies that bind human TIGIT and IL-15/IL-15rα complexes
The C-terminus of the variable domain antibody that binds to human TIGIT was fused to the Fc fragment of murine antibody IgG2a (mFc, SEQ ID NO: 5) or to the Fc fragment of human antibody IgG1 (IgG 1-Fc, SEQ ID NO: 6). The C-terminus of hIL-15Rα (SEQ ID NO: 8) was fused to the Fc fragment of murine antibody IgG2a (mFc, SEQ ID NO: 5) or to the Fc fragment of human antibody IgG1 (IgG 1-Fc, SEQ ID NO: 6).
The synthesized single domain antibody gene encoding the binding human TIGIT is cloned into a suitable eukaryotic expression vector (such as pcdna3.1 from invitrogen corporation, etc.) using conventional molecular biology techniques to express the recombinant single domain antibody. The IL-15/IL-15Rα complex is expressed by co-transfection using conventional molecular biology techniques by cloning the IL-15 (hIL-15, SEQ ID NO: 7) and Fc-segment-containing IL-15Rα genes, respectively, into appropriate eukaryotic expression vectors. The prepared recombinant protein expression plasmid is transfected into HEK293 cells (such as HEK293F of the company Invitrogen) by using liposome (such as 293fectin of the company Invitrogen, etc.) or other cationic transfection reagent (such as PEI, etc.), and cultured under serum-free suspension culture condition for 3-4 days. The culture supernatant is then harvested by centrifugation or the like and purified in one step using a ProteinA/G affinity column (e.g., mabSelect SURE, GE, etc.). The recombinant antibody preservation buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., hitrap desaulting from GE). If necessary, the sample may be sterilized by filtration and then stored in aliquots at-20 ℃.
Example 2 screening of camel immune repertoire
2.1 Preparation of camel immunity and immune antibody repertoire
1 Healthy adult Bactrian camel is selected, and background serum is collected before immunization. Taking 1mg hTIGIT-His recombinant protein for primary immunization, emulsifying with Freund's complete adjuvant, and performing subcutaneous multipoint injection; boosting at intervals of two weeks, taking 1mg hTIGIT-His recombinant protein, emulsifying with Freund's incomplete adjuvant, performing subcutaneous multipoint injection, performing 5 times of boosting, and collecting blood before each immunization to analyze antibody titer; no adjuvant is added in seventh immunization, 1mg hTIGIT-His recombinant protein is taken as antigen, the subcutaneous multipoint injection is used for impact immunization, and 200mL of peripheral blood is collected for lymphocyte separation after 3 days.
200ML of camel peripheral blood lymphocytes are isolated by using a camel peripheral blood lymphocyte separation kit (Solarbio, CAT#P5750); extracting lymphocyte total RNA by using a cell total RNA extraction kit (CAT#DP430, tiangen Biochemical technology (Beijing)) Co., ltd; using the extracted total RNA as a template, synthesizing a camel heavy chain variable region by using a first-strand cDNA synthesis kit (Thermo scientific, CAT#K1621), wherein a reverse transcription primer adopts a gene specific primer, and a primer pairing region is positioned in a heavy chain constant region CH2 structural domain of an antibody, and the specific sequence is PCal-CH2R: TCCTTCCCCGTCAGCCAGTCCT (SEQ ID NO: 31). The synthesized cDNA is immediately stored at the temperature of-70 ℃ for standby; then primers were synthesized using the cDNA obtained by reverse transcription as a template, reference 9, and camel VHH gene was isolated by nested PCR amplification. Finally, the amplified VHH gene was cloned into vector pADSCFV-S (see Chinese patent application No. 201510097117.0, the entire contents of which are incorporated herein by reference), and a VHH library was constructed. The library capacity of the antibody library reaches 1.4E+08, and the accuracy is 90%.
2.2 Screening of anti-human TIGIT Single-Domain antibodies
Referring to the literature (see Chinese patent application No. 201510097117.0 for experimental technological process), recombinant protein hTIGIT prepared in example 1 is taken as an antigen, the camel immune library constructed in the above is screened by utilizing a solid phase screening strategy 10, three rounds of screening are carried out in a combined, eluted, neutralized, infected and amplified mode, and finally a single-domain antibody with different sequences and capable of specifically binding to recombinant protein hTIGIT is obtained, wherein the single-domain antibody comprises clone N9C9 (the amino acid sequence is shown as SEQ ID NO: 9).
2.3 Affinity analysis of anti-human TIGIT single domain antibodies
The affinity of anti-TIGIT single domain antibodies to bind TIGIT was determined by surface plasmon resonance technique using Biacore T200. The relevant reagents and consumables such as amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (14100530) and 10 XHBS-EP (BR 100669) at pH7.4 were purchased from GE HEALTHCARE. Carboxylated CM5 chip surfaces were activated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, EDC) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) according to the instructions in the kit, anti-human IgG (Fc) antibody (capture antibody) was diluted to 25. Mu.g/mL with 10mM sodium acetate (pH 5.0), followed by injection at a flow rate of 10. Mu.L/min to achieve a coupling of approximately up to 10000 Response Units (RU). After injection of the capture antibody, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, the anti-TIGIT single domain antibodies were diluted to 0.5-1 μg/mL, injected 10 μl/min, ensuring that about 150RU of single domain antibody was captured by the anti-human Fc antibody. The recombinant protein TIGIT-His was then subjected to a series of concentration gradients (e.g., 0.25nM, 0.74nM, 2.22nM, 6.67nM and 20 nM) at 25 ℃ for 30 μl/min from low to high concentrations for 120s binding time and 1200s dissociation time, and the chip surface was regenerated by injecting 3M MgCl 2 s at 10 μl/min. Binding rate (K a) and dissociation rate (K d) were calculated by fitting the binding and dissociation sensorgrams using Biacore T200 evaluation software version 3.2.1, 1 binding model. The dissociation equilibrium constant (K D) is calculated at a ratio K d/Ka. The fitting results are shown in table 1.
TABLE 1 affinity constant for binding of anti-human TIGIT Single Domain antibodies to human TIGIT recombinant proteins
Ka(M-1s-1) Kd(s-1) KD(M)
N9C9-Fc 1.111E+7 5.088E-3 4.58E-10
Example 3 humanization of N9C9 Single Domain antibodies
Humanization of 3.1N9C9 Single-Domain antibodies
Humanization studies were performed on N9C9 to reduce its immunogenicity. The humanization protocol employs a classical frame grafting strategy 11. The amino acid sequence of N9C9 was compared to human antibody germline gene sequences in the IMGT database, the appropriate germline gene sequences were selected to provide framework regions 1 to 3 (fr1+fr2+fr3) of the antibody, and the appropriate J region gene sequences were selected to provide framework region 4 (FR 4). This template may be selected based on a variety of factors, such as: the relative total length of the antibody, the size of the CDRs, the amino acid residues at the junction between the antibody Framework Regions (FR) and the hypervariable regions (CDRs), the homology of the sequence as a whole, and the like. The template selected may be a mixture of sequences or may be a consensus template in order to maintain as much of the appropriate conformation of the parental Complementarity Determining Regions (CDRs) as possible. Meanwhile, 4 hot spot amino acids 37F/44E/45R/47F in FR2 of the humanized antibody are subjected to back mutation in consideration of the characteristics of solubility, stability, expression yield and the like of the humanized antibody, and finally 2 humanized molecules N9C9-h1 (SEQ ID NO: 13) and N9C9-h2 (SEQ ID NO: 14) are obtained.
3.2. Affinity analysis of humanized N9C9 antibodies
Referring to example 2.3, affinity analysis was performed on N9C9 humanized monoclonal antibody with Biacore T200, and the analytical data are shown in table 2.
TABLE 2 affinity constants of N9C9 and humanized molecules binding to human TIGIT recombinant proteins
Ka(M-1s-1) Kd(s-1) KD(M)
N9C9-Fc 6.79E+6 1.813E-3 2.67E-10
N9C9-h1-Fc 4.424E+6 4.447E-3 1.005E-9
N9C9-h2-Fc 4.526E+6 4.711E-3 1.041E-9
3.3 Analysis of the ability of humanized N9C9 antibodies to bind to cell surface hTIGIT
Taking CHO-hTIGIT (CHO cell membrane stably expressing full-length hTIGIT, SEQ ID NO: 1) in logarithmic phase, centrifuging at 1000r/min for 5 min, discarding culture supernatant, re-suspending cells at a density of 2×10≡6 cells/mL by using PBS, plating 100 μl per well to 96-well V-bottom cell plates, preparing the antibody N9C9 and humanized molecule N9C9-h1/N9C9-h2 to be detected as 33nM initial concentration, performing 3-fold gradient dilution, setting 9 dilution concentration points altogether, uniformly mixing 100 μl per well with the cells, and placing in a refrigerator at 4 ℃ for incubation for 1 hour; centrifuging the cell plate at 1500r/min for 5 min, discarding the supernatant, re-suspending and washing the cells by using 200 mu L of PBS (phosphate buffered saline) per well, repeatedly washing for three times, adding FITC (FITC) fluorescence-labeled goat anti-human IgG secondary antibody (CAT#ZF-0512) of the golden bridge biotechnology company in Beijing), and incubating at 4 ℃ for 30min in a refrigerator for 100 mu L per well; antibody binding levels were detected using a flow cytometer (ACEA Biosciences, 2060R) after washing the cells three times with PBS. The results of fig. 1 show that: N9C9-Fc, N9C9-h1-Fc and N9C9-h2-Fc were all capable of binding CHO-hTIGIT.
EXAMPLE 4 Activity assay of IL-15/IL-15Rα Complex
HEK-Blue TM IL-2 is an engineering cell line developed by InvivoGen company based on HEK293 cells, stably expresses IL-2Rα, IL-2Rβ and IL-2Rγ, and carries JAK3 and STAT5 genes; and the SEAP reporter gene is integrated. When IL-2 or IL-15 acts on this cell line, the intracellular JAK/STAT5 signaling pathway is activated, and the SEAP reporter gene is expressed and secreted into the cell supernatant, and the activity of IL-2 or IL-15 is assayed by measuring SEAP concentration in the cell supernatant.
Cells were resuspended to 5X 10 5/mL in DMEM medium containing 10% serum, added to 96-well flat bottom cell culture plates (100. Mu.L/well), hIL-15/IL-15Rα complex, DP47-IgG1 antibody (negative control, prepared with reference to U.S. patent application No. 20160200833A1, wherein the amino acid sequences of the heavy and light chain variable regions are shown as SEQ ID NOS: 32 and 33, respectively) at an initial concentration of 1. Mu.g/mL, and diluted at 4-fold gradient, 100. Mu.L/well was added to the cells, and the light absorbance at 630nm was read after 24 hours. The results of fig. 2 show that: the hIL-15/IL-15Rα complex was able to effectively activate the HEK-Blue TM IL-2 intracellular signaling pathway with an EC 50 value of 0.62ng/mL.
Example 5: preparation of N9C9-IL-15/IL-15Rα recombinant proteins
N9C9-h1 (SEQ ID NO: 16) was constructed from two single domain antibodies N9C9-h1 joined by a GS flexible peptide (SEQ ID NO: 15), and mutants of the Fc fragment of the C-terminal fusion murine antibody IgG2a of N9C9-h1 and 2X 9C9-h1 (mIgG 2a-K, SEQ ID NO: 17) or of the Fc fragment of the murine antibody IgG1 (mIgG 1-K, SEQ ID NO: 19) or of the Fc fragment of the human antibody IgG1 (IgG 1m3-K, SEQ ID NO: 21) or of the Fc fragment of the human antibody IgG1 (IgG 1-K, SEQ ID NO: 22). The C-terminal fusion of IL-15Rα (SEQ ID NO: 8) with the Fc fragment of the murine antibody IgG2a (mIgG 2a-H, SEQ ID NO: 18) or with the Fc fragment of the murine antibody IgG1 (mIgG 1-H, SEQ ID NO: 20) or with the Fc fragment of the human antibody IgG1 (IgG 1m3-H, SEQ ID NO: 23) or with the Fc fragment of the human antibody IgG1 (IgG 1-H, SEQ ID NO: 24).
Referring to example 1.2, eukaryotic expression vectors of each recombinant protein were constructed, and recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2xN9C9-h1+hIL-15/IL-15 Rα were expressed in combination.
Example 6: analysis of N9C9-h1+hIL-15/IL-15Rα recombinant protein Activity
6.1 Affinity analysis of recombinant protein N9C9-h1+hIL-15/IL-15Rα with TIGIT and IL-2Rβ
Referring to example 2.3, affinity analysis was performed on N9C9-h1+IL-15/IL-15Rα recombinant protein using Biacore T200, and the analytical data are shown in tables 3 and 4.
TABLE 3 affinity constants for binding of N9C9-h1 and recombinant protein N9C9-h1+hIL-15/IL-15Rα to human TIGIT
Ka(M-1s-1) Kd(s-1) KD(M)
N9C9-h1-Fc 3.432E+6 9.829E-3 2.864E-9
N9C9-h1+hIL-15/IL-15Rα 4.098E+6 1.011E-2 2.467E-9
TABLE 4 affinity constants of hIL-15/IL-15Rα Complex and recombinant protein N9C9_h1+hIL-15/IL-15 Rα binding to human IL-2Rβ
Ka(M-1s-1) Kd(s-1) KD(M)
IL-15/IL-15Rα 2.245E+6 3.940E-3 1.755E-9
N9C9-h1+hIL-15/IL-15Rα 1.957E+6 3.804E-3 1.944E-9
6.2 Analysis of cell binding Activity of recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2 x N9C9-h1+hIL-15/IL-15Rα
Referring to example 3.3, the binding capacity of recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2xN9C9-h1+hIL-15/IL-15 Rα to CHO-hTIGIT was analyzed. The results are shown in FIG. 3: the binding capacity of the recombinant protein 2 x N9C9-h1+hIL-15/IL-15Rα to CHO-hTIGIT is higher than that of the recombinant protein N9C9-h1+hIL-15/IL-15Rα to CHO-hTIGIT.
6.3 Activity analysis of recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2 x N9C9-h1+hIL-15/IL-15Rα
IL-2 dependent NK92 cells proliferate normally under stimulation of IL-2 or IL-15. NK92 cells were incubated for 24 hours with IL-2 removed, then NK92 cells were plated 5X 10 4 per well into 96 well plates, hIL-15/IL-15Rα complex, N9C9-h1+hIL-15/IL-15Rα, 2X N9C9-h1+hIL-15/IL-15Rα and DP47 antibodies (negative control, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region were shown as SEQ ID NO:32 and 33, respectively) at an initial concentration of 16nM, diluted with a 5-fold gradient, and mixed well with the above cells for 48 hours, and assayed for NK92 proliferation using a cell titration-Glo kit (Promega, CAT#G7571). The results are shown in FIG. 4: recombinant proteins N9C9-h1+hIL-15/IL-15Rα and 2 x N9C9-h1+hIL-15/IL-15Rα showed better ability to promote NK cell proliferation than hIL-15/IL-15Rα complex.
Example 7: in vivo Activity analysis of recombinant proteins
Mice colon cancer cells MC38-hPDL1 stably expressing human PDL1 were inoculated subcutaneously into hTIGIT humanized C57 mice (Baioerse (Beijing) pharmaceutical technologies Co., ltd.) and appropriate mice were selected into groups according to tumor volume and body weight 7 days after tumor cell inoculation, in this example tumor volumes of 50mm 3-86mm3, mice weights of 19+ -2 g were selected into groups, randomly allocated to 7 experimental groups of 8 mice each, average tumor volumes of 66+ -1 mm 3, average body weights of 19.5+ -1 g were administered according to the protocol shown in FIG. 5, and tumor proliferation and body weight changes of the mice were monitored twice weekly. The experiment was completed for 6 times, and the tumor proliferation and physiological state of the mice were continuously monitored for one week after stopping the drug, and the tumor volume proliferation results of the mice are shown in fig. 5, wherein the recombinant protein N9C9-h1+hIL-15/IL-15Rα shows excellent tumor inhibition effect, which is superior to that of the hIL-15/IL-15Rα complex alone and the antibody N9C9-h1 alone, and is also superior to that of the combined group of the hIL-15/IL-15Rα complex and the antibody N9C9-h 1.
All patents, patent application publications, and non-patent documents mentioned and/or listed in this disclosure are incorporated herein by reference in their entirety. While exemplary embodiments of the application have been described above, modifications and improvements to the described exemplary embodiments of the application can be made by those skilled in the art without departing from the spirit and scope of the application, and the resulting variations or equivalents thereof also fall within the scope of the application.
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5.Yu X,Harden K,Gonzalez LC,et al.The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells.Nat Immunol.2009Jan;10(1):48-57.
6.Sloan KE,Eustace BK,Stewart JK,et al.CD155/PVR plays a key role in cell motility during tumor cell invasion and migration.BMC Cancer.2004Oct 7;4:73.
7.Wu L,Mao L,Liu JF,et al.Blockade of TIGIT/CD155 Signaling Reverses T-cell Exhaustion and Enhances Antitumor Capability in Head and Neck Squamous Cell Carcinoma.Cancer Immunol Res.2019Oct;7(10):1700-1713.
8.Chauvin JM,Ka M,Pagliano O,et al.IL15 Stimulation with TIGIT Blockade Reverses CD155-mediated NK-Cell Dysfunction in Melanoma.Clin Cancer Res.2020Oct 15;26(20):5520-5533.
9.Methods Mol Biol.2018；1701：169-187.
10. Phage display: general guidelines, (meclokson, t.) (mecloman, lowman, h.b.) code; ma Lan, et al, chemical industry Press, 2008.5.
11.J Immunol，2002Jul 15；169(2)：1119-25.
Sequence(s)
SEQ ID NO:1
MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQD
QLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLE
SSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSA
PSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG
SEQ ID NO:2
MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRV
APGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP
SEQ ID NO:3
AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNL
ILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISW
EISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEF
TTWSPWSQPLAFRTKPAALGKDT
SEQ ID NO:4
HHHHHH
SEQ ID NO:5
PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN
VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR
APQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM
YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
SEQ ID NO:6
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:7
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVEN
LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO:8
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
DPALVHQRPAPPSTVTTAGVTPQPESLSPSGKE
SEQ ID NO:9
QVQLVESGGGLVQAGGSLRLSCAASGLTFSPYAMGWFRQAPGKEREFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTVYLQMNSLKSEDTAVYFCAADQWRSVVAGRKPDYWGQGTQVTVSS
SEQ ID NO:10
GLTFSPYAMG
SEQ ID NO:11
SIRGSFDSTDYADSVKG
SEQ ID NO:12
DQWRSVVAGRKPDY
SEQ ID NO:13
EVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSS
SEQ ID NO:14
QVQLVESGGGVVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKEREFVASIRGSFDSTDYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSS
SEQ ID NO:15
GGGGSGGGGSGGGGS
SEQ ID NO:16
EVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVAS
IRGSFDSTDYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDY
WGQGTLVTVSS
SEQ ID NO:17
PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN
VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR
APQVYVLPPCEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF
MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
SEQ ID NO:18
PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN
VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVR
APQVCVLPPPEEEMTKKQVTLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM
VSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
SEQ ID NO:19
PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT
AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI
PPPKKQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMKTDGSYFVYSKLNVQ
KSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:20
PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT
AQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTI
PPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYDNTQPIMDTDGSYFVYSDLNVQK
SNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:21
EPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP
REPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:22
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:23
EPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP
REPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:24
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:25
EVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSSASE
PKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP
REPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:26
EVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVAS
IRGSFDSTDYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDY
WGQGTLVTVSSASEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PASIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:27
EVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSSASE
PKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:28
EVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVASIRGSFDSTDYADSV
KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDYWGQGTLVTVSSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGLTFSPYAMGWFRQAPGKERVFVAS
IRGSFDSTDYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADQWRSVVAGRKPDY
WGQGTLVTVSSASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:29
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
DPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEASEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK
SEQ ID NO:30
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
DPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEASEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK
SEQ ID NO:31
TCCTTCCCCGTCAGCCAGTCCT
SEQ ID NO:32
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADS
VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSS
SEQ ID NO:33
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGS
GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIK。

Claims (10)

1. A recombinant protein comprising a complex of a T cell immunoglobulin and an ITIM domain protein (TIGIT) binding moiety and IL-15 ra.
2. The recombinant protein according to claim 1, wherein
The TIGIT binding moiety is in the form of a single domain antibody; preferably, the TIGIT binding moiety comprises 1 or 2 TIGIT single domain antibodies; more preferably, the TIGIT binding moiety comprises HCDR1 as shown in SEQ ID No. 10, HCDR2 as shown in SEQ ID No. 11 and HCDR3 as shown in SEQ ID No. 12;
wherein the amino acid sequence of HCDR is defined according to Kabat.
3. The recombinant protein of claim 1 or 2, wherein the TIGIT binding moiety comprises the amino acid sequence set forth in SEQ ID NO: 9. 13, 14 or 16.
4. The recombinant protein of any one of claims 1-3, comprising:
SEQ ID NO: 13. 7 and 8;
SEQ ID NO: 14. 7 and 8; or alternatively
SEQ ID NO: 16. 7 and 8;
preferably, the recombinant protein comprises SEQ ID NO: 13. 7 and 8.
5. The recombinant protein according to any one of claims 1-4, wherein said TIGIT binding moiety and a complex of IL-15 and IL-15 ra are linked by an antibody heavy chain constant region Fc fragment comprising a first Fc fragment and a second Fc fragment, wherein
Amino acids 354 and 366 of the first Fc fragment are C and W, respectively, or amino acids 349, 366, 368 and 407 of the first Fc fragment are C, S, A and V, respectively; and the amino acids at positions 354 and 366 of the second Fc fragment are C and W, respectively, or the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; preferably, the amino acids at positions 354 and 366 of the first Fc fragment are C and W, respectively, and the amino acids at positions 349, 366, 368 and 407 of the second Fc fragment are C, S, A and V, respectively; and/or
Amino acids 234, 235 and 331 of the first and second Fc fragments are F, E and S, respectively;
wherein the amino acid position of the antibody constant region is determined according to EU numbering.
6. The recombinant protein according to claim 5, wherein one of said first Fc fragment and said second Fc fragment is linked to said TIGIT binding moiety and the other of said first Fc fragment and said second Fc fragment is linked to one of IL-15 and IL-15 ra.
7. A single domain antibody that binds human T cell immunoglobulin and ITIM domain protein (TIGIT) comprising
As set forth in SEQ ID NO: the HCDR1 shown in figure 10,
As set forth in SEQ ID NO:11, and
As set forth in SEQ ID NO:12, HCDR3;
preferably, the single domain antibody that binds to human TIGIT comprises the amino acid sequence as set forth in SEQ ID NO: 9. 13, 14 or 16;
wherein the amino acid sequence of HCDR is defined according to Kabat.
8. A nucleic acid molecule encoding the recombinant protein of any one of claims 1-6 or the single domain antibody of claim 7.
9. A pharmaceutical composition comprising the recombinant protein of any one of claims 1-6 or the single domain antibody of claim 7, and a pharmaceutically acceptable excipient, diluent or carrier.
10. Use of the recombinant protein of any one of claims 1-6, the single domain antibody of claim 7 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for preventing or treating a tumor; preferably, the tumor is selected from: melanoma, colon cancer, renal cell carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, soft tissue sarcoma, bladder cancer, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma.
CN202211578437.4A 2022-12-09 2022-12-09 Recombinant proteins comprising IL-15/IL-15Rα complexes and uses thereof Pending CN118165119A (en)

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