WO2024056097A1 - 基于nkg2d的细胞接合器分子在清除衰老细胞中的应用 - Google Patents

基于nkg2d的细胞接合器分子在清除衰老细胞中的应用 Download PDF

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WO2024056097A1
WO2024056097A1 PCT/CN2023/119470 CN2023119470W WO2024056097A1 WO 2024056097 A1 WO2024056097 A1 WO 2024056097A1 CN 2023119470 W CN2023119470 W CN 2023119470W WO 2024056097 A1 WO2024056097 A1 WO 2024056097A1
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cells
nkg2d
binding domain
amino acid
use according
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French (fr)
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赵旭东
杨东
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四川大学华西医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to the field of biomedicine. Specifically related to the application of NKG2D-CD3 and NKG2D-CD16 cell adapter molecules in clearing senescent cells and treating related diseases.
  • Cellular senescence is a process in which cells enter a state of permanent cell cycle termination in response to various stresses and occurs throughout the entire life cycle of an organism. Although cellular aging and individual aging are related, they are two completely different concepts. Even in older individuals, the number of senescent cells is very low. When an organism's immune system is strong enough to eliminate senescent cells in time, senescent cells can temporarily exist in the body and play beneficial functions such as promoting body tissue regeneration, wound repair, and reducing the production of cancer cells. However, when an organism's immune function declines or it is continuously injured and cannot remove senescent cells in time, the balance between the production and removal of senescent cells is broken, and senescent cells begin to accumulate in the organism.
  • Accumulated senescent cells will not only directly affect the normal functions of tissues and organs, but also continuously secrete a variety of inflammatory factors to cause tissue and organ damage, causing fatty liver, liver fibrosis, pulmonary fibrosis, diabetes, osteoporosis, muscle atrophy, and Atherosclerosis and other diseases. Due to factors such as changes in modern lifestyles and poor eating habits, the incidence of these diseases in young individuals is gradually increasing and has become the focus of social attention. Preclinical data show that clearing accumulated senescent cells in human disease models such as mice, rats and monkeys can delay, prevent or alleviate more than 40 related diseases.
  • Bispecific antibodies have been one of the hot topics in the field of drug research and development in recent years. A large number of biotechnology companies have invested in their development and have made breakthrough progress in the fields of cancer, autoimmune diseases, and inflammatory diseases.
  • Bispecific cell engager is a type of bispecific antibody that selectively recruits immune effector cells (Bispecific T Cell Engager, BiTE for T cells; Bispecific NK cell Engager, BiKE for NK cells). Around target cells, it establishes immune synapses and activates immune effector cells, which then secrete effector molecules such as telomerase and perforin to eliminate target cells. Compared with traditional monoclonal antibodies, bispecific cell adapters have higher sensitivity and specificity.
  • the object of the present invention is to provide NKG2D-CD3 and NKG2D-CD16 cell adapter molecule specificity A method to effectively and efficiently remove senescent cells.
  • a cell adapter molecule targeting NKG2D ligand and immune cells for the preparation of a medicine, the medicine being used for:
  • the cell adapter molecule includes a first binding domain and a second binding domain
  • the first binding domain specifically binds to NKG2D ligand
  • the second binding domain specifically binds to immune cell surface membrane proteins.
  • the first binding domain is the NKG2D extracellular domain.
  • the first binding domain includes the polypeptide shown in SEQ ID NO: 1, or a polypeptide that has more than 80% similarity with the sequence shown in SEQ ID NO: 1 and can bind to the NKG2D ligand .
  • the second binding domain specifically binds to CD3 or CD16.
  • the cell adapter molecule has a structure selected from the following formula (I) or (II) from the N-terminus to the C-terminus:
  • Each "-" is independently a connecting peptide or peptide bond
  • S is none or signal peptide sequence
  • D 1 is the first binding domain
  • L 1 is no or connected peptide
  • D 2 is the second binding domain
  • T is None or Tagged protein.
  • the second binding domain has a structure selected from the following group: single domain antibody (sdAb), single chain antibody (scFv), antigen-binding fragment (Fab fragment), ligand, or its Polymers, or combinations thereof.
  • sdAb single domain antibody
  • scFv single chain antibody
  • Fab fragment antigen-binding fragment
  • ligand or its Polymers, or combinations thereof.
  • the second binding domain is a CD3 binding domain
  • the CD3 binding domain can specifically bind to CD3 on the surface of T lymphocytes.
  • the CD3 binding domain has the amino acid sequence shown in SEQ ID NO:2, or has at least 80%, 85%, 90%, or 91% of the amino acid sequence shown in SEQ ID NO:2. , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity of the amino acid sequence.
  • the second binding domain is a CD16 binding domain
  • the CD16 binding domain can specifically bind to CD16 on the surface of natural killer cells.
  • the CD16 binding domain has the amino acid sequence shown in SEQ ID NO: 3 or 4. sequence, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity of amino acid sequences.
  • the CD16 binding domain is a human IgG Fc fragment.
  • the human IgG FC fragment has the amino acid sequence shown in SEQ ID NO:5, or has an amino acid sequence shown in SEQ ID NO:5 that is at least 80%, 85%, 90%, 91%, Amino acid sequences with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the marker protein T is selected from: His tag and FLAG tag.
  • the connecting peptide is a glycine-serine connecting peptide.
  • the connecting peptide is represented by formula (G 4 S)n, where n is 1, 2, 3, 4 or 5.
  • the senescent cells are selected from the following group: lung cells, adipocytes, kidney cells, muscle cells, or combinations thereof.
  • the senescent cells are human embryonic lung cells HEL1.
  • the senescent cells are natural or artificially induced senescence.
  • the artificial senescence induction method includes: DNA damage-induced senescence, P16 overexpression-induced senescence, telomere shortening-induced senescence, or a combination thereof.
  • the expression of NKG2D ligand is up-regulated in the senescent cells.
  • the upregulation of NKG2D ligand expression means that the ratio of the expression amount of NKG2D ligand in senescent cells (F1) to the expression amount in normal cells (F0) (i.e., F1/F0) ⁇ 1.5, preferably ⁇ 2 , more preferably ⁇ 2.5.
  • the medicine is used to prevent and/or treat diseases caused by accumulation of senescent cells.
  • the NKG2D ligands include (but are not limited to) MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the senile disease is selected from the following group: heart failure, atherosclerosis, diabetes, cardiac hypertrophy, osteoporosis, tissue/organ fibrosis, Alzheimer's disease, Parkinson's syndrome Organ degenerative diseases caused by cell aging such as arthritis, arthritis, or a combination thereof.
  • composition which pharmaceutical composition includes:
  • the other anti-aging drugs include other drugs that can specifically eliminate senescent cells.
  • component (b) includes a small molecule compound capable of specifically clearing senescent cells, preferably selected from the following group: dasatinib, quercetin, ABT263, ABT737, piperonamide, or its combination.
  • a pharmaceutical composition which pharmaceutical composition includes:
  • component (b) includes a ligand capable of upregulating cell surface NKG2D ligand expression, preferably selected from the group consisting of etoposide, cisplatin, vincristine, paclitaxel or a combination thereof.
  • the pharmaceutical composition is a liquid pharmaceutical composition.
  • the pharmaceutical composition is an injection.
  • Figure 1 shows NKG2D-CD3 protein preparation.
  • A Schematic diagram of the structure of NKG2D-CD3 and CD19-CD3 vectors.
  • B 293T cells were transfected with NKG2D-CD3 and CD19-CD3 vectors, and the culture supernatant was collected, purified, and stained with Coomassie brilliant blue.
  • C His antibody was used to detect the expression of NKG2D-CD3 and CD19-CD3 in the purified protein.
  • Figure 2 shows the upregulation of NKG2D ligand expression in senescent cells induced by overexpression of P16.
  • A ⁇ -gal staining of HEL1-P16 cells after inducing P16 overexpression with tetracycline (DOX).
  • B After HEL1-P16 cells were overexpressed with tetracycline (DOX) to induce P16, realtime PCR was used to detect the expression of NKG2D ligand.
  • C After HEL1-P16 cells were induced with tetracycline (DOX) to overexpress P16, flow cytometry was performed to detect NKG2D ligand expression.
  • Figure 3 shows that NKG2D-CD3 promotes T cell killing of senescent cells induced by overexpression of P16.
  • A After incubation of NKG2D-CD3 and T cells, the binding rate was detected by flow cytometry.
  • B After incubation of NKG2D-CD3 with DOX-induced senescence HEL1-P16 cells, flow cytometry was used to detect the binding rate.
  • C 8 hours after NKG2D-CD3 was added to the co-culture system of T cells and senescent cells, the death rate of senescent cells was detected.
  • Figure 4 shows DNA damage-induced upregulation of NKG2D ligand expression in senescent cells.
  • A ⁇ -gal staining of HEL1 cells after treatment with Etoposide to induce DNA damage.
  • B After HEL1 cells were treated with Etoposide to induce DNA damage, real-time PCR detected the expression of NKG2D ligand MICA.
  • C HEL1 cells were treated with Etoposide to induce DNA damage and then utilized by flow cytometry. NKG2D-FC fusion protein was used to detect total NKG2D ligand expression.
  • Figure 5 shows that NKG2D-CD3 promotes T cell killing of DNA damage-induced senescent cells.
  • A After incubation of NKG2D-CD3 and CD19-CD3 antibodies with DMSO or Etoposide-treated HEL1 cells, flow cytometry was used to detect the binding rate.
  • B NKG2D-CD3 or CD19-CD3 antibody was added to the co-culture system of T cells and HEL1 cells treated with DMSO or Etoposide, and the half inhibition rate (IC50) of HEL1 cells was detected after 8 hours.
  • Figure 6 shows NKG2D-CD16 and NKG2D-FC protein preparation.
  • A Schematic diagram of the structures of NKG2D-CD16, NKG2D-FC and control vectors.
  • B NKG2D-CD16, NKG2D-FC and control vector were transfected into 293T cells. After the culture supernatant was collected and purified, His antibody was used to detect the expression of NKG2D-NKp46 and control protein in the purified protein.
  • Figure 7 shows that NKG2D-CD16 and NKG2D-FC proteins promote NK cell killing of senescent cells.
  • A After NKG2D-CD16, NKG2D-FC and control vector were incubated with NK cells respectively, the binding rate was detected by flow cytometry.
  • B After incubating NKG2D-CD16 and NKG2D-FC with young cells and Etoposide-induced senescent cells respectively, the binding rate was detected by flow cytometry.
  • C NKG2D-CD16, NKG2D-FC and control vector were added to the NK cells and senescent cell co-culture system for 8 hours respectively, and the death rate of senescent cells was detected.
  • NKG2D-CD16, NKG2D-FC and control vector were added to the co-culture system of NK cells and senescent cells for 8 hours respectively.
  • the supernatant of the culture system was aspirated and the expression of IFN- ⁇ was detected by enzyme-linked immunosorbent (ELISA).
  • the bispecific cell adapter molecule provided by the invention consists of three parts: an NKG2D extracellular domain targeting NKG2D ligand on the surface of senescent cells, a connecting segment, and a CD3 or CD16 domain that binds immune cells.
  • the adapter molecule passes through NKG2D
  • the extracellular domain connects senescent cells and bridges immune cells, thereby bridging effector immune cells to the surface of senescent cells to achieve the purpose of eliminating senescent cells.
  • treatment refers to the administration of an internal or external therapeutic agent, including the bispecific cell engagers of the present invention, and compositions thereof to a patient having one or more symptoms of a disease for which it is known that the Therapeutic agents have a therapeutic effect on these symptoms.
  • a therapeutic agent is administered to a patient in an amount effective to relieve one or more symptoms of the disease (a therapeutically effective amount).
  • the terms “optionally” or “optionally” mean that the subsequently described event or circumstance may occur but does not necessarily occur.
  • “optionally including 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a specific sequence may be present but are not required to be present, and may be 1, 2 or 3.
  • Sequence identity means the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, deletions and other mutations.
  • sequence identity between the sequence described in the present invention and its identical sequence may be at least 85%, 90% or 95%, preferably at least 95%.
  • Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.
  • the first binding domain of the cell adapter used in the present invention specifically binds NKG2D ligand (NKG2DL).
  • NKG2D-NKG2DL signal chain is an important mechanism for the body's immune system to exert its immune surveillance function.
  • Bispecific cell adapter molecules NKG2D-CD3 and NKG2D-CD16 constructed based on the NKG2D extracellular domain have also gradually attracted attention in the anti-tumor field.
  • In vivo NKG2D-CD3 can inhibit tumor growth and prolong the survival of model mice by targeting tumor cells and immunosuppressive cells.
  • the expression of NKG2D ligand is also significantly up-regulated in senescent cells.
  • the first binding domain of the cell adapter of the invention is preferably derived from the extracellular domain of NKG2D.
  • the NKG2D extracellular domain includes the polypeptide shown in SEQ ID NO:1, or has a similarity of more than 80% (preferably more than 85%, more preferably more preferably) with the sequence shown in SEQ ID NO:1 More than 90%) and a polypeptide that can bind to NKG2D ligand.
  • the second binding domain of the cell adapter used in the present invention specifically binds to immune cell surface membrane proteins.
  • the immune cell surface membrane protein is preferably CD3 or CD16.
  • the second binding domain contained in the cell adapter molecule of the present invention can be an antibody, an antigen-binding fragment of an antibody, or any other polypeptide with the ability to specifically bind to CD3 or CD16.
  • the antibody referred to herein may be an immunologically active antibody fragment, such as Fab or (Fab')2 fragment; antibody heavy chain; antibody light chain.
  • the antibody used in the present invention is in the form of a single chain antibody.
  • the single chain antibody (scFv) contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and has all The smallest fragment of an antibody that has an antigen-binding site.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the antibody used in the present invention is in the form of a single domain antibody.
  • the single domain antibody (VhH) consists of only one heavy chain variable region of the cloned antibody and is the smallest antigen with complete functionality. Combine fragments.
  • antibodies bind with an affinity (KD) of about less than 10 "7 M, such as about less than 10 "8 M, 10 "9 M, or 10 "10 M or less.
  • the second binding domain is a CD3 binding domain
  • the CD3 binding domain can specifically bind to CD3 on the surface of T lymphocytes.
  • the CD3 binding domain has the amino acid sequence shown in SEQ ID NO: 2, or has an amino acid sequence shown in SEQ ID NO: 2 that is at least 80%, 85%, 90%, 91%, 92%, 93%, Amino acid sequences with 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • the second binding domain is a CD16 binding domain
  • the CD16 binding domain can specifically bind to CD16 on the surface of natural killer cells.
  • the CD16 binding domain is an anti-CD16 scFv or single domain antibody, which has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or has the same amino acid sequence as SEQ ID NO: 3 or SEQ ID NO: 3 or SEQ ID NO: 4.
  • the amino acid sequence shown in ID NO:4 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity of the amino acid sequence.
  • the CD16 binding domain is a human IgG FC fragment, which has the amino acid sequence shown in SEQ ID NO: 5, or has at least 80% or 85% of the amino acid sequence shown in SEQ ID NO: 5. Amino acid sequences that have %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.
  • bispecific cell engager molecule As used herein, the terms “bispecific cell engager molecule”, “bispecific cell engager”, “cell engager”, “BiTE”, “bispecific antibody” are used interchangeably and all refer to the invention.
  • a cell adapter molecule capable of simultaneously binding NKG2D ligands and proteins expressed by immune cells is provided.
  • Bispecific cell adapter molecules are composed of two protein or peptide sequences (antibodies being the most common) that bind different target proteins.
  • the function of the BiTE of the present invention is determined by the NKG2D receptor extracellular domain segment and the CD3 or CD16 protein binding segment.
  • the antibody of the present invention can simultaneously bind to NKG2D ligand and immune cell surface membrane protein CD3 or CD16, connect senescent cells through the ligand segment, and simultaneously At this time, immune cells are connected through anti-immune cell surface membrane protein segments, thereby effectively bridging effector immune cells and senescent cells, and more effectively exerting the effect of clearing senescent cells.
  • bispecific refers to a molecule that contains at least two binding domains with different binding specificities. Each binding domain is capable of specifically binding to a target molecule.
  • a bispecific cell engager is a polymer molecule with two or more peptides.
  • the binding domain comprises the antigen-binding domain, or variable region, or CDR of an antibody.
  • the binding domain comprises a ligand or fragment thereof that specifically binds to the target protein.
  • At least two targeting domains of the cell adapter molecules of the invention are optionally linked by a linker peptide.
  • the preferred connecting peptide sequence is (G 4 S) 3 , but is not limited thereto.
  • the bispecific cell adapter molecule is a single-chain polypeptide, which includes the NKG2D extracellular domain segment as the first binding domain, the linker segment, and the antibody as the second binding domain.
  • CD3 or CD16 single chain antibody wherein the anti-CD3 or CD16 single chain antibody is a conventional single chain antibody in this field, which includes a heavy chain variable region and a light chain variable region.
  • the first binding domain of the BiTE of the present invention is preferably the NKG2D extracellular domain segment
  • the first binding domain can also be selected as an antibody that specifically binds NKG2D, or an antigen-binding fragment thereof, As long as it can obtain the cell joining effect of the present invention.
  • the antibodies of the invention may be chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting NKG2D ligand and CD3 (eg, human NKG2D ligand and CD3).
  • the antibodies of the invention may be chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting NKG2D ligand and CD16 (eg, human NKG2D ligand and CD16).
  • the BiTE of the present invention also includes conservative variants thereof, which means that compared with the amino acid sequence of the BiTE of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, optimally Up to three amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
  • the amino acid sequence also includes a sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence, preferably having a homology or sequence identity of at least 80%, preferably at least 85%, and more Preferably it is at least 90%, most preferably at least 95% of the amino acid sequence.
  • the preferred method of determining identity is to obtain the greatest match between the sequences tested.
  • Methods for determining identity are compiled in publicly available computer programs.
  • Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990).
  • the BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD. 20894; Altschul, S. et al., 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, and more preferably 1-33%. , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of added, deleted, modified and/or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, even more preferably For 1-2 pieces.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned cell adapter or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition which pharmaceutical composition includes:
  • component (b) includes a small molecule compound capable of specifically clearing senescent cells, preferably selected from the group consisting of: dasatinib, quercetin, ABT263, ABT737, piperonamide, or its combination.
  • composition comprising:
  • component (b) includes a ligand capable of upregulating cell surface NKG2D ligand expression, preferably selected from the group consisting of etoposide, cisplatin, vincristine, paclitaxel or combinations thereof.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intramuscular, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
  • the injection administration preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
  • the pharmaceutical composition is in various conventional dosage forms in this field, preferably in the form of solid, semi-solid or liquid, and can be in the form of aqueous solution, non-aqueous solution or suspension, and more preferably in the form of tablets, capsules, granules , injections or infusions, etc.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. Additionally, the cell engagers of the present invention may be used with other therapeutic agents.
  • the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in this field, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in this field, preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of the pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.
  • the dosage of the pharmaceutical composition is an effective amount
  • the effective amount is an amount that can alleviate or delay the progression of diseases, degenerative or damaging conditions.
  • the effective amount can be determined on an individual basis and will be based in part on considerations of the condition to be treated and the results sought. One skilled in the art can determine an effective amount by using such factors as the above on an individual basis and using no more than routine experimentation.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the present invention provides uses of targeting NKG2D ligand cell engager molecules and pharmaceutical compositions of the present invention for preventing and/or treating age-related diseases.
  • the present invention also provides the use of the NKG2D-targeting cell adapter molecule of the present invention and the pharmaceutical composition of the present invention for preparing a medicine, which is used to (i) clear senescent cells; (ii) delay Individual aging; and/or (iii) prevention and/or treatment of age-related diseases.
  • the NKG2D ligand in the senescent cells is up-regulated by more than 1.5 times compared with normal cells.
  • senile diseases include: muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, cardiac hypertrophy, osteoporosis, tissue/organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis Organ degenerative diseases caused by cellular senescence, or combinations thereof.
  • the universal cell engager molecules of the present invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is human.
  • the present invention also provides compositions and methods for use in vivo to enhance immune responses against targeted antigens in patients.
  • compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the characteristics of the patient's condition, the type and severity of the disease - although appropriate dosages may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by the physician, taking into account the patient Individual differences in (subject's) age, weight, size of aged tissue, degree of aging, and disease.
  • the bispecific cell adapter molecules constructed in the present invention target NKG2D ligands and immune cells at the same time, and can be directly infused into the body, or carried by cells in the body (such as NK cells, T lymphocytes, CAR-T cells, etc.) and carried in the body.
  • the antibody protein is continuously expressed in the body, allowing the bispecific cell adapter molecule to exert a killing effect in the body. Its main advantages include:
  • Bispecific cell adapter molecules targeting NKG2D ligand-positive senescent cells can effectively bridge senescent cells and immune cells, with stable combination and strong killing power.
  • NKG2D ligand is an important target for natural immune cells to eliminate senescent cells and tumor cells, and its expression on the surface of normal cells is strictly regulated; the NKG2D-NKG2DL signal chain has experienced long-term natural selection and is highly safety. And there are currently a large number of immunotherapies targeting NKG2D ligands in clinical trials, and no serious treatment-related side effects have been found.
  • SEQ ID NO:2CD3 binding domain (derived from CD3 monoclonal antibody OKT3)
  • HEK-293T cells were cultured in 15 cm culture dishes for virus packaging. When the HEK-293T cell confluence is about 90% for transfection, prepare 2ml OPTIMEM dissolved plasmid mixture (core plasmid 20ug, pCMV ⁇ R8.9 10ug, PMD2.G 4ug); add 2ml OPTIMEM and 68ul lipo8000 in another centrifuge tube . After standing at room temperature for 5 minutes, add the plasmid complex to the liposome complex and let stand at room temperature for 20 minutes. The above mixture was added dropwise to HEK-293T cells, incubated at 37°C for 6 hours and then the medium was removed. Re-add pre-warmed complete medium.
  • 2ml OPTIMEM dissolved plasmid mixture core plasmid 20ug, pCMV ⁇ R8.9 10ug, PMD2.G 4ug
  • 2ml OPTIMEM and 68ul lipo8000 in another centrifuge tube . After standing at
  • the above virus was used to infect CHO cells, and 1ug/ml puromycin was added 24h later for selection for 48h. After the screened CHO cells were cultured for 7 days, the culture supernatant was collected and filtered with a 0.22um filter membrane, and His-tagged antibodies were obtained from the expression supernatant using an affinity chromatography column.
  • NKG2D-CD3 bispecific antibody After passing through the cation exchange column, the NKG2D-CD3 bispecific antibody is obtained, and finally the PBS buffer is used for liquid exchange and concentration.
  • the SDS-PAGE electrophoresis of purified NKG2D-CD3 protein is shown in Figure 1B.
  • the Western Blot results are shown in 1C, and the molecular weight is consistent with the theory.
  • the lentivirus overexpressing the p16 protein in the Tet-on system is infected with the cells at a multiplicity of infection (MOI) of 50-100, and added to the stock solution at a ratio of 1:1000 with a concentration of 8mg/mL polybrene to improve infection efficiency;
  • MOI multiplicity of infection
  • Fluorescence real-time quantitative PCR was used to detect NKG2D ligand expression. The specific operation was carried out according to the instructions of the Thermo powerup TM SYBR Green Master Mix (A25742) kit. Program: 50°C, 2min; 95°C, 2min; 95°C, 15s (40 pieces Cycle); 60°C, 1min (40 cycles); 12°C, forever;
  • NKG2D ligand expression was detected by flow cytometry, and the results are shown in Figures 2C and 4C.
  • Example 3 NKG2D-CD3 protein promotes T cell killing of senescent cells
  • NKG2D-CD3 protein promotes T cell killing of senescent cells
  • NKG2D-CD16 and NKG2D-FC proteins promote the killing of senescent cells by NK cells
  • NKG2D-CD16 and NKG2D-FC promote NK cells to kill senescent cells
  • the senescent cells and NK cells induced by P16 overexpression were seeded in a 96-well plate at an effect-to-target ratio of 2:1 (senescent cells are target cells and NK cells are effector cells), and then different concentrations of NKG2D-CD16 or NKG2D-FC were added.

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Abstract

提供一种NKG2D-CD3和NKG2D-CD16细胞接合器分子在清除衰老细胞和治疗衰老细胞累积相关疾病中的用途，所述细胞接合器分子包括NKG2D胞外域、连接段，以及CD3或CD16结合域，通过桥接衰老细胞和免疫细胞高效地诱导NKG2D配体高表达的衰老细胞死亡，可用于清除表达NKG2D配体的衰老细胞和治疗衰老相关疾病。

Description

基于NKG2D的细胞接合器分子在清除衰老细胞中的应用 技术领域
本发明涉及生物医药领域。具体涉及NKG2D-CD3和NKG2D-CD16细胞接合器分子在清除衰老细胞和治疗相关疾病中的应用。
背景技术
细胞衰老是细胞应对各种压力而进入永久性细胞周期终止状态的过程，贯穿于生物体的整个生命周期。细胞衰老与个体衰老虽有关联，但却是两个完全不同的概念。即使在老年个体中，衰老细胞的数量也是非常少的。当生物体的免疫系统足够强大可以及时清除衰老细胞时，衰老细胞可短暂存在于体内并发挥促进身体组织再生，伤口修复，减少癌细胞产生等有益功能。然而当生物体免疫功能下降或者持续受到伤害导致无法及时清除衰老细胞时，衰老细胞产生和清除的平衡被打破，衰老细胞开始在生物体内累积。累积的衰老细胞不仅会直接影响组织和器官的正常功能，同时还会不断分泌多种炎症因子造成组织器官损伤，引起脂肪肝、肝纤维化、肺纤维化、糖尿病、骨质疏松、肌肉萎缩以及动脉粥样硬化等多种疾病。由于现代生活方式的改变和不良的饮食习惯等因素，这些疾病在年轻个体中的发病率呈逐渐上升趋势，已成为社会关注焦点。临床前数据表明，清除小鼠、大鼠和猴子等人类疾病模型体内累积的衰老细胞，可使超过40种相关病症得到了延迟、预防或缓解。此外，通过清除衰老细胞治疗肾脏纤维化、糖尿病、肥胖、肾功能不全、衰老相关骨质疏松、骨关节炎等疾病的临床试验也在进行中。目前，清除衰老细胞的方法主要集中在寻找能够选择性清除衰老细胞的小分子化合物方面，例如达沙替尼、槲皮素、ABT263等，但这些化合物或清除衰老细胞效果欠佳，或存在明显的毒副作用。例如ABT-263可引起短暂性血小板减少症和中性粒细胞减少症；达沙替尼可能导致严重的肺动脉高压。因此，本领域迫切需要开发更安全有效清除衰老细胞的技术手段。双特异性抗体是近年来药物研发领域的热点之一，大量生物科技公司投入到其开发中，目前已在癌症、自身免疫性疾病和炎症性疾病等领域取得了突破性进展。双特异性细胞接合器是双特异性抗体中的一种，通过选择性募集免疫效应细胞(用于T细胞的为Bispecific T Cell Engager,BiTE；用于NK细胞的为Bispecific NK cell Engager,BiKE)到靶细胞周围，建立免疫突触并激活免疫效应细胞，进而分泌端粒酶和穿孔素等效应分子清除靶细胞。相对于传统单抗，双特异性细胞接合器具有更高的灵敏度和特异性。
因此，本领域有需要开发一种安全有效清除衰老细胞的技术手段。
发明内容
本发明的目的是提供NKG2D-CD3和NKG2D-CD16细胞接合器分子特异性 地、高效地清除衰老细胞的方法。
在本发明的第一方面，提供了一种靶向NKG2D配体和免疫细胞的细胞接合器分子的用途，用于制备一药物，所述药物用于：
(i)清除衰老细胞；
(ii)预防和/或治疗衰老细胞累积相关疾病；
(iii)延缓个体衰老；
所述细胞接合器分子包括第一结合结构域和第二结合结构域，
其中，所述第一结合结构域特异性结合NKG2D配体，所述第二结合结构域特异性结合免疫细胞表面膜蛋白。
在另一优选例中，所述的第一结合结构域为NKG2D胞外域。
在另一优选例中，所述的第一结合结构域包括SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示序列具有80％以上相似度并且可结合NKG2D配体的多肽。
在另一优选例中，所述的第二结合结构域特异性结合CD3或CD16。
在另一优选例中，所述的细胞接合器分子从N端到C端具有选自下式(I)或(II)的结构：
S-D1-L1-D2-T    (I)；或
S-D2-L1-D1-T   (II)，
式中，
各“-”独立地为连接肽或肽键；
S是无或信号肽序列；
D1是第一结合结构域；
L1是无或连接肽；
D2是第二结合结构域；
T是无或标记蛋白。
在另一优选例中，所述的第二结合结构域具有选自下组的结构：单域抗体(sdAb)、单链抗体(scFv)、抗原结合片段(Fab片段)、配体，或其多聚体，或其组合。
在另一优选例中，所述的第二结合结构域为CD3结合域，所述CD3结合域可特异性结合T淋巴细胞表面CD3。
在另一优选例中，所述CD3结合域具有SEQ ID NO:2所示的氨基酸序列，或具有与SEQ ID NO:2所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
在另一优选例中，所述的第二结合结构域为CD16结合域，所述CD16结合域可特异性结合自然杀伤细胞表面CD16。
在另一优选例中，所述CD16结合域具有SEQ ID NO:3或4所示的氨基酸序 列，或具有与SEQ ID NO:3所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
在另一优选例中，所述的CD16结合域为人IgG FC片段。
在另一优选例中，人IgG FC片段具有SEQ ID NO:5所示的氨基酸序列，或具有与SEQ ID NO:5所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
在另一优选例中，所述的标记蛋白T选自：His标签、FLAG标签。
在另一优选例中，所述连接肽是甘氨酸-丝氨酸连接肽。
在另一优选例中，所述连接肽由式(G4S)n表示，其中n为1、2、3、4或5。
在另一优选例中，所述的衰老细胞的选自下组：肺细胞、脂肪细胞、肾细胞、肌肉细胞，或其组合。
在另一优选例中，所述的衰老细胞为人胚肺细胞HEL1。
在另一优选例中，所述的衰老细胞是自然的或人工诱导衰老的。
在另一优选例中，所述的人工诱导衰老方法包括：DNA损伤诱导衰老、过表达P16诱导衰老、端粒缩短诱导衰老，或其组合。
在另一优选例中，所述的衰老细胞中NKG2D配体表达上调。
在另一优选例中，所述NKG2D配体表达上调是指衰老细胞NKG2D配体的表达量(F1)与正常细胞表达量(F0)之比(即F1/F0)≥1.5，优选地≥2，更优选地≥2.5。
在另一优选例中，所述的药物用于预防和/或治疗衰老细胞累积导致的疾病。
在另一优选例中，所述的NKG2D配体包括(但不限于)MICA，MICB，ULBP1,ULBP2,ULBP3,ULBP4,ULBP5,ULBP6。
在另一优选例中，所述的老年性疾病选自下组：心衰、动脉粥样硬化、糖尿病、心肌肥厚、骨质疏松、组织/器官纤维化、阿尔茨海默病、帕金森综合征、关节炎等细胞衰老导致的器官退行性疾病，或其组合。
在本发明的第二方面，提供了一种药物组合物，所述药物组合物包括：
(a)靶向NKG2D配体和免疫细胞的细胞接合器分子；
(b)除(a)之外的其他清除衰老细胞药物；和
(c)药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，在组分(b)中，所述的其他抗衰老药物包括其他能够特异性地清除衰老细胞的药物。
在另一优选例中，组分(b)包括能够特异性清除衰老细胞的小分子化合物，优选地选自下组：达沙替尼、槲皮素、ABT263、ABT737、荜茇酰胺，或其组合。
在本发明的第三方面，提供了一种药物组合物，所述药物组合物包括：
(a)靶向NKG2D配体和免疫细胞的细胞接合器分子；
(b)能够上调NKG2D配体表达的药物；和
(c)药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，组分(b)包括能够上调细胞表面NKG2D配体表达，优选地选自下组：依托泊苷、顺铂、长春新碱、紫杉醇或其组合。
在另一优选例中，所述药物组合物是液态的药物组合物。
在另一优选例中，所述药物组合物是注射剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示了NKG2D-CD3蛋白制备。(A)NKG2D-CD3和CD19-CD3载体结构示意图。(B)NKG2D-CD3和CD19-CD3载体转染293T细胞，收集培养液上清并进行纯化后，考马斯亮蓝染色。(C)利用His抗体检测纯化蛋白中NKG2D-CD3和CD19-CD3表达。
图2显示了过表达P16诱导的衰老细胞中NKG2D配体表达上调。(A)HEL1-P16细胞用四环素(DOX)诱导P16过表达后，细胞β-gal染色。(B)HEL1-P16细胞用四环素(DOX)诱导P16过表达后，realtime PCR检测NKG2D配体表达。(C)HEL1-P16细胞用四环素(DOX)诱导P16过表达后，流式细胞术检测NKG2D配体表达。
图3显示了NKG2D-CD3促进T细胞对过表达P16诱导衰老细胞的杀伤。(A)NKG2D-CD3与T细胞孵育后，流式细胞术检测结合率。(B)NKG2D-CD3与DOX诱导衰老的HEL1-P16细胞孵育后，流式细胞术检测结合率。(C)NKG2D-CD3加入到T细胞和衰老细胞共培养体系中8h后，检测衰老细胞死亡率。(D)NKG2D-CD3加入到T细胞和衰老细胞共培养体系中8h后，吸取培养体系上清，酶联免疫吸附(ELISA)检测IFN-γ表达。(E)NKG2D-CD3对过表达P16诱导衰老细胞的半抑制率(IC50)检测。
图4显示了DNA损伤诱导的衰老细胞中NKG2D配体表达上调。(A)HEL1细胞用依托泊苷(Etoposide)处理诱导DNA损伤后，细胞β-gal染色。(B)HEL1细胞用Etoposide处理诱导DNA损伤后，realtime PCR检测NKG2D配体MICA表达。(C)HEL1细胞用Etoposide处理诱导DNA损伤后，通过流式细胞术利用 NKG2D-FC融合蛋白检测总NKG2D配体表达。
图5显示了NKG2D-CD3促进T细胞对DNA损伤诱导衰老细胞的杀伤。(A)NKG2D-CD3和CD19-CD3抗体分别与DMSO或Etoposide处理的HEL1细胞孵育后，流式细胞术检测结合率。(B)NKG2D-CD3或CD19-CD3抗体加入到T细胞与DMSO或Etoposide处理HEL1细胞共培养体系中，8小时后HEL1细胞的半抑制率(IC50)检测。
图6显示了NKG2D-CD16和NKG2D-FC蛋白制备。(A)NKG2D-CD16、NKG2D-FC和对照载体结构示意图。(B)NKG2D-CD16、NKG2D-FC和对照载体转染293T细胞，收集培养液上清并进行纯化后，利用His抗体检测纯化蛋白中NKG2D-NKp46和对照蛋白表达。
图7显示了NKG2D-CD16和NKG2D-FC蛋白促进NK细胞对衰老细胞的杀伤。(A)NKG2D-CD16、NKG2D-FC和对照载体分别与NK细胞孵育后，流式细胞术检测结合率。(B)NKG2D-CD16和NKG2D-FC分别与年轻细胞和Etoposide诱导衰老细胞孵育后，流式细胞术检测结合率。(C)NKG2D-CD16、NKG2D-FC和对照载体分别加入到NK细胞和衰老细胞共培养体系中8h后，检测衰老细胞死亡率。(D)NKG2D-CD16、NKG2D-FC和对照载体分别加入到NK细胞和衰老细胞共培养体系中8h后，吸取培养体系上清，酶联免疫吸附(ELISA)检测IFN-γ表达。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种利用双特异性细胞接合器技术高特异性和高效地清除受试者中衰老细胞和治疗老年相关疾病的方法。
本发明所提供的双特异性细胞接合器分子由三部分组成：靶向衰老细胞表面NKG2D配体的NKG2D胞外域、连接段、以及结合免疫细胞的CD3或CD16结构域，该接合器分子通过NKG2D胞外域连接衰老细胞，同时桥接免疫细胞，从而将效应性免疫细胞桥接到衰老细胞表面，达到清除衰老细胞的目的。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属 领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的的双特异性细胞接合器及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
本发明所述的“序列同一性”表示当具有适当的替换、插入或缺失等突变的情况下最佳比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其具有同一性的序列之间的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,100％。
第一结合结构域
本发明所用的细胞接合器的第一结合结构域特异性结合NKG2D配体(NKG2DL)。
NKG2D-NKG2DL信号链是机体免疫系统发挥免疫监视功能的重要机制。基于NKG2D胞外域构建的双特异性细胞接合器分子NKG2D-CD3和NKG2D-CD16亦在抗肿瘤领域逐渐受到关注。研究表明NKG2D-CD3和NKG2D-CD16可分别激活T细胞和NK细胞，在体外高效杀伤NKG2D配体高表达的急性髓细胞性白血病、淋巴瘤、软组织肉瘤和黑色素瘤等肿瘤细胞。在体内NKG2D-CD3可通过靶向肿瘤细胞和免疫抑制细胞抑制肿瘤生长并延长模型小鼠生存期。NKG2D配体在衰老细胞中表达也显著上调。
本发明细胞接合器的第一结合结构域优选地来自NKG2D的胞外域。在优选的实施方式中，所述的NKG2D胞外域包括SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示序列具有80％以上相似度(优选地85％以上，更优选地90％以上)并且可结合NKG2D配体的多肽。
第二结合结构域
本发明所用的细胞接合器的第二结合结构域特异性结合免疫细胞表面膜蛋白。 所述免疫细胞表面膜蛋白优选地为CD3或CD16。
本发明细胞接合器分子中所包含的第二结合结构域可以是抗体、或抗体的抗原结合片段、或其他任意的具有特异性结合CD3或CD16能力的多肽。
本文所称的抗体可以是具有免疫活性的抗体片段，如Fab或(Fab')2片段；抗体重链；抗体轻链。在一种优选的实施方式中，本发明所用的抗体为单链抗体形式，所述单链抗体(scFv)含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。在另一种优选的实施方式中，本发明所用的抗体为单域抗体形式，所述单域抗体(VhH)仅由克隆抗体的一个重链可变区组成，是具有完整功能的最小的抗原结合片段。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-7M，例如大约小于10-8M、10-9M或l0-10M或更小的亲和力(KD)结合。
在本发明的优选实施方式中，所述的第二结合结构域为CD3结合域，所述CD3结合域可特异性结合T淋巴细胞表面CD3。所述CD3结合域具有SEQ ID NO:2所示的氨基酸序列，或具有与SEQ ID NO:2所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
在本发明的另一个优选实施方式中，所述的第二结合结构域为CD16结合域，所述CD16结合域可特异性结合自然杀伤细胞表面CD16。在一个实施方式中，所述CD16结合域为抗CD16的scFv或单域抗体，其具有SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列，或具有与SEQ ID NO:3或SEQ ID NO:4所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。在另一个实施方式中，所述的CD16结合域为人IgG FC片段，其具有SEQ ID NO:5所示的氨基酸序列，或具有与SEQ ID NO:5所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
双特异性细胞接合器分子
如本文所用，术语“双特异性细胞接合器分子”、“双特异性细胞接合器”、“细胞接合器”、“BiTE”、“双特异性抗体”可互换使用，均指本发明第一方面提供能够同时结合NKG2D配体和免疫细胞表达蛋白的细胞接合器分子。
双特异性细胞接合器分子由两种结合不同靶蛋白的蛋白或多肽序列(抗体最为常见)连接而成。在优选的实施方式中，本发明的BiTE的功能是由NKG2D受体胞外域段以及CD3或CD16蛋白结合段决定的。本发明的抗体可以同时结合NKG2D配体和免疫细胞表面膜蛋白CD3或CD16，通过配体段连接衰老细胞，同 时通过抗免疫细胞表面膜蛋白段连接免疫细胞，从而有效将效应性免疫细胞与衰老细胞桥接，更有效地发挥清除衰老细胞的效果。
如本文所用，术语“双特异性”是指包含至少两个具有不同结合特异性的结合结构域的分子。每个结合结构域都能够与靶分子特异性结合。在一些实施方式中，双特异性细胞接合器是具有两个或更多个肽的聚合物分子。在一些实施方式中，结合结构域包含抗体的抗原结合结构域，或可变区，或CDR。在一些实施方式中，结合结构域包含与靶蛋白特异性结合的配体或其片段。
本发明的细胞接合器分子的至少两个靶向结构域可选地由连接肽相连。优选的连接肽序列为(G4S)3，但不限于此。
在本发明的一个优选例中，所述的双特异性细胞接合器分子为单链多肽，其包括作为第一结合结构域的NKG2D胞外域段、连接段，以及作为第二结合结构域的抗CD3或CD16单链抗体，其中抗CD3或CD16单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区。
同时，本领域技术人员应当理解，虽然本发明BiTE的第一结合结构域优选为NKG2D胞外域段，但该第一结合结构域也可以选择为特异性结合NKG2D的抗体、或其抗原结合片段，只要其能够获得本发明的细胞接合效果。
本发明抗体可以是靶向NKG2D配体和CD3(例如人NKG2D配体和CD3)的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
本发明抗体可以是靶向NKG2D配体和CD16(例如人NKG2D配体和CD16)的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明中，本发明的BiTE还包括其保守性变异体，指与本发明BiTE的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1

并且，所述的氨基酸序列还包括经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列，优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明上述内容中，更优选地，所述添加、缺失、修饰和/或取代的氨基酸数量，可以是1-7个，更优选为1-5个，更优选为1-3个，更优选为1-2个。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的细胞接合器或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。
在本发明的一个实施方式中，提供了一种药物组合物，所述药物组合物包括：
(a)结合NKG2D和免疫细胞表面膜蛋白的细胞接合器分子；
(b)除(a)之外的其他清除衰老细胞药物；和
(c)药学上可接受的载体、稀释剂或赋形剂。
在优选的实施方式中，组分(b)包括能够特异性清除衰老细胞的小分子化合物，优选地选自下组：达沙替尼、槲皮素、ABT263、ABT737、荜茇酰胺，或其组合。
在本发明的另一个实施方式中，提供了一种药物组合物，所述药物组合物包括：
(a)结合NKG2D和免疫细胞表面膜蛋白的细胞接合器分子；
(b)能够上调NKG2D配体表达的药物；和
(c)药学上可接受的载体、稀释剂或赋形剂。
在优选的实施方式中，组分(b)包括能够上调细胞表面NKG2D配体表达，优选地选自下组：依托泊苷、顺铂、长春新碱、紫杉醇或其组合。
通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：肌肉内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、动脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的细胞接合器还可与其他治疗剂一起使用。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳 的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
治疗性应用
本发明提供了靶向NKG2D配体细胞接合器分子和本发明的药物组合物的用途，用于预防和/或治疗老年性疾病。
并且，本发明还提供了本发明的靶向NKG2D的细胞接合器分子和本发明的药物组合物的用途，用于制备一药物，所述药物用于(i)清除衰老细胞；(ii)延缓个体衰老；和/或(iii)预防和/或治疗老年性疾病。
在优选的实施方式中，所述衰老细胞中NKG2D配体较正常细胞上调1.5倍以上。
其中，老年性疾病包括：肌肉萎缩症、脂肪肝、心衰、动脉粥样硬化、糖尿病、心肌肥厚、骨质疏松、组织/器官纤维化、阿尔茨海默病、帕金森综合征、关节炎等细胞衰老导致的器官退行性疾病，或其组合。
本发明的通用型细胞接合器分子也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
除了就离体免疫细胞而言使用基于细胞的疫苗之外，本发明也提供了用于体内以增强针对患者中靶向抗原的免疫应答的组合物和方法。
本发明的药物组合物可以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症的特征、疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗衰老有效量”、“衰老性疾病-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、衰老组织大小、衰老程度和病症的个体差异。
本发明的主要优点
本发明构建的双特异性细胞接合器分子同时靶向NKG2D配体和免疫细胞，通过直接输注入体内，或利用体内细胞(如NK细胞、T淋巴细胞、CAR-T细胞等)携带并在体内持续表达该抗体蛋白，从而使该双特异性细胞接合器分子在体内发挥杀伤作用。其主要优点包括：
1)高靶向性：针对NKG2D配体阳性衰老细胞的双特异性细胞接合器分子，能够有效地桥接衰老细胞和免疫细胞，结合稳定，杀伤力强。
2)高安全性：NKG2D配体是天然免疫细胞清除衰老细胞和肿瘤细胞的重要靶点，其在正常细胞表面表达受到严格调控；NKG2D-NKG2DL信号链经历了长期的自然选择形成，具有高度的安全性。并且目前已有大量针对NKG2D配体的免疫疗法正在进行临床实验，尚未发现严重的治疗相关副作用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明实施例中涉及的序列如下所示。
SEQ ID NO:1NKG2D胞外域
SEQ ID NO:2CD3结合域(源于CD3单克隆抗体OKT3)
SEQ ID NO:3CD16结合域scFv片段(引用自专利US11167029B2)
SEQ ID NO:4CD16结合域VhH片段(引用自GenBank ABS29544.1)

SEQ ID NO:5IgG抗体FC片段(源于马吉妥昔单抗)
实施例1 NKG2D-CD3蛋白制备
1.1载体构建
将如图1A所示结构的目的基因核苷酸序列合成后，通过EcoR I和Swa I酶切位点克隆至慢病毒载体pCDH-CMV-MCS-EF1-Puro(参见Myeloid Leukemia.Mol Ther,2016.24(9):p.1615-26.)。克隆后的载体经过酶切和测序验证正确后，转化感受态大肠杆菌(Stbl3)并扩大培养，然后用QIAGEN公司的无内毒素中抽试剂盒抽提，并用Hind III酶切鉴定。
1.2病毒包装
在15cm培养皿中培养HEK-293T细胞用于病毒包装。待HEK-293T细胞汇合度在90％左右进行转染，准备2ml OPTIMEM溶解的质粒混合物(核心质粒20ug、pCMVΔR8.9 10ug、PMD2.G 4ug)；在另一离心管中2ml OPTIMEM以及68ul的lipo8000。室温静置5min后，将质粒复合物加入脂质体复合物中，室温静置20min。将上述混合物滴加入HEK-293T细胞中，37℃孵育6小时后去除培养基。重新加入预热的完全培养基。收集48小时和72小时病毒上清后，于4℃3000rpm离心20分钟。用0.45um滤膜过滤后，于25000rpm 4℃离心2.5小时进行病毒浓缩。浓缩的病毒用30ul病毒溶解液过夜溶解后，病毒滴度用QPCR检测。结果显示，病毒滴度达到要求。
1.3蛋白制备和纯化
取上述病毒感染CHO细胞，24h后加入1ug/ml嘌呤霉素筛选48h。筛选后的CHO细胞继续培养至7d后，收集培养液上清并用0.22um滤膜过滤，利用亲和层析柱从表达上清中获得带His标签的抗体。平衡缓冲液为0.5M Nacl、20mM磷酸钠缓冲液900ml，PH＝7.4；洗脱缓冲液为0.5M咪唑、0.5M Nacl、20mM磷酸钠缓冲液900ml,PH＝7.4。通过阳离子交换柱后获得NKG2D-CD3双特性抗体，最后用PBS缓冲液进行换液浓缩。纯化后NKG2D-CD3蛋白SDS-PAGE电泳如图1B所示。Western Blot结果如1C所示，分子量大小与理论一致。
实施例2衰老细胞中NKG2D配体表达上调
2.1 Tet-on系统过表达p16蛋白的细胞衰老模型构建
(1)将3×105个细胞分别铺于10cm皿，第二天贴壁后细胞密度在20％左右；
(2)待细胞贴壁后，将Tet-on系统过表达p16蛋白的慢病毒以50-100的感染复数(multiplicity of infection,MOI)分别感染细胞，并按照1:1000的比例加入原液浓度为8mg/mL的polybrene以提高感染效率；
(3)24h后以相同的病毒量进行二次感染；
(4)感染病毒4天后，加入终浓度为3μg/mL的嘌呤霉素筛选；
(5)将构建好的过表达p16蛋白的细胞传于孔板或培养皿中，24h贴壁后加入1μg/mL的dox诱导p16蛋白的表达；
(6)诱导8天后，用SA-βgal染色试剂盒(CS0030,Sigma)对细胞进行衰老染色，结果如图2A所示，90％以上细胞呈阳性，表明此时细胞已衰老。
2.2 DNA损伤药物诱导细胞衰老模型的构建
(1)铺细胞于10cm皿中，使贴壁后的密度在50％左右；
(2)24小时后加入Etoposide(sigma,E1383)，使其终浓度为50μM；
(3)36h后更换为新鲜培养基；
(4)继续培养细胞，其间每三天更换一次培养基。8天后细胞出现衰老表型。用SA-βgal染色试剂盒(CS0030,Sigma)对细胞进行衰老鉴定,结果如图4A所示，90％以上细胞呈阳性，表明此时细胞已衰老。
2.3 NKG2D配体转录水平表达检测
(1)按上述方法准备衰老细胞后，在10cm皿中依据细胞密度加入1～2mL Trizol，冰上放置5min，枪头吹打混匀；
(2)吸取1mL各孔裂解液加入1.5mL EP管中，加入氯仿200μL，用力振摇15s；室温放置5min离心(4℃，12000g，15min)；
(3)在新的EP管中加入450μL的异丙醇；
(4)小心吸取离心后的上层无色液体，加入含异丙醇的EP管中，混匀，室温孵育10min，离心(4℃，12000g，10min)；
(5)弃上清，加入1mL RNase free水配置的75％乙醇洗RNA，离心(4℃，7500g，5min)；
(6)小心去上清，倒扣5min晾干，用枪头吸去管壁的液体；
(7)加入30μL RNase Free水溶解，溶解后立即放在冰上，测定浓度
(8)以提取的RNA为模板，使用Thermo Scientific RevertAidTM First Strand cDNA Synthesis Kit试剂盒将2μg RNA反转录为cDNA。反应体系如下所示：
(9)按以上体系将反应物加入PCR管中，PCR仪内65℃，5min，立即放于冰上，再向管中加入以下成份：
轻轻混匀并瞬时离心，置于PCR仪中进行如下反应：25℃，5min；42℃，1h；70℃，5min；
(10)荧光实时定量PCR检测NKG2D配体表达，具体操作根据Thermo powerupTM SYBR Green Master Mix(A25742)试剂盒说明书进行，程序：50℃，2min；95℃，2min；95℃，15s(40个循环)；60℃，1min(40个循环)；12℃，forever；
(11)导出Excel格式的数据，计算NKG2D配体的相对表达量，结果如图2B和4B所示，NKG2D配体表达显著上调。
2.4 NKG2D配体膜表达水平表达检测
(1)胰酶消化收集HEIP细胞，1xPBS洗三次后，用200ul 1xPBS(含2％FBS)重悬，调整浓度细胞浓度为1x106cell/ml；
(2)向重悬的细胞中加入NKG2D配体抗体，混匀，放置于冰上孵育120min，期间每隔10min涡旋一次细胞，以500g的速度离心5min，弃上清；
(3)加入1ml 1xPBS(含2％FBS)重悬细胞，500g离心5min；
(4)重复步骤；
(5)流式仪检测NKG2D配体表达，结果如图2C和4C所示。
实施例3 NKG2D-CD3蛋白促进T细胞对衰老细胞的杀伤
3.1人体外周血T淋巴细胞分离
(1)将人体外周血转移至50mL离心管中，添加RosetteSepTM Cocktail至血液中(50μL/mL血液)；
(2)充分混匀后，室温孵育20min；
(3)配制稀释液：将1640培养基与1*PBS按体积比1:2混合；
(4)准备梯度离心管，加入15mL梯度分离液Ficol Lymphoprep；
(5)将稀释液与孵育血样按1:1混合；
(6)将稀释血样轻柔转移至分离液上，1200g离心20min；
(7)将离心后的全部上清液快速转移至新的离心管；
(8)用25mL的稀释液与上清混合均匀，300g离心10min；
(9)重复操作上述步骤；
(10)加入2mL T细胞完全培养基重悬T细胞，计数并用于后续实验。
3.2 NKG2D-CD3与衰老细胞和T淋巴细胞结合检测
(1)用200ul 1xPBS(含2％FBS)重悬T细胞和衰老细胞；
(2)向重悬的细胞中加入终浓度为100ug/ml的NKG2D-CD3或对照抗体，混匀，放置于冰上孵育120min，期间每隔10min涡旋一次细胞，以500g的速度离心5min，弃上清；
(3)加入1ml 1xPBS(含2％FBS)重悬细胞，500g离心5min；
(4)重复步骤；
(5)加入anti-His抗体并混匀，放置于冰上孵育60min，期间每隔10min涡旋一次细胞，以500g的速度离心5min，弃上清；
(6)加入荧光标记的的羊抗兔二抗，室温孵育30min后，以500g的速度离心5min，弃上清；
(7)加入1ml 1xPBS(含2％FBS)重悬细胞，500g离心5min；
(8)重复步骤7；
(9)流式仪检测结合检测率，结果如图3A，3B和5A所示，表明NKG2D-CD3与衰老细胞和T淋巴细胞均可有效结合。
3.3 NKG2D-CD3蛋白促进T细胞对衰老细胞的杀伤
将T细胞与衰老细胞按效靶比2:1(T细胞为效应细胞；衰老细胞为靶细胞)接种于96孔板中，然后分别加入NKG2D-CD3或CD19-CD3蛋白共孵育8小时(细胞培养液：advance 1640培养基(Gibco)+10％胎牛血清(Gibco)+1％青霉素、链霉素(Gibco))，显微镜先计算活细胞数目并计算T细胞杀伤率。杀伤效率＝(Blank组靶细胞数量-共培养组靶细胞数量)/Blank组靶细胞数量*100％。结果如图3C，3E，和5B所示，相对于对照组，NKG2D-CD3显著促进T细胞对衰老细胞杀伤作用，并呈现出剂量依赖性。
收集培养体系中上清，用ELISA试剂盒检测IFN-γ浓度，并用GraphPad Prism软件进行统计分析。结果如图3D所示，相对于对照组，加入NKG2D-CD3后IFN-γ分泌显著增加。
实施例4 NKG2D-CD16和NKG2D-FC蛋白制备
将如图6A所示结构的目的基因核苷酸序列合成后，通过EcoR I和Swa I酶切位点克隆至慢病毒载体pCDH-CMV-MCS-EF1-Puro。克隆后的载体经过酶切和测序验证正确后，转化感受态大肠杆菌(Stbl3)并扩大培养，并用QIAGEN公司的无内毒素中抽试剂盒抽提，然后用于病毒包装，包装过程如实施例1所描述。将包装好的病毒感染CHO细胞进行蛋白制备。用His标签抗体检测制备的NKG2D-CD16和NKG2D-FC蛋白，结果如图6B所示，蛋白大小与预期一致。
实施例5 NKG2D-CD16和NKG2D-FC蛋白促进NK细胞对衰老细胞的杀伤
5.1 NKG2D-CD16和NKG2D-FC与NK细胞和衰老细胞结合检测
(1)用200ul 1xPBS(含2％FBS)重悬NK细胞或衰老细胞；
(2)向重悬的细胞中分别加入终浓度为100ug/ml的NKG2D-CD16和NKG2D-FC蛋白，混匀，放置于冰上孵育120min，期间每隔10min涡旋一次细胞，以500g的速度离心5min，弃上清；
(3)加入1ml 1xPBS(含2％FBS)重悬细胞，500g离心5min；
(4)重复步骤；
(5)加入anti-His抗体并混匀，放置于冰上孵育60min，期间每隔10min涡旋一次细胞，以500g的速度离心5min，弃上清；
(6)加入荧光标记的的羊抗兔二抗，室温孵育30min后，以500g的速度离心5min，弃上清；
(7)加入1ml 1xPBS(含2％FBS)重悬细胞，500g离心5min；
(8)重复步骤7；
(9)流式仪检测结合率，结果如图7A和7B所示。结果表明NKG2D-CD16和NKG2D-FC均可与NK细胞或衰老细胞结合。
5.2 NKG2D-CD16和NKG2D-FC促进NK细胞对衰老细胞杀伤
将P16过表达诱导的衰老细胞与NK细胞按效靶比2:1(衰老细胞为靶细胞，NK细胞为效应细胞)接种于96孔板中，然后分别加入不同浓度NKG2D-CD16或NKG2D-FC蛋白共孵育8小时，利用显微镜计算活细胞数目并计算T细胞杀伤率。杀伤效率＝(Blank组靶细胞数量-共培养组靶细胞数量)/Blank组靶细胞数量。结果如图7C所示，相对于对照组，NKG2D-CD16和NKG2D-FC均显著促进NK细胞对衰老细胞杀伤作用。收集培养体系中上清，用ELISA试剂盒检测IFN-γ浓度，并用GraphPad Prism软件进行统计分析。结果如图7D所示，相对于对照组，加入NKG2D-CD16或NKG2D-FC后IFN-γ分泌显著增加。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (19)

  1. 一种靶向NKG2D配体和免疫细胞的细胞接合器分子的用途，其特征在于，用于制备一药物，所述药物用于：
    (i)清除衰老细胞；
    (ii)预防和/或治疗衰老细胞累积相关疾病；
    (iii)延缓个体衰老；
    所述细胞接合器分子包括第一结合结构域和第二结合结构域，
    其中，所述第一结合结构域特异性结合NKG2D配体，所述第二结合结构域特异性结合免疫细胞表面膜蛋白。
  2. 如权利要求1所述的用途，其特征在于，所述的第一结合结构域为NKG2D胞外域。
  3. 如权利要求1所述的用途，其特征在于，所述的第一结合结构域包括氨基酸序列如SEQ ID NO:1所示的多肽，或氨基酸序列与SEQ ID NO:1所示序列具有80％以上相似度并且可结合NKG2D配体的多肽。
  4. 如权利要求1所述的用途，其特征在于，所述的第二结合结构域特异性结合CD3或CD16。
  5. 如权利要求1所述的用途，其特征在于，所述的第二结合结构域具有选自下组的结构：单域抗体(sdAb)、单链抗体(scFv)、抗原结合片段(Fab片段)、配体，或其多聚体，或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述的第二结合结构域为CD3结合域，所述CD3结合域可特异性结合T淋巴细胞表面CD3。
  7. 如权利要求6所述的用途，其特征在于，所述CD3结合域具有SEQ ID NO:2所示的氨基酸序列，或具有与SEQ ID NO:2所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
  8. 如权利要求1所述的用途，其特征在于，所述的第二结合结构域为CD16结合域，所述CD16结合域特异性结合自然杀伤细胞表面CD16。
  9. 如权利要求8所述的用途，其特征在于，所述CD16结合域具有SEQ ID NO:3或4所示的氨基酸序列，或具有与SEQ ID NO:3或4所示的氨基酸序列至少有80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
  10. 如权利要求8所述的用途，其特征在于，所述的CD16结合域为人IgG FC片段。
  11. 如权利要求10所述的用途，其特征在于，人IgG FC片段具有SEQ ID NO:5所示的氨基酸序列，或具有与SEQ ID NO:5所示的氨基酸序列至少有80％、 85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同源性或序列相同性的氨基酸序列。
  12. 如权利要求1所述的用途，其特征在于，所述的细胞接合器分子从N端到C端具有选自下式(I)或(II)的结构：
    S-D1-L1-D2-T  (I)；或
    S-D2-L1-D1-T  (II)，
    式中，
    各“-”独立地为连接肽或肽键；
    S是无或信号肽序列；
    D1是第一结合结构域；
    L1是无或连接肽；
    D2是第二结合结构域；
    T是无或标记蛋白。
  13. 如权利要求1所述的用途，其特征在于，所述的衰老细胞的选自下组：肺细胞、脂肪细胞、肾细胞、肌肉细胞，或其组合。
  14. 如权利要求1所述的用途，其特征在于，所述的衰老细胞中NKG2D配体表达上调。
  15. 如权利要求1所述的用途，其特征在于，所述NKG2D配体表达上调是指衰老细胞NKG2D配体的表达量(F1)与正常细胞表达量(F0)之比(即F1/F0)≥1.5，优选地≥2，更优选地≥2.5。
  16. 如权利要求1所述的用途，其特征在于，所述的NKG2D配体选自下组：MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4、ULBP5、ULBP6、或其组合。
  17. 如权利要求1所述的用途，其特征在于，所述的老年性疾病选自下组：心衰、动脉粥样硬化、糖尿病、心肌肥厚、骨质疏松、组织/器官纤维化、阿尔茨海默病、帕金森综合征、关节炎、或其组合。
  18. 一种药物组合物，所述药物组合物包括：
    (a)靶向NKG2D和免疫细胞的细胞接合器分子；
    (b)除(a)之外的其他清除衰老细胞药物；和
    (c)药学上可接受的载体、稀释剂或赋形剂。
  19. 一种药物组合物，所述药物组合物包括：
    (a)靶向NKG2D和免疫细胞的细胞接合器分子；
    (b)能够上调NKG2D配体表达的药物；和
    (c)药学上可接受的载体、稀释剂或赋形剂。
PCT/CN2023/119470 2022-09-16 2023-09-18 基于nkg2d的细胞接合器分子在清除衰老细胞中的应用 WO2024056097A1 (zh)

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