[go: up one dir, main page]

TW202449142A - Conjugation complex - Google Patents

Conjugation complex Download PDF

Info

Publication number
TW202449142A
TW202449142A TW113112850A TW113112850A TW202449142A TW 202449142 A TW202449142 A TW 202449142A TW 113112850 A TW113112850 A TW 113112850A TW 113112850 A TW113112850 A TW 113112850A TW 202449142 A TW202449142 A TW 202449142A
Authority
TW
Taiwan
Prior art keywords
binding partner
cell
molecule
cells
active substance
Prior art date
Application number
TW113112850A
Other languages
Chinese (zh)
Inventor
松本悟
見城江利也
西本豊
竹中智哉
石井友美子
喬瑟夫 西恩 哈里森
Original Assignee
日商武田藥品工業股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日商武田藥品工業股份有限公司 filed Critical 日商武田藥品工業股份有限公司
Publication of TW202449142A publication Critical patent/TW202449142A/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Nanotechnology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention provides a complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell, the ligand being added to the outer surface of the delivery vehicle, wherein the delivery vehicle is comprised of an anchor molecule, a first binding partner is covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.

Description

接合複合物Bonding complex

本發明係關於一種用於將活性物質遞送至靶細胞之複合物、一種用於產生該複合物之方法、一種用於將活性物質遞送至包含該複合物之靶細胞的組合物，及其類似者。本發明亦關於一種用於使靶細胞活化及/或增殖之方法，及一種用於在靶細胞內部遞送活性物質之方法。The present invention relates to a complex for delivering an active substance to a target cell, a method for producing the complex, a composition for delivering an active substance to a target cell comprising the complex, and the like. The present invention also relates to a method for activating and/or proliferating a target cell, and a method for delivering an active substance inside a target cell.

使遺傳物質進入哺乳動物細胞中的非病毒遞送系統之研究及開發正快速進展，尤其在細胞療法領域中，包括使用表現自癌症抗原特異性殺手T細胞衍生之嵌合抗原受體(CAR)或T細胞受體(TCR)之T細胞的癌症免疫療法。目前之CAR-T細胞療法，諸如在美國批准之Kymriah (商品名)及Yescarta (商品名)，一般包括藉由使用病毒載體(諸如慢病毒載體或逆轉錄病毒載體)將CAR基因引入自患者離體收集之T細胞中來產生CAR-T細胞，及向患者投與CAR-T細胞。然而，此方法存在之問題係，由於細胞培養及病毒載體製備之成本，生產成本變高，因為多個步驟需要在長時段內進行，諸如T細胞之活化/增殖、病毒載體之製備、基因至T細胞之轉移及其類似步驟。Research and development of non-viral delivery systems for genetic material into mammalian cells are progressing rapidly, especially in the field of cell therapy, including cancer immunotherapy using T cells expressing chimeric antigen receptors (CAR) or T cell receptors (TCR) derived from cancer antigen-specific killer T cells. Current CAR-T cell therapies, such as Kymriah (trade name) and Yescarta (trade name) approved in the United States, generally involve generating CAR-T cells by introducing CAR genes into T cells collected ex vivo from a patient using a viral vector (such as a lentiviral vector or a retroviral vector), and administering the CAR-T cells to the patient. However, a problem with this method is that the production cost becomes high due to the cost of cell culture and viral vector preparation, because multiple steps need to be performed over a long period of time, such as activation/proliferation of T cells, preparation of viral vectors, gene transfer to T cells and the like.

作為一種在不使用病毒載體之情況下將CAR引入T細胞中之方法，已報導將CAR離體或活體內轉染至T細胞中係使用以下物質：奈米粒子(專利文件1、非專利文件1)，該等奈米粒子含有編碼CAR之質體DNA及陽離子型聚合物之聚集體且塗佈有與抗CD3抗體片段接合之非陽離子型聚合物；或奈米載劑(專利文件2)，該奈米載劑含有將編碼CAR之DNA囊封於孔隙中的中孔二氧化矽且塗佈有表面經抗CD3抗體改質之脂質。As a method for introducing CAR into T cells without using a viral vector, it has been reported that the following substances are used to transfect CAR into T cells in vitro or in vivo: nanoparticles (Patent Document 1, Non-Patent Document 1), which contain plasmid DNA encoding CAR and aggregates of cationic polymers and are coated with non-cationic polymers conjugated to anti-CD3 antibody fragments; or nanocarriers (Patent Document 2), which contain mesoporous silica that encapsulates DNA encoding CAR in the pores and is coated with lipids whose surfaces are modified with anti-CD3 antibodies.

除此之外，已報導用於藉由將所關注之siRNA囊封於「脂質奈米粒子(LNP)」中而將siRNA遞送至靶細胞的技術，該等「脂質奈米粒子(LNP)」不具有內部孔隙結構且包含陽離子型脂質、非陽離子型輔助脂質及用於遞送至靶細胞之配體。舉例而言，已報導藉由使用抗CD4抗體片段作為靶向配體將針對CD45之siRNA離體或活體內轉染至T細胞中(專利文件3、非專利文件2)。In addition, a technology for delivering siRNA to target cells by encapsulating the siRNA of interest in "lipid nanoparticles (LNPs)" has been reported, which do not have an internal pore structure and include cationic lipids, non-cationic auxiliary lipids, and ligands for delivery to target cells. For example, it has been reported that siRNA targeting CD45 is transfected into T cells in vitro or in vivo by using an anti-CD4 antibody fragment as a targeting ligand (Patent Document 3, Non-Patent Document 2).

此外，專利文件4描述用於將活性成分(諸如核酸或其類似物)引入各種細胞(包括T細胞)、組織及器官中的陽離子型脂質。In addition, Patent Document 4 describes cationic lipids for introducing active ingredients (such as nucleic acids or their analogs) into various cells (including T cells), tissues and organs.

另一方面，作為一種用於使T細胞活化/增殖之方法，已報導一種使用固定有抗CD3/CD28抗體之珠粒或奈米尺寸之基質珠粒來使細胞毒性免疫細胞活化及/或增殖的方法(專利文件5及6)。On the other hand, as a method for activating/proliferating T cells, a method for activating and/or proliferating cytotoxic immune cells using beads or nano-sized matrix beads to which anti-CD3/CD28 antibodies are immobilized has been reported (Patent Documents 5 and 6).

吾人先前開發了一種基於脂質奈米粒子(LNP)之技術，從而以類似方式同時執行使細胞毒性免疫細胞活化/增殖及將基因引入T細胞中之步驟(專利文件7)。We have previously developed a lipid nanoparticle (LNP)-based technology to simultaneously perform the steps of activating/proliferating cytotoxic immune cells and introducing genes into T cells in a similar manner (Patent Document 7).

然而，標靶特異性及轉染效率並不令人滿意。為了改良LNP之標靶特異性，需要重新檢驗用於連接LNP與對靶細胞具有特異性之配體的化學技術。已知二苯并環辛炔(DBCO)基及疊氮基允許用活細胞、整個生物體及非活樣品進行無銅點擊化學。在DBCO-疊氮化物反應之前，DBCO基團必須與對靶細胞具有特異性之配體接合。然而，除DBCO衍生物(諸如NHS-酯-DBCO)與配體之莫耳比為至少5:1至至少20:1 (專利文件8)以外，尚未引入精細地控制DBCO與配體之接合的先前技術，諸如配體分子上之DBCO接合的確切位點或每一個配體分子之DBCO接合數目。 [文件清單] However, the target specificity and transfection efficiency are not satisfactory. In order to improve the target specificity of LNPs, the chemical techniques used to link LNPs to ligands that are specific for target cells need to be re-examined. It is known that dibenzocyclooctyne (DBCO) groups and azido groups allow copper-free click chemistry with living cells, whole organisms and non-living samples. Before the DBCO-azido reaction, the DBCO group must be conjugated to a ligand that is specific for the target cell. However, except for the molar ratio of DBCO derivatives (such as NHS-ester-DBCO) to ligands of at least 5:1 to at least 20:1 (Patent Document 8), no prior art has been introduced to precisely control the binding of DBCO to ligands, such as the exact site of DBCO binding on the ligand molecule or the number of DBCO binding per ligand molecule. [Document List]

[專利文件][Patent Documents]

專利文件1：US 2017/0296676Patent Document 1: US 2017/0296676

專利文件2：US 2016/0145348Patent document 2: US 2016/0145348

專利文件3：WO 2016/189532Patent document 3: WO 2016/189532

專利文件4：WO 2016/021683Patent Document 4: WO 2016/021683

專利文件5：美國專利第6,352,694號Patent Document 5: U.S. Patent No. 6,352,694

專利文件6：US 2014/0087462Patent Document 6: US 2014/0087462

專利文件7：WO 2020/080475Patent Document 7: WO 2020/080475

專利文件8：WO2021/113519 [非專利文件] Patent document 8: WO2021/113519 [Non-patent document]

非專利文件1：Nature Nanotechnology 12, 813-820 (2017)Non-patent document 1: Nature Nanotechnology 12, 813-820 (2017)

非專利文件2：ACS Nano, 2015, 9(7), 6706-6716Non-patent document 2: ACS Nano, 2015, 9(7), 6706-6716

[技術問題][Technical issues]

本發明之目標為提供一種用於將活性物質遞送至靶細胞或組織之藥劑，其具有增強之標靶特異性及更高之轉染效率，從而使得由複合物轉導之靶細胞具有活性物質預期將展現之更高活性。本發明之一些其他目標為提供一種用於產生複合物之方法；及一種用於使靶細胞活化及/或增殖之方法，該方法包含使藥劑與包含靶細胞之細胞群體或組織接觸之步驟。出人意料地，本發明人發現，藉由精細地控制配體之接合(諸如配體分子上之接合的確切位點或每一個配體分子之接合數目)不僅顯著增強複合物之物理化學特徵，亦顯著增強標靶特異性及轉染效率，尤其對於包含脂質奈米粒子之複合物而言。 [問題之解決方案] The object of the present invention is to provide an agent for delivering an active substance to a target cell or tissue, which has enhanced target specificity and higher transfection efficiency, so that the target cells transduced by the complex have a higher activity expected to be exhibited by the active substance. Some other objects of the present invention are to provide a method for producing a complex; and a method for activating and/or proliferating a target cell, the method comprising the step of contacting the agent with a cell population or tissue containing the target cell. Unexpectedly, the inventors have found that by precisely controlling the binding of the ligand (such as the exact binding site on the ligand molecule or the number of binding per ligand molecule), not only the physicochemical properties of the complex are significantly enhanced, but also the target specificity and transfection efficiency are significantly enhanced, especially for the complex containing lipid nanoparticles. [Solution to the problem]

為了實現上文所提及之目標，本發明人已進行充分研究，且成功地提供用於將活性物質遞送至靶細胞或組織之藥劑，該藥劑呈包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體的複合物形式。此外，本發明人意外地發現，與先前技術之藥劑相比，有效負載遺傳物質在活體外及活體內於更高百分比之靶細胞中的表現可藉由以下有效地實現：將所添加之配體佈置於遞送媒介之外表面，其中複合物包含錨分子，將第一結合搭配物佈置為以1至4個第一結合搭配物分子/一個配體分子的比率與配體共價鍵結；將第二結合搭配物佈置為與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且將第一結合搭配物佈置為與第二結合搭配物共價鍵結，從而完成本發明。或者，與先前技術之藥劑相比，有效負載遺傳物質在活體外及活體內於更高百分比之靶細胞中的表現可藉由以下有效地實現：將所添加之配體佈置於遞送媒介之外表面，其中複合物包含錨分子；第一結合搭配物藉由分選酶識別模體與配體共價鍵結；第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。或者，與先前技術之藥劑相比，有效負載遺傳物質在活體外及活體內於更高百分比之靶細胞中的表現可藉由以下有效地實現：將所添加之配體佈置於遞送媒介之外表面，其中複合物包含錨分子；第一結合搭配物與配體之C端共價鍵結；第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。To achieve the above-mentioned object, the present inventors have conducted intensive studies and have successfully provided an agent for delivering an active substance to a target cell or tissue in the form of a complex comprising a delivery vehicle for the active substance and a ligand having specificity for the target cell. Furthermore, the inventors unexpectedly discovered that, compared to the agents of the prior art, the expression of effectively loaded genetic substances in a higher percentage of target cells in vitro and in vivo can be effectively achieved by: disposing the added ligand on the outer surface of the delivery medium, wherein the complex comprises an anchor molecule, disposing the first binding partner to covalently bond to the ligand at a ratio of 1 to 4 first binding partner molecules per one ligand molecule; disposing the second binding partner to covalently bond to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and disposing the first binding partner to covalently bond to the second binding partner, thereby completing the present invention. Alternatively, compared to agents of the prior art, efficient expression of a loaded genetic substance in a higher percentage of target cells in vitro and in vivo can be effectively achieved by: disposing an added ligand on the outer surface of a delivery medium, wherein the complex comprises an anchor molecule; the first binding partner is covalently bonded to the ligand via a sortase recognition motif; the second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner. Alternatively, compared to agents of the prior art, expression of an effectively loaded genetic substance in a higher percentage of target cells in vitro and in vivo can be effectively achieved by: disposing the added ligand on the outer surface of the delivery medium, wherein the complex comprises an anchor molecule; the first binding partner is covalently bonded to the C-terminus of the ligand; the second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner.

因此，本發明提供以下內容。Therefore, the present invention provides the following contents.

[1]一種用於遞送活性物質之複合物，其包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體，將配體添加至遞送媒介之外表面，其中遞送媒介包含錨分子，其中：第一結合搭配物係以1至4個第一結合搭配物分子/一個配體分子之比率與配體共價鍵結；第二結合搭配物與錨分子共價鍵結，從而將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。[1] A complex for delivering an active substance, comprising a delivery medium for the active substance and a ligand specific to a target cell, wherein the ligand is added to the outer surface of the delivery medium, wherein the delivery medium comprises an anchor molecule, wherein: a first binding partner is covalently bonded to the ligand at a ratio of 1 to 4 first binding partner molecules per one ligand molecule; a second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner.

[1a]  一種用於遞送活性物質之複合物，其包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體，將配體添加至遞送媒介之外表面，其中遞送媒介包含錨分子，其中：第一結合搭配物藉由分選酶識別模體與配體共價鍵結；第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。[1a] A complex for delivering an active substance, comprising a delivery medium for the active substance and a ligand specific to a target cell, wherein the ligand is added to the outer surface of the delivery medium, wherein the delivery medium comprises an anchor molecule, wherein: a first binding partner is covalently bonded to the ligand via a sortase recognition motif; a second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner.

[2]    一種用於遞送活性物質之複合物，其包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體，將配體添加至遞送媒介之外表面，其中遞送媒介包含錨分子，第一結合搭配物與配體之C端共價鍵結；第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。[2]    A complex for delivering an active substance, comprising a delivery medium for the active substance and a ligand specific to a target cell, wherein the ligand is added to the outer surface of the delivery medium, wherein the delivery medium comprises an anchor molecule, a first binding partner is covalently bonded to the C-terminus of the ligand; a second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner.

[3]    如[1]、[1a]或[2]之複合物，其中用於活性物質之遞送媒介為脂質奈米粒子或脂質體。[3]    A complex as in [1], [1a] or [2], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[4]    如[3]之複合物，其中第一結合搭配物為包含親偶極物之分子，且其中第二結合搭配物為包含1,3-偶極子之分子。[4]    A complex as in [3], wherein the first binding partner is a molecule comprising an apolarophile, and wherein the second binding partner is a molecule comprising a 1,3-polarophile.

[5]    如[4]之複合物，其中第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。[5]    A complex as in [4], wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group.

[6]    如[4]之複合物，其中第二結合搭配物為包含疊氮基之分子。[6]    A complex as in [4], wherein the second binding partner is a molecule comprising an azido group.

[7]    如[1]、[1a]或[2]之複合物，其中將活性物質遞送至其中之靶細胞為免疫細胞。[7]    A complex as described in [1], [1a] or [2], wherein the target cell to which the active substance is delivered is an immune cell.

[8]    如[7]之複合物，其中免疫細胞為細胞毒性細胞。[8]    A complex as in [7], wherein the immune cell is a cytotoxic cell.

[9]    如[8]之複合物，其中細胞毒性細胞為NK細胞或T細胞。[9]    A complex as in [8], wherein the cytotoxic cell is a NK cell or a T cell.

[10]  如[8]之複合物，其中對細胞毒性細胞具有特異性之配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD8、CD7、CD16及CD56。[10] A complex as described in [8], wherein the ligand specific for cytotoxic cells comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD8, CD7, CD16 and CD56.

[11]  如[1]、[1a]或[2]之複合物，其中活性物質為核酸。[11] A complex as described in [1], [1a] or [2], wherein the active substance is a nucleic acid.

[12]  如[11]之複合物，其中核酸包含編碼嵌合抗原受體(CAR)及/或T細胞受體(TCR)之核酸。[12] A complex as in [11], wherein the nucleic acid comprises nucleic acid encoding a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR).

[13]  一種藥劑，其包含如[12]之複合物。[13] A medicament comprising the complex as described in [12].

[14]  一種用於產生如[1]、[1a]或[2]之複合物的方法，其包含：(1)使第一結合搭配物與配體以一個配體分子/一個第一結合搭配物分子之比率共價鍵結之步驟；(2)使第二結合搭配物與錨分子共價鍵結之步驟；及(3)藉由化學反應使第一結合搭配物與第二結合搭配物以一個第二結合搭配物分子/一個第一結合搭配物分子之比率鍵結之步驟。[14] A method for producing a complex as described in [1], [1a] or [2], comprising: (1) a step of covalently bonding a first binding partner to a ligand at a ratio of one ligand molecule to one first binding partner molecule; (2) a step of covalently bonding a second binding partner to an anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner at a ratio of one second binding partner molecule to one first binding partner molecule by a chemical reaction.

[15]  如[14]之方法，其中用於活性物質之遞送媒介為脂質奈米粒子或脂質體。[15] A method as in [14], wherein the delivery vehicle for the active substance is lipid nanoparticles or liposomes.

[16]如[15]之方法，其中第一結合搭配物為包含親偶極物之分子，且其中第二結合搭配物為包含1,3-偶極子之分子。[16] The method of [15], wherein the first binding partner is a molecule comprising an apolarophile, and wherein the second binding partner is a molecule comprising a 1,3-polarophile.

[17]  如[16]之方法，其中第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。[17] A method as in [16], wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group.

[18]  如[16]之方法，其中第二結合搭配物為包含疊氮基之分子。[18] A method as in [16], wherein the second binding partner is a molecule comprising an azido group.

[19]  如[14]之方法，其中將活性物質遞送至其中之標靶T細胞為免疫細胞。[19] A method as in [14], wherein the target T cells to which the active substance is delivered are immune cells.

[20]  如[19]之方法，其中免疫細胞為細胞毒性細胞。[20] A method as described in [19], wherein the immune cells are cytotoxic cells.

[21]  如[20]之方法，其中細胞毒性細胞為NK細胞或T細胞。[21] The method of [20], wherein the cytotoxic cells are NK cells or T cells.

[22]如[20]之方法，其中對細胞毒性細胞具有特異性之配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD8、CD7、CD16及CD56。[22] The method of [20], wherein the ligand specific for cytotoxic cells comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of CD3, CD8, CD7, CD16 and CD56.

[23]  如[14]之方法，其中活性物質為核酸。[23] A method as described in [14], wherein the active substance is a nucleic acid.

[24]  一種用於將活性物質遞送至靶細胞之組合物，其包含如[1]、[1a]或[2]之複合物。[24] A composition for delivering an active substance to target cells, comprising a complex as described in [1], [1a] or [2].

[25]  如[24]之組合物，其中用於活性物質之遞送媒介為脂質奈米粒子或脂質體。[25] A composition as described in [24], wherein the delivery vehicle for the active substance is lipid nanoparticles or liposomes.

[26]  如[25]之組合物，其中第一結合搭配物為包含伸烷基之分子，且其中第二結合搭配物為1,3-偶極子分子。[26] A composition as in [25], wherein the first binding partner is a molecule comprising an alkylene group, and wherein the second binding partner is a 1,3-dipole molecule.

[27]  如[26]之組合物，其中第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。[27] A composition as in [26], wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group.

[28]  如[26]之組合物，其中第二結合搭配物為包含疊氮基之分子。[28] A composition as in [26], wherein the second binding partner is a molecule comprising an azido group.

[29]  如[24]之組合物，其中將活性物質遞送至其中之靶細胞為免疫細胞。[29] A composition as described in [24], wherein the target cells to which the active substance is delivered are immune cells.

[30]  如[29]之組合物，其中免疫細胞為細胞毒性細胞。[30] A composition as described in [29], wherein the immune cells are cytotoxic cells.

[31]  如[30]之組合物，其中細胞毒性細胞為NK細胞或T細胞。[31] A composition as described in [30], wherein the cytotoxic cells are NK cells or T cells.

[32]  如[30]之組合物，其中對細胞毒性細胞具有特異性之配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD8、CD7、CD16及CD56。[32] A composition as described in [30], wherein the ligand specific for cytotoxic cells comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD8, CD7, CD16 and CD56.

[33]  如[24]之組合物，其中活性物質為核酸。[33] A composition as described in [24], wherein the active substance is a nucleic acid.

[34]  如[33]之組合物，其進一步包含培養基或鹽水。[34] A composition as described in [33], further comprising a culture medium or saline.

[35]  一種用於使靶細胞活化及/或增殖之方法，其包含：使如[1]、[1a]或[2]之複合物與包含靶細胞之細胞群體接觸之步驟，其中複合物包含至少一種對靶細胞具有特異性之配體。[35] A method for activating and/or proliferating target cells, comprising: contacting a complex as described in [1], [1a] or [2] with a cell population comprising target cells, wherein the complex comprises at least one ligand specific for the target cells.

[36]  如[35]之方法，其中用於活性物質之遞送媒介為脂質奈米粒子或脂質體。[36] A method as in [35], wherein the delivery vehicle for the active substance is lipid nanoparticles or liposomes.

[37]  如[36]之方法，其中第一結合搭配物為包含親偶極物之分子，且其中第二結合搭配物為包含1,3-偶極子之分子。[37] A method as in [36], wherein the first binding partner is a molecule comprising a dipolephile, and wherein the second binding partner is a molecule comprising a 1,3-dipolephile.

[38]  如[37]之方法，其中第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。[38] A method as in [37], wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group.

[39]  如[37]之方法，其中第二結合搭配物為包含疊氮基之分子。[39] A method as in [37], wherein the second binding partner is a molecule comprising a hydrazine group.

[40]  如[35]之方法，其中將活性物質遞送至其中之靶細胞為免疫細胞。[40] A method as in [35], wherein the target cells to which the active substance is delivered are immune cells.

[41]  如[40]之方法，其中免疫細胞為細胞毒性細胞。[41] A method as in [40], wherein the immune cells are cytotoxic cells.

[42]  如[41]之方法，其中細胞毒性細胞為NK細胞或T細胞。[42] The method of [41], wherein the cytotoxic cells are NK cells or T cells.

[43]  如[41]之方法，其中對細胞毒性細胞具有特異性之配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD8、CD7、CD16及CD56。[43] A method as described in [41], wherein the ligand specific for cytotoxic cells comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD8, CD7, CD16 and CD56.

[44]  如[41]之方法，其中將超過一種配體添加至遞送媒介之外表面。[44] A method as in [41], wherein more than one ligand is added to the outer surface of the delivery medium.

[45]  如[35]之方法，其中活性物質為核酸。[45] A method as in [35], wherein the active substance is a nucleic acid.

[46]  如[45]之方法，其中核酸包含編碼CAR及/或TCR之核酸。[46] A method as in [45], wherein the nucleic acid comprises nucleic acid encoding CAR and/or TCR.

[47]  如[35]之方法，其中使複合物與包含靶細胞之細胞群體接觸之步驟係離體進行。[47] A method as in [35], wherein the step of contacting the complex with a cell population comprising target cells is performed in vitro.

[48]  一種用於在靶細胞內部遞送活性物質之方法，其包含：使如[1]、[1a]或[2]之複合物與包含靶細胞之細胞群體接觸之步驟，其中活性物質既不包括編碼CAR亦不包括編碼TCR之任何核酸。[48] A method for delivering an active substance inside a target cell, comprising: a step of contacting a complex as described in [1], [1a] or [2] with a cell population comprising target cells, wherein the active substance does not include any nucleic acid encoding neither CAR nor TCR.

[49]  如[48]之方法，其中用於活性物質之遞送媒介為脂質奈米粒子或脂質體。[49] A method as in [48], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[50]  如[49]之方法，其中第一結合搭配物為包含親偶極物之分子，且其中第二結合搭配物為包含1,3-偶極子之分子。[50] A method as in [49], wherein the first binding partner is a molecule comprising a dipolephile, and wherein the second binding partner is a molecule comprising a 1,3-dipolephile.

[51]  如[50]之方法，其中第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。[51] A method as in [50], wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group.

[52]  如[50]之方法，其中第二結合搭配物為包含疊氮基之分子。[52] A method as in [50], wherein the second binding partner is a molecule comprising a hydrazine group.

[53]  如[48]之方法，其中將活性物質遞送至其中之靶細胞為免疫細胞。[53] A method as in [48], wherein the target cells to which the active substance is delivered are immune cells.

[54]  如[53]之方法，其中免疫細胞為細胞毒性細胞。[54] A method as in [53], wherein the immune cells are cytotoxic cells.

[55]  如[54]之方法，其中細胞毒性細胞為NK細胞或T細胞。[55] The method of [54], wherein the cytotoxic cells are NK cells or T cells.

[56]  如[55]之方法，其中對細胞毒性細胞具有特異性之配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD8、CD7、CD16及CD56。[56] A method as in [55], wherein the ligand specific for cytotoxic cells comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD8, CD7, CD16 and CD56.

[57]  如[54]之方法，其中將超過一種配體添加至遞送媒介之外表面。[57] A method as in [54], wherein more than one ligand is added to the outer surface of the delivery medium.

[58]如[48]之方法，其中活性物質為核酸。[58] The method of [48], wherein the active substance is a nucleic acid.

[59]  如[58]之方法，其中核酸包含抑制細胞毒性細胞活化抑制因子之表現之核酸，及/或編碼細胞毒性細胞活化促進因子之核酸。[59] The method of [58], wherein the nucleic acid comprises a nucleic acid that inhibits the expression of a cytotoxic cell activation inhibitor and/or a nucleic acid that encodes a cytotoxic cell activation promoting factor.

[60]  如[48]之方法，其中步驟係離體進行。[60] A method as in [48], wherein the step is performed in vitro.

[61]  一種靶細胞，其係藉由如[48]之方法將活性物質遞送至其中。[61] A target cell to which an active substance is delivered by a method such as [48].

[62]  一種藥劑，其包含如[61]之靶細胞。[62] A pharmaceutical agent comprising a target cell as described in [61].

[63]  如[62]之藥劑，其進一步包含培養基或鹽水。 [本發明之有利作用] [63]  The drug of [62] further comprises a culture medium or saline. [Beneficial effects of the present invention]

根據本發明，用於遞送活性物質之複合物包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體。與先前技術之細胞療法藥劑相比，複合物可如下在活體外及活體內於更高百分比之靶細胞中表現有效負載遺傳物質：藉由佈置以1至4個第一結合搭配物分子/一個配體分子之比率與配體共價鍵結之第一結合搭配物，或藉由佈置藉由分選酶識別模體與配體共價鍵結之第一結合搭配物，或藉由佈置與配體之C端共價鍵結之第一結合搭配物，且第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介。According to the present invention, the complex for delivering active substances comprises a delivery medium for active substances and a ligand having specificity for target cells. Compared with the cell therapy agents of the prior art, the complex can effectively carry genetic substances in a higher percentage of target cells in vitro and in vivo by disposing a first binding partner covalently bound to the ligand at a ratio of 1 to 4 first binding partner molecules per one ligand molecule, or by disposing a first binding partner covalently bound to the ligand via a sortase recognition motif, or by disposing a first binding partner covalently bound to the C-terminus of the ligand, and the second binding partner covalently bound to an anchor molecule, thereby fixing the second binding partner to the delivery medium.

本說明書中所描述之生物序列資料係以標準化電子格式(「序列表XML」)呈現為本發明之單獨部分。即，序列表係由表示分選酶識別模體之五個胺基酸殘基之單一胺基酸序列(SEQ ID NO.:1)，或由來自金黃色葡萄球菌(Staphylococcus aureus)之分選酶A (StrA)識別之共通胺基酸序列組成。胺基酸序列為：Leu-Pro-Xxx-Thr-Gly或LPXTG，其中Xxx或X為任何胺基酸且Gly或G不能為游離羧酸酯。The biological sequence data described in this specification is presented as a separate part of the present invention in a standardized electronic format ("Sequence Listing XML"). That is, the sequence listing consists of a single amino acid sequence (SEQ ID NO.: 1) representing the five amino acid residues of the sortase recognition motif, or a common amino acid sequence recognized by the sortase A (StrA) from Staphylococcus aureus. The amino acid sequence is: Leu-Pro-Xxx-Thr-Gly or LPXTG, where Xxx or X is any amino acid and Gly or G cannot be a free carboxylate.

1.1. 本發明之複合物The complex of the present invention

在一個態樣中，本發明提供一種用於遞送活性物質之複合物，其包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體， 將配體添加至遞送媒介之外表面，其中 遞送媒介包含錨分子， 第一結合搭配物係以1至4個第一結合搭配物分子/一個配體分子之比率與配體共價鍵結； 第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；及 第一結合搭配物與第二結合搭配物共價鍵結。(下文亦稱為「本發明之複合物」)。 在另一態樣中，本發明提供一種用於遞送活性物質之複合物，其包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體， 將配體添加至遞送媒介之外表面， 遞送媒介包含錨分子，其中：第一結合搭配物藉由分選酶識別模體與配體共價鍵結；第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。 在另一態樣中，本發明提供一種用於遞送活性物質之複合物，其包含用於活性物質之遞送媒介及對靶細胞具有特異性之配體， 將配體添加至遞送媒介之外表面， 遞送媒介包含錨分子，其中：第一結合搭配物與配體之C端共價鍵結；第二結合搭配物與錨分子共價鍵結，由此將第二結合搭配物固定至遞送媒介；且第一結合搭配物與第二結合搭配物共價鍵結。 在本說明書中，複合物係指由(a)用於活性物質之遞送媒介及(b)對靶細胞具有特異性之配體構成的分子總成。複合物充當用於將活性物質特異性遞送至靶細胞之媒介。 下文闡釋構成元素(a)及(b)。 1.  (a) 用於活性物質之遞送媒介 In one embodiment, the present invention provides a complex for delivering an active substance, comprising a delivery medium for the active substance and a ligand specific to a target cell, the ligand is added to the outer surface of the delivery medium, wherein the delivery medium comprises an anchor molecule, the first binding partner is covalently bonded to the ligand at a ratio of 1 to 4 first binding partner molecules per one ligand molecule; the second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner. (hereinafter also referred to as the "complex of the present invention"). In another aspect, the present invention provides a complex for delivering an active substance, which comprises a delivery medium for the active substance and a ligand specific to a target cell, wherein the ligand is added to the outer surface of the delivery medium, the delivery medium comprises an anchor molecule, wherein: the first binding partner is covalently bonded to the ligand via a sortase recognition motif; the second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner. In another aspect, the present invention provides a complex for delivering an active substance, which comprises a delivery medium for the active substance and a ligand specific to a target cell. The ligand is added to the outer surface of the delivery medium. The delivery medium comprises an anchor molecule, wherein: a first binding partner is covalently bonded to the C-terminus of the ligand; a second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner. In this specification, a complex refers to a molecular assembly composed of (a) a delivery medium for an active substance and (b) a ligand specific to a target cell. The complex serves as a medium for specific delivery of an active substance to a target cell. The components (a) and (b) are explained below. 1. (a) Delivery medium for active substances

用於活性物質之遞送媒介可為包含錨分子之任何分子總成，該錨分子可充當活性物質之保護遮罩或媒介且配體透過第一及第二結合搭配物與其連接。用於活性物質之遞送媒介之實例包括脂質奈米粒子、脂質體、陽離子型聚合物(例如，聚乙亞胺、聚離胺酸、聚鳥胺酸、聚葡萄胺糖、去端肽膠原(atelocollagen)、魚精蛋白等)、陽離子型聚合物囊封於脂質體中之媒介及其類似物。或者，亦可使用胞泌體，其為來源於活的生物體之組分。The delivery vehicle for the active substance can be any molecular assembly comprising an anchor molecule that can act as a protective mask or medium for the active substance and to which the ligand is linked via the first and second binding partners. Examples of delivery vehicles for active substances include lipid nanoparticles, liposomes, cationic polymers (e.g., polyethyleneimine, polylysine, polyguanine, polyglucosamine, atelocollagen, protamine, etc.), cationic polymer encapsulated vehicles in liposomes, and the like. Alternatively, exosomes, which are components derived from living organisms, can also be used.

錨分子為脂質分子，其經共價修飾以包含第二結合搭配物(1,3-偶極子)。錨分子適用於與接合至第一結合搭配物(親偶極物)之配體共價鍵結。第一及第二結合搭配物能夠不僅在活體外且在活體內以生物學上安全之方式在彼此之間形成特異性化學鍵，而無任何不良副反應。錨分子可為脂質分子中之任一或多者。錨分子可為非陽離子型脂質。「非陽離子型脂質」意謂除陽離子型脂質以外之脂質，且係在所選擇之pH (諸如生理pH及其類似pH)下不具有淨正電荷之脂質。用於本發明之脂質奈米粒子中之非陽離子型脂質的實例包括磷脂、類固醇、PEG脂質及其類似物。An anchor molecule is a lipid molecule that is covalently modified to include a second binding partner (1,3-dipole). An anchor molecule is suitable for covalent bonding with a ligand that is attached to a first binding partner (dipolephile). The first and second binding partners are capable of forming specific chemical bonds between each other in a biologically safe manner not only in vitro but also in vivo without any adverse side reactions. An anchor molecule can be any one or more of the lipid molecules. An anchor molecule can be a non-cationic lipid. "Non-cationic lipid" means a lipid other than a cationic lipid, and is a lipid that does not have a net positive charge at a selected pH (such as physiological pH and pH similar thereto). Examples of non-cationic lipids used in the lipid nanoparticles of the present invention include phospholipids, steroids, PEG lipids and the like.

本發明之複合物包含遞送媒介及配體。遞送媒介係由錨分子及其他脂質分子聚集而成，其中可囊封活性物質且保護其免受酶或其他使活性物質降解之化學物質(諸如，當活性物質為蛋白質時之蛋白酶，及當活性物質為核酸時之核酸酶)的攻擊。遞送媒介較佳為具有內部脂質核心之脂質奈米粒子(LNP)，而非一般細胞及脂質體之脂質雙層。因此，遞送媒介之外表面處於水相中，且遞送媒介之內部核心處於較少水相或非水相中。當活性物質為親水性物質時，假定親水性活性物質係由脂質囊封，其中脂質之親水性部分面向親水性活性物質。藉由指定將配體添加至遞送媒介之外表面，所有配體皆暴露於諸如培養基或鹽水之水相及靶細胞之外表面，而不損失埋於遞送媒介中之任何配體，以使得配體與靶細胞或確切而言靶細胞之細胞表面分子之間的特異性相互作用促進複合物之靶細胞特異性併入。The complex of the present invention comprises a delivery vehicle and a ligand. The delivery vehicle is an aggregate of anchor molecules and other lipid molecules, in which the active substance can be encapsulated and protected from attack by enzymes or other chemicals that degrade the active substance (e.g., proteases when the active substance is a protein, and nucleases when the active substance is a nucleic acid). The delivery vehicle is preferably a lipid nanoparticle (LNP) with an internal lipid core, rather than the lipid bilayer of ordinary cells and liposomes. Therefore, the outer surface of the delivery vehicle is in the aqueous phase, and the inner core of the delivery vehicle is in a less aqueous phase or a non-aqueous phase. When the active substance is a hydrophilic substance, it is assumed that the hydrophilic active substance is encapsulated by the lipid, wherein the hydrophilic part of the lipid faces the hydrophilic active substance. By specifically adding the ligand to the external surface of the delivery medium, all of the ligand is exposed to the aqueous phase such as culture medium or saline and the external surface of the target cell without losing any ligand buried in the delivery medium, so that specific interactions between the ligand and the target cell or, more precisely, the cell surface molecules of the target cell promote target cell-specific incorporation of the complex.

在本申請案中，負載於本發明之遞送媒介中之活性物質可為一或多種所選擇之藥物。在一個實施例中，遞送媒介含有單一藥物組分。在另一實施例中，遞送媒介負載有多種藥物組分。如本文中所使用，「藥物」意謂包含於本文中所描述之遞送媒介內的任何治療性、預防性或診斷性化合物或試劑。在一個實施例中，藥物為可與水混溶之化合物。負載於本發明之遞送媒介中之活性物質可為任何天然存在或以合成方式產生之分子，包括分子質量不超過2,500道爾頓之低分子量化合物、分子質量在遞送媒介之尺寸限制內的生物聚合物，諸如碳水化合物、多肽、蛋白質、核酸或其任何衍生物。In the present application, the active substance carried in the delivery vehicle of the present invention may be one or more selected drugs. In one embodiment, the delivery vehicle contains a single drug component. In another embodiment, the delivery vehicle carries multiple drug components. As used herein, "drug" means any therapeutic, preventive or diagnostic compound or reagent contained in the delivery vehicle described herein. In one embodiment, the drug is a water-miscible compound. The active substance carried in the delivery vehicle of the present invention may be any naturally occurring or synthetically produced molecule, including low molecular weight compounds with a molecular mass of no more than 2,500 Daltons, biopolymers with a molecular mass within the size limit of the delivery vehicle, such as carbohydrates, polypeptides, proteins, nucleic acids or any derivatives thereof.

負載於本發明之遞送媒介中之活性物質可為核酸，其包括聚核苷酸，該聚核苷酸包含去氧核糖核苷酸、核糖核苷酸及/或其在核鹼基、磷酸二酯主鏈及/或糖部分中具有修飾之非天然類似物中之任一者。當活性物質為核酸時，其可編碼蛋白質或抑制蛋白質之表現之反義聚核苷酸。 The active substance carried in the delivery vehicle of the present invention may be a nucleic acid, including a polynucleotide comprising a deoxyribonucleotide, a ribonucleotide and/or any of its non-natural analogs with modifications in the nucleobase, the phosphodiester backbone and/or the sugar moiety. When the active substance is a nucleic acid, it may encode a protein or an antisense polynucleotide that inhibits the expression of a protein.

在本發明之一個實施例中，在負載於遞送媒介中之核酸中編碼的蛋白質可為在經遞送或轉導之細胞中發揮治療或預防作用之任何蛋白質。為了遞送至免疫細胞，較佳細胞毒性細胞，在負載於遞送媒介中之核酸中編碼的蛋白質可為嵌合抗原受體(CAR)及/或T細胞受體(TCR)。由於細胞毒性細胞應經活化以殺傷由CAR及/或TCR識別之細胞，因此活性物質藉由活化靶細胞以攻擊及殺傷病理性變化之細胞(諸如惡性細胞或排出病理性沉積物以引起纖維化之細胞)而充當癌症或任何其他疾病之治療劑。除免疫細胞以外，複合物之靶細胞包括其病理性變化可藉由外源基因或蛋白質之異位表現補救的各種體細胞或生殖系細胞，諸如中樞及周邊神經系統、感覺系統、消化系統、呼吸系統、泌尿系統、心血管系統、生殖系統、骨骼、肌肉、皮膚之細胞及血細胞。各種體細胞及生殖系細胞中之各者的配體可使用諸如CellMarker資料庫之網際網路資源鑑別(Xinxin Zhang等人, Nucleic Acids Research, 2019,第47卷, Database issue D721-D728)。在一個實施例中，可在本發明之複合物中併入超過一種配體。 In one embodiment of the present invention, the protein encoded in the nucleic acid carried in the delivery vector can be any protein that exerts a therapeutic or preventive effect in the delivered or transduced cells. For delivery to immune cells, preferably cytotoxic cells, the protein encoded in the nucleic acid carried in the delivery vector can be a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). Since cytotoxic cells should be activated to kill cells recognized by CAR and/or TCR, the active substance acts as a therapeutic agent for cancer or any other disease by activating target cells to attack and kill pathologically changed cells (such as malignant cells or cells that excrete pathological deposits to cause fibrosis). In addition to immune cells, target cells of the complex include various somatic cells or germ line cells whose pathological changes can be rescued by ectopic expression of exogenous genes or proteins, such as cells of the central and peripheral nervous systems, sensory systems, digestive systems, respiratory systems, urinary systems, cardiovascular systems, reproductive systems, bones, muscles, skin, and blood cells. Ligands for various somatic cells and germline cells can be identified using internet resources such as the CellMarker database (Xinxin Zhang et al., Nucleic Acids Research, 2019, Vol. 47, Database issue D721-D728). In one embodiment, more than one ligand can be incorporated into the complex of the present invention.

負載於本發明之遞送媒介中之活性物質既不限於CAR亦不限於TCR。當活性物質為編碼既非CAR亦非TCR之蛋白質的核酸時，在靶細胞中表現之蛋白質充當用於感染之疫苗或用於蛋白質之病理性損失或減少之補充療法。當活性物質為反義RNA或siRNA或其他抑制互補RNA之轉錄及/或轉譯之核酸時，反義RNA及其他核酸充當抑制劑以減輕互補RNA或其經轉譯之蛋白質之病理性增加。The active substance carried in the delivery vehicle of the present invention is neither limited to CAR nor TCR. When the active substance is a nucleic acid encoding a protein that is neither CAR nor TCR, the protein expressed in the target cell serves as a vaccine for infection or as a supplemental therapy for pathological loss or reduction of the protein. When the active substance is antisense RNA or siRNA or other nucleic acids that inhibit the transcription and/or translation of complementary RNA, the antisense RNA and other nucleic acids serve as inhibitors to reduce the pathological increase of complementary RNA or its translated protein.

當活性物質為包含引導RNA及/或其相關位點特異性核酸酶或編碼核酸酶之核酸的基因體編輯系統時，引導RNA及/或核酸酶充當治療劑以將所關注之基因中之基因體編輯修飾引入靶細胞之基因體中。引導RNA包含與所關注之基因互補之引導序列。核酸酶與靶細胞中之引導RNA締合，與靶細胞基因體中之互補序列處的所關注之基因結合，且使基因體DNA之磷酸二酯鍵斷裂或自基因體DNA中裂解一大塊DNA。視引導RNA之設計而定，與引導RNA締合之核酸酶可誘導基因體DNA之單股斷裂或雙股斷裂。位點特異性核酸酶可選自由以下組成之群：大範圍核酸酶、鋅指核酸酶(ZFN)、CRISPR/Cas9蛋白、CRISPR-Cpf1蛋白及TAL效應核酸酶(TALEN)。When the active substance is a genome editing system comprising a guide RNA and/or its associated site-specific nuclease or a nucleic acid encoding the nuclease, the guide RNA and/or the nuclease acts as a therapeutic agent to introduce genome editing modifications in the gene of interest into the genome of a target cell. The guide RNA comprises a guide sequence that is complementary to the gene of interest. The nuclease binds to the guide RNA in the target cell, binds to the gene of interest at the complementary sequence in the genome of the target cell, and cleaves the phosphodiester bonds of the genome DNA or cleaves a large piece of DNA from the genome DNA. Depending on the design of the guide RNA, the nuclease bound to the guide RNA can induce single-strand breaks or double-strand breaks in the genome DNA. The site-specific nuclease may be selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a CRISPR/Cas9 protein, a CRISPR-Cpf1 protein and a TAL effector nuclease (TALEN).

因此，在本發明之一個實施例中，活性物質為上文關於基因體編輯所描述之引導RNA及/或位點特異性核酸酶。較佳地，活性物質為引導RNA及衍生自CRISPR系統之位點特異性核酸酶。Therefore, in one embodiment of the present invention, the active substance is the guide RNA and/or site-specific nuclease described above for genome editing. Preferably, the active substance is the guide RNA and the site-specific nuclease derived from the CRISPR system.

CRISPR或規律間隔成簇短回文重複序列(Clustered Regularly Interspaced Short Palindromic Repeat)係指含有在大致40%之定序細菌及90%之定序古菌之基因體中發現的多個短的直接重複序列之基因座。CRISPR系統為參與對抗侵入噬菌體及質體之防禦的微生物核酸酶系統，其提供後天性免疫之形式。微生物宿主中之CRISPR基因座含有CRISPR相關(Cas)基因以及能夠程式化CRISPR介導之核酸裂解之特異性的非編碼RNA元件的組合。將外源DNA之短片段(稱為間隔子)併入CRISPR重複序列之間的基因體中，且充當過去暴露之「記憶體」。Cas9與sgRNA (在本文中亦互換地稱為「gRNA」)之3'端形成複合物，且蛋白質-RNA對藉由sgRNA序列之5'端與經預定義之20 bp DNA序列(稱為原型間隔子)之間的互補鹼基配對來識別其基因體標靶。此複合物經由在crRNA內編碼之區域(亦即，在病原體基因體內之原型間隔子及原型間隔子相鄰模體(PAM))導引至病原體DNA之同源基因座。非編碼CRISPR陣列在直接重複序列內轉錄及裂解成含有個別間隔子序列之短crRNA，其將Cas核酸酶導引至標靶位點(原型間隔子)。藉由簡單交換所表現之sgRNA的20 bp識別序列，可將Cas9核酸酶導引至新的基因體標靶。CRISPR間隔子用於以類似於真核生物中之RNAi的方式來識別外源基因元件且使其緘默。CRISPR or Clustered Regularly Interspaced Short Palindromic Repeat refers to a locus containing multiple short direct repeat sequences found in the genome of approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in the defense against invading phages and plastids, providing a form of acquired immunity. The CRISPR locus in a microbial host contains a combination of CRISPR-associated (Cas) genes and non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage. Short fragments of foreign DNA, called spacers, are incorporated into the genome between the CRISPR repeat sequences and serve as a "memory" of past exposures. Cas9 forms a complex with the 3' end of the sgRNA (also interchangeably referred to herein as "gRNA"), and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predefined 20 bp DNA sequence, called a protospacer. This complex is guided to the homologous locus of the pathogen DNA via the region encoded within the crRNA, i.e., the protospacer and protospacer adjacent motif (PAM) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within the direct repeat sequence into short crRNAs containing individual spacer sequences, which guide the Cas nuclease to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed sgRNA, the Cas9 nuclease can be directed to a new genomic target. CRISPR spacers are used to recognize and silence foreign genetic elements in a manner similar to RNAi in eukaryotes.

已知三種類別之CRISPR系統(I型、II型及III型效應子系統)。II型效應子系統在四個依序步驟中使用單一效應酶Cas9進行所靶向之DNA雙股斷裂，以使dsDNA裂解。與需要充當複合物之多個不同效應子的I型及III型效應子系統相比，II型效應子系統可在諸如真核細胞之替代性情形中起作用。II型效應子系統係由長的前crRNA (其自含有間隔子之CRISPR基因座轉錄)、Cas9蛋白及tracrRNA (其參與前crRNA加工)組成。tracrRNA與分離前crRNA之間隔子的重複區域雜交，因此藉由內源性RNA酶III啟始dsRNA裂解。此裂解之後為在各間隔子內藉由Cas9進行之第二裂解事件，產生保持與tracrRNA及Cas9相關之成熟crRNA，形成Cas9：crRNA-tracrRNA複合物。Three classes of CRISPR systems are known (Type I, Type II, and Type III effector systems). Type II effector systems use a single effector enzyme, Cas9, to perform targeted DNA double strand breaks in four sequential steps to cleave dsDNA. Compared to Type I and Type III effector systems, which require multiple different effectors to function as a complex, Type II effector systems can function in alternative contexts such as eukaryotic cells. Type II effector systems consist of a long pre-crRNA (which is transcribed from a spacer-containing CRISPR locus), a Cas9 protein, and a tracrRNA (which participates in pre-crRNA processing). The tracrRNA hybridizes with the repetitive region of the spacer separating the pre-crRNA, thereby initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event by Cas9 within each spacer, generating a mature crRNA that remains associated with tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex.

據證實，化膿性鏈球菌(Streptococcus pyogenes)之II型效應子系統的經工程改造形式在用於基因體工程改造之人類細胞中起作用。在此系統中，Cas9蛋白藉由以合成方式重建之「引導RNA」(「gRNA」，在本文中亦可互換地用作嵌合單引導RNA (「sgRNA」))而導引至基因體標靶位點，該引導RNA為一般不需要RNA酶III及crRNA加工之crRNA-tracrRNA融合物。本文提供DNA靶向系統，其用於基因體編輯及治療遺傳疾病。本發明所揭示之DNA靶向系統可經設計以靶向任何基因，包括參與遺傳疾病、衰老、組織再生或傷口癒合之基因。DNA靶向系統包括編碼Cas9蛋白或Cas9融合蛋白之聚核苷酸及一或多個gRNA。在一些實施例中，編碼Cas9蛋白或Cas9融合蛋白之聚核苷酸為mRNA。mRNA可為經修飾之mRNA。經修飾之mRNA可包括一或多個選自N端NLS、C端NLS、HA標籤及尿苷取代之修飾。Cas9融合蛋白可例如包括具有對於Cas9為內源性之不同活性的域，諸如反式活化域。An engineered version of the type II effector system of Streptococcus pyogenes has been shown to function in human cells for genome engineering. In this system, the Cas9 protein is guided to a genome target site by a synthetically reconstructed "guide RNA" ("gRNA", also interchangeably referred to herein as a chimeric single guide RNA ("sgRNA")), which is a crRNA-tracrRNA fusion that generally does not require RNase III and crRNA processing. Provided herein is a DNA targeting system for genome editing and treatment of genetic diseases. The DNA targeting system disclosed herein can be designed to target any gene, including genes involved in genetic diseases, aging, tissue regeneration, or wound healing. The DNA targeting system includes a polynucleotide encoding a Cas9 protein or a Cas9 fusion protein and one or more gRNAs. In some embodiments, the polynucleotide encoding the Cas9 protein or Cas9 fusion protein is mRNA. The mRNA may be a modified mRNA. The modified mRNA may include one or more modifications selected from N-terminal NLS, C-terminal NLS, HA tag and uridine substitution. The Cas9 fusion protein may, for example, include a domain with a different activity than endogenous to Cas9, such as a transactivation domain.

標靶基因(例如，所關注之任何基因)可參與細胞分化或其中基因活化可能需要之任何其他方法，或可具有突變，諸如讀框轉移突變或無意義突變。若標靶基因具有引起過早終止密碼子、異常剪接受體位點或異常剪接供體位點之突變，則DNA靶向系統可經設計以識別及結合在過早終止密碼子、異常剪接受體位點或異常剪接供體位點之上游或下游的核苷酸序列。DNA靶向系統亦可用於藉由靶向剪接受體及供體來破壞正常之基因剪接，以誘導過早終止密碼子之跳過或恢復經破壞之讀框。DNA靶向系統可或可不介導基因體之蛋白質編碼區之脫靶變化。Cas9分子及Cas9融合蛋白。The target gene (e.g., any gene of interest) may be involved in cell differentiation or any other process in which gene activation may be required, or may have a mutation, such as a frame shift mutation or a nonsense mutation. If the target gene has a mutation that causes a premature stop codon, an abnormal splice acceptor site, or an abnormal splice donor site, the DNA targeting system may be designed to recognize and bind to nucleotide sequences upstream or downstream of the premature stop codon, abnormal splice acceptor site, or abnormal splice donor site. The DNA targeting system may also be used to disrupt normal gene splicing by targeting splice acceptors and donors to induce skipping of premature stop codons or restore a disrupted reading frame. The DNA targeting system may or may not mediate off-target changes in the protein coding region of the genome. Cas9 molecules and Cas9 fusion proteins.

本發明之DNA靶向系統包含編碼Cas9蛋白或Cas9融合蛋白之mRNA。Cas9蛋白為一種核酸內切酶，其使核酸裂解且由CRISPR基因座編碼且涉及II型CRISPR系統。Cas9蛋白可來自任何細菌或古菌物種，包括但不限於：化膿性鏈球菌、金黃色葡萄球菌(S. aureus)、燕麥嗜酸菌(Acidovorax avenae)、胸膜肺炎放線桿菌(Actinobacillus pleuropneumoniae)、產琥珀酸放線桿菌(Actinobacillus succinogenes)、豬放線桿菌(Actinobacillus suis)、放線菌(Actinomyces sp.)、反硝化嗜脂環菌(cycliphilus denitrificans)、少食胺基單胞菌(Aminomonas paucivorans)、蠟樣芽胞桿菌(Bacillus cereus)、史氏芽孢桿菌(Bacillus smithii)、蘇力菌(Bacillus thuringiensis)、擬桿菌(Bacteroides sp.)、海洋芽殖小梨形菌(Blastopirellula marina)、根瘤菌(Bradyrhizobium sp.)、側孢短芽孢桿菌(Brevibacillus latemsporus)、大腸彎曲桿菌(Campylobacter coli)、空腸彎曲桿菌(Campylobacter jejuni)、紅嘴鷗彎曲桿菌(Campylobacter lari)、普尼側螺旋菌候選種(Candidatus Puniceispirillum)、解纖維素梭菌(Clostridiu cellulolyticum)、產氣莢膜芽胞梭菌(Clostridium perfringens)、擁擠棒狀桿菌(Corynebacterium accolens)、白喉棒狀桿菌(Corynebacterium diphtheria)、馬氏棒狀桿菌(Corynebacterium matruchotii)、絲貝玫瑰桿菌(Dinoroseobacter shibae)、細長真桿菌(Eubacterium dolichum)、γ變形桿菌(gamma proteobacterium)、重氮營養葡糖酸醋桿菌(Gluconacetobacler diazotrophicus)、副流感嗜血桿菌(Haemophilus parainfluenzae)、生痰嗜血桿菌(Haemophilus sputorum)、加拿大螺旋桿菌(Helicobacter canadensis)、同性戀螺旋桿菌(Helicobacter cinaedi)、鼬鼠螺旋桿菌(Helicobacter mustelae)、營養泥桿菌(Ilyobacler polytropus)、金氏金氏菌(Kingella kingae)、捲曲乳桿菌(Lactobacillus crispatus)、伊氏李斯特氏菌(Listeria ivanovii)、產單核球李斯特氏菌(Listeria monocytogenes)、李斯特氏菌(Listeriaceae bacterium)、甲基孢囊菌屬(Methylocystis sp.)、甲烷氧化菌(Methylosinus trichosporium)、羞怯動彎桿菌(Mobiluncus mulieris)、桿狀奈瑟氏菌(Neisseria bacilliformis)、灰色奈瑟球菌(Neisseria cinerea)、淺黃奈瑟氏菌(Neisseria flavescens)、乳糖奈瑟氏菌(Neisseria lactamica)、奈瑟氏菌(Neisseria sp.)、瓦茨瓦爾西奈瑟氏菌(Neisseria wadsworthii)、亞硝化單胞菌(Nitrosomonas sp.)、食清潔劑細小棒菌(Parvibaculum lavamentivorans)、多殺性巴氏桿菌(Pasteurella multocida)、琥珀酸考拉桿菌(Phascolarctobacterium succinatutens)、丁香羅爾斯頓菌(Ralstonia syzygii)、沼澤紅假單胞菌(Rhodopseudomonas palustris)、小紅卵菌(Rhodovulum sp.)、米氏西蒙斯氏菌(Simonsiella muelleri)、鞘脂單胞菌(Sphingomonas sp.)、威尼亞芽孢乳桿菌(Sporolactobacillus vineae)、路鄧葡萄球菌(Staphylococcus lugdunensis)、鏈球菌(Streptococcus sp.)、罕見小球菌(Subdoligranulum sp.)、運動替斯崔納菌(Tislrella mobilis)、密螺旋體菌(Treponema sp.)或艾森氏蠕形桿菌(Verminephrobacter eiseniae)。在某些實施例中，Cas9分子為化膿性鏈球菌Cas9分子(在本文中亦稱為「SpCas9」)。在某些實施例中，Cas9分子為金黃色葡萄球菌Cas9分子(在本文中亦稱為「SaCas9」)。在一些實施例中，Cas9分子為突變型Cas9分子。Cas9蛋白可經突變以使得核酸酶活性不活化。在一些實施例中，Cas9分子為不具有核酸內切酶活性之去活化或不活化之Cas9蛋白(dCas9或iCas9)。關於使核酸酶活性不活化之化膿性鏈球菌Cas9序列的例示性突變包括：D10A、E762A、H840A、N854A、N863A及/或D986A。關於使核酸酶活性不活化之金黃色葡萄球菌Cas9序列的例示性突變包括D10A及N580A。The DNA targeting system of the present invention comprises mRNA encoding Cas9 protein or Cas9 fusion protein. Cas9 protein is an endonuclease that cleaves nucleic acids and is encoded by the CRISPR locus and is involved in the type II CRISPR system. Cas9 protein can be derived from any bacterial or archaeal species, including but not limited to: Streptococcus pyogenes, Staphylococcus aureus, Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, thuringiensis), Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridiu cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria), Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacler diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacler polytropus, Kingella kingae), Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans), Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tislrella mobilis, Treponema sp.) or Verminephrobacter eiseniae. In some embodiments, the Cas9 molecule is a Streptococcus purulentis Cas9 molecule (also referred to herein as "SpCas9"). In some embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule (also referred to herein as "SaCas9"). In some embodiments, the Cas9 molecule is a mutant Cas9 molecule. The Cas9 protein can be mutated to inactivate the nuclease activity. In some embodiments, the Cas9 molecule is a deactivated or inactivated Cas9 protein (dCas9 or iCas9) that does not have endonuclease activity. Exemplary mutations of the Streptococcus purulentis Cas9 sequence that inactivate the nuclease activity include: D10A, E762A, H840A, N854A, N863A and/or D986A. Exemplary mutations of the S. aureus Cas9 sequence that inactivate nuclease activity include D10A and N580A.

編碼Cas9分子之mRNA可為合成核酸序列。舉例而言，合成核酸分子可經化學修飾。合成核酸序列可經密碼子最佳化，例如至少一種非常見密碼子或較常見密碼子已經常見密碼子置換。舉例而言，合成核酸可導引經最佳化(例如，經最佳化以在例如本文中所描述之哺乳動物表現系統中表現)之信使mRNA之合成。在本發明之各種實施例中，在投與至個體後，與Cas9具有交叉反應性之體液反應有限或無體液反應。The mRNA encoding the Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. The synthetic nucleic acid sequence can be codon-optimized, such as by replacing at least one uncommon codon or a more common codon with a common codon. For example, the synthetic nucleic acid can direct the synthesis of a messenger mRNA that is optimized (e.g., optimized for expression in a mammalian expression system such as described herein). In various embodiments of the invention, upon administration to a subject, there is limited or no humoral response to cross-reactivity with Cas9.

本文可互換地使用的「重複可變二殘基」或「RVD」係指TALE DNA結合域之DNA識別模體(亦稱為「RVD模組」)內的一對相鄰胺基酸殘基，其包括33至35個胺基酸。RVD確定RVD模組之核苷酸特異性。RVD模組可經組合以產生RVD陣列。如本文中所使用，「RVD陣列長度」係指對應於TALEN標靶區域內由TALEN (亦即，結合區)識別之核苷酸序列之長度的RVD模組之數目。 1. (a-1) 脂質奈米粒子 "Repeating variable di-residues" or "RVDs" used interchangeably herein refer to a pair of adjacent amino acid residues within the DNA recognition motif (also referred to as an "RVD module") of the TALE DNA binding domain, which includes 33 to 35 amino acids. The RVD determines the nucleotide specificity of the RVD module. RVD modules can be combined to produce RVD arrays. As used herein, "RVD array length" refers to the number of RVD modules corresponding to the length of the nucleotide sequence recognized by the TALEN (i.e., the binding region) within the TALEN target region. 1. (a-1) Lipid Nanoparticles

在一個實施例中，用於活性物質之遞送媒介為脂質奈米粒子。在本說明書中，「脂質奈米粒子(LNP)」意謂在由陽離子型脂質及非陽離子型脂質構成的分子總裝體中平均直徑小於1 μm且不含小孔結構(例如中孔物質)之粒子。 1. (a-1.1) 陽離子型脂質 In one embodiment, the delivery vehicle for the active substance is a lipid nanoparticle. In this specification, "lipid nanoparticle (LNP)" means a particle with an average diameter of less than 1 μm and no pore structure (such as mesoporous material) in a molecular assembly composed of cationic lipids and non-cationic lipids. 1. (a-1.1) Cationic lipids

在本說明書中，「陽離子型脂質」意謂在低pH環境中，諸如在生理pH下具有淨正電荷之脂質。用於本發明中之脂質奈米粒子中的陽離子型脂質不受特定限制。舉例而言，可提及WO 2016/021683、WO 2015/011633、WO 2011/153493、WO 2013/126803、WO 2010/054401、WO 2010/042877、WO 2016/104580、WO 2015/005253、WO 2014/007398、WO 2017/117528、WO 2017/075531、WO 2017/00414、WO 2015/199952、US 2015/0239834、WO2019/131839及其類似者中所描述之陽離子型脂質及其類似物。In this specification, "cationic lipid" means a lipid having a net positive charge in a low pH environment, such as at physiological pH. The cationic lipid used in the lipid nanoparticles of the present invention is not particularly limited. By way of example, cationic lipids and the like described in WO 2016/021683, WO 2015/011633, WO 2011/153493, WO 2013/126803, WO 2010/054401, WO 2010/042877, WO 2016/104580, WO 2015/005253, WO 2014/007398, WO 2017/117528, WO 2017/075531, WO 2017/00414, WO 2015/199952, US 2015/0239834, WO2019/131839 and the like may be mentioned.

或者，可提及Dong等人(Proc Natl Acad Sci U S A. 2014年4月15日; 111(15):5753)中所描述之合成陽離子型脂質(例如，K-E12、H-A12、Y-E12、G-O12、K-A12、R-A12、cKK-E12、cPK-E12、PK1K-E12、PK500-E12、cQK-E12、cKK-A12、KK-A12、PK-4K-E12、cWK-E12、PK500-O12、PK1K-O12、cYK-E12、cDK-E12、cSK-E12、cEK-E12、cMK-E12、cKK-O12、cIK-E12、cKK-E10、cKK-E14及cKK-E16，較佳為cKK-E12、cKK-E14)，及Love KT等人(Proc Natl Acad Sci U S A. 2010年5月25日; 107(21):9915)中所描述之合成陽離子型脂質(例如，C14-98、C18-96、C14-113、C14-120、C14-120、C14-110、C16-96及C12-200，較佳為014-110、C16-96及C12-200)。Alternatively, the synthetic cationic lipids described in Dong et al. (Proc Natl Acad Sci U S A. 2014 Apr 15; 111(15):5753) (e.g., K-E12, H-A12, Y-E12, G-O12, K-A12, R-A12, cKK-E12, cPK-E12, PK1K-E12, PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K-E12) may be mentioned. , cWK-E12, PK500-O12, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-O12, cIK-E12, cKK-E10, cKK-E14 and cKK-E16, preferably cKK-E12 and cKK-E14), and Love Synthetic cationic lipids (e.g., C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96, and C12-200, preferably C14-110, C16-96, and C12-200) described in KT et al. (Proc Natl Acad Sci U S A. 2010 May 25; 107(21):9915).

在一個較佳實施例中，可提及由以下通式表示且描述於WO 2016/021683中之陽離子型脂質。In a preferred embodiment, the cationic lipid represented by the following general formula and described in WO 2016/021683 can be mentioned.

其中 W為式-NR1R2或式-N+R3R4R5(Z-)， R1及R2各自獨立地為C1-4烷基或氫原子， R3、R4及R5各自獨立地為C1-4烷基， Z-為陰離子， X為視情況經取代之C1-6伸烷基， YA、YB及YC各自獨立地為視情況經取代之次甲基， LA、LB及LC各自獨立地為視情況經取代之亞甲基或鍵，及 RA1、RA2、RB1、RB2、RC1及RC2各自獨立地為視情況經取代之C4-10烷基， 或其鹽。 Wherein W is a formula -NR1R2 or a formula -N+R3R4R5(Z-), R1 and R2 are each independently a C1-4 alkyl group or a hydrogen atom, R3, R4 and R5 are each independently a C1-4 alkyl group, Z- is an anion, X is a C1-6 alkylene group which may be substituted, YA, YB and YC are each independently a methylene group which may be substituted, LA, LB and LC are each independently a methylene group or a bond which may be substituted, and RA1, RA2, RB1, RB2, RC1 and RC2 are each independently a C4-10 alkyl group which may be substituted, or a salt thereof.

陽離子型脂質之更多細節闡釋於WO2019/131770中，其內容以全文引用之方式併入本文中。Further details of cationic lipids are explained in WO2019/131770, the contents of which are incorporated herein by reference in their entirety.

本發明之脂質奈米粒子中存在的陽離子型脂質與總脂質之比率(莫耳%)為例如約10%至約80%、較佳約20%至約70%、更佳約40%至約60%；然而，該比率不限於此等。 亦可僅使用一種上文所提及之陽離子型脂質，或可組合使用其兩種或更多種類別。當使用多種陽離子型脂質時，整個陽離子型脂質之比率較佳如上文所提及。 1. (a-1.2)非陽離子型脂質 The ratio of cationic lipids to total lipids in the lipid nanoparticles of the present invention (molar %) is, for example, about 10% to about 80%, preferably about 20% to about 70%, and more preferably about 40% to about 60%; however, the ratio is not limited thereto. Only one of the cationic lipids mentioned above may be used, or two or more types thereof may be used in combination. When multiple cationic lipids are used, the ratio of the entire cationic lipid is preferably as mentioned above. 1. (a-1.2) Non-cationic lipids

在本發明書中，「非陽離子型脂質」意謂除陽離子型脂質以外之脂質，且係在所選擇之pH (諸如生理pH及其類似pH)下不具有淨正電荷之脂質。用於本發明之脂質奈米粒子中之非陽離子型脂質的實例包括磷脂、類固醇、PEG脂質及其類似物。In the present invention, "non-cationic lipids" means lipids other than cationic lipids, and are lipids that do not have a net positive charge at a selected pH (such as physiological pH and the like). Examples of non-cationic lipids used in the lipid nanoparticles of the present invention include phospholipids, steroids, PEG lipids and the like.

當活性物質為核酸，諸如編碼CAR或TCR之聚核苷酸時，磷脂不受特定限制，只要其穩定地維持核酸且不抑制與細胞膜(質膜及胞器膜)之融合即可。舉例而言，可提及磷脂醯膽鹼、磷脂醯乙醇胺、磷脂醯絲胺酸、磷脂醯肌醇、磷脂酸、棕櫚醯油醯磷脂醯膽鹼、溶血磷脂醯膽鹼、溶血磷脂醯乙醇胺、二棕櫚醯磷脂醯膽鹼、二油醯磷脂醯膽鹼、二硬脂醯磷脂醯膽鹼、二亞油醯磷脂醯膽鹼及其類似物。When the active substance is a nucleic acid, such as a polynucleotide encoding a CAR or TCR, the phospholipid is not particularly limited as long as it stably maintains the nucleic acid and does not inhibit fusion with the cell membrane (plasma membrane and organelle membrane). For example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, disalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearylphosphatidylcholine, dilinoleylphosphatidylcholine and the like can be mentioned.

較佳之磷脂包括二硬脂醯磷脂醯膽鹼(DSPC)、二油醯磷脂醯膽鹼(DOPC)、二棕櫚醯磷脂醯膽鹼(DPPC)、二油醯磷脂醯甘油(DOPG)、棕櫚醯油醯磷脂醯甘油(POPG)、二棕櫚醯磷脂醯甘油(DPPG)、二油醯磷脂醯乙醇胺(DOPE)、棕櫚醯油醯磷脂醯膽鹼(POPC)、軟脂醯油醯磷脂醯乙醇胺(POPE)及二油醯磷脂醯乙醇胺4-(N-順丁烯二醯亞胺甲基)-環己烷-1-甲酸酯(DOPE-mal)，更佳為DOPC、DPPC、POPC及DOPE。Preferred phospholipids include distearyl phospholipid acyl choline (DSPC), dioleoyl phospholipid acyl choline (DOPC), dimalmitoyl phospholipid acyl choline (DPPC), dioleoyl phospholipid acyl glycerol (DOPG), palmitoyl phospholipid acyl glycerol (POPG), dimalmitoyl phospholipid acyl glycerol (DPPG), dioleoyl phospholipid acyl ethanol Preferably, the present invention is used as the alkyl ester of DOPE, DPPC, POPC and DOPE.

本發明之脂質奈米粒子中存在的磷脂與總脂質之比率(莫耳%)可為例如約0%至約90%、較佳約5%至約30%、更佳約8%至約15%。 可僅使用一種上文所提及之磷脂，或可組合使用其兩種或更多種類別。當使用多種磷脂時，整個磷脂之比率較佳如上文所提及。 The ratio (molar %) of phospholipids to total lipids present in the lipid nanoparticles of the present invention may be, for example, about 0% to about 90%, preferably about 5% to about 30%, and more preferably about 8% to about 15%. Only one of the phospholipids mentioned above may be used, or two or more types thereof may be used in combination. When multiple phospholipids are used, the ratio of the entire phospholipids is preferably as mentioned above.

作為類固醇，可提及膽固醇、5α-膽甾烷醇、5β-糞甾烷醇、膽固醇基-(2'-羥基)-乙基醚、膽固醇基-(4'-羥基)-丁基醚、6-酮膽甾烷醇、5α-膽甾烷、膽甾烯酮、5α-膽甾烷酮、5β-膽甾烷酮及癸酸膽固醇酯，較佳為膽固醇。As the steroid, there may be mentioned cholesterol, 5α-cholestanol, 5β-naphthalene-stanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone and cholesterol decanoate, preferably cholesterol.

當存在類固醇時，本發明之脂質奈米粒子中存在的類固醇與總脂質之比率(莫耳%)可為例如約10%至約60%、較佳約12%至約58%、更佳約20%至約55%。 可僅使用一種上文所提及之類固醇，或可組合使用其兩種或更多種類別。當使用多種類固醇時，整個類固醇之比率較佳如上文所提及。 When a steroid is present, the ratio (molar %) of the steroid present in the lipid nanoparticles of the present invention to the total lipid may be, for example, about 10% to about 60%, preferably about 12% to about 58%, and more preferably about 20% to about 55%. Only one of the steroids mentioned above may be used, or two or more types thereof may be used in combination. When multiple steroids are used, the ratio of the entire steroid is preferably as mentioned above.

在本說明書中，「PEG脂質」意謂聚乙二醇(PEG)及脂質之任何複合物。PEG脂質不受特定限制，只要其具有抑制本發明之脂質奈米粒子聚集之作用即可。舉例而言，與二烷基氧基丙基接合之PEG (PEG-DAA)、與二醯甘油接合之PEG (PEG-DAG) (例如，SUNBRIGHT GM-020或GS-020 (NOF CORPORATION))、與諸如磷脂醯乙醇胺之磷脂接合之PEG (PEG-PE)、與神經醯胺接合之PEG (PEG-Cer)、與以下接合之PEG：膽固醇(PEG-膽固醇)、或其衍生物、或其混合物、mPEG2000-1,2-二-O-烷基-sn3-碳醯甘油酯(PEG-C-DOMG)、1-[8'-(1,2-二肉豆蔻醯基-3-丙氧基)-甲醯胺-3',6-二氧雜辛基]胺基甲醯基-ω-甲基-聚(乙二醇) (2KPEG-DMG)及其類似物。較佳之PEG脂質包括PEG-DGA、PEG-DAA、PEG-PE、PEG-Cer及其混合物，更佳包括選自由以下組成之群的PEG-DAA接合物：PEG-二癸基氧基丙基接合物、PEG-二月桂基氧基丙基接合物、PEG-二肉豆蔻基氧基丙基接合物、PEG-二棕櫚基氧基丙基接合物、PEG-二硬脂醯基氧基丙基接合物及其混合物。In this specification, "PEG lipid" means any complex of polyethylene glycol (PEG) and lipid. The PEG lipid is not particularly limited as long as it has the effect of inhibiting the aggregation of the lipid nanoparticles of the present invention. For example, PEG conjugated to a dialkyloxypropyl group (PEG-DAA), PEG conjugated to a diacylglycerol (PEG-DAG) (e.g., SUNBRIGHT GM-020 or GS-020 (NOF CORPORATION)), PEG conjugated to a phospholipid such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide (PEG-Cer), PEG conjugated to cholesterol (PEG-cholesterol), or a derivative thereof, or a mixture thereof, mPEG2000-1,2-di-O-alkyl-sn3-carbonylglyceride (PEG-C-DOMG), 1-[8'-(1,2-dimyristoyl-3-propoxy)-formamide-3',6-dioxaoctyl]aminoformyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG), and the like. Preferred PEG lipids include PEG-DGA, PEG-DAA, PEG-PE, PEG-Cer and mixtures thereof, and more preferably include PEG-DAA conjugates selected from the group consisting of PEG-didecyloxypropyl conjugates, PEG-dilauryloxypropyl conjugates, PEG-dimyristyloxypropyl conjugates, PEG-dipalmityloxypropyl conjugates, PEG-distearyloxypropyl conjugates and mixtures thereof.

除甲氧基以外，用於結合稍後描述之T細胞靶向配體的順丁烯二醯亞胺基、N-羥基丁二醯亞胺基及其類似基團可用作PEG之自由端。舉例而言，SUNBRIGHT DSPE-0201MA或SUNBRIGHT DSPE-0201MA (NOF)可用作具有用於結合T細胞靶向配體之官能基的PEG脂質(在本說明書中有時稱為「末端反應性PEG脂質」)。In addition to the methoxy group, a cis-butylenediimide group, an N-hydroxysuccinimidyl group, and the like for binding to a T cell targeting ligand described later can be used as the free end of PEG. For example, SUNBRIGHT DSPE-0201MA or SUNBRIGHT DSPE-0201MA (NOF) can be used as a PEG lipid having a functional group for binding to a T cell targeting ligand (sometimes referred to as a "terminal reactive PEG lipid" in this specification).

在一個實施例中，具有與第二結合搭配物(例如疊氮化物)共價鍵結之錨分子(例如PEG脂質)的遞送媒介如下製備。將脂質之混合物(陽離子型脂質:DPPC:膽固醇:SUNBRIGHT GS-020:DSPE-PEG (2000)-疊氮化物= 60:10.6:27:1.4:1，莫耳%)溶解於90% EtOH/10%乙酸鹽緩衝液(25 mM，pH 4.0)中以獲得14 mg/mL之脂質溶液。作為陽離子型脂質，使用以下化合物：WO2016/021683中所描述之3-戊基辛酸3-((4-(二甲基胺基)丁醯基)氧基)-2,2-雙(((3-戊基辛醯基)氧基)甲基)丙酯(用於靶向T細胞之LNP)；或WO2020/032184中所描述之2-(((4,5-二丁基壬醯基)氧基)甲基)-2-(((5-(二甲基胺基)戊醯基)氧基)甲基)丙烷-1,3-二基二癸酸酯(用於靶向NK細胞之LNP)。In one embodiment, a delivery vehicle having an anchor molecule (e.g., PEG lipid) covalently bonded to a second binding partner (e.g., an azide) is prepared as follows: A mixture of lipids (cationic lipid: DPPC: cholesterol: SUNBRIGHT GS-020: DSPE-PEG (2000)-azide = 60:10.6:27:1.4:1, mol%) is dissolved in 90% EtOH/10% acetate buffer (25 mM, pH 4.0) to obtain a 14 mg/mL lipid solution. As cationic lipids, the following compounds were used: 3-pentyloctanoic acid 3-((4-(dimethylamino)butyryl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl ester described in WO2016/021683 (for LNPs targeting T cells); or 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyldidecanoate described in WO2020/032184 (for LNPs targeting NK cells).

本發明之脂質奈米粒子中存在的PEG脂質與總脂質之比率(莫耳%)可為例如約0%至約20%、較佳約0.1%至約5%、更佳約0.7%至約2%。 上文所提及之總PEG脂質中之末端反應性PEG脂質的比率(莫耳%)為例如約10%至約100%、較佳約20%至約100%、更佳約30%至約100%。 可僅使用一種上文所提及之PEG脂質，或可組合使用其兩種或更多種類別。當使用多種PEG脂質時，整個PEG脂質之比率較佳如上文所提及。 1. (a-2)脂質體 The ratio (molar %) of the PEG lipid to the total lipid present in the lipid nanoparticles of the present invention may be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, and more preferably about 0.7% to about 2%. The ratio (molar %) of the terminal reactive PEG lipid in the total PEG lipid mentioned above is, for example, about 10% to about 100%, preferably about 20% to about 100%, and more preferably about 30% to about 100%. Only one of the PEG lipids mentioned above may be used, or two or more types thereof may be used in combination. When multiple PEG lipids are used, the ratio of the entire PEG lipid is preferably as mentioned above. 1. (a-2) Liposomes

脂質體被稱為由包覆內部孔隙結構或水性隔室之外部脂質雙層形成的微觀球形粒子。作為活性物質之遞送媒介，廣泛使用藉由混合經開發為轉染試劑之各種陽離子型脂質(例如，DOTMA、DOTAP、DDAB、DMRIE等)及促進自胞內體釋放之膜融合中性脂質(例如，DOPE、膽固醇等)而製備之脂質體。亦可使用其中將諸如PEG、pH反應性膜融合肽、膜滲透促進肽及其類似物之功能分子添加至脂質體之表面的脂質體。Liposomes are referred to as microscopic spherical particles formed by an outer lipid bilayer covering an inner pore structure or aqueous compartment. As a delivery vehicle for active substances, liposomes prepared by mixing various cationic lipids developed as transfection reagents (e.g., DOTMA, DOTAP, DDAB, DMRIE, etc.) and membrane fusogenic neutral lipids (e.g., DOPE, cholesterol, etc.) that promote release from endosomes are widely used. Liposomes in which functional molecules such as PEG, pH-responsive membrane fusogenic peptides, membrane permeability-promoting peptides, and the like are added to the surface of the liposomes can also be used.

由於尺寸以及疏水性及親水性特徵，脂質體為用於藥物遞送之有前景的系統。脂質體特性因脂質組成、表面電荷、尺寸及製備方法而顯著不同。此外，雙層組分之選擇確定雙層之『剛性』或『流動性』及電荷。舉例而言，來自天然來源之不飽和磷脂醯膽鹼物種(蛋或大豆磷脂醯膽鹼)產生滲透性高得多且不太穩定之雙層，而具有長醯基鏈之飽和磷脂(例如，二棕櫚醯磷脂醯膽鹼)形成剛性、相當不可滲透之雙層結構。本文中適用之脂質體可使用此項技術中已知之技術製備。參見Akbarzadeh等人, Nanoscale Res Lett. Feb 2013; 8(1): 102，其以引用之方式併入本文中。在一個實施例中，脂質體包含膽固醇。已觀測到，脂質體組合物中之膽固醇的量可影響脂質體之遞送。因此，膽固醇之量可變化。在一個實施例中，脂質體中之膽固醇的量按脂質膜組合物計為約10%至50%。在一個實施例中，脂質體之膽固醇含量為約25% (膽固醇莫耳數/脂質之總莫耳數)。在一個實施例中，脂質體之膽固醇含量為約40% (膽固醇莫耳數/脂質之總莫耳數)。在一個實施例中，脂質體之膽固醇含量為至少25% (膽固醇莫耳數/脂質之總莫耳數)。在一個實施例中，脂質體之膽固醇含量為至少40% (膽固醇莫耳數/脂質之總莫耳數)。Liposomes are promising systems for drug delivery due to their size and hydrophobic and hydrophilic properties. Liposome properties vary significantly depending on lipid composition, surface charge, size, and method of preparation. In addition, the choice of bilayer components determines the "rigidity" or "fluidity" and charge of the bilayer. For example, unsaturated phospholipid acylcholine species from natural sources (egg or soybean phospholipid acylcholine) produce much more permeable and less stable bilayers, while saturated phospholipids with long acyl chains (e.g., dipalmitoyl phospholipid acylcholine) form rigid, fairly impermeable bilayer structures. Liposomes useful herein can be prepared using techniques known in the art. See Akbarzadeh et al., Nanoscale Res Lett. Feb 2013; 8(1): 102, which is incorporated herein by reference. In one embodiment, the liposomes comprise cholesterol. It has been observed that the amount of cholesterol in the liposome composition can affect the delivery of the liposomes. Therefore, the amount of cholesterol can vary. In one embodiment, the amount of cholesterol in the liposomes is about 10% to 50% of the lipid membrane composition. In one embodiment, the cholesterol content of the liposomes is about 25% (mole number of cholesterol/total mole number of lipids). In one embodiment, the cholesterol content of the liposomes is about 40% (mole number of cholesterol/total mole number of lipids). In one embodiment, the cholesterol content of the liposomes is at least 25% (mole number of cholesterol/total mole number of lipids). In one embodiment, the cholesterol content of the liposomes is at least 40% (mole number of cholesterol/total mole number of lipids).

本發明之活性物質之例示性遞送媒介為脂質奈米粒子。An exemplary delivery vehicle for the active substances of the present invention is lipid nanoparticles.

1. (b) 對靶細胞具有特異性之配體能夠使本發明之複合物靶向靶細胞之配體不受特定限制，只要其可特異性地識別在靶細胞中特異性或高度表現之表面分子即可。在實施例中，配體包含蛋白質或肽。在免疫細胞作為靶細胞之實施例中，其包括含有針對CD3、CD4、CD7、CD8、CD16、CD28或CD56之抗體之一或多個抗原結合域的配體，且更佳地，其包括含有抗CD3抗體、抗CD16抗體、抗CD28抗體及/或抗CD56抗體之抗原結合域的配體。用於活體內遞送至細胞毒性T細胞之尤其較佳實例為僅含有抗CD3抗體之抗原結合域的配體。用於活體內遞送至NK細胞之尤其較佳實例為僅含有抗CD56抗體之抗原結合域的配體或僅含有抗CD16抗體之抗原結合域的配體。此處，「抗原結合域」與構成上文所提及之CAR之抗原結合域同義。然而，由於CAR需要製備為編碼其之核酸，因此存在限制且在許多情況下一般使用單鏈抗體。因為本發明之複合物中含有呈蛋白質形式的作為T細胞靶向配體之抗原結合域，因此不僅可較佳地使用單鏈抗體，且亦可使用任何其他抗體片段，諸如完整抗體分子、Fab、F(ab')2、Fab'、Fv、還原抗體(rIgG)、dsFv、scFv、雙功能抗體、三功能抗體、HCAb、VHH及其類似者。較佳使用不具有Fc部分之Fab或Fab'。Fab或Fab'尤其較佳用於活體內遞送至標靶免疫細胞。對於本發明之複合物，靶細胞之配體需要使用轉肽酶與包含親偶極物(諸如DBCO)之分子接合。因此，靶細胞之配體為諸如IgG或其抗原結合域之免疫球蛋白，其藉由重組DNA技術產生為配體融合蛋白，以便併入對應於如上文中所提及之分選酶識別模體的寡肽。 1. (b) Ligands specific for target cells The ligands that enable the complex of the present invention to target cells are not particularly limited, as long as they can specifically recognize surface molecules that are specifically or highly expressed in target cells. In embodiments, the ligands include proteins or peptides. In embodiments where immune cells are target cells, they include ligands that contain one or more antigen binding domains of antibodies against CD3, CD4, CD7, CD8, CD16, CD28 or CD56, and more preferably, they include ligands that contain antigen binding domains of anti-CD3 antibodies, anti-CD16 antibodies, anti-CD28 antibodies and/or anti-CD56 antibodies. A particularly preferred example for in vivo delivery to cytotoxic T cells is a ligand containing only the antigen binding domain of an anti-CD3 antibody. A particularly preferred example for in vivo delivery to NK cells is a ligand containing only the antigen binding domain of an anti-CD56 antibody or a ligand containing only the antigen binding domain of an anti-CD16 antibody. Here, "antigen binding domain" is synonymous with the antigen binding domain constituting the CAR mentioned above. However, since CAR needs to be prepared as a nucleic acid encoding it, there are limitations and single-chain antibodies are generally used in many cases. Because the complex of the present invention contains an antigen-binding domain in the form of a protein that serves as a T cell targeting ligand, not only single-chain antibodies can be preferably used, but any other antibody fragments, such as complete antibody molecules, Fab, F(ab')2, Fab', Fv, reduced antibodies (rIgG), dsFv, scFv, bifunctional antibodies, trifunctional antibodies, HCAbs, VHHs and the like can also be used. It is preferred to use Fab or Fab' without an Fc portion. Fab or Fab' is particularly preferred for in vivo delivery to target immune cells. For the complex of the present invention, the ligand of the target cell needs to be conjugated to a molecule containing a polar dipeptide (such as DBCO) using a transpeptidase. Thus, the ligand of the target cell is an immunoglobulin such as IgG or its antigen binding domain, which is produced by recombinant DNA technology as a ligand fusion protein in order to incorporate an oligopeptide corresponding to the sortase recognition motif as mentioned above.

當免疫細胞靶向配體為完整抗體分子時，可使用可商購之抗CD3、CD4、CD7、CD8、CD16、CD28或CD56抗體等，或可自產生抗體之細胞的培養物中分離配體。另一方面，當配體為前述抗原結合域(抗體片段)中之任一者時，以與獲得編碼抗原結合域(構成該CAR)之核酸相同的方式分離編碼抗原結合域(諸如抗CD3、CD4、CD7、CD8、CD16、CD28或CD56抗體等)之核酸，且可使用其以重組方式產生抗原結合域。When the immune cell targeting ligand is a complete antibody molecule, commercially available anti-CD3, CD4, CD7, CD8, CD16, CD28 or CD56 antibodies, etc., can be used, or the ligand can be isolated from a culture of cells producing the antibody. On the other hand, when the ligand is any of the aforementioned antigen-binding domains (antibody fragments), nucleic acids encoding antigen-binding domains (such as anti-CD3, CD4, CD7, CD8, CD16, CD28 or CD56 antibodies, etc.) are isolated in the same manner as nucleic acids encoding antigen-binding domains (constituting the CAR), and can be used to recombinantly produce antigen-binding domains.

在本發明之複合物中，靶向免疫細胞之配體藉由以下方式與遞送媒介之外表面結合：首先在遞送媒介中製備與配體共價鍵結之第一結合搭配物及與錨分子共價鍵結之第二結合搭配物；且接著第一結合搭配物與第二結合搭配物共價鍵結。In the complex of the present invention, the ligand targeting immune cells is bound to the outer surface of the delivery medium by first preparing a first binding partner covalently bound to the ligand and a second binding partner covalently bound to the anchor molecule in the delivery medium; and then the first binding partner is covalently bound to the second binding partner.

在一些實施例中，使用包含親偶極物及1,3-偶極子之分子之間的點擊化學反應產生第一結合搭配物與第二結合搭配物之間的共價鍵。因為化學反應不涉及銅作為催化劑，所以確保了用於遞送活性物質之複合物的安全性。在本發明中，包含親偶極物之分子經選擇以與配體共價鍵結且1,3-偶極子經選擇以與錨分子共價鍵結。錨分子與其他脂質分子聚集在一起以構成遞送媒介。吾人發現，此組合較佳為與配體共價鍵結之1,3-偶極子(疊氮化物)及與錨分子共價鍵結之包含親偶極物(二苯并環辛炔(DBCO)基)之分子的反向組合，因為具有反向組合之複合物呈現相對較高之多分散性指數，該多分散性指數經測定不足以用於醫藥調配物。雖然不希望受理論束縛，但相對較高之多分散性指數很可能歸因於具有錨分子及其他脂質分子之聚集體之遞送媒介與龐大疏水性DBCO基團之間的疏水性相互作用。In some embodiments, a click chemistry reaction between molecules comprising an exopolephile and a 1,3-exopole is used to generate a covalent bond between the first binding partner and the second binding partner. Because the chemical reaction does not involve copper as a catalyst, the safety of the complex for the delivery of active substances is ensured. In the present invention, the molecule comprising an exopolephile is selected to covalently bond with the ligand and the 1,3-exopole is selected to covalently bond with the anchor molecule. The anchor molecule is aggregated with other lipid molecules to form a delivery vehicle. We found that this combination is preferably the reverse combination of the 1,3-dipole (azide) covalently bonded to the ligand and the molecule containing the dipolephile (dibenzocyclooctyne (DBCO) group) covalently bonded to the anchor molecule, because the complex with the reverse combination exhibited a relatively high polydispersity index, which was determined to be insufficient for use in pharmaceutical formulations. Although not wishing to be bound by theory, the relatively high polydispersity index is likely due to the hydrophobic interaction between the delivery vehicle with the aggregate of the anchor molecule and other lipid molecules and the bulky hydrophobic DBCO group.

第一結合搭配物與配體之共價鍵結係以1至4個第一結合搭配物分子/一個配體分子之比率進行；或第一結合搭配物與配體之共價鍵結係藉由分選酶識別模體進行；或第一結合搭配物與配體之羧基端(C端)共價鍵結。基於第一結合搭配物及配體適當地選擇每一個配體分子之第一結合搭配物分子之比率。比率可為1、2、3或4，較佳為1或2，更佳為1。當配體為Fab時，比率較佳為1。當配體為完整抗體時，比率較佳為2或4。The covalent bonding of the first binding partner to the ligand is carried out at a ratio of 1 to 4 first binding partner molecules per one ligand molecule; or the covalent bonding of the first binding partner to the ligand is carried out via a sortase recognition motif; or the first binding partner is covalently bonded to the carboxyl terminus (C-terminus) of the ligand. The ratio of the first binding partner molecules per ligand molecule is appropriately selected based on the first binding partner and the ligand. The ratio may be 1, 2, 3 or 4, preferably 1 or 2, more preferably 1. When the ligand is a Fab, the ratio is preferably 1. When the ligand is a complete antibody, the ratio is preferably 2 or 4.

可設計第一結合搭配物與配體之間的共價鍵在配體上之確切數目及位點，因為在配體上接合第一結合搭配物或包含親偶極物之分子的確切位點係由以下定義：製備配體之融合蛋白或配體融合蛋白，其在羧基端併入稱為分選酶識別模體之胺基酸序列；將配體融合蛋白與包含親偶極物之分子的衍生物混合；且用分選酶對其進行處理以接合DBCO。The exact number and location of the covalent bonds between the first binding partner and the ligand on the ligand can be designed, because the exact location on the ligand where the first binding partner or the molecule comprising the exotropophile is attached is defined by: making a fusion protein of the ligand or a ligand fusion protein that incorporates an amino acid sequence called a sortase recognition motif at the carboxyl terminus; mixing the ligand fusion protein with a derivative of the molecule comprising the exotropophile; and treating it with a sortase to attach DBCO.

最常用之蛋白質修飾分選酶係來自金黃色葡萄球菌之分選酶A (StrA)，其藉由識別以下之模體來介導轉肽酶反應：在融合蛋白之羧基端附近的Leu-Pro-Xxx-Thr-Gly (LPXTG，其中X為任何胺基酸且Gly不能為游離羧酸酯；在本發明之序列表中列舉為SEQ ID NO: 1)及其對應物分子，其必須在其胺基端具有游離甘胺酸殘基。在本發明中，當配體與包含諸如DBCO之親偶極物的分子接合時，對應物分子可為在其胺基端具有甘胺酸之任何DBCO衍生物，例如5-(甘胺醯基甘胺醯基-β-丙胺醯基)-11,12-二氫-5,6-二氫二苯并[b,f]氮雜環辛。藉由轉肽酶反應，用DBCO衍生物之胺基端之甘胺酸更換配體蛋白之分選酶識別模體之最後一個甘胺酸，得到羧基端與DBCO接合之配體融合蛋白。在本發明之轉肽酶反應中，可使用除金黃色葡萄球菌之StrA以外的分選酶，諸如金黃色葡萄球菌之StrB，其識別具有五個胺基酸之不同識別模體。分選酶及其識別模體見於例如Jacobitz, AW等人, Adv Protein Chem Struct Biol. 2017; 109: 223-264中。用於使DBCO基團與融合蛋白接合的經純化之重組分選酶可商購自許多供應商，諸如Funakoshi Co., Ltd. (Tokyo)。The most commonly used protein modification sortase is sortase A (StrA) from Staphylococcus aureus, which mediates the transpeptidase reaction by recognizing the following motif: Leu-Pro-Xxx-Thr-Gly (LPXTG, wherein X is any amino acid and Gly cannot be a free carboxylate; listed as SEQ ID NO: 1 in the sequence listing of the present invention) near the carboxyl terminus of the fusion protein and its counterpart molecule, which must have a free glycine residue at its amino terminus. In the present invention, when the ligand is conjugated to a molecule containing an exophile such as DBCO, the counterpart molecule may be any DBCO derivative having glycine at its amino terminus, such as 5-(glyaminoglyaminoglycinyl-β-propylamino)-11,12-dihydro-5,6-dihydrodibenzo[b,f]azepine. By a transpeptidase reaction, the glycine at the amino terminus of the DBCO derivative is replaced with the last glycine of the sortase recognition motif of the ligand protein to obtain a ligand fusion protein conjugated to DBCO at the carboxyl terminus. In the transpeptidase reaction of the present invention, a sortase other than StrA of Staphylococcus aureus, such as StrB of Staphylococcus aureus, which recognizes a different recognition motif having five amino acids, may be used. Sortases and their recognition motifs are found, for example, in Jacobitz, AW et al., Adv Protein Chem Struct Biol. 2017; 109: 223-264. Purified recombinant sortases for conjugating DBCO groups to fusion proteins are commercially available from a number of suppliers, such as Funakoshi Co., Ltd. (Tokyo).

第二結合搭配物與錨分子之共價鍵結係藉由已知方法進行。在一個實施例中，作為第二結合搭配物之疊氮化物與錨分子可藉由疊氮試劑與經活化之錨分子的親核加成反應而共價鍵結。在一個實施例中，錨分子及與疊氮化物接合之分子可透過諸如但不限於醯胺、酯、胺基甲酸酯、碳酸酯之連接子結構共價鍵結。與第二結合搭配物鍵結之錨分子可用於藉由使分子與其他脂質分子聚集來製備遞送媒介。The covalent bonding of the second binding partner and the anchor molecule is performed by known methods. In one embodiment, the azide as the second binding partner and the anchor molecule can be covalently bonded by a nucleophilic addition reaction of an azide reagent and an activated anchor molecule. In one embodiment, the anchor molecule and the molecule bound to the azide can be covalently bonded through a linker structure such as but not limited to amide, ester, carbamate, carbonate. The anchor molecule bonded to the second binding partner can be used to prepare a delivery vehicle by aggregating the molecule with other lipid molecules.

對於本發明之複合物，向其中遞送活性物質之靶細胞可為免疫細胞。免疫細胞涵蓋參與人類或其他哺乳動物細胞之免疫功能的所有類型之細胞，即負責後天性免疫中之細胞免疫的T細胞，負責先天性免疫之NK細胞、NKT細胞、單核球、巨噬細胞、樹突狀細胞及其類似細胞，及作為具有NK細胞特性之T細胞的NKT細胞。For the complex of the present invention, the target cell to which the active substance is delivered may be an immune cell. Immune cells include all types of cells involved in the immune function of human or other mammalian cells, namely T cells responsible for cellular immunity in acquired immunity, NK cells, NKT cells, monocytes, macrophages, dendritic cells and the like responsible for innate immunity, and NKT cells which are T cells with NK cell characteristics.

本發明之複合物可例如藉由US9,404,127中所描述之方法產生。將包含錨分子之遞送媒介與疊氮化物一起培育以將疊氮化物固定於遞送媒介上。陽離子型脂質、磷脂、膽固醇、PEG脂質及疊氮化物之混合比(莫耳比)為例如40至60:0至20:0至50:0至5:1，但比率不限於此。上文所提及之混合可使用移液管、微流體混合系統(例如，Asia微流系統(Syrris)或Nanoassemblr (Precision Nanosystems))進行。可藉由凝膠過濾、透析或無菌過濾對所獲得之脂質粒子進行純化。總脂質組分在有機溶劑溶液中之濃度較佳為0.5至100 mg/mL。The complex of the present invention can be produced, for example, by the method described in US9,404,127. A delivery medium containing an anchor molecule is incubated with an azide to fix the azide on the delivery medium. The mixing ratio (molar ratio) of the cationic lipid, phospholipid, cholesterol, PEG lipid and azide is, for example, 40 to 60:0 to 20:0 to 50:0 to 5:1, but the ratio is not limited thereto. The mixing mentioned above can be performed using a pipette, a microfluidic mixing system (e.g., Asia microfluidic system (Syrris) or Nanoassemblr (Precision Nanosystems)). The obtained lipid particles can be purified by gel filtration, dialysis or sterile filtration. The concentration of total lipid components in the organic solvent solution is preferably 0.5 to 100 mg/mL.

作為有機溶劑，可列舉例如甲醇、乙醇、1-丙醇、2-丙醇、1-丁醇、三級丁醇、丙酮、乙腈、N,N-二甲基甲醯胺、二甲亞碸或其混合物。有機溶劑可含有0至20%之水或緩衝溶液。作為緩衝溶液，可列舉酸性緩衝溶液(例如，乙酸鹽緩衝溶液、檸檬酸鹽緩衝溶液)或中性緩衝溶液(例如，4-(2-羥基乙基)-1-哌𠯤乙烷磺酸(HEPES)緩衝溶液、參(羥基甲基)胺基甲烷(Tris)緩衝溶液、磷酸鹽緩衝溶液、磷酸鹽緩衝鹽水(PBS))。Examples of the organic solvent include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, or a mixture thereof. The organic solvent may contain 0 to 20% of water or a buffer solution. As the buffer solution, there can be mentioned an acidic buffer solution (e.g., an acetic acid buffer solution, a citrate buffer solution) or a neutral buffer solution (e.g., a 4-(2-hydroxyethyl)-1-piperidiniumethanesulfonic acid (HEPES) buffer solution, a tris(hydroxymethyl)aminomethane (Tris) buffer solution, a phosphate buffer solution, a phosphate buffered saline (PBS)).

當以上文所描述產生遞送媒介分散液時，含有上文所提及之脂質的分散液可藉由將活性物質添加至水或緩衝溶液中來產生。以使得活性成分於水或緩衝溶液中之濃度為0.05至2.0 mg/mL之方式添加活性物質為較佳的。When the delivery vehicle dispersion is produced as described above, the dispersion containing the above-mentioned lipid can be produced by adding the active substance to water or a buffer solution. It is preferred to add the active substance in such a manner that the concentration of the active ingredient in water or a buffer solution is 0.05 to 2.0 mg/mL.

當複合物進一步含有對靶細胞具有特異性之配體時，其可藉由以下來產生：首先製備在羧基端附近併入分選酶識別模體之配體融合蛋白，接著將配體融合蛋白與包含親偶極物之分子的衍生物一起培育，從而使得包含親偶極物之分子與配體融合蛋白之羧基端共價連接。When the complex further contains a ligand specific for a target cell, it can be produced by first preparing a ligand fusion protein incorporating a sortase recognition motif near the carboxyl terminus, and then incubating the ligand fusion protein with a derivative of a molecule containing an exophile, thereby covalently linking the molecule containing an exophile to the carboxyl terminus of the ligand fusion protein.

將與包含親偶極物之分子接合的配體融合蛋白及與疊氮基接合之遞送媒介混合，從而使得對於靶向小鼠T細胞之複合物，配體融合蛋白與疊氮化物之莫耳濃度比在1:20、1:10或1:20至2:1 (配體融合蛋白:疊氮化物)之範圍內；對於靶向人類T細胞之複合物為1:100、1:20或1:100至2:1 (配體融合蛋白:疊氮化物)；且對於人類NK細胞為1:40、1:20或1:40至2:1 (配體融合蛋白:疊氮化物)。遞送媒介可囊封活性物質。反應可在溫和條件下進行，諸如在25℃下進行24小時。複合物可儲存於-80℃下。The ligand fusion protein conjugated to the molecule comprising the exophile and the delivery vehicle conjugated to the azide are mixed so that the molar concentration ratio of the ligand fusion protein to the azide is in the range of 1:20, 1:10, or 1:20 to 2:1 (ligand fusion protein: azide) for complexes targeting mouse T cells; 1:100, 1:20, or 1:100 to 2:1 (ligand fusion protein: azide) for complexes targeting human T cells; and 1:40, 1:20, or 1:40 to 2:1 (ligand fusion protein: azide) for human NK cells. The delivery vehicle can encapsulate the active substance. The reaction can be carried out under mild conditions, such as at 25°C for 24 hours. The complex can be stored at -80°C.

當超過一種配體或兩種或更多種不同配體併入本發明之複合物中時，兩種或更多種配體分別與包含親偶極物之分子接合，且與具有與疊氮基共價鍵結之錨分子的遞送媒介以1:1至1:10之所需混合比混合。When more than one ligand or two or more different ligands are incorporated into the complex of the present invention, the two or more ligands are separately conjugated to a molecule containing an aptophile and mixed with a delivery vehicle having an anchor molecule covalently bonded to an azido group at a desired mixing ratio of 1:1 to 1:10.

複合物之物理化學特徵可自熟習此項技術者已知之任何態樣確定。物理化學特徵之項目包括粒徑、ζ電位、多分散性指數(polydispersity index；PDI)。PDI定義為粒徑分佈之標準差(σ)除以平均粒徑。PDI用於估計粒子溶液之平均均勻性，且較大PDI值對應於粒子樣品中之較大尺寸分佈。複合物之PDI可小於1.0、0.9、0.8、0.7、0.6、0.5、0.4、0.3、0.2、0.1，較佳小於0.5、0.4、0.3、0.2、0.1，更佳小於0.2、0.1。The physicochemical characteristics of the complex can be determined in any manner known to those skilled in the art. Items of physicochemical characteristics include particle size, zeta potential, and polydispersity index (PDI). PDI is defined as the standard deviation (σ) of the particle size distribution divided by the average particle size. PDI is used to estimate the average uniformity of a particle solution, and a larger PDI value corresponds to a larger size distribution in the particle sample. The PDI of the complex can be less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, preferably less than 0.5, 0.4, 0.3, 0.2, 0.1, and more preferably less than 0.2, 0.1.

2.2. 本發明之藥劑The drug of the present invention

本發明提供一種藥劑，其包含本發明之複合物。包含本發明之複合物的藥劑可用於預防或治療表現由本發明之複合物遞送之活性物質的各種疾病，且可投與至例如哺乳動物(例如，小鼠、大鼠、倉鼠、兔、貓、狗、牛、綿羊、猴、人類)，較佳為人類。因此，在本發明之一個實施例中，提供本發明之藥劑，其用於預防或治療表現由本發明之複合物或確切而言由配體識別之抗原的腫瘤。另外，包含本發明之複合物的藥劑可用於預防或治療由基因表現之缺乏或減少引起的遺傳或表觀遺傳病症，其係藉由促進尤其血液、血管、內臟器官(包括但不限於消化道及/或呼吸道及器官)、周邊及/或中樞神經系統、感覺器官、肌肉、骨軟骨、皮膚及其他身體器官及組織中之基因表現來實現。The present invention provides a medicament comprising the complex of the present invention. The medicament comprising the complex of the present invention can be used to prevent or treat various diseases expressing the active substance delivered by the complex of the present invention, and can be administered to, for example, mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans), preferably humans. Therefore, in one embodiment of the present invention, a medicament of the present invention is provided for preventing or treating a tumor expressing an antigen recognized by the complex of the present invention or, more precisely, by a ligand. In addition, the medicaments comprising the complex of the present invention can be used to prevent or treat genetic or epigenetic diseases caused by lack or reduction of gene expression by promoting gene expression in the blood, blood vessels, internal organs (including but not limited to the digestive tract and/or respiratory tract and organs), peripheral and/or central nervous system, sensory organs, muscles, bone cartilage, skin and other body organs and tissues.

包含本發明之複合物的本發明之藥劑的劑量例如每1 kg體重每劑量按活性物質之量計在0.001 mg至10 mg範圍內。舉例而言，當投與人類患者時，對於體重為60 kg之患者而言，劑量在0.0001至50 mg範圍內。上文所提及之劑量為實例，且可根據所使用之活性物質之類型、投藥途徑、投藥之個體或患者的年齡、體重、症狀等適當地選擇劑量。The dosage of the medicament of the present invention comprising the complex of the present invention is, for example, in the range of 0.001 mg to 10 mg per 1 kg body weight in terms of the amount of the active substance. For example, when administered to a human patient, for a patient weighing 60 kg, the dosage is in the range of 0.0001 to 50 mg. The dosages mentioned above are examples, and the dosage can be appropriately selected according to the type of active substance used, the route of administration, the age, weight, symptoms, etc. of the individual or patient being administered.

藉由投與至哺乳動物(例如人類或其他哺乳動物(例如，小鼠、大鼠、倉鼠、兔、貓、狗、牛、綿羊、猴)，較佳為人類)，包含本發明之複合物的本發明之藥劑可引入活性物質，且當活性物質為核酸時，可誘導在核酸中編碼之蛋白質在動物體內之靶細胞中的表現。當活性物質為CAR或外源TCR，且靶細胞為活體內免疫細胞或細胞毒性細胞時，靶細胞特異性識別表現CAR或外源TCR所靶向之表面抗原的癌細胞及其類似細胞且殺傷病變細胞，由此展現針對疾病之預防或治療作用。By administering to mammals (e.g., humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys), preferably humans), the medicament of the present invention comprising the complex of the present invention can introduce active substances, and when the active substance is a nucleic acid, it can induce the expression of the protein encoded in the nucleic acid in the target cells in the animal. When the active substance is CAR or exogenous TCR, and the target cell is an immune cell or cytotoxic cell in vivo, the target cell specifically recognizes cancer cells and similar cells expressing the surface antigen targeted by CAR or exogenous TCR and kills the diseased cells, thereby exhibiting a preventive or therapeutic effect against the disease.

本發明提供一種藥劑，其包含由本發明之複合物遞送之靶細胞，且表現活性物質。包含本發明之靶細胞的藥劑可用於預防或治療各種疾病，且可例如藉由移植包含本發明之靶細胞的藥劑而投與至哺乳動物(例如，小鼠、大鼠、倉鼠、兔、貓、狗、牛、綿羊、猴、人類)，較佳為人類。因此，在本發明之一個實施例中，提供本發明之藥劑，其用於預防或治療表現由本發明之複合物或確切而言由配體識別之抗原的腫瘤。另外，包含本發明之靶細胞的藥劑可用於預防或治療由特定細胞之缺乏或減少引起的遺傳或表觀遺傳病症，其係藉由將包含本發明之靶細胞的藥劑移植至尤其血液、血管、內臟器官(包括但不限於消化道及/或呼吸道及器官)、周邊及/或中樞神經系統、感覺器官、肌肉、骨軟骨、皮膚及其他身體器官及組織中來實現。The present invention provides a medicament comprising a target cell delivered by the complex of the present invention and expressing an active substance. The medicament comprising the target cell of the present invention can be used to prevent or treat various diseases, and can be administered to mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans), preferably humans, by transplanting the medicament comprising the target cell of the present invention. Therefore, in one embodiment of the present invention, a medicament of the present invention is provided for preventing or treating a tumor expressing an antigen recognized by the complex of the present invention or, more precisely, by a ligand. In addition, the agent comprising the target cells of the present invention can be used to prevent or treat genetic or epigenetic diseases caused by the lack or reduction of specific cells, which is achieved by transplanting the agent comprising the target cells of the present invention into the blood, blood vessels, internal organs (including but not limited to the digestive tract and/or respiratory tract and organs), peripheral and/or central nervous system, sensory organs, muscles, bone cartilage, skin and other body organs and tissues.

包含本發明之複合物的本發明之藥劑的劑量例如每1 kg體重每劑量按活性物質之量計在0.001 mg至10 mg範圍內。舉例而言，當投與人類患者時，對於體重為60 kg之患者而言，劑量在0.0001至50 mg範圍內。上文所提及之劑量為實例，且可根據所使用之活性物質之類型、投藥途徑、投藥之個體或患者的年齡、體重、症狀等適當地選擇劑量。The dosage of the medicament of the present invention comprising the complex of the present invention is, for example, in the range of 0.001 mg to 10 mg per 1 kg body weight in terms of the amount of the active substance. For example, when administered to a human patient, for a patient weighing 60 kg, the dosage is in the range of 0.0001 to 50 mg. The dosages mentioned above are examples, and the dosage can be appropriately selected according to the type of active substance used, the route of administration, the age, weight, symptoms, etc. of the individual or patient being administered.

藉由投與至哺乳動物(例如人類或其他哺乳動物(例如，小鼠、大鼠、倉鼠、兔、貓、狗、牛、綿羊、猴)，較佳為人類)，包含本發明之靶細胞的本發明之藥劑可在動物體內起作用。當活性物質為CAR或外源TCR，且靶細胞為活體內免疫細胞或細胞毒性細胞時，靶細胞特異性識別表現CAR或外源TCR所靶向之表面抗原的癌細胞及其類似細胞且殺傷病變細胞，由此展現針對疾病之預防或治療作用。By administering to mammals (e.g., humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys), preferably humans), the medicament of the present invention comprising the target cells of the present invention can act in the animal body. When the active substance is CAR or exogenous TCR, and the target cell is an immune cell or cytotoxic cell in vivo, the target cell specifically recognizes cancer cells and similar cells expressing the surface antigen targeted by CAR or exogenous TCR and kills the diseased cells, thereby exhibiting a preventive or therapeutic effect against the disease.

含有本發明之靶細胞作為活性成分之藥劑較佳非經腸投與至個體。非經腸投與方法包括靜脈內、動脈、肌內、腹膜內及皮下投與。雖然根據個體之病況、體重、年齡等選擇劑量，但對60 公斤體重之個體進行投與通常可實現每劑量1×10 6至1×10 10個細胞，較佳1×10 7至1×10 9個細胞，更佳5×10 7至5×10 8個細胞。藥劑可以單次劑量或多次劑量形式投與。含有本發明之靶細胞作為活性成分之本發明藥劑可呈適用於非經腸投與之已知形式，諸如注射或輸注劑。視需要，藥劑可含有醫藥學上可接受之賦形劑。醫藥學上可接受之賦形劑包括上文所描述之賦形劑。藥劑可含有鹽水、磷酸鹽緩衝鹽水(PBS)、培養基等以穩定地維持細胞。培養基不受特定限制，且其實例包括但不限於RPMI、AIM-V、X-VIVO10及其類似者。另外，出於穩定之目的，可將醫藥學上可接受之載劑(例如人類血清白蛋白)、防腐劑及其類似物添加至藥劑中。 The agent containing the target cells of the present invention as an active ingredient is preferably administered to an individual parenterally. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal and subcutaneous administration. Although the dosage is selected according to the individual's condition, weight, age, etc., administration to an individual weighing 60 kg can generally achieve 1×10 6 to 1×10 10 cells per dose, preferably 1×10 7 to 1×10 9 cells, and more preferably 5×10 7 to 5×10 8 cells. The agent can be administered in a single dose or multiple doses. The medicament of the present invention containing the target cells of the present invention as an active ingredient may be in a known form suitable for parenteral administration, such as an injection or an infusion. If necessary, the medicament may contain a pharmaceutically acceptable formulation. Pharmaceutically acceptable formulations include the formulations described above. The medicament may contain saline, phosphate buffered saline (PBS), a culture medium, etc. to stably maintain the cells. The culture medium is not particularly limited, and examples thereof include but are not limited to RPMI, AIM-V, X-VIVO10 and the like. In addition, for the purpose of stability, a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative and the like may be added to the medicament.

3.3. 用於產生本發明之複合物的方法Method for producing the complex of the present invention

本發明提供一種用於產生本發明之複合物的方法，其包含：(1)使第一結合搭配物與配體以1至4個第一結合搭配物分子/一個配體分子之比率共價鍵結之步驟；(2)使第二結合搭配物與錨分子共價鍵結之步驟；及(3)藉由化學反應使第一結合搭配物與第二結合搭配物以一個第二結合搭配物分子/一個第一結合搭配物分子之比率鍵結之步驟。或者，本發明提供一種用於產生本發明之複合物的方法，其包含：(1)藉由分選酶識別模體使第一結合搭配物與配體共價鍵結之步驟；(2)使第二結合搭配物與錨分子共價鍵結之步驟；及(3)藉由化學反應使第一結合搭配物與第二結合搭配物以一個第二結合搭配物分子/一個第一結合搭配物分子之比率鍵結之步驟。或者，本發明提供一種用於產生本發明之複合物的方法，其包含：(1)使第一結合搭配物與配體之C端共價鍵結之步驟；(2)使第二結合搭配物與錨分子共價鍵結之步驟；及(3)藉由化學反應使第一結合搭配物與第二結合搭配物以一個第二結合搭配物分子/一個第一結合搭配物分子之比率鍵結之步驟。 使第一結合搭配物與配體共價鍵結之步驟(1)可藉由以下來進行：使用習知重組DNA技術產生及純化配體與分選酶識別模體肽之融合蛋白(「配體融合蛋白」)，隨後使用分選酶、轉肽酶使配體融合蛋白與包含親偶極物之分子接合，隨後使用習知蛋白質純化技術(諸如凝膠過濾或親和力層析)純化。 使第二結合搭配物與錨分子共價鍵結之步驟(2)係藉由已知方法進行。在一個實施例中，作為第二結合搭配物之疊氮化物與錨分子可藉由疊氮試劑與經活化之錨分子的親核加成反應而共價鍵結。在一個實施例中，錨分子及與疊氮化物接合之分子可透過諸如但不限於醯胺、酯、胺基甲酸酯、碳酸酯之連接子結構共價鍵結。與第二結合搭配物鍵結之錨分子可用於藉由使分子與其他脂質分子聚集來製備遞送媒介。 使第一結合搭配物與第二結合搭配物鍵結之步驟(3)係藉由例如以下來進行：使包含親偶極物之分子的標靶融合蛋白接合物與經疊氮化之脂質奈米粒子或遞送媒介在25℃下反應24小時。 The present invention provides a method for producing the complex of the present invention, which comprises: (1) a step of covalently bonding a first binding partner to a ligand at a ratio of 1 to 4 first binding partner molecules/one ligand molecule; (2) a step of covalently bonding a second binding partner to an anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner at a ratio of one second binding partner molecule/one first binding partner molecule by a chemical reaction. Alternatively, the present invention provides a method for producing the complex of the present invention, comprising: (1) a step of covalently bonding a first binding partner to a ligand by using a sortase recognition motif; (2) a step of covalently bonding a second binding partner to an anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner at a ratio of one second binding partner molecule to one first binding partner molecule by a chemical reaction. Alternatively, the present invention provides a method for producing the complex of the present invention, comprising: (1) a step of covalently bonding a first binding partner to the C-terminus of a ligand; (2) a step of covalently bonding a second binding partner to an anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner at a ratio of one second binding partner molecule to one first binding partner molecule by chemical reaction. The step (1) of covalently bonding the first binding partner to the ligand can be performed by using known recombinant DNA technology to produce and purify a fusion protein of the ligand and a sortase recognition motif peptide ("ligand fusion protein"), then using a sortase or a transferase to bind the ligand fusion protein to a molecule containing an affinity partner, and then purifying using known protein purification techniques (such as gel filtration or affinity analysis). The step (2) of covalently bonding the second binding partner to the anchor molecule is performed by a known method. In one embodiment, the azide as the second binding partner and the anchor molecule can be covalently bonded by a nucleophilic addition reaction between an azide reagent and an activated anchor molecule. In one embodiment, the anchor molecule and the molecule conjugated to the azide can be covalently bonded via a linker structure such as, but not limited to, amide, ester, carbamate, carbonate. The anchor molecule bound to the second binding partner can be used to prepare a delivery vehicle by aggregating the molecule with other lipid molecules. The step (3) of binding the first binding partner to the second binding partner is performed, for example, by reacting a target fusion protein conjugate containing a molecule of an amphophile with azide-polymerized lipid nanoparticle or a delivery vehicle at 25°C for 24 hours.

4.4. 本發明之組合物Composition of the present invention

本發明提供一種組合物，其包含本發明之複合物。包含本發明之複合物的組合物可用於在靶細胞內部遞送活性物質，不僅用於醫學用途，諸如用於預防或治療各種疾病，且亦可用於任何非醫學用途，諸如用於標記靶細胞之實驗工具，或用於獸醫學或食品生產之工具。包含本發明之複合物的組合物亦可包含培養基或鹽水。The present invention provides a composition comprising the complex of the present invention. The composition comprising the complex of the present invention can be used to deliver active substances inside target cells, not only for medical purposes, such as for preventing or treating various diseases, but also for any non-medical purposes, such as experimental tools for marking target cells, or tools for veterinary medicine or food production. The composition comprising the complex of the present invention may also contain a culture medium or saline.

5.5. 用於使本發明之靶細胞活化及Used to activate the target cells of the present invention and // 或增殖的方法or proliferation method

在一個態樣中，本發明提供一種用於使靶細胞活化及/或增殖之方法，其包含：使本發明之複合物與包含靶細胞之細胞群體接觸的步驟，其中複合物包含至少一種對靶細胞具有特異性之配體。In one aspect, the present invention provides a method for activating and/or proliferating target cells, comprising: contacting a complex of the present invention with a cell population comprising target cells, wherein the complex comprises at least one ligand specific to the target cells.

在另一態樣中，靶細胞可為免疫細胞。如本文中所使用，「免疫細胞」不受特定限制，只要其為能夠藉由一些作用機制來破壞諸如癌細胞及其類似細胞之靶細胞(病原細胞)的細胞(亦即免疫效應細胞)即可。其實例包括負責後天性免疫中之細胞免疫的T細胞，負責先天性免疫之NK細胞、單核球、巨噬細胞、樹突狀細胞等，及作為具有NK細胞特性之T細胞的NKT細胞。在一個較佳實施例中，免疫細胞可為T細胞。自活的生物體收集之T細胞在本發明中亦稱為「離體T細胞」。In another aspect, the target cell may be an immune cell. As used herein, "immune cell" is not particularly limited, as long as it is a cell (i.e., immune effector cell) that can destroy target cells (pathogenic cells) such as cancer cells and similar cells by some mechanism of action. Examples include T cells responsible for cellular immunity in acquired immunity, NK cells, monocytes, macrophages, dendritic cells, etc. responsible for innate immunity, and NKT cells as T cells with NK cell characteristics. In a preferred embodiment, the immune cell may be a T cell. T cells collected from living organisms are also referred to as "ex vivo T cells" in the present invention.

在另一較佳實施例中，免疫細胞可負責先天性免疫，諸如NK細胞、巨噬細胞、樹突狀細胞及其類似細胞。即使HLA類型匹配，但T細胞仍被視為由同種異體(allo)移植引起GVHD之風險相當大，而allo-NK細胞等則被視為不引起GVHD。因此，各種HLA型allo離體免疫細胞之製備准許使用現成的細胞。CAR-NK細胞描述於例如US2016/0096892、Mol Ther. 25(8): 1769-1781 (2017)及其類似者中，CAR-樹突狀細胞、CAR-巨噬細胞及其類似細胞描述於例如WO 2017/019848、eLIFE.2018 e36688及其類似者中。In another preferred embodiment, the immune cells may be responsible for innate immunity, such as NK cells, macrophages, dendritic cells and the like. Even if the HLA type is matched, T cells are still considered to have a considerable risk of causing GVHD by allogeneic (allo) transplantation, while allo-NK cells and the like are considered not to cause GVHD. Therefore, the preparation of various HLA-type allo ex vivo immune cells allows the use of ready-made cells. CAR-NK cells are described in, for example, US2016/0096892, Mol Ther. 25(8): 1769-1781 (2017) and the like, and CAR-dendritic cells, CAR-macrophages and the like are described in, for example, WO 2017/019848, eLIFE.2018 e36688 and the like.

在另一態樣中，本發明提供一種組合物，其用於誘導含有本發明之脂質奈米粒子的CAR或外源TCR之表現。In another aspect, the present invention provides a composition for inducing the expression of CAR or exogenous TCR containing the lipid nanoparticles of the present invention.

6.6. 用於在本發明之靶細胞內部遞送活性物質的方法Method for delivering active substances inside target cells of the present invention

本發明提供一種用於將活性物質遞送至靶細胞內部之方法，其包含使本發明之複合物與包含靶細胞之細胞群體接觸之步驟，其中活性物質既不包括編碼CAR亦不包括編碼TCR之任何核酸。本發明可包含抑制細胞毒性細胞活化抑制因子之表現之核酸，及/或編碼細胞毒性細胞活化促進因子之核酸。本發明可離體或在活體內進行。 The present invention provides a method for delivering an active substance to the interior of a target cell, comprising the step of contacting the complex of the present invention with a cell population comprising target cells, wherein the active substance does not include any nucleic acid encoding CAR or TCR. The present invention may include a nucleic acid that inhibits the expression of a cytotoxic cell activation inhibitor, and/or a nucleic acid that encodes a cytotoxic cell activation promoting factor. The present invention may be performed in vitro or in vivo.

除非另外指示，否則當與可量測數值變數結合使用時，措辭「約」或「大致」係指變數之指示值，及在指示值之實驗誤差內或在指示值之±10百分比內(以更大者為準)的所有變數值。 [實例] Unless otherwise indicated, the term "about" or "approximately" when used in conjunction with a measurable numerical variable refers to the indicated value of the variable and all values of the variable that are within the experimental error of the indicated value or within ±10 percent of the indicated value, whichever is greater. [Example]

1.1. 分析條件Analysis conditions 1.11.1 LNPLNP 物理特性之評估Evaluation of physical properties

藉由DLS、RiboGreen分析法及HPLC測定LNP之物理特性。The physical properties of LNPs were determined by DLS, RiboGreen analysis and HPLC.

1.1.11.1.1 尺寸量測Dimension measurement

藉由Zetasizer Nano ZS (Malvern Panalytical)測定粒徑及PDI。The particle size and PDI were measured by Zetasizer Nano ZS (Malvern Panalytical).

1.1.21.1.2 核酸濃度之量測。Measurement of nucleic acid concentration.

將LNP溶解於0.5% Triton X-100中，且用Quant-it™ RiboGreen RNA分析套組(Thermo Fisher Scientific)定量地測定mRNA濃度。不含Triton X-100之mRNA濃度經量測作為未囊封於LNP中之mRNA的濃度。自此等量測計算經囊封之mRNA之百分比。LNPs were dissolved in 0.5% Triton X-100 and the mRNA concentration was quantitatively determined using the Quant-it™ RiboGreen RNA Assay Kit (Thermo Fisher Scientific). The mRNA concentration without Triton X-100 was measured as the concentration of mRNA not encapsulated in LNPs. The percentage of encapsulated mRNA was calculated from these measurements.

1.1.31.1.3 抗體濃度之量測Measurement of Antibody Concentration

如下進行HPLC分析，且定量地測定與LNP結合之抗體之濃度。亦測定未與LNP結合之抗體(殘餘抗體)之濃度。自此等量測計算殘餘抗體相對於所添加之抗體之濃度的百分比。 量測儀器：Thermo Scientific Dionex UltiMate 3000 移動相A：0.2%三氟乙酸(TFA)/水 移動相B：0.2% TFA/乙腈 偵測器：UV280 nm 管柱：Agilent PLRP-S，5 μm，1000A，2.1 mm × 50 mm HPLC analysis was performed as follows and the concentration of antibody bound to LNP was quantitatively determined. The concentration of antibody not bound to LNP (residual antibody) was also determined. From these measurements, the percentage of residual antibody relative to the concentration of added antibody was calculated. Measuring instrument: Thermo Scientific Dionex UltiMate 3000 Mobile phase A: 0.2% trifluoroacetic acid (TFA)/water Mobile phase B: 0.2% TFA/acetonitrile Detector: UV280 nm Column: Agilent PLRP-S, 5 μm, 1000A, 2.1 mm × 50 mm

2.  LNP2. LNP 之製備Preparation 2.1 Fab-DBCO2.1 Fab-DBCO 之製備Preparation

藉由以下兩個步驟製備Fab-DBCO。藉由LC/MS測定分子量及DBCO/抗體比率(DAR)且藉由BCA方法測定蛋白質濃度。物理特性評估之結果展示於表1中。 (1)在C端具有LPETGG-His6之Fab之製備 使用電穿孔儀器(Maxcyte)將編碼Fab之各種基因的質體pMG2.2載體引入CHOZN細胞中，且使用EX-CELL Advanced CHO Feed 1 (含葡萄糖)培養細胞六至八天。接著，藉由用cOmplete Ni管柱及Superdex 200純化來製備經LPETGG-His標記之Fab。 (2) Fab-DBCO之製備 將經LPETGG-His標記之Fab與CaCl 2及5-(甘胺醯基甘胺醯基-β-丙胺醯基)-11,12-二氫-5,6-二氫二苯并[b,f]氮雜環辛混合，且接著進行分選酶A處理(Protein Science, 89, 15.3.1-15.3.19)。在室溫下反應四小時，隨後使用Ni管柱純化且進行透析，獲得Fab-DBCO。 Fab-DBCO was prepared by the following two steps. The molecular weight and DBCO/antibody ratio (DAR) were determined by LC/MS and the protein concentration was determined by the BCA method. The results of the physical property evaluation are shown in Table 1. (1) Preparation of Fab with LPETGG-His6 at the C-terminus The plasmid pMG2.2 vector encoding various genes of Fab was introduced into CHOZN cells using an electroporator (Maxcyte), and the cells were cultured using EX-CELL Advanced CHO Feed 1 (containing glucose) for six to eight days. Then, LPETGG-His-tagged Fab was prepared by purification using a cOmplete Ni column and Superdex 200. (2) Preparation of Fab-DBCO The LPETGG-His-labeled Fab was mixed with CaCl 2 and 5-(glycinamidoylglycinamidoyl-β-propylamino)-11,12-dihydro-5,6-dihydrodibenzo[b,f]azepine and then treated with sortase A (Protein Science, 89, 15.3.1-15.3.19). The reaction was allowed to proceed at room temperature for four hours, and then the mixture was purified using a Ni column and dialyzed to obtain Fab-DBCO.

Fab-DBCO之清單Fab-DBCO List

[表1] 名稱 純系 靶細胞 DAR* 濃度 (mg/mL) CD3Fab-DBCO 145-2C11 小鼠T Cell 1 11.02 CD3Fab-DBCO OKT3 人類T Cell 1 7.02 CD7Fab-DBCO 格瑞尼單抗(Grisnilimab) 人類T/NK Cell 1 11.66 CD8Fab-DBCO OKT-8 人類T Cell 1 7.63 CD16Fab-DBCO 3G8 人類NK Cell 1 10.87 CD56Fab-DBCO huN901 人類NK Cell 1 14.10 *DAR：DBCO/抗體比率 [Table 1] Name Pure Target cells DAR* Concentration (mg/mL) CD3Fab-DBCO 145-2C11 Mouse T Cell 1 11.02 CD3Fab-DBCO OKT3 Human T Cell 1 7.02 CD7Fab-DBCO Grisnilimab Human T/NK Cell 1 11.66 CD8Fab-DBCO OKT-8 Human T Cell 1 7.63 CD16Fab-DBCO 3G8 Human NK Cell 1 10.87 CD56Fab-DBCO huN901 Human NK Cell 1 14.10 *DAR: DBCO/antibody ratio

2.22.2 疊氮化物Nitride -LNP-LNP 之製備Preparation

將脂質之混合物(陽離子型脂質:DPPC:膽固醇:SUNBRIGHT GS-020:DSPE-PEG (2000)-疊氮化物= 60:10.6:27:1.4:1，莫耳%)溶解於90% EtOH/10%乙酸鹽緩衝液(25 mM，pH 4.0)中以獲得14 mg/mL之脂質溶液。作為陽離子型脂質，使用以下化合物：WO2016/021683中所描述之3-戊基辛酸3-((4-(二甲基胺基)丁醯基)氧基)-2,2-雙(((3-戊基辛醯基)氧基)甲基)丙酯(下文亦稱為「化合物A」；用於靶向T細胞之LNP)；或WO2020/032184中所描述之2-(((4,5-二丁基壬醯基)氧基)甲基)-2-(((5-(二甲基胺基)戊醯基)氧基)甲基)丙烷-1,3-二基二癸酸酯(下文亦稱為「化合物B」；用於靶向NK細胞之LNP)。將編碼具有CD28及CD3ε作為胞內信號轉導域的靶向CD19之CAR的mRNA溶解於10 mM 2-𠰌啉基乙烷磺酸(MES)緩衝液(pH 5.5)中以獲得0.3 mg/mL之核酸溶液。使用Nanoassemblr TM(Precision Nanosystems)將以上脂質溶液及核酸溶液以3 mL/min:6 mL/min (脂質溶液:核酸溶液)之流動速率比混合，以獲得包含組合物之分散液。使用Slide-A-Lyzer透析(MWCO：20 k，Thermo Fisher Scientific)，在室溫下用水透析以上經分散之液體一小時且在4℃下用PBS透析24小時。接著，藉由使用Amicon Ultra (MWCO：30K，Merck)之超濾來濃縮分散液且使用0.2 µm針筒過濾器過濾。用含20%蔗糖之PBS將mRNA濃度調節為350 µg/mL之最終濃度，且儲存於4℃下。 2.3 Fab-DBCO 與疊氮化物 -LNP 之結合反應 The lipid mixture (cationic lipid: DPPC: cholesterol: SUNBRIGHT GS-020: DSPE-PEG (2000)-azide = 60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH/10% acetate buffer (25 mM, pH 4.0) to obtain a 14 mg/mL lipid solution. As cationic lipids, the following compounds were used: 3-pentyloctanoic acid 3-((4-(dimethylamino)butyryl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl ester described in WO2016/021683 (hereinafter also referred to as "Compound A"; used for LNPs targeting T cells); or 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyldidecanoate described in WO2020/032184 (hereinafter also referred to as "Compound B"; used for LNPs targeting NK cells). The mRNA encoding the CD19-targeted CAR with CD28 and CD3ε as intracellular signaling domains was dissolved in 10 mM 2-phenoxyethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.3 mg/mL nucleic acid solution. The above lipid solution and nucleic acid solution were mixed at a flow rate ratio of 3 mL/min:6 mL/min (lipid solution:nucleic acid solution) using Nanoassemblr TM (Precision Nanosystems) to obtain a dispersion containing the composition. The above dispersed liquid was dialyzed with water at room temperature for one hour and with PBS at 4°C for 24 hours using Slide-A-Lyzer dialysis (MWCO: 20 k, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using an Amicon Ultra (MWCO: 30K, Merck) and filtered using a 0.2 µm syringe filter. The mRNA concentration was adjusted to a final concentration of 350 µg/mL with PBS containing 20% sucrose and stored at 4°C. 2.3 Conjugation reaction of Fab-DBCO with azide -LNP

將各種Fab-DBCO溶液中之各者與LNP分散液混合，從而使得針對靶向小鼠T細胞之LNP而言，Fab-DBCO與疊氮化物之莫耳濃度為1/20或1/10；對於靶向人類T細胞之LNP為1/100或1/20；且對於靶向人類NK細胞之LNP為1/20。在25℃下進行反應持續24小時且儲存於-80℃下。物理特性評估之結果展示於表2、表3及表4中。 2.4 MAL-LNP 之製備 2.4.1 順丁烯二醯亞胺 -LNP-CD19CAR 之製備 Each of the various Fab-DBCO solutions was mixed with the LNP dispersion so that the molar concentration of Fab-DBCO to azide was 1/20 or 1/10 for LNPs targeting mouse T cells; 1/100 or 1/20 for LNPs targeting human T cells; and 1/20 for LNPs targeting human NK cells. The reaction was carried out at 25°C for 24 hours and stored at -80°C. The results of the physical property evaluation are shown in Tables 2, 3 and 4. 2.4 Preparation of MAL-LNP 2.4.1 Preparation of butylenediimide -LNP-CD19CAR

將脂質之混合物(陽離子型脂質:DPPC:膽固醇:SUNBRIGHT GS-020:DSPE-PEG-順丁烯二醯亞胺(MW 2,000) = 60:10.6:27:1.4:1，莫耳比)溶解於90% EtOH/10%乙酸鹽緩衝液(25 mM，pH 4.0)中以獲得14 mg/mL之脂質溶液。化合物A係用作陽離子型脂質。將編碼具有CD28及CD3ε作為胞內信號轉導域的靶向CD19之CAR的mRNA溶解於10 mM MES緩衝液(pH 5.5)中以獲得0.3 mg/mL之核酸溶液。使用Nanoassemblr TM(Precision Nanosystems)將以上脂質溶液及核酸溶液以3 mL/min:6 mL/min之流動速率混合，以獲得包含組合物之分散液。使用Slide-A-Lyzer透析(MWCO：20 k，Thermo Fisher Scientific)，在室溫下用水透析以上經分散之液體一小時且在4℃下用PBS透析24小時。接著，藉由使用Amicon Ultra (MWCO：30K，Merck)之超濾來濃縮分散液且使用0.2 µm針筒過濾器過濾。用含20%蔗糖之PBS將mRNA濃度調節為350 µg/mL之最終濃度，且儲存於4℃下。 2.4.2 F(ab') 2 之減少 A mixture of lipids (cationic lipids: DPPC: cholesterol: SUNBRIGHT GS-020: DSPE-PEG-cis-butylenediimide (MW 2,000) = 60:10.6:27:1.4:1, molar ratio) was dissolved in 90% EtOH/10% acetate buffer (25 mM, pH 4.0) to obtain a 14 mg/mL lipid solution. Compound A was used as a cationic lipid. mRNA encoding a CD19-targeting CAR having CD28 and CD3ε as intracellular signaling domains was dissolved in 10 mM MES buffer (pH 5.5) to obtain a 0.3 mg/mL nucleic acid solution. The above lipid solution and nucleic acid solution were mixed at a flow rate of 3 mL/min:6 mL/min using Nanoassemblr TM (Precision Nanosystems) to obtain a dispersion containing the composition. The above dispersed liquid was dialyzed against water at room temperature for one hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer dialysis (MWCO: 20 k, Thermo Fisher Scientific). Then, the dispersion was concentrated by ultrafiltration using Amicon Ultra (MWCO: 30K, Merck) and filtered using a 0.2 µm syringe filter. The mRNA concentration was adjusted to a final concentration of 350 µg/mL with PBS containing 20% sucrose and stored at 4°C. 2.4.2 Reduction of F(ab') 2

將抗小鼠CD3 F(ab') 2溶液(Bio X Cell)與PBS及2-巰基乙胺鹽酸鹽(2-MEA)混合以分別將F(ab') 2及2-MEA之濃度調節為1.5 mg/mL及50 mM。混合後，在光屏蔽條件下，於40℃進行反應持續90分鐘。藉由使用Zeba自旋去鹽管柱(MWCO 7K，Thermo Fisher Scientific)重複純化三次而自反應混合物中移除2-MEA，以獲得Fab'溶液。分別藉由在280 nm下之吸光度及與N-(7-二甲基胺基-4-甲基-3-香豆素基)順丁烯二醯亞胺(DACM)之螢光比色反應來測定蛋白質及硫醇基之濃度。所獲得之抗小鼠CD3 Fab'之蛋白質及硫醇基濃度分別測定為1.1 mg/mL及34.3 µM。 2.4.3 Fab' 與順丁烯二醯亞胺 -LNP 之結合反應 Anti-mouse CD3 F(ab') 2 solution (Bio X Cell) was mixed with PBS and 2-methylethylamine hydrochloride (2-MEA) to adjust the concentrations of F(ab') 2 and 2-MEA to 1.5 mg/mL and 50 mM, respectively. After mixing, the reaction was carried out at 40°C for 90 minutes under light shielding conditions. 2-MEA was removed from the reaction mixture by repeated purification three times using a Zeba spin desalination column (MWCO 7K, Thermo Fisher Scientific) to obtain a Fab' solution. The concentrations of protein and thiol groups were determined by absorbance at 280 nm and fluorescent colorimetric reaction with N-(7-dimethylamino-4-methyl-3-coumarinyl) cis-butylenediimide (DACM), respectively. The protein and thiol group concentrations of the anti-mouse CD3 Fab' obtained were determined to be 1.1 mg/mL and 34.3 µM, respectively. 2.4.3 Binding reaction of Fab' with cis-butylene diimide -LNP

將抗小鼠CD3 Fab'溶液與順丁烯二醯亞胺-LNP分散液混合，從而使得還原抗體之莫耳濃度為順丁烯二醯亞胺之莫耳濃度的1/20，且使其在室溫下靜置一小時。此後，使混合物仍在4℃下靜置18小時且儲存於-80℃下。物理特性評估之結果展示於表2中。The anti-mouse CD3 Fab' solution was mixed with the cis-imide-LNP dispersion so that the molar concentration of the reduced antibody was 1/20 of that of cis-imide, and allowed to stand at room temperature for one hour. Thereafter, the mixture was allowed to stand at 4°C for 18 hours and stored at -80°C. The results of the physical property evaluation are shown in Table 2.

靶向小鼠T細胞之LNP之特性Characteristics of LNPs targeting mouse T cells

[表2] 名稱 批次編號 粒徑 (nm) PDI 核酸濃度 (μg/mL) 囊封之百分比(%) 與LNP結合之抗體之濃度(μg/mL) 殘餘抗體 (%) CD3-DBCO-LNP-1 220829YY-11-Y65 110.6 0.17 302 91 187 9 CD3 DBCO-LNP-2 220829YY-11-Y70 94.7 0.11 315 90 363 10 CD3 DBCO-LNP-3 221212MS-69-Y162 85.5 0.05 301 96 343 12 CD3-MAL-LNP 220411AX-01-01 120.2 0.08 202 97 116 31 [Table 2] Name Batch number Particle size (nm) PDI Nucleic acid concentration (μg/mL) Percentage of encapsulation (%) Concentration of antibody bound to LNP (μg/mL) Residual antibodies (%) CD3-DBCO-LNP-1 220829YY-11-Y65 110.6 0.17 302 91 187 9 CD3 DBCO-LNP-2 220829YY-11-Y70 94.7 0.11 315 90 363 10 CD3 DBCO-LNP-3 221212MS-69-Y162 85.5 0.05 301 96 343 12 CD3-MAL-LNP 220411AX-01-01 120.2 0.08 202 97 116 31

靶向人類T細胞之LNP之特性Characteristics of LNPs targeting human T cells

[表3] 名稱 批次編號 粒徑 (nm) PDI 核酸濃度(μg/mL) 囊封之百分比 (%) 與LNP結合之抗體之濃度(μg/mL) 殘餘抗體 (%) CDS DBCO-LNP 220328AX-01-00-YN2 110.8 0.09 318 95 36 13 CDS DBCO-LNP 220328AX-01-00-YN1 93.7 0.18 320 95 184 18 CDS DBCO-LNP 220106AX-01-00-YN6 105.3 0.04 265 98 36 7 CDS DBCO-LNP 220106AX-01-00-YN3 86.1 0.08 227 99 177 13 [Table 3] Name Batch number Particle size (nm) PDI Nucleic acid concentration (μg/mL) Percentage of encapsulation (%) Concentration of antibody bound to LNP (μg/mL) Residual antibodies (%) CDS DBCO-LNP 220328AX-01-00-YN2 110.8 0.09 318 95 36 13 CDS DBCO-LNP 220328AX-01-00-YN1 93.7 0.18 320 95 184 18 CDS DBCO-LNP 220106AX-01-00-YN6 105.3 0.04 265 98 36 7 CDS DBCO-LNP 220106AX-01-00-YN3 86.1 0.08 227 99 177 13

靶向人類NK細胞之LNP之特性Characteristics of LNPs targeting human NK cells

[表4] 名稱 批次編號 粒徑 (nm) PDI 核酸濃度 (μg/mL) 囊封之百分比(%) 與LNP結合之抗體之濃度(μg/mL) 殘餘抗體 (%) CD16 DBCO-LNP 220801YD-8-Y27 118.5 0.04 245 98 188 9 CD56 DBCO-LNP 220801YD-8-Y28 129.1 0.14 240 94 245 9 CD7 DBCO-LNP 220801YD-8-Y30 113.0 0.06 238 97 184 14 CD7 DBCO-LNP 221003YY-43-Y130 106.7 0.08 294 95 176 17 CD16 DBCO-LNP 220906YY-23-Y81 106.3 0.05 386 96 192 11 [Table 4] Name Batch number Particle size (nm) PDI Nucleic acid concentration (μg/mL) Percentage of encapsulation (%) Concentration of antibody bound to LNP (μg/mL) Residual antibodies (%) CD16 DBCO-LNP 220801YD-8-Y27 118.5 0.04 245 98 188 9 CD56 DBCO-LNP 220801YD-8-Y28 129.1 0.14 240 94 245 9 CD7 DBCO-LNP 220801YD-8-Y30 113.0 0.06 238 97 184 14 CD7 DBCO-LNP 221003YY-43-Y130 106.7 0.08 294 95 176 17 CD16 DBCO-LNP 220906YY-23-Y81 106.3 0.05 386 96 192 11

3.3. 使用靶向小鼠Using targeted mice TT 細胞之Cell LNPLNP 的小鼠Mouse TT 細胞中之活體外In vitro in cells CARCAR 表現。Performance. 3.13.1 實驗設計Experimental design

自C57BL/6NJcl小鼠收穫脾臟且使用ACK裂解緩衝液(Lonza) 裂解紅血球以獲得小鼠脾細胞。將小鼠脾細胞用於測試CD3-MAL-LNP且將自小鼠脾細胞中分離之T細胞用於測試CD3-DBCO-LNP。將所獲得之細胞分散於補充有10% FBS、1%青黴素-鏈黴素、50 µM 2-巰基乙醇、25 mM HEPES及100 U/mL rhIL-2之RPMI 1640培養基中。將細胞以3 × 10 6個細胞/孔接種至24孔盤中。向孔中添加LNP以使得mRNA之最終濃度為3 µg/mL。在37℃下培養細胞48小時。用標記物之經標記之抗體試劑處理細胞(CD45、活/死、CD90.2、B220、CD4、CD8、CD19-PE)且使用LSRFortessa流式細胞儀(BD Biosciences)分析CD19CAR陽性細胞在CD90.2陽性T細胞中之比率。表5展示CAR陽性細胞之比率。 Spleens were harvested from C57BL/6NJcl mice and red blood cells were lysed using ACK lysis buffer (Lonza) to obtain mouse spleen cells. Mouse spleen cells were used to test CD3-MAL-LNP and T cells isolated from mouse spleen cells were used to test CD3-DBCO-LNP. The obtained cells were dispersed in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 50 µM 2-hydroxyethanol, 25 mM HEPES and 100 U/mL rhIL-2. The cells were seeded into a 24-well plate at 3 × 10 6 cells/well. LNPs were added to the wells to give a final concentration of 3 µg/mL of mRNA. Cells were cultured at 37°C for 48 hours. Cells were treated with labeled antibody reagents of markers (CD45, live/dead, CD90.2, B220, CD4, CD8, CD19-PE) and the ratio of CD19CAR-positive cells in CD90.2-positive T cells was analyzed using an LSRFortessa flow cytometer (BD Biosciences). Table 5 shows the ratio of CAR-positive cells.

3.23.2 結果result

如表5中所示，與CD3-MAL-LNP相比，CD3-DBCO-LNP在活體外展示更高之CAR表現。As shown in Table 5, CD3-DBCO-LNPs displayed higher CAR expression in vitro compared to CD3-MAL-LNPs.

小鼠脾臟T細胞中之活體外CD19CAR陽性細胞之比率The ratio of CD19CAR positive cells in mouse spleen T cells in vitro

[表5] 名稱 批次編號 CAR陽性T細胞(%) CD3-DBCO-LNP-1 220829YY-1LY65 37.7 CD3-DBCO-LNP-2 220829YY-11-Y70 49.7 CD3-MAL-LNP 220411AXBL01 4.5 [Table 5] Name Batch number CAR-positive T cells (%) CD3-DBCO-LNP-1 220829YY-1LY65 37.7 CD3-DBCO-LNP-2 220829YY-11-Y70 49.7 CD3-MAL-LNP 220411AXBL01 4.5

4.4. 動物實驗Animal experiments (1)(1) 使用靶向小鼠Using targeted mice TT 細胞之Cell LNPLNP 的小鼠Mouse TT 細胞中之活體內In vivo in cells CARCAR 表現Performance 4.14.1 實驗設計Experimental design

將含有0.375 mg/kg或1 mg/kg RNA之10 mL/kg LNP以24小時之間隔投與至C57BL/6NJcl小鼠之尾部靜脈中兩次。在投與後收穫脾臟。在自脾臟中分離免疫細胞後，用標記物之經標記之抗體試劑處理細胞(CD45、CD90.2、CD4、CD8、活/死、CD19-PE)且使用LSRFortessa流式細胞儀(BD Biosciences)分析CD19CAR陽性細胞在CD90.2陽性T細胞中之比率。表6展示CAR陽性細胞之比率。 4.2 結果 10 mL/kg LNP containing 0.375 mg/kg or 1 mg/kg RNA was administered twice into the tail vein of C57BL/6NJcl mice at 24-hour intervals. Spleens were harvested after administration. After isolating immune cells from the spleen, cells were treated with labeled antibody reagents of markers (CD45, CD90.2, CD4, CD8, live/dead, CD19-PE) and the ratio of CD19CAR-positive cells in CD90.2-positive T cells was analyzed using LSRFortessa flow cytometer (BD Biosciences). Table 6 shows the ratio of CAR-positive cells. 4.2 Results

與CD3-MAL-LNP相比，CD3-DBCO-LNP展示更高之CAR表現，儘管前者之劑量低於後者。Compared with CD3-MAL-LNP, CD3-DBCO-LNP showed higher CAR expression, although the former was administered at a lower dose than the latter.

小鼠脾臟T細胞中之活體內CD19CAR陽性細胞之比率The ratio of CD19CAR positive cells in mouse spleen T cells in vivo

[表6] 名稱 批次編號 劑量 CAR陽性T細胞(%) CD3-DBCO-LNP-3 221212MS-69-Y162 0375 mg/kg 11.9 CD3-MAL-LNP 220411AX-01-01 1 mgZkg 2.0 [Table 6] Name Batch number Dosage CAR-positive T cells (%) CD3-DBCO-LNP-3 221212MS-69-Y162 0375 mg/kg 11.9 CD3-MAL-LNP 220411AX-01-01 1 mgZkg 2.0

5.5. 用經培養之細胞進行之實驗Experiments with cultured cells (1)(1) 使用靶向人類Use targeted humans TT 細胞之Cell LNPLNP 的of 人類Human TT 細胞中之活體外In vitro in cells CARCAR 表現Performance 5.15.1 實驗設計Experimental design

使用EasySep™人類T細胞分離套組(STEMCELL Technologies)自PBMC (Hemacare)中分離T細胞。將T細胞分散於補充有IL-2 (10 ng/mL，Miltenyi)之X-VIVO15 (Lonza)培養基中。在將T細胞以1 × 10 6個細胞/孔接種至24孔盤後，添加包含LNP之培養基以使得最終mRNA濃度為2 µg/mL。在37℃下培養72小時後，用標記物之經標記之抗體試劑處理細胞(CD45、活/死、CD3、CD8、CD4、CD19-PE)且使用LSRFortessa流式細胞儀(BD Biosciences)分析CAR陽性細胞在CD3陽性T細胞、CD4陽性T細胞及CD8陽性T細胞中之比率。表7展示CAR陽性細胞之比率。 T cells were isolated from PBMC (Hemacare) using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies). T cells were dispersed in X-VIVO15 (Lonza) medium supplemented with IL-2 (10 ng/mL, Miltenyi). After T cells were seeded at 1 × 10 6 cells/well in a 24-well plate, medium containing LNPs was added to give a final mRNA concentration of 2 µg/mL. After culturing at 37°C for 72 hours, cells were treated with labeled antibody reagents of markers (CD45, live/dead, CD3, CD8, CD4, CD19-PE) and the ratio of CAR-positive cells among CD3-positive T cells, CD4-positive T cells, and CD8-positive T cells was analyzed using LSRFortessa flow cytometer (BD Biosciences). Table 7 shows the ratio of CAR-positive cells.

5.2 結果CD3-DBCO-LNP在所有CD3陽性T細胞、CD4陽性T細胞及CD8陽性T細胞中誘導高CAR表現。CD8-DBCO-LNP在CD8陽性T細胞中選擇性地誘導CAR表現，此表明CAR表現對標靶T細胞具有特異性。 5.2 Results CD3-DBCO-LNP induced high CAR expression in all CD3-positive T cells, CD4-positive T cells, and CD8-positive T cells. CD8-DBCO-LNP selectively induced CAR expression in CD8-positive T cells, indicating that CAR expression was specific to the target T cells.

人類T細胞中之活體外CD19 CAR陽性細胞之比率The ratio of CD19 CAR-positive cells in human T cells in vitro

[表7] 名稱 批次編號 CD3陽性T細胞中之CAR陽性細胞(%) CD4陽性T細胞中之CAR陽性細胞(%) CD8陽性T細胞中之CAR陽性細胞(%) CD3-DBCO-LNP 220328AX-01-00-YN2 90.1 90.0 83.9 CD8-DBCO-LNP 220328AX-01-00-YN1 17.6 1.0 82.7 [Table 7] Name Batch number CAR-positive cells among CD3-positive T cells (%) CAR-positive cells among CD4-positive T cells (%) CAR-positive cells among CD8-positive T cells (%) CD3-DBCO-LNP 220328AX-01-00-YN2 90.1 90.0 83.9 CD8-DBCO-LNP 220328AX-01-00-YN1 17.6 1.0 82.7

5.25.2 使用靶向人類Use targeted humans NKNK 細胞之Cell LNPLNP 的人類Human NKNK 細胞中之活體外In vitro in cells CARCAR 表現Performance 5.2.15.2.1 實驗設計Experimental design

使用EasySep™人類NK細胞分離套組(STEMCELL Technologies)自PBMC (Hemacare)中分離NK細胞。在補充有5% AB血清(Sigma-Aldrich)、167 ng/mL人類IL-2 IS (Miltenyi)及28 ng/mL人類IL-15 (Miltenyi)之NK MACS ®培養基(Miltenyi)中培養NK細胞。在將T細胞以1 × 10 6個細胞/孔接種至24孔盤後，添加包含LNP之培養基以使得最終mRNA濃度為2 µg/mL。在添加LNP後的3天，用標記物之經標記之抗體試劑處理細胞且使用LSRFortessa流式細胞儀(BD Biosciences)分析CAR陽性細胞在CD56陽性NK細胞中之比率。表8展示CAR陽性細胞之比率。 5.2.2 結果 NK cells were isolated from PBMC (Hemacare) using the EasySep™ Human NK Cell Isolation Kit (STEMCELL Technologies). NK cells were cultured in NK MACS ® medium (Miltenyi) supplemented with 5% AB serum (Sigma-Aldrich), 167 ng/mL human IL-2 IS (Miltenyi), and 28 ng/mL human IL-15 (Miltenyi). After T cells were seeded at 1 × 10 6 cells/well in a 24-well plate, medium containing LNPs was added to give a final mRNA concentration of 2 µg/mL. Three days after the addition of LNPs, cells were treated with labeled antibody reagents and the ratio of CAR-positive cells in CD56-positive NK cells was analyzed using LSRFortessa flow cytometer (BD Biosciences). Table 8 shows the ratio of CAR-positive cells. 5.2.2 Results

所有靶向人類NK細胞之LNP皆在NK細胞中誘導CAR表現。All LNPs targeting human NK cells induced CAR expression in NK cells.

人類NK細胞中之活體外CD19CAR陽性細胞之比率The ratio of CD19CAR-positive cells in human NK cells in vitro

[表8] 名稱 批次編號 CD56陽性NK細胞中之CAR陽性細胞之比率 CD16-DBCO-LNP 220801YD-8-Y27 14.4 CD56-DBCO-LNP 220801YD-8-Y28 42.4 CD7-DBCO-LNP 220801YD-8-Y30 65.1 [Table 8] Name Batch number The ratio of CAR-positive cells in CD56-positive NK cells CD16-DBCO-LNP 220801YD-8-Y27 14.4 CD56-DBCO-LNP 220801YD-8-Y28 42.4 CD7-DBCO-LNP 220801YD-8-Y30 65.1

6.  動物實驗(2) 6.1 使用靶向人類 T 細胞之 LNP 的人類 T 細胞中之活體內 CAR 表現 6.1.1 實驗設計 6. Animal Experiments (2) 6.1 In vivo CAR Expression in Human T Cells Using LNPs Targeting Human T Cells 6.1.1 Experimental Design

使用EasySep™人類T細胞分離套組(STEMCELL Technologies)自人類PBMC (Hemacare)中分離T細胞。在37℃下在5% CO 2氛圍中在補充有人類IL-2 IS (Miltenyi)及T Cell TransAct (Miltenyi)之TheraPEAK™ X-VIVO™ 15無血清造血細胞培養基(Lonza)中培養T細胞四天。接著，將T細胞懸浮於PBS中且向NSG小鼠(The Jackson Laboratory)植入1 × 10 7個細胞/小鼠之細胞。以0.375 mg/kg之劑量將LNP反覆投與至尾部靜脈中。在投與LNP後的48小時，收穫肺。使用Dri腫瘤及組織解離試劑(Dri Tumor & Tissue Dissociation Reagent) (BD)分離細胞，用標記物之經標記之抗體試劑處理細胞(CD45、活/死、CD3、CD4、CD8)且使用LSRFortessa流式細胞儀(BD Biosciences)分析CD19CAR陽性細胞在T細胞、CD4陽性T細胞及CD8陽性T細胞中之比率。表9展示CAR陽性細胞之比率。 6.1.2 結果 T cells were isolated from human PBMC (Hemacare) using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies). T cells were cultured in TheraPEAK™ X-VIVO™ 15 serum-free hematopoietic cell culture medium (Lonza) supplemented with human IL-2 IS (Miltenyi) and T Cell TransAct (Miltenyi) at 37°C in a 5% CO2 atmosphere for four days. T cells were then suspended in PBS and implanted into NSG mice (The Jackson Laboratory) at 1 × 107 cells/mouse. LNPs were repeatedly administered into the tail vein at a dose of 0.375 mg/kg. Lungs were harvested 48 hours after LNP administration. Cells were isolated using Dri Tumor & Tissue Dissociation Reagent (BD), treated with labeled antibody reagents of markers (CD45, live/dead, CD3, CD4, CD8), and the ratio of CD19CAR-positive cells in T cells, CD4-positive T cells, and CD8-positive T cells was analyzed using LSRFortessa flow cytometer (BD Biosciences). Table 9 shows the ratio of CAR-positive cells. 6.1.2 Results

CD3-DBCO-LNP在所有CD3陽性T細胞、CD4陽性T細胞及CD8陽性T細胞中誘導高CAR表現。CD8-DBCO-LNP在CD8陽性T細胞中選擇性地誘導CAR表現，此表明CAR表現對標靶T細胞具有特異性。CD3-DBCO-LNP induced high CAR expression in all CD3-positive T cells, CD4-positive T cells, and CD8-positive T cells. CD8-DBCO-LNP selectively induced CAR expression in CD8-positive T cells, indicating that CAR expression was specific to the target T cells.

自肺中分離之人類T細胞中之活體內CD19CAR陽性細胞之比率The ratio of CD19CAR-positive cells in vivo in human T cells isolated from lung

[表9] 名稱 批次編號 CD3陽性T細胞中之CAR陽性細胞(%) CD4陽性T細胞中之CAR陽性細胞(%) CD8陽性T細胞中之CAR陽性細胞(%) CD3-DBCO-LNP 220106AX-01-00-YN6 78.9 92.2 73.7 CD8-DBCO-LNP 220106AX-01-00-YN3 15.0 12.6 61.1 [Table 9] Name Batch number CAR-positive cells among CD3-positive T cells (%) CAR-positive cells among CD4-positive T cells (%) CAR-positive cells among CD8-positive T cells (%) CD3-DBCO-LNP 220106AX-01-00-YN6 78.9 92.2 73.7 CD8-DBCO-LNP 220106AX-01-00-YN3 15.0 12.6 61.1

6.26.2 使用靶向人類Use targeted humans NKNK 細胞之Cell LNPLNP 的人類Human NKNK 細胞中之活體內In vivo in cells CARCAR 表現Performance 6.2.16.2.1 實驗設計Experimental design

使用EasySep™人類NK細胞分離套組(STEMCELL Technologies)自人類PBMC (Hemacare)中分離NK細胞。在補充有5% AB血清(Sigma-Aldrich)、167 ng/mL人類IL-2 IS (Miltenyi)及28 ng/mL人類IL-15 (Miltenyi)之NK MACS ®培養基(Miltenyi)中培養細胞。接著，將細胞懸浮於PBS中且向NOG-hIL15-Tg小鼠(In-Vivo Science Inc.)植入1 × 10 7個細胞/小鼠之細胞。以0.8 mg/kg之劑量將LNP反覆投與至尾部靜脈中。在投與LNP後的48小時，收穫肺。使用Dri腫瘤及組織解離試劑(BD)分離細胞，且用標記物之經標記之抗體試劑處理細胞(CD45、活/死、CD56、CD19CAR)且使用LSRFortessa流式細胞儀(BD Biosciences)分析CD19CAR陽性細胞在CD56陽性NK細胞中之比率。表9展示CAR陽性細胞之比率。表10展示CAR陽性細胞之比率。 NK cells were isolated from human PBMC (Hemacare) using the EasySep™ Human NK Cell Isolation Kit (STEMCELL Technologies). Cells were cultured in NK MACS® medium (Miltenyi) supplemented with 5% AB serum (Sigma-Aldrich), 167 ng/mL human IL-2 IS (Miltenyi), and 28 ng/mL human IL-15 (Miltenyi). Cells were then suspended in PBS and implanted into NOG-hIL15-Tg mice (In-Vivo Science Inc.) at 1 × 10 7 cells/mouse. LNPs were repeatedly administered into the tail vein at a dose of 0.8 mg/kg. Lungs were harvested 48 hours after LNP administration. Cells were isolated using Dri tumor and tissue dissociation reagent (BD), and cells were treated with labeled antibody reagents of markers (CD45, live/dead, CD56, CD19CAR) and the ratio of CD19CAR-positive cells in CD56-positive NK cells was analyzed using LSRFortessa flow cytometer (BD Biosciences). Table 9 shows the ratio of CAR-positive cells. Table 10 shows the ratio of CAR-positive cells.

6.2.26.2.2 結果result

所有靶向人類NK細胞之LNP皆在NK細胞中誘導CAR表現。All LNPs targeting human NK cells induced CAR expression in NK cells.

自肺中分離之人類NK細胞中之活體內CD19CAR陽性細胞之比率In vivo ratio of CD19CAR-positive cells in human NK cells isolated from lung

[表10] 名稱 批次編號 CD56陽性NK細胞中之CAR陽性細胞(%) CD16-DBCO-LNP 220801YD-8-Y27 71.9 CD56-DBCO-LNP 220801YD-8-Y28 32.4 CD7-DBCO-LNP 220801YD-8-Y30 81.8 [Table 10] Name Batch number CAR-positive cells among CD56-positive NK cells (%) CD16-DBCO-LNP 220801YD-8-Y27 71.9 CD56-DBCO-LNP 220801YD-8-Y28 32.4 CD7-DBCO-LNP 220801YD-8-Y30 81.8

7.7. 動物實驗Animal experiments (3)(3) 7.17.1 攜帶腫瘤之小鼠中靶向人類Targeting humans in tumor-bearing mice TT 細胞之Cell LNPLNP 的功效Benefits 7.1.17.1.1 實驗設計Experimental design

將NALM6-Luc腫瘤細胞懸浮於PBS中且以0.5 × 10 6個細胞/小鼠自NSG小鼠(The Jackson Laboratory)之尾部靜脈中植入。腫瘤植入後的兩天，在腹部投與螢光素後使用活體內成像儀器(IVIS LUMINA II)藉由發光成像來測定腫瘤體積。根據腫瘤體積，使用EXSUS將攜帶腫瘤之小鼠分成五隻動物之小組，且將T細胞植入動物中。自T細胞植入之第3天，將LNP在室溫下解凍，藉由用PBS、水或PBS-20%蔗糖稀釋至預定濃度來製備且以0.375 mg/kg之劑量反覆地投與至尾部靜脈中。在第一次投與後的九天，使用IVIS測定腫瘤體積。表11展示使用發光強度作為指標之腫瘤體積測定之結果。 7.1.2 結果 NALM6-Luc tumor cells were suspended in PBS and implanted at 0.5 × 10 6 cells/mouse from the tail vein of NSG mice (The Jackson Laboratory). Two days after tumor implantation, tumor volume was determined by luminescence imaging using an in vivo imaging instrument (IVIS LUMINA II) after abdominal administration of luciferin. Based on tumor volume, tumor-bearing mice were divided into groups of five animals using EXSUS, and T cells were implanted into the animals. On day 3 from T cell implantation, LNPs were thawed at room temperature, prepared by diluting with PBS, water, or PBS-20% sucrose to a predetermined concentration, and repeatedly administered into the tail vein at a dose of 0.375 mg/kg. Nine days after the first administration, the tumor volume was measured using IVIS. Table 11 shows the results of tumor volume measurement using luminescence intensity as an indicator. 7.1.2 Results

據證實，CD3-DBCO-LNP及CD8-DBCO-LNP具有抗腫瘤作用。It has been demonstrated that CD3-DBCO-LNP and CD8-DBCO-LNP have anti-tumor effects.

使用發光強度作為指標測定NALM6-Luc小鼠中之腫瘤體積(平均值±標準誤差)Tumor volume in NALM6-Luc mice measured using luminescence intensity as an indicator (mean ± SD)

[表11] 名稱 批次編號 在第一次LNP投與後的9天 (總通量(p/s)) 對照 - 7.2x10 8± 2.3x10 8 CD3-DBCO-LNP 220328AX-01-00-YN2 4.0x10 6± 5.0x10 6 CD8-DBCO-LNP 220328AX-01-00-YN1 2.3x10 7± 1.8x10 7 [Table 11] Name Batch number 9 days after the first LNP administration (total flux (p/s)) Comparison - 7.2x10 8 ± 2.3x10 8 CD3-DBCO-LNP 220328AX-01-00-YN2 4.0x10 6 ± 5.0x10 6 CD8-DBCO-LNP 220328AX-01-00-YN1 2.3x10 7 ± 1.8x10 7

7.27.2 攜帶腫瘤之小鼠中靶向人類Targeting humans in tumor-bearing mice NKNK 細胞之Cell LNPLNP 的功效Benefits 7.2.17.2.1 實驗設計Experimental design

將表現螢光素酶之NALM6-Luc腫瘤細胞懸浮於PBS中且以0.5 × 10 6個細胞/小鼠自NOG-hIL-15 Tg小鼠(InVivo science)之尾靜脈植入。移植腫瘤後，腹部投與螢光素且接著使用活體內成像儀器(IVIS LUMINA II)藉由發光成像來測定腫瘤體積。根據腫瘤體積，使用分配軟體將攜帶腫瘤之小鼠分成五隻動物之小組，且將NK細胞植入動物中。在LNP投與當天，將LNP在室溫下解凍，藉由用PBS、水或PBS-20%蔗糖稀釋至預定濃度來製備且以0.375 mg/kg或0.8 mg/kg之劑量反覆地投與至尾部靜脈中。表12及表13展示使用發光強度作為指標之腫瘤體積測定之結果。 7.2.2 結果 NALM6-Luc tumor cells expressing luciferase were suspended in PBS and implanted from the tail vein of NOG-hIL-15 Tg mice (InVivo science) at 0.5 × 10 6 cells/mouse. After tumor implantation, luciferin was administered abdominally and then tumor volume was determined by luminescence imaging using an in vivo imaging instrument (IVIS LUMINA II). Tumor-bearing mice were divided into groups of five animals using a distribution software according to tumor volume, and NK cells were implanted into the animals. On the day of LNP administration, LNP was thawed at room temperature, prepared by diluting with PBS, water or PBS-20% sucrose to a predetermined concentration and repeatedly administered into the tail vein at a dose of 0.375 mg/kg or 0.8 mg/kg. Tables 12 and 13 show the results of tumor volume determination using luminescence intensity as an indicator. 7.2.2 Results

據證實，CD16-DBCO-LNP及CD7-DBCO-LNP具有抗腫瘤作用。It has been demonstrated that CD16-DBCO-LNP and CD7-DBCO-LNP have anti-tumor effects.

使用發光強度作為指標測定投與CD16-DBCO-LNP之動物中之腫瘤體積(平均值±標準誤差)Tumor volume in animals administered with CD16-DBCO-LNP was measured using luminescence intensity as an indicator (mean ± SD)

[表12] 名稱 批次編號 在第一次LNP投與後的20天 (總通量(p/s)) 對照 - 8.4x10 9± 2.7x10 9 CD16-DBCO-LNP 220906YY-23-Y81 3.8x10 9± 9.4x10 8 [Table 12] Name Batch number 20 days after the first LNP administration (Total flux (p/s)) Comparison - 8.4x10 9 ± 2.7x10 9 CD16-DBCO-LNP 220906YY-23-Y81 3.8x10 9 ± 9.4x10 8

使用發光強度作為指標測定投與CD7-DBCO-LNP之動物中之腫瘤體積(平均值±標準誤差)Tumor volume in animals administered with CD7-DBCO-LNP was measured using luminescence intensity as an indicator (mean ± SD)

[表13] 名稱 批次編號 在第一次LNP投與後的14天 (總通量(p/s)) 對照 - 3.5x10 9± 9 2x10 8 CD7-DBCOLNP 221003YY-43-Y130 2.0x10 8± 1.5x10 8 [Table 13] Name Batch number 14 days after the first LNP administration (total flux (p/s)) Comparison - 3.5x10 9 ± 9 2x10 8 CD7-DBCOLNP 221003YY-43-Y130 2.0x10 8 ± 1.5x10 8

如本說明書及所附申請專利範圍中所使用，除非上下文另外清晰地指示，否則諸如「一(a)」、「一(an)」及「該(the)」之單數冠詞可指單一對象或複數個對象。因此，舉例而言，提及含有「化合物」之組合物可包括單一化合物或兩種或更多種化合物。以上描述意欲為說明性而非限制性的。熟習此項技術者在閱讀以上描述後將顯而易知許多實施例。因此，本發明之範疇應參考所附申請專利範圍來判定且包括此申請專利範圍所授權之等效物的完整範疇。本揭露中所引用之所有文章及參考文獻的揭示內容(包括專利、專利申請案及公開案)皆以全文引用之方式併入本文中且用於所有目的。 [序列表] 093495_seq.xml As used in this specification and the appended claims, singular articles such as "a", "an", and "the" may refer to a single object or plural objects unless the context clearly indicates otherwise. Thus, for example, a reference to a composition containing a "compound" may include a single compound or two or more compounds. The above description is intended to be illustrative and not limiting. Many embodiments will become apparent to those skilled in the art after reading the above description. Therefore, the scope of the invention should be determined with reference to the appended claims and includes the full scope of equivalents authorized by the claims. The disclosures of all articles and references cited in this disclosure (including patents, patent applications, and publications) are incorporated herein by reference in their entirety and for all purposes. [Sequence Listing] 093495_seq.xml

TW202449142A_113112850_SEQL.xmlTW202449142A_113112850_SEQL.xml

Claims (63)

一種用於遞送活性物質之複合物，其包含用於該活性物質之遞送媒介及對靶細胞具有特異性之配體， 該配體係被添加至該遞送媒介之外表面， 其中該遞送媒介包含錨分子， 第一結合搭配物係以1至4個該第一結合搭配物分子/一個該配體分子之比率與該配體共價鍵結； 第二結合搭配物與該錨分子共價鍵結，由此將該第二結合搭配物固定至該遞送媒介；及 該第一結合搭配物與該第二結合搭配物共價鍵結。 A complex for delivering an active substance, comprising a delivery medium for the active substance and a ligand specific to a target cell, wherein the ligand is added to the outer surface of the delivery medium, wherein the delivery medium comprises an anchor molecule, wherein a first binding partner is covalently bonded to the ligand at a ratio of 1 to 4 first binding partner molecules per one ligand molecule; a second binding partner is covalently bonded to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner is covalently bonded to the second binding partner. 一種用於遞送活性物質之複合物，其包含用於該活性物質之遞送媒介及對靶細胞具有特異性之配體， 該配體被添加至該遞送媒介之外表面， 其中該遞送媒介包含錨分子， 第一結合搭配物與該配體之C端共價鍵結； 第二結合搭配物與該錨分子共價鍵結，由此將該第二結合搭配物固定至該遞送媒介；及 該第一結合搭配物與該第二結合搭配物共價鍵結。 A complex for delivering an active substance, comprising a delivery medium for the active substance and a ligand specific to a target cell, the ligand being added to the outer surface of the delivery medium, wherein the delivery medium comprises an anchor molecule, a first binding partner covalently bonds to the C-terminus of the ligand; a second binding partner covalently bonds to the anchor molecule, thereby fixing the second binding partner to the delivery medium; and the first binding partner covalently bonds to the second binding partner. 如請求項1或2之複合物，其中用於該活性物質之該遞送媒介為脂質奈米粒子或脂質體。The complex of claim 1 or 2, wherein the delivery vehicle for the active substance is lipid nanoparticles or liposomes. 如請求項3之複合物，其中該第一結合搭配物為包含親偶極物之分子，且其中該第二結合搭配物為包含1,3-偶極子之分子。The complex of claim 3, wherein the first binding partner is a molecule comprising an exopole, and wherein the second binding partner is a molecule comprising a 1,3-exopole. 如請求項4之複合物，其中該第一結合搭配物為包含二苯并環辛炔(dibenzocyclooctyne；DBCO)基之分子。The complex of claim 4, wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group. 如請求項4之複合物，其中該第二結合搭配物為包含疊氮基之分子。The complex of claim 4, wherein the second binding partner is a molecule comprising an azido group. 如請求項1之複合物，其中該活性物質所遞送至其中之該靶細胞為免疫細胞。The complex of claim 1, wherein the target cell to which the active substance is delivered is an immune cell. 如請求項7之複合物，其中該免疫細胞為細胞毒性細胞。The complex of claim 7, wherein the immune cell is a cytotoxic cell. 如請求項8之複合物，其中該細胞毒性細胞為NK細胞或T細胞。The complex of claim 8, wherein the cytotoxic cell is a NK cell or a T cell. 如請求項8之複合物，其中對該細胞毒性細胞具有特異性之該配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD7、CD16、CD8及CD56。The complex of claim 8, wherein the ligand specific for the cytotoxic cell comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of CD3, CD7, CD16, CD8 and CD56. 如請求項1之複合物，其中該活性物質為核酸。The complex of claim 1, wherein the active substance is a nucleic acid. 如請求項11之複合物，其中該核酸包含編碼嵌合抗原受體(chimeric antigen receptor；CAR)及/或T細胞受體(T cell receptor；TCR)之核酸。The complex of claim 11, wherein the nucleic acid comprises a nucleic acid encoding a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). 一種藥劑，其包含如請求項12之複合物。A medicament comprising the complex of claim 12. 一種用於產生如請求項1之複合物的方法，該方法包含： (1)    使該第一結合搭配物與該配體以1至4個該第一結合搭配物分子/一個該配體分子之比率共價鍵結之步驟； (2)    使該第二結合搭配物與該錨分子共價鍵結之步驟；及 (3)    藉由化學反應使該第一結合搭配物與該第二結合搭配物以一個該第二結合搭配物分子/一個該第一結合搭配物分子之比率鍵結之步驟。 A method for producing a complex as claimed in claim 1, the method comprising: (1)    a step of covalently bonding the first binding partner to the ligand at a ratio of 1 to 4 molecules of the first binding partner/one molecule of the ligand; (2)    a step of covalently bonding the second binding partner to the anchor molecule; and (3)    a step of bonding the first binding partner to the second binding partner at a ratio of one molecule of the second binding partner/one molecule of the first binding partner by a chemical reaction. 如請求項14之方法，其中用於該活性物質之該遞送媒介為脂質奈米粒子或脂質體。The method of claim 14, wherein the delivery vehicle for the active substance is lipid nanoparticles or liposomes. 如請求項15之方法，其中該第一結合搭配物為包含親偶極物之分子，且其中該第二結合搭配物為包含1,3-偶極子之分子。The method of claim 15, wherein the first binding partner is a molecule comprising an exopole, and wherein the second binding partner is a molecule comprising a 1,3-exopole. 如請求項16之方法，其中該第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。The method of claim 16, wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group. 如請求項16之方法，其中該第二結合搭配物為包含疊氮基之分子。The method of claim 16, wherein the second binding partner is a molecule comprising an azido group. 如請求項14之方法，其中該活性物質所遞送至其中之該靶細胞為免疫細胞。The method of claim 14, wherein the target cell to which the active substance is delivered is an immune cell. 如請求項19之方法，其中該免疫細胞為細胞毒性細胞。The method of claim 19, wherein the immune cell is a cytotoxic cell. 如請求項20之方法，其中該細胞毒性細胞為NK細胞或T細胞。The method of claim 20, wherein the cytotoxic cell is a NK cell or a T cell. 如請求項20之方法，其中對該細胞毒性細胞具有特異性之該配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD7、CD16、CD8及CD56。The method of claim 20, wherein the ligand specific for the cytotoxic cell comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD7, CD16, CD8 and CD56. 如請求項14之方法，其中該活性物質為核酸。The method of claim 14, wherein the active substance is a nucleic acid. 一種用於將活性物質遞送至靶細胞之組合物，其包含如請求項1之複合物。A composition for delivering an active substance to a target cell, comprising the complex of claim 1. 如請求項24之組合物，其中用於該活性物質之該遞送媒介為脂質奈米粒子或脂質體。The composition of claim 24, wherein the delivery vehicle for the active substance is lipid nanoparticles or liposomes. 如請求項25之組合物，其中該第一結合搭配物為包含親偶極物之分子，且其中該第二結合搭配物為包含1,3-偶極子之分子。The composition of claim 25, wherein the first binding partner is a molecule comprising an exopole, and wherein the second binding partner is a molecule comprising a 1,3-exopole. 如請求項26之組合物，其中該第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。The composition of claim 26, wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group. 如請求項26之組合物，其中該第二結合搭配物為包含疊氮基之分子。The composition of claim 26, wherein the second binding partner is a molecule comprising an azido group. 如請求項24之組合物，其中將該活性物質遞送至其中之該靶細胞為免疫細胞。The composition of claim 24, wherein the target cell to which the active substance is delivered is an immune cell. 如請求項29之組合物，其中該免疫細胞為細胞毒性細胞。The composition of claim 29, wherein the immune cell is a cytotoxic cell. 如請求項30之組合物，其中該細胞毒性細胞為NK細胞或T細胞。The composition of claim 30, wherein the cytotoxic cell is a NK cell or a T cell. 如請求項30之組合物，其中對該細胞毒性細胞具有特異性之該配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD7、CD16、CD8及CD56。The composition of claim 30, wherein the ligand specific for the cytotoxic cell comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD7, CD16, CD8 and CD56. 如請求項24之組合物，其中該活性物質為核酸。The composition of claim 24, wherein the active substance is a nucleic acid. 如請求項33之組合物，其進一步包含培養基、鹽水或緩衝液。The composition of claim 33, further comprising a culture medium, saline or a buffer solution. 一種用於使靶細胞活化及/或增殖之方法，該方法包含： 使如請求項1之複合物與包含該靶細胞之細胞群體接觸之步驟，其中該複合物包含至少一種對該靶細胞具有特異性之配體。 A method for activating and/or proliferating a target cell, the method comprising: A step of contacting the complex of claim 1 with a cell population comprising the target cell, wherein the complex comprises at least one ligand specific to the target cell. 如請求項35之方法，其中用於該活性物質之該遞送媒介為脂質奈米粒子或脂質體。The method of claim 35, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome. 如請求項36之方法，其中該第一結合搭配物為包含親偶極物之分子，且其中該第二結合搭配物為包含1,3-偶極子之分子。The method of claim 36, wherein the first binding partner is a molecule comprising an exopole, and wherein the second binding partner is a molecule comprising a 1,3-exopole. 如請求項37之方法，其中該第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。The method of claim 37, wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group. 如請求項37之方法，其中該第二結合搭配物為包含疊氮基之分子。The method of claim 37, wherein the second binding partner is a molecule comprising an azido group. 如請求項35之方法，其中該活性物質所遞送至其中之該靶細胞為免疫細胞。The method of claim 35, wherein the target cell to which the active substance is delivered is an immune cell. 如請求項40之方法，其中該免疫細胞為細胞毒性細胞。The method of claim 40, wherein the immune cell is a cytotoxic cell. 如請求項41之方法，其中該細胞毒性細胞為NK細胞或T細胞。The method of claim 41, wherein the cytotoxic cell is a NK cell or a T cell. 如請求項41之方法，其中對該細胞毒性細胞具有特異性之該配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD7、CD16、CD8及CD56。The method of claim 41, wherein the ligand specific for the cytotoxic cell comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD7, CD16, CD8 and CD56. 如請求項41之方法，其中超過一種配體係經添加至該遞送媒介之外表面。The method of claim 41, wherein more than one ligand is added to the outer surface of the delivery medium. 如請求項35之方法，其中該活性物質為核酸。The method of claim 35, wherein the active substance is a nucleic acid. 如請求項45之方法，其中該核酸包含編碼CAR及/或TCR之核酸。The method of claim 45, wherein the nucleic acid comprises nucleic acid encoding CAR and/or TCR. 如請求項35之方法，其中使該複合物與包含該靶細胞之該細胞群體接觸之該步驟係離體進行。The method of claim 35, wherein the step of contacting the complex with the cell population comprising the target cell is performed in vitro. 一種用於在靶細胞內部遞送活性物質之方法，該方法包含： 使如請求項1之複合物與包含該靶細胞之細胞群體接觸之步驟，其中該活性物質既不包括編碼CAR亦不包括編碼TCR之任何核酸。 A method for delivering an active substance inside a target cell, the method comprising: A step of contacting the complex of claim 1 with a cell population comprising the target cell, wherein the active substance does not include any nucleic acid encoding neither CAR nor TCR. 如請求項48之方法，其中用於該活性物質之該遞送媒介為脂質奈米粒子或脂質體。The method of claim 48, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome. 如請求項49之方法，其中該第一結合搭配物為包含親偶極物之分子，且其中該第二結合搭配物為包含1,3-偶極子之分子。The method of claim 49, wherein the first binding partner is a molecule comprising an exopole, and wherein the second binding partner is a molecule comprising a 1,3-exopole. 如請求項50之方法，其中該第一結合搭配物為包含二苯并環辛炔(DBCO)基之分子。The method of claim 50, wherein the first binding partner is a molecule comprising a dibenzocyclooctyne (DBCO) group. 如請求項50之方法，其中該第二結合搭配物為包含疊氮基之分子。The method of claim 50, wherein the second binding partner is a molecule comprising an azido group. 如請求項48之方法，其中該活性物質所遞送至其中之該靶細胞為免疫細胞。The method of claim 48, wherein the target cell to which the active substance is delivered is an immune cell. 如請求項53之方法，其中該免疫細胞為細胞毒性細胞。The method of claim 53, wherein the immune cell is a cytotoxic cell. 如請求項54之方法，其中該細胞毒性細胞為NK細胞或T細胞。The method of claim 54, wherein the cytotoxic cell is a NK cell or a T cell. 如請求項55之方法，其中對該細胞毒性細胞具有特異性之該配體包含至少一種選自由以下組成之群的抗體或其抗原結合片段：CD3、CD7、CD16、CD8及CD56。The method of claim 55, wherein the ligand specific for the cytotoxic cell comprises at least one antibody or antigen-binding fragment thereof selected from the group consisting of: CD3, CD7, CD16, CD8 and CD56. 如請求項54之方法，其中超過一種配體係經添加至該遞送媒介之外表面。 The method of claim 54, wherein more than one ligand is added to the outer surface of the delivery medium. 如請求項48之方法，其中該活性物質為核酸。The method of claim 48, wherein the active substance is a nucleic acid. 如請求項58之方法，其中該核酸包含抑制細胞毒性細胞活化抑制因子之表現之核酸，及/或編碼細胞毒性細胞活化促進因子之核酸。The method of claim 58, wherein the nucleic acid comprises a nucleic acid that inhibits the expression of a cytotoxic cell activation inhibitor and/or a nucleic acid encoding a cytotoxic cell activation promoting factor. 如請求項48之方法，其中該步驟係離體進行。The method of claim 48, wherein the step is performed in vitro. 一種靶細胞，其係藉由如請求項48之方法將活性物質遞送至其中。A target cell into which an active substance is delivered by the method of claim 48. 一種藥劑，其包含如請求項61之靶細胞。A medicament comprising the target cell of claim 61. 如請求項62之藥劑，其進一步包含培養基或鹽水。The pharmaceutical preparation of claim 62, further comprising a culture medium or saline.
TW113112850A 2023-04-07 2024-04-03 Conjugation complex TW202449142A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363494889P 2023-04-07 2023-04-07
US63/494,889 2023-04-07

Publications (1)

Publication Number Publication Date
TW202449142A true TW202449142A (en) 2024-12-16

Family

ID=90825510

Family Applications (1)

Application Number Title Priority Date Filing Date
TW113112850A TW202449142A (en) 2023-04-07 2024-04-03 Conjugation complex

Country Status (3)

Country Link
AR (1) AR132276A1 (en)
TW (1) TW202449142A (en)
WO (1) WO2024210160A1 (en)

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6352694B1 (en) 1994-06-03 2002-03-05 Genetics Institute, Inc. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells
PL2350043T3 (en) 2008-10-09 2014-09-30 Tekmira Pharmaceuticals Corp Improved amino lipids and methods for the delivery of nucleic acids
WO2010054406A1 (en) 2008-11-10 2010-05-14 Alnylam Pharmaceuticals, Inc. Novel lipids and compositions for the delivery of therapeutics
SG186085A1 (en) 2010-06-03 2013-01-30 Alnylam Pharmaceuticals Inc Biodegradable lipids for the delivery of active agents
US9006417B2 (en) 2010-06-30 2015-04-14 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
PT2817287T (en) 2012-02-24 2018-12-28 Arbutus Biopharma Corp Trialkyl cationic lipids and methods of use thereof
TW201408625A (en) 2012-07-06 2014-03-01 Kyowa Hakko Kirin Co Ltd Cationic lipid
EP2711418B1 (en) 2012-09-25 2017-08-23 Miltenyi Biotec GmbH Method for polyclonal stimulation of T cells by flexible nanomatrices
WO2014153114A1 (en) 2013-03-14 2014-09-25 Fred Hutchinson Cancer Research Center Compositions and methods to modify cells for therapeutic objectives
TW201534578A (en) 2013-07-08 2015-09-16 Daiichi Sankyo Co Ltd Novel lipid
CN105555757A (en) 2013-07-23 2016-05-04 普洛体维生物治疗公司 Compositions and methods for delivering messenger RNA
CN110003066B (en) 2013-11-18 2021-09-03 阿克丘勒斯治疗公司 Ionizable cationic lipids for RNA delivery
IL298516B2 (en) 2014-06-25 2025-03-01 Acuitas Therapeutics Inc Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
SG10201913765YA (en) 2014-07-21 2020-03-30 Novartis Ag Treatment of cancer using a cd33 chimeric antigen receptor
ES2799409T3 (en) 2014-08-07 2020-12-17 Takeda Pharmaceuticals Co Cationic lipid
US10081598B2 (en) 2014-12-26 2018-09-25 Eisai R&D Management Co., Ltd. Cationic lipid
EP3303598B1 (en) 2015-05-26 2025-03-19 Ramot at Tel-Aviv University Ltd. Targeted lipid particles for systemic delivery of nucleic acid molecules to leukocytes
CN105636090A (en) 2015-06-30 2016-06-01 宇龙计算机通信科技(深圳)有限公司 Business detection method, business detection system, terminal and base station
IL315940A (en) 2015-07-28 2024-11-01 Univ Pennsylvania Modified monocytes/macrophage expressing chimeric antigen receptors and uses thereof
RS63986B1 (en) 2015-10-28 2023-03-31 Acuitas Therapeutics Inc Novel lipids and lipid nanoparticle formulations for delivery of nucleic acids
EP3397613A1 (en) 2015-12-30 2018-11-07 Acuitas Therapeutics Inc. Lipids and lipid nanoparticle formulations for delivery of nucleic acids
US10188749B2 (en) 2016-04-14 2019-01-29 Fred Hutchinson Cancer Research Center Compositions and methods to program therapeutic cells using targeted nucleic acid nanocarriers
CN118634318A (en) * 2017-12-27 2024-09-13 武田药品工业株式会社 Nucleic acid-containing lipid nanoparticles and uses thereof
PL3733641T3 (en) 2017-12-28 2024-07-15 Takeda Pharmaceutical Company Limited Cationic lipids
WO2020032184A1 (en) 2018-08-10 2020-02-13 武田薬品工業株式会社 Cationic lipid
WO2020080475A1 (en) 2018-10-18 2020-04-23 武田薬品工業株式会社 Method for activation/proliferation of t cells
WO2021113519A1 (en) 2019-12-04 2021-06-10 The Trustees Of The University Of Pennsylvania Nanoparticle compositions and methods of use

Also Published As

Publication number Publication date
WO2024210160A1 (en) 2024-10-10
AR132276A1 (en) 2025-06-11

Similar Documents

Publication Publication Date Title
JP7600208B2 (en) Nanomaterials containing constrained lipids and uses thereof - Patents.com
JP7395483B2 (en) Peptides and nanoparticles for intracellular delivery of mRNA
JP2024160243A (en) mRNA combination therapy for the treatment of cancer
CN110446506B (en) Nanosilosome-microbubble conjugate and composition for improving or treating hair loss comprising same
JP2021523110A (en) Nanoparticles for gene expression and their use
CA3026112A1 (en) Cpf1 complexes with reduced indel activity
CN109476718A (en) Combinations of mRNAs encoding immunomodulatory polypeptides and uses thereof
KR20210027389A (en) Compositions and methods for genome editing by insertion of donor polynucleotides
EP2264167A1 (en) Double-stranded lipid-modified rna having high rna interference effect
WO2007102481A1 (en) Vector for nuclear transport of substance
WO2020262150A1 (en) Lipid nanoparticle
Zhu et al. Guanidinium-rich lipopeptide-based nanoparticle enables efficient gene editing in skeletal muscles
US11802146B2 (en) Polynucleotides encoding anti-chikungunya virus antibodies
Xu et al. Effective delivery of CRISPR/dCas9-SAM for multiplex gene activation based on mesoporous silica nanoparticles for bladder cancer therapy
TW202417019A (en) Engineered polynucleotides for cell selective expression
EP2910564B1 (en) Weakly acidic ph-responsive peptide and liposome containing same
TW202449142A (en) Conjugation complex
EP4565279A1 (en) Hybrid peptide dendrimer systems and extrahepatic delivery
WO2024039896A1 (en) Cationic peptide/protein-modified exosomes for applications in drug delivery
JP2025521199A (en) Lipid microbubbles for targeted delivery of active ingredients
JP2024528983A (en) mRNAs encoding chimeric metabolic reprogramming polypeptides and uses thereof
KR20220168607A (en) Nanoparticles targeting immune cells and encapsulated with mRNA of interest or DNA encoding the same, use thereof, and method for preparing the same
CN117460537A (en) Nucleic acid delivery
KR20220138309A (en) Structures targeting immune cells and encapsulating the mRNA of interest, its use, and its preparation method
WO2025137646A1 (en) Gene editing methods and compositions for treating cystic fibrosis