技术领域Technical field
本发明涉及生物技术领域，具体涉及一种基于结构优化蛋白活性的抗CD19/CD22/CD3三特异性抗体以及该类抗体的用途。The present invention relates to the field of biotechnology, and specifically relates to an anti-CD19/CD22/CD3 trispecific antibody based on structure-optimized protein activity and the use of such antibodies.
背景技术Background technique
近年来，肿瘤免疫治疗的进步极大拓展了人们对恶性肿瘤的治疗手段。常见的免疫治疗包括免疫检查点阻断治疗或者利用基因工程手段改造T细胞使得T细胞对肿瘤细胞具有更高且持续的杀伤能力。尽管免疫治疗对一些恶性肿瘤治疗有不错的效果，但大多数恶性肿瘤对免疫治疗依旧没有反应，急需开发新的更有效的免疫治疗方法(O′Donnell JS，et al.Cancer immunoediting and resistance to T cell-based immunotherapy.NatRev Clin Oncol.2019 Mar；16(3)：151-167.；Chandran SS，et al.T cell receptor-based cancer immunotherapy：Emerging efficacy and pathways ofresistance.Immunol Rev.2019 Jul；290(1)：127-147.)。其中一种提高免疫治疗效率的方法是双特异性抗体，分别可特异性识别靶细胞表面的抗原和T细胞表面的CD3分子，将T细胞引导至特定的恶性肿瘤细胞靶点发挥细胞杀伤作用，已经展现不错的前景(Huehls AM，etal.Bispecific T-cell engagers for cancer immunotherapy.Immunol Cell Biol.2015Mar；93(3)：290-6.；Trabolsi A，et al.T Cell-Activating Bispecific Antibodies inCancer Therapy.J Immunol.2019 Aug 1；203(3)：585-592.)。自1985年Brennan等人将2个单克隆抗体用化学偶联的方式构建了第一个双特异性抗体后(Brennan M，etal.Preparation of bispecific antibodies by chemical recombination ofmonoclonal immunoglobulin G1 fragments.Science.1985 Jul 5；229(4708)：81-3.)，不同靶点、不同形式的双特异性抗体得以广泛研究，但是由于重链错配、抗体失活和非人类相关免疫原性等问题，限制了双特异性抗体更广泛的临床应用(Thakur A，et al.Bispecificantibody based therapeutics：Strengths and challenges.Blood Rev.2018 Jul；32(4)：339-347.)。随着基因工程技术的快速发展，人们可以通过重组DNA等手段控制双特异性抗体的大小、亲和力、半衰期、稳定性、溶解性等特性，并且可以构建嵌合的或人源化的Abs，以满足不同需求(Du J，et al.Engineering Bifunctional Antibodies withConstant Region Fusion Architectures.J Am Chem Soc.2017 Dec 27；139(51)：18607-18615.；Hollander N.Bispecific antibodies for cancertherapy.Immunotherapy.2009 Mar；1(2)：211-22.)。靶向CD3/CD19的Blinatumomab和靶向CD3/EpCAM的Catumaxomab是目前仅有的被批准上市用于肿瘤免疫治疗的双特异性抗体，分别用于治疗急性淋巴细胞白血病和恶性腹水(May MB，et al.Blinatumomab：A novel，bispecific，T-cell engaging antibody.Am J Health Syst Pharm.2016 Jan 1；73(1)：e6-e13.；Bokemeyer C.Catumaxomab--trifunctional anti-EpCAM antibody used totreat malignant ascites.Expert Opin Biol Ther.2010 Aug；10(8)：1259-69.)。为了克服肿瘤免疫逃逸和T细胞的活化限制，近年来三特异性抗体的设计和开发得到重视(RuncieK，et al.Bi-specific and tri-specific antibodies-the next big thing in solidtumor therapeutics.Mol Med.2018 Sep 24；24(1)：50.)。2017年报道的一种三特异性抗体SAR441236能够独立地结合HIV包膜的3种不同表位，并在动物模型上验证有一定的抗病毒能力(Mullard A.Trispecific antibodies take to the clinic.Nat Rev DrugDiscov.2020 Oct；19(10)：657-658.)。研发团队设想将多特异性抗体策略，应用到肿瘤免疫治疗领域，通过靶向淋巴细胞表面更多的分子，传递更多的激活型号，使活化T细胞能更好的特异性消除肿瘤细胞。In recent years, advances in tumor immunotherapy have greatly expanded people’s treatment options for malignant tumors. Common immunotherapy includes immune checkpoint blockade therapy or the use of genetic engineering to modify T cells so that T cells have higher and sustained killing capabilities against tumor cells. Although immunotherapy has good effects on some malignant tumors, most malignant tumors still do not respond to immunotherapy, and there is an urgent need to develop new and more effective immunotherapy methods (O'Donnell JS, et al. Cancer immunoediting and resistance to T cell-based immunotherapy. Nat Rev Clin Oncol. 2019 Mar; 16(3): 151-167.; Chandran SS, et al. T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance. Immunol Rev. 2019 Jul; 290 ( 1):127-147.). One of the methods to improve the efficiency of immunotherapy is bispecific antibodies, which can specifically recognize antigens on the surface of target cells and CD3 molecules on the surface of T cells, guiding T cells to specific malignant tumor cell targets to exert cell killing effects. Has shown good prospects (Huehls AM, et al. Bispecific T-cell engagers for cancer immunotherapy. Immunol Cell Biol. 2015 Mar; 93 (3): 290-6.; Trabolsi A, et al. T Cell-Activating Bispecific Antibodies in Cancer Therapy .J Immunol.2019 Aug 1;203(3):585-592.). Since 1985, Brennan et al. constructed the first bispecific antibody by chemically coupling two monoclonal antibodies (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science. 1985 Jul 5;229(4708):81-3.), bispecific antibodies with different targets and different forms have been extensively studied, but problems such as heavy chain mismatching, antibody inactivation, and non-human related immunogenicity have limited Wider clinical applications of bispecific antibodies (Thakur A, et al. Bispecific antibody based therapeutics: Strengths and challenges. Blood Rev. 2018 Jul; 32(4): 339-347.). With the rapid development of genetic engineering technology, people can control the size, affinity, half-life, stability, solubility and other characteristics of bispecific antibodies through recombinant DNA and other means, and can construct chimeric or humanized Abs to Meet different needs (Du J, et al. Engineering Bifunctional Antibodies with Constant Region Fusion Architectures. J Am Chem Soc. 2017 Dec 27; 139(51): 18607-18615.; Hollander N. Bispecific antibodies for cancer therapy. Immunotherapy. 2009 Mar; 1(2):211-22.). Blinatumomab targeting CD3/CD19 and Catumaxomab targeting CD3/EpCAM are currently the only bispecific antibodies approved for tumor immunotherapy and are used to treat acute lymphoblastic leukemia and malignant ascites respectively (May MB, et al. al. Blinatumomab: A novel, bispecific, T-cell engaging antibody. Am J Health Syst Pharm. 2016 Jan 1; 73(1): e6-e13.; Bokemeyer C. Catumaxomab--trifunctional anti-EpCAM antibody used to treat malignant ascites .Expert Opin Biol Ther. 2010 Aug; 10(8): 1259-69.). In order to overcome tumor immune evasion and T cell activation limitations, the design and development of trispecific antibodies has received attention in recent years (RuncieK, et al. Bi-specific and tri-specific antibodies-the next big thing in solidtumor therapeutics. Mol Med. 2018 Sep 24;24(1):50.). A trispecific antibody SAR441236 reported in 2017 can independently bind to three different epitopes of the HIV envelope and has been verified to have certain antiviral capabilities in animal models (Mullard A. Trispecific antibodies take to the clinic. Nat Rev DrugDiscov. 2020 Oct; 19(10): 657-658.). The R&D team envisions applying the multi-specific antibody strategy to the field of tumor immunotherapy, by targeting more molecules on the surface of lymphocytes and delivering more activation models, so that activated T cells can better specifically eliminate tumor cells.
T细胞结合三特异性抗体(T cell-engaging trispecific antibodies，TsAbs)代表了一种非常有效的将激活的细胞毒性T细胞重定向到肿瘤的方式(Runcie K，et al.Bi-specific and tri-specific antibodies-the next big thing in solid tumortherapeutics.Mol Med.2018 Sep 24；24(1)：50.；Mullard A.Trispecific antibodiestake to the clinic.Nat Rev Drug Discov.2020 Oct；19(10)：657-658.)。CD3作为T细胞受体的一部分，表达于成熟T细胞，能够转导TCR识别抗原所产生的活化信号。TsAbs能够同时结合表面肿瘤抗原和T细胞受体的CD3ε亚基，在T细胞和肿瘤细胞之间提供一个物理连接，从而有效的激活静止的T细胞杀伤肿瘤细胞，达到治疗肿瘤的效果。因为T细胞双特异性旁路TCR抗原识别和T细胞活化的共刺激要求，它们消除了对肿瘤特异性免疫的需要，并克服了肿瘤微环境中T细胞面临的许多障碍。Wu等科研人员开发出来一种新型三特异性抗体，可通过T细胞受体共刺激改善并增强T细胞靶向能力及信号转导的能力，持续性激活T细胞。与双抗相比，这种三特异性抗体增设了靶向T细胞表面CD28蛋白，从而驱动蛋白Bcl-xL的表达，Bcl-xL的存在则能够阻止T细胞凋亡，而且在小鼠的多发性骨髓瘤模型中，该三特异性抗体也能够增强抵抗骨髓瘤细胞系的能力(Wu L，et al.Trispecific antibodiesenhance the therapeutic efficacy of tumor-directed T cells through T cellreceptor co-stimulation.Nature Cancer 1，86-98.)。T cell-engaging trispecific antibodies (TsAbs) represent a very efficient way to redirect activated cytotoxic T cells to tumors (Runcie K, et al. Bi-specific and tri- specific antibodies-the next big thing in solid tumortherapeutics.Mol Med.2018 Sep 24;24(1):50.; Mullard A.Trispecific antibodies take to the clinic.Nat Rev Drug Discov.2020 Oct;19(10):657- 658.). As part of the T cell receptor, CD3 is expressed on mature T cells and can transduce activation signals generated by TCR recognition of antigens. TsAbs can simultaneously bind to surface tumor antigens and the CD3ε subunit of the T cell receptor, providing a physical connection between T cells and tumor cells, thereby effectively activating quiescent T cells to kill tumor cells and achieve the effect of treating tumors. Because T cell bispecificities bypass the costimulatory requirements of TCR antigen recognition and T cell activation, they eliminate the need for tumor-specific immunity and overcome many of the obstacles faced by T cells in the tumor microenvironment. Researchers such as Wu developed a new type of trispecific antibody that can improve and enhance T cell targeting and signal transduction capabilities through T cell receptor costimulation, and continuously activate T cells. Compared with double antibodies, this trispecific antibody additionally targets the CD28 protein on the surface of T cells, thereby driving the expression of the protein Bcl-xL. The presence of Bcl-xL can prevent T cell apoptosis, and in mice with multiple In the myeloma model, the trispecific antibody can also enhance the ability to resist myeloma cell lines (Wu L, et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cellreceptor co-stimulation. Nature Cancer 1, 86-98.).
TsAbs的开发对于解决目前双特异性抗体临床使用的局限性，具有重要的应用前景，然而，针对TsAbs所涉及的不同抗体，其结合抗原表位所在细胞表面的位置不同，意味着其结构设计需要进行个性化的调整。通常来说，TsAbs介导T细胞和肿瘤细胞的结合涉及到免疫突触的形成，免疫突触是在T细胞活化过程中，T细胞与肿瘤细胞或者抗原呈递细胞之间形成的一个高度复杂，有序的超分子结构域，是被一圈整合素家族的黏附分子所包围的中心簇TCR与抗原复合物结合的一种特殊结构(Wu L，et al.Trispecific antibodiesenhance the therapeutic efficacy of tumor-directed T cells through T cellreceptor co-stimulation.Nature Cancer 1，86-98.；Yokosuka T，et al.Theimmunological synapse，TCR microclusters，and T cell activation.Curr TopMicrobiol Immunol.2010；340：81-107.)。在免疫突触形成过程中，由于TCR和抗原复合物的结构缘故，T细胞和靶细胞之间的距离约在15nm。磷酸化酶CD45和CD148由于具有远长于15nm的外功能区而被排挤在突触外，从而使无负调控因子情况下的TCR诱导的酪氨酸磷酸化过程顺利进行。研究表明具有更短的外功能区的突变型CD45蛋白削弱了T细胞相关信号，这也验证了上述的分子排挤作用在T细胞信号中的重要性(Basu R，et al.Cytotoxic TCells Use Mechanical Force to Potentiate Target Cell Killing.Cell.2016 Mar24；165(1)：100-110.；Basu R，et al.Mechanical Communication at the ImmunologicalSynapse.Trends Cell Biol.2017 Apr；27(4)：241-254.)。TsAbs与不同抗原之间的空间距离对于相应的T细胞免疫突触形成以及T细胞信号激活可能也同样重要，只是这一空间距离取决完全不同的结构因素，包括抗原表位的位置以及抗体和T细胞膜之间的连接区域。The development of TsAbs has important application prospects for solving the limitations of the current clinical use of bispecific antibodies. However, the different antibodies involved in TsAbs have different locations on the cell surface where the antigen epitopes are bound, which means that their structural design requires Make personalized adjustments. Generally speaking, TsAbs-mediated binding of T cells and tumor cells involves the formation of an immune synapse. The immune synapse is a highly complex formation between T cells and tumor cells or antigen-presenting cells during T cell activation. The ordered supramolecular domain is a special structure in which the central cluster TCR, surrounded by a circle of adhesion molecules of the integrin family, binds to the antigen complex (Wu L, et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cellreceptor co-stimulation. Nature Cancer 1, 86-98.; Yokosuka T, et al. The immunological synapse, TCR microclusters, and T cell activation. Curr Top Microbiol Immunol. 2010; 340: 81-107.). During the formation of the immune synapse, due to the structure of the TCR and antigen complex, the distance between T cells and target cells is approximately 15 nm. The phosphorylase CD45 and CD148 are excluded from the synapse due to their outer functional domains that are much longer than 15 nm, allowing the TCR-induced tyrosine phosphorylation process to proceed smoothly in the absence of negative regulators. Studies have shown that mutant CD45 proteins with shorter outer functional domains weaken T cell-related signaling, which also verifies the importance of the above-mentioned molecular crowding out in T cell signaling (Basu R, et al. Cytotoxic TCells Use Mechanical Force to Potentiate Target Cell Killing.Cell.2016 Mar24;165(1):100-110.;Basu R, et al.Mechanical Communication at the ImmunologicalSynapse.Trends Cell Biol.2017 Apr;27(4):241-254.) . The spatial distance between TsAbs and different antigens may also be equally important for the corresponding T cell immune synapse formation and T cell signaling activation, but this spatial distance depends on completely different structural factors, including the location of the antigenic epitope and the antibody and T The connecting area between cell membranes.
综上所述，为了实现TsAbs介导T细胞对抗原表达肿瘤细胞的最佳杀伤效果，需要对不同的抗体连接方式以及不同的连接位置进行考察，通过结构设计的优化和筛选，建立一种基于介导T细胞靶向肿瘤杀伤的最佳TsAbs。In summary, in order to achieve the best killing effect of TsAbs-mediated T cells on antigen-expressing tumor cells, it is necessary to investigate different antibody connection methods and different connection positions, and establish a method based on structural design optimization and screening. Optimum TsAbs for mediating T-cell targeted tumor killing.
发明内容Contents of the invention
多靶点抗体药物成为解决上述类似问题的关键，因此，为寻找更有效的治疗手段，本发明提供了一种抗CD19/CD22/CD3三特异性抗体，这将给患者提供更多的用药选择。Multi-target antibody drugs have become the key to solving similar problems mentioned above. Therefore, in order to find more effective treatment methods, the present invention provides an anti-CD19/CD22/CD3 trispecific antibody, which will provide patients with more medication options. .
本发明第一个方面，提供了一种基于结构优化设计的CD19/CD22/CD3三特异性抗体，所述CD19/CD22/CD3三特异性抗体包含对CD19/CD3具有结合特异性的如SEQ ID NO：1或SEQ ID NO：2或SEQ ID NO：3所示的重组重链氨基酸序列，和对CD22/CD3具有结合特异性的如SEQ ID NO：4或SEQ ID NO：5或SEQ ID NO：6所示的重组轻链氨基酸序列。A first aspect of the present invention provides a CD19/CD22/CD3 trispecific antibody designed based on structural optimization. The CD19/CD22/CD3 trispecific antibody contains a binding specificity for CD19/CD3 such as SEQ ID The recombinant heavy chain amino acid sequence shown in NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3, and having binding specificity for CD22/CD3 such as SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO : The recombinant light chain amino acid sequence shown in 6.
优选地，所述CD19/CD22/CD3(SO)三特异性抗体包含对CD19/CD3具有结合特异性的如SEQ ID NO：3所示的重组重链氨基酸序列，和对CD22/CD3具有结合特异性的如SEQ IDNO：6所示的重组轻链氨基酸序列。Preferably, the CD19/CD22/CD3 (SO) trispecific antibody comprises the recombinant heavy chain amino acid sequence shown in SEQ ID NO: 3 with binding specificity for CD19/CD3, and has binding specificity for CD22/CD3 The specific recombinant light chain amino acid sequence shown in SEQ ID NO: 6.
本发明第二个方面，提供了一种三特异性抗体分子的构建方法。所述三特异性抗体的构建方法是以第一结构域——单克隆抗体的Fab作为结构基础，同时增加了靶向2种肿瘤特异性或相关抗原的抗体片段，分别为第二结构域和第三结构域，第二结构域与第三结构域通过连接肽(Linker)可连接至Fab片段。In a second aspect, the present invention provides a method for constructing trispecific antibody molecules. The construction method of the trispecific antibody is based on the first structural domain, the Fab of a monoclonal antibody, as the structural basis, and at the same time, antibody fragments targeting two tumor-specific or related antigens are added, namely the second structural domain and the The third domain, the second domain and the third domain can be connected to the Fab fragment through a linker.
具体地，所述构建方法包括：Specifically, the construction method includes:
(1)将第二结构域连接于第一结构域Fab的重链结构域；(1) Connect the second domain to the heavy chain domain of the first domain Fab;
(2)将第三结构域连接于第一结构域Fab的轻链结构域；(2) Connect the third domain to the light chain domain of the first domain Fab;
(3)重链和轻链通过CH1和CL中的二硫键结合形成异源二聚体的三特异性抗体。(3) The heavy chain and light chain are combined through the disulfide bond in CH1 and CL to form a heterodimer trispecific antibody.
步骤(1)所述连接方式包括可分别通过刚性连接肽将第二结构域连接于Fab重链可变区(VH)结构域N端，可标记为HNT，刚性连接肽可以为PD Linker；或通过卷曲螺旋连接肽(Coiled Coil Linker)将第二结构域连接于Fab重链恒定区(CH1)结构域的184S-187L之间，并且缺失185S 186G，可标记为H184-187；或通过柔性连接肽将第二结构域连接于Fab重链恒定区(CH1)结构域C末端，可标记为HCT。The connection method in step (1) includes connecting the second domain to the N-terminus of the Fab heavy chain variable region (VH) domain through a rigid linking peptide, which can be labeled as HNT, and the rigid linking peptide can be a PD Linker; or The second domain is connected between 184S-187L of the Fab heavy chain constant region (CH1) domain through a coiled coil linker, and 185S 186G is deleted, which can be marked as H184-187; or through a flexible connection The peptide connects the second domain to the C-terminus of the Fab heavy chain constant region (CH1) domain and can be labeled HCT.
步骤(2)所述连接方式包括可分别通过刚性连接肽将第三结构域连接于在Fab轻链可变区(VL)结构域N端，可标记为LNT，刚性连接肽可以为PD Linker；或通过卷曲螺旋连接肽(Coiled Coil Linker)将第三结构域连接于Fab轻链恒定区(CL)结构域171S-173D之间，并缺失172K，可标记为L171-173；或通过柔性连接肽第三结构域连接于在Fab轻链恒定区(CL)结构域C末端，可标记为LCT。The connection method in step (2) includes connecting the third domain to the N-terminus of the Fab light chain variable region (VL) domain through a rigid linking peptide, which can be labeled as LNT, and the rigid linking peptide can be a PD Linker; Or connect the third domain to the Fab light chain constant region (CL) domain 171S-173D through a coiled coil linker, and delete 172K, which can be labeled L171-173; or use a flexible linker peptide The third domain is connected to the C-terminus of the Fab light chain constant region (CL) domain and can be labeled LCT.
本发明所述刚性连接肽PD linker具有如SEQ ID NO：11所示的氨基酸序列，所述卷曲螺旋连接肽(Coiled Coil Linker)具有如SEQ ID NO：12、13所示的氨基酸序列，所述柔性连接肽为(G4S)3。The rigid linking peptide PD linker of the present invention has the amino acid sequence shown in SEQ ID NO: 11, and the coiled coil linker peptide (Coiled Coil Linker) has the amino acid sequence shown in SEQ ID NO: 12 and 13. The flexible connecting peptide is (G4S)3.
步骤(1)、步骤(2)所述第一结构域、第二结构域、第三结构域各自独立地具有针对选择HER2、VEGFR1/2、CD3、CD19、CD22、EGFR、EGFRvIII、HER3、HER4、IGF1R、c-Met、MUC-1、MUC-16、IL13R、Mesothelin、Trop-2、GPC2、GPC3、GD2、CEA、PSMA、PSCA、EpCAM、CD79、ROR1、AXL、CD133、CD171、BCMA、CD20、CD123、Claudin 6、Claudin 18.2、CD38、CD30、CD33、CD138、CD56、CS1、CLL1、CD7、CD4、CD8、Lewis Y、ALK、KRAS突变体、MYD88突变体、IDH1突变体、P53突变体、NY-ESO-1、NKG2D、CD16、CD56、CD64、PD-1、PD-L1、B7-H3、B7-H4、TGF-beta、CTLA-4、LAG-3、TIM-3、TIGHT、VISTA、ICOS、GITR、CD28、4-1BB、OX40、CD27、CD24、CD47、CXCR4、DLL3、Integrin等等相关靶点抗原的结合特异性。The first domain, the second domain, and the third domain described in step (1) and step (2) each independently have the ability to select HER2, VEGFR1/2, CD3, CD19, CD22, EGFR, EGFRvIII, HER3, and HER4 , IGF1R, c-Met, MUC-1, MUC-16, IL13R, Mesothelin, Trop-2, GPC2, GPC3, GD2, CEA, PSMA, PSCA, EpCAM, CD79, ROR1, AXL, CD133, CD171, BCMA, CD20 , CD123, Claudin 6, Claudin 18.2, CD38, CD30, CD33, CD138, CD56, CS1, CLL1, CD7, CD4, CD8, Lewis Y, ALK, KRAS mutant, MYD88 mutant, IDH1 mutant, P53 mutant, NY-ESO-1, NKG2D, CD16, CD56, CD64, PD-1, PD-L1, B7-H3, B7-H4, TGF-beta, CTLA-4, LAG-3, TIM-3, TIGHT, VISTA, Binding specificity of related target antigens such as ICOS, GITR, CD28, 4-1BB, OX40, CD27, CD24, CD47, CXCR4, DLL3, Integrin, etc.
步骤(1)与步骤(2)所述第二结构域、第三结构域可以为Adnectin(人纤连蛋白)、亲和体、anticalin(抗运载蛋白)、双环肽、DARPin(天然锚蛋白重复序列)、Fynomer、Kunitz型结构域、E7免疫蛋白、淋巴细胞受体可变区、单域抗体、全抗体、抗体片段、单链抗体以及核酸适配体等形式。The second domain and the third domain described in steps (1) and (2) can be Adnectin (human fibronectin), affibody, anticalin (anticalin), bicyclic peptide, DARPin (natural ankyrin repeats). sequence), Fynomer, Kunitz type domain, E7 immune protein, lymphocyte receptor variable region, single domain antibody, whole antibody, antibody fragment, single chain antibody and nucleic acid aptamer, etc.
所述第一结构域Fab片段可以来源包括但不局限于抗CD3单克隆抗体。The first domain Fab fragment may be derived from sources including, but not limited to, anti-CD3 monoclonal antibodies.
本发明第三个方面，公开了一种靶向免疫效应T细胞抗原CD3的介导T细胞重定位到肿瘤细胞的二聚体型三特异性抗体分子的构建方式。所述三特异性抗体的构建方法是以抗CD3单克隆抗体(克隆SP34)的Fab作为结构基础，第二结构域为抗CD19单链抗体(singlechain variable fragment，scFv)，第三结构域抗CD22单价纳米抗体(VHH)，两者分别通过Linker连接至SP34 Fab片段。构建方法包括以下步骤：The third aspect of the present invention discloses a construction method of a dimeric trispecific antibody molecule that targets the immune effector T cell antigen CD3 and mediates the relocation of T cells to tumor cells. The construction method of the trispecific antibody is based on the Fab of anti-CD3 monoclonal antibody (clone SP34) as the structural basis, the second domain is anti-CD19 single chain antibody (singlechain variable fragment, scFv), and the third domain is anti-CD22 Monovalent Nanobody (VHH), both of which are connected to the SP34 Fab fragment via Linker. The build method includes the following steps:
(1)抗CD19-scFv连接于SP34 Fab结构的重链结构域。(1) Anti-CD19-scFv is connected to the heavy chain domain of the SP34 Fab structure.
(2)抗CD22-VHH连接于SP34 Fab结构的轻链结构域。(2) Anti-CD22-VHH is connected to the light chain domain of the SP34 Fab structure.
(3)本发明中重链和轻链通过CH1和CL中的二硫键结合形成异源二聚体的抗CD3Fab(SP34)，即抗CD19/CD22/CD3三特异性抗体。(3) In the present invention, the heavy chain and the light chain are combined through the disulfide bond in CH1 and CL to form a heterodimer anti-CD3 Fab (SP34), that is, an anti-CD19/CD22/CD3 trispecific antibody.
优选地，步骤(1)可通过以下方式将抗CD19-scFv连接于SP34 Fab结构的重链结构域：Preferably, step (1) can connect the anti-CD19-scFv to the heavy chain domain of the SP34 Fab structure in the following manner:
(a)通过刚性连接肽PD Linker将抗CD19-scFv连接于SP34 Fab重链可变区(VH)结构域N端1E；(a) Connect the anti-CD19-scFv to the N-terminal 1E of the SP34 Fab heavy chain variable region (VH) domain through a rigid linker peptide PD Linker;
(b)通过螺旋卷曲连接肽Coiled Coil Linker将抗CD19-scFv嵌合于SP34 Fab重链恒定区(CH1)结构域184S-187L之间，并且缺失185S、186G；(b) The anti-CD19-scFv is chimeric between the SP34 Fab heavy chain constant region (CH1) domain 184S-187L through the coiled coil linker, and 185S and 186G are deleted;
(c)通过柔性连接肽(G4S)3 Linker将抗CD19-scFv连接于SP34 Fab重链恒定区(CH1)结构域C端228C。(c) Anti-CD19-scFv was connected to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S)3 Linker.
以上(a)-(c)所述方式制备得到的CD19/CD3双特异性抗体可以分别标记为CD19/CD3 HNT、CD19/CD3 H184-187、CD19/CD3 HCT。The CD19/CD3 bispecific antibodies prepared in the manner described in (a) to (c) above can be labeled as CD19/CD3 HNT, CD19/CD3 H184-187, and CD19/CD3 HCT respectively.
进一步优选地，步骤(1)可通过以下方式将抗CD19-scFv连接于SP34 Fab结构的重链结构域：Further preferably, step (1) can connect the anti-CD19-scFv to the heavy chain domain of the SP34 Fab structure in the following manner:
(a)通过螺旋卷曲连接肽Coiled Coil Linker将抗CD19-scFv嵌合于SP34 Fab重链恒定区(CH1)结构域184S-187L之间，并且缺失185S、186G；(a) Anti-CD19-scFv is chimeric between the SP34 Fab heavy chain constant region (CH1) domain 184S-187L through the coiled coil linker, and 185S and 186G are deleted;
(b)通过柔性连接肽(G4S)3 Linker将抗CD19-scFv连接于SP34 Fab重链恒定区(CH1)结构域C端228C。(b) Connect the anti-CD19-scFv to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S)3 Linker.
更进一步优选地，步骤(1)可通过以下方式将抗CD19-scFv连接于SP34 Fab结构的重链结构域：More preferably, step (1) can connect the anti-CD19-scFv to the heavy chain domain of the SP34 Fab structure in the following manner:
(a)通过柔性连接肽(G4S)3 Linker将抗CD19-scFv连接于SP34 Fab重链恒定区(CH1)结构域C端228C。(a) Anti-CD19-scFv was connected to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S)3 Linker.
优选地，步骤(2)可通过以下方式将抗CD22-VHH连接于SP34 Fab结构的轻链结构域：Preferably, step (2) can connect the anti-CD22-VHH to the light chain domain of the SP34 Fab structure in the following manner:
(a)通过刚性连接肽PD Linker将抗CD22-VHH连接于SP34 Fab轻链可变区(VL)结构域N端1Q；(a) Connect anti-CD22-VHH to the N-terminal 1Q of the SP34 Fab light chain variable region (VL) domain through a rigid linker peptide PD Linker;
(b)通过螺旋卷曲连接肽Coiled Coil Linker将抗CD22-VHH嵌合于SP34 Fab轻链恒定区(CL)结构域171S-173D之间，并缺失172K；(b) The anti-CD22-VHH is chimeric between the 171S-173D domain of the SP34 Fab light chain constant region (CL) through the coiled coil linker, and 172K is deleted;
(c)通过柔性连接肽(G4S)3 Linker将抗CD22-VHH连接于SP34 Fab轻链恒定区(CL)结构域C端217C。(c) Anti-CD22-VHH was connected to the C-terminal 217C of the SP34 Fab light chain constant region (CL) domain through a flexible linker peptide (G4S)3 Linker.
以上(a)-(c)所述方式制备得到的CD22/CD3双特异性抗体可以分别标记为CD22/CD3 LNT、CD22/CD3 L171-173、CD22/CD3 LCT。The CD22/CD3 bispecific antibodies prepared in the manner described in (a) to (c) above can be labeled as CD22/CD3 LNT, CD22/CD3 L171-173, and CD22/CD3 LCT respectively.
进一步优选地，步骤(2)可通过以下方式将抗CD22-VHH连接于SP34 Fab结构的轻链结构域：Further preferably, step (2) can connect the anti-CD22-VHH to the light chain domain of the SP34 Fab structure in the following manner:
(a)通过螺旋卷曲连接肽Coiled Coil Linker将抗CD22-VHH嵌合于SP34 Fab轻链恒定区(CL)结构域171S-173D之间，并缺失172K；(a) The anti-CD22-VHH is chimeric between the 171S-173D domain of the SP34 Fab light chain constant region (CL) through the coiled coil linker, and 172K is deleted;
(b)通过柔性连接肽(G4S)3 Linker将抗CD22-VHH连接于SP34 Fab轻链恒定区(CL)结构域C端217C。(b) Anti-CD22-VHH is connected to the C-terminal 217C of the SP34 Fab light chain constant region (CL) domain through a flexible linker peptide (G4S)3 Linker.
更进一步优选地，步骤(2)可通过以下方式将抗CD22-VHH连接于SP34 Fab结构的轻链结构域：More preferably, step (2) can connect the anti-CD22-VHH to the light chain domain of the SP34 Fab structure in the following manner:
(a)通过柔性连接肽(G4S)3 Linker将抗CD22-VHH连接于SP34 Fab轻链恒定区(CL)结构域C端217C。(a) Anti-CD22-VHH is connected to the C-terminal 217C of the SP34 Fab light chain constant region (CL) domain through a flexible linker peptide (G4S)3 Linker.
在一个优选实施例中，所述CD19/CD22/CD3(SO)三特异性抗体是以抗CD3 Fab结构为基础，通过以下方法进行构建：In a preferred embodiment, the CD19/CD22/CD3 (SO) trispecific antibody is based on the anti-CD3 Fab structure and is constructed by the following method:
(1)通过柔性连接肽(G4S)3 Linker将抗CD19-scFv连接于SP34 Fab重链恒定区(CH1)结构域C端228C；(1) Connect the anti-CD19-scFv to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH1) domain through a flexible linker peptide (G4S)3 Linker;
(2)通过柔性连接肽(G4S)3 Linker将抗CD22-VHH连接于SP34 Fab轻链恒定区(CL)结构域C端217C；(2) Connect the anti-CD22-VHH to the C-terminal 217C of the SP34 Fab light chain constant region (CL) domain through a flexible linker peptide (G4S)3 Linker;
(3)重链(HC)和轻链(LC)通过CH1和CL中的二硫键结合形成异源二聚体的抗CD3Fab(SP34)，即抗CD19/CD22/CD3(SO)三特异性抗体。(3) The heavy chain (HC) and the light chain (LC) combine through the disulfide bond in CH1 and CL to form a heterodimer anti-CD3Fab (SP34), that is, anti-CD19/CD22/CD3 (SO) trispecific Antibody.
本发明的第四个方面，还提供了本发明制备所述三特异性抗体的方法。其包括：A fourth aspect of the present invention also provides a method for preparing the trispecific antibody of the present invention. These include:
(1)获得三特异性抗体的融合基因，构建三特异性抗体的表达载体；(1) Obtain the fusion gene of the trispecific antibody and construct the expression vector of the trispecific antibody;
(2)通过基因工程方法将上述表达载体转染至宿主细胞中；(2) Transfect the above expression vector into host cells through genetic engineering methods;
(3)在允许产生所述三特异性抗体的条件下培养上述宿主细胞；(3) Cultivate the above host cells under conditions that allow the production of the trispecific antibody;
(4)分离纯化产生的所述抗体蛋白。(4) Separate and purify the produced antibody protein.
步骤(1)所述表达载体可以为真核细胞表达载体pFuse、pSeqtag、pCMV、pcDNA、pFastBac1、pPIC9K等和原核表达载体pET、pGEX、pMAL、pQE、pTrc、pBV、pTXB，优选真核表达载体。The expression vector in step (1) can be eukaryotic expression vectors pFuse, pSeqtag, pCMV, pcDNA, pFastBacl, pPIC9K, etc. and prokaryotic expression vectors pET, pGEX, pMAL, pQE, pTrc, pBV, pTXB, preferably eukaryotic expression vectors .
步骤(2)所述宿主细胞可以为大肠杆菌、苏云金芽孢杆菌、毕赤酵母、昆虫细胞、293悬浮细胞以及中国昆虫卵巢细胞，优选293悬浮细胞。The host cell in step (2) can be Escherichia coli, Bacillus thuringiensis, Pichia pastoris, insect cells, 293 suspension cells and Chinese insect ovary cells, preferably 293 suspension cells.
本发明的第五个方面，提供了所述抗体在制造用于治疗癌症的药物中的应用。所述癌症包括但不限于乳腺癌、结肠直肠癌、肛门癌、胰腺癌、胆囊癌、胆管癌、头颈癌、鼻咽癌、皮肤癌、黑素瘤、卵巢癌、前列腺癌、尿道癌、肺癌、非小细胞肺癌、小细胞肺癌、脑肿瘤、神经胶质瘤、成神经细胞瘤、食道癌、胃癌、肝癌、肾癌、膀胱癌、宫颈癌、子宫内膜癌、甲状腺癌、眼癌、肉瘤、骨癌、白血病、骨髓瘤或淋巴瘤。所述包括表达CD19、CD22等的肿瘤细胞。A fifth aspect of the present invention provides the use of the antibody in the manufacture of a medicament for treating cancer. The cancers include, but are not limited to, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethra cancer, lung cancer , non-small cell lung cancer, small cell lung cancer, brain tumors, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, Sarcoma, bone cancer, leukemia, myeloma or lymphoma. This includes tumor cells expressing CD19, CD22, etc.
在一个实施例中，可用于治疗同时表达CD19/CD22阳性肿瘤的用途，靶向CD19/CD22/CD3三特异性抗体，在体外能够显著杀伤CD19/CD22双阳性、或者CD19和CD22单阳性肿瘤细胞，而对CD19/CD22双阴性细胞没有非特异性杀伤效果。同时体外的肿瘤杀伤效果也显著高于临床获批的CD19/CD3双特异性抗体博纳吐单抗(Blinatumomab)。In one embodiment, a trispecific antibody targeting CD19/CD22/CD3 can significantly kill CD19/CD22 double-positive or CD19 and CD22 single-positive tumor cells in vitro. , but has no non-specific killing effect on CD19/CD22 double-negative cells. At the same time, the tumor killing effect in vitro is also significantly higher than that of the clinically approved CD19/CD3 bispecific antibody Blinatumomab.
本发明的第六个方面，提供了用于治疗肿瘤的方法，其包括以有效治疗所述肿瘤的用量，向有此需要的受试者施用本申请的三特异性抗体。A sixth aspect of the present invention provides a method for treating tumors, which includes administering the trispecific antibody of the present application to a subject in need thereof in an amount effective to treat the tumor.
本发明的技术方案，取得了有益的技术效果：The technical solution of the present invention has achieved beneficial technical effects:
1、本发明所述三特异性抗体药物，首次实现了同时靶向T细胞、肿瘤细胞以及肿瘤血管的多靶点治疗，基于Fab和VHH结构基础的三特异性抗体，相比于带Fc区域的抗体表达纯化更加简便，在实体肿瘤微环境中穿透能力更强，基于Fab和VHH结构基础的三特异性抗体，相比于普通的单链抗体串联结构，其在体内的半衰期更长，更有利于减少给药次数及剂量。1. The trispecific antibody drug of the present invention realizes for the first time a multi-target treatment targeting T cells, tumor cells and tumor blood vessels at the same time. The trispecific antibody based on the Fab and VHH structural basis, compared with the Fc region The antibody expression and purification is simpler and has stronger penetration ability in the solid tumor microenvironment. Trispecific antibodies based on Fab and VHH structures have a longer half-life in the body than ordinary single-chain antibody tandem structures. It is more conducive to reducing the frequency and dosage of administration.
2、将VHH或者scFv抗体分子通过不同连接肽Linker连接于Fab的不同位置有着不同的作用效果：在构建本发明中所述三特异性抗体时，将结构域之间的连接和功能区分需经过综合考虑。在Fab的N端添加结构域时，需要重视的是Fab结构域本身的靶向性，为了有效隔离2个不同结构域，本发明使用的是刚性连接肽Linker，保证了2端抗体的靶向性；在Fab的C端添加结构域时，需要重视的是添加的结构域能够正常表达，同时保证结构域的独立性，因此本发明使用的是柔性连接肽Linker；本发明中也提供了将结构域通过螺旋卷曲连接肽(Coiled Coil Linker)嵌合于轻/重链恒定区内的方法，更能有效保证3个结构域之间的独立性和靶向性。通过在Fab不同位置引入VHH或者scFv，可以有效调整靶向肿瘤细胞和T细胞之间的距离，从而调整形成有效免疫突触，避免免疫抑制信号对T细胞发挥活化杀伤效果的影响。基于此，我们发现了在CD3靶向Fab引入CD19和CD22靶向抗体的最佳位点是Fab的C端。2. Connecting VHH or scFv antibody molecules to different positions of the Fab through different linking peptide Linkers has different effects: when constructing the trispecific antibody described in the present invention, the connection and functional differentiation between the domains need to be Considering. When adding a domain to the N-terminus of Fab, what needs to be paid attention to is the targeting of the Fab domain itself. In order to effectively isolate two different domains, the present invention uses a rigid connecting peptide Linker to ensure the targeting of the antibody at both ends. property; when adding a domain to the C-terminal of Fab, it is important to note that the added domain can be expressed normally while ensuring the independence of the domain, so the present invention uses a flexible linking peptide Linker; the present invention also provides a The structural domain is embedded in the constant region of the light/heavy chain through a coiled coil linker, which can effectively ensure the independence and targeting of the three domains. By introducing VHH or scFv at different positions of the Fab, the distance between the targeted tumor cells and T cells can be effectively adjusted, thereby adjusting the formation of an effective immune synapse and avoiding the impact of immunosuppressive signals on the activation and killing effect of T cells. Based on this, we found that the best site to introduce CD19 and CD22-targeting antibodies into CD3-targeting Fab is the C-terminus of the Fab.
3、通过文献总结和前期试验成果，成功选择靶向CD19、CD22和CD3的抗体克隆，并且通过分子克隆技术在真核表达系统中分别进行了表达，经过体外验证，确认所选抗体均可以和目的抗原具有很好的结合特异性和亲和力。3. Through literature review and preliminary test results, we successfully selected antibody clones targeting CD19, CD22 and CD3, and expressed them in eukaryotic expression systems through molecular cloning technology. After in vitro verification, it was confirmed that the selected antibodies can be used with The target antigen has good binding specificity and affinity.
4、选择了合适的表达系统并成功表达了不同结构的双特异性抗体，以抗CD3抗体片段Fab为基础支架，分别构建CD19/CD3和CD22/CD3的双特异性抗体，通过结构优化和活性比较，确认这些双特异性抗体能够表现出良好的结合稳定性以及介导T细胞免疫杀伤靶细胞效果。4. An appropriate expression system was selected and bispecific antibodies with different structures were successfully expressed. Using the anti-CD3 antibody fragment Fab as the basic scaffold, bispecific antibodies for CD19/CD3 and CD22/CD3 were constructed respectively. Through structural optimization and activity Comparison confirmed that these bispecific antibodies can exhibit good binding stability and mediate T cell immune killing of target cells.
5、通过结构优化和活性验证，明确CD19/CD22/CD3(SO)三特异性抗体的优化分子结构，进一步通过真核表达体系获得结构稳定的蛋白，在体外展现出稳定的靶点结合特异性和亲和力，同时能够有效介导T细胞对血液肿瘤细胞的杀伤效果。相比于目前有疗效的双特异性抗体博纳吐单抗(Blinatumomab)，所述CD19/CD22/CD3(SO)三特异性抗体可以同时靶向CD19/CD22双阳性和单阳性肿瘤，从而有效防止双特异性抗体针对单靶点识别造成的免疫逃逸，具有协同靶向治疗，增强免疫治疗效果的功能。5. Through structural optimization and activity verification, the optimized molecular structure of the CD19/CD22/CD3(SO) trispecific antibody was clarified, and a structurally stable protein was further obtained through a eukaryotic expression system, which demonstrated stable target binding specificity in vitro. and affinity, and can effectively mediate the killing effect of T cells on blood tumor cells. Compared with the currently effective bispecific antibody Blinatumomab, the CD19/CD22/CD3 (SO) trispecific antibody can simultaneously target CD19/CD22 double-positive and single-positive tumors, thereby effectively It prevents immune evasion caused by bispecific antibody recognition of a single target, has the function of synergizing targeted therapy and enhancing the effect of immunotherapy.
附图说明Description of the drawings
图1双特异性抗体结构设计以及基于结构优化活性设计的三特异性抗体示意图。Figure 1 Schematic diagram of bispecific antibody structural design and trispecific antibody design based on structure optimization and activity.
图2不同结构CD19/CD3双特异性抗体对CD19阳性K562-CD19肿瘤细胞的结合能力比较。Figure 2 Comparison of the binding abilities of CD19/CD3 bispecific antibodies with different structures to CD19-positive K562-CD19 tumor cells.
图3不同结构CD19/CD3双特异性抗体对CD19阴性K562肿瘤细胞的结合能力比较。Figure 3 Comparison of the binding abilities of CD19/CD3 bispecific antibodies with different structures to CD19-negative K562 tumor cells.
图4不同CD19/CD3双特异性抗体对CD19/CD22双阳性Nalm6肿瘤细胞的体外杀伤活性验证。Figure 4 Verification of the in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19/CD22 double-positive Nalm6 tumor cells.
图5不同CD19/CD3双特异性抗体对CD19阳性CD22阴性Nalm6-KO22肿瘤细胞的体外杀伤活性验证。Figure 5 Verification of the in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19-positive CD22-negative Nalm6-KO22 tumor cells.
图6不同CD19/CD3双特异性抗体对CD19阴性CD22阳性Nalm6-KO19肿瘤细胞的体外杀伤活性验证。Figure 6 Verification of the in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19-negative CD22-positive Nalm6-KO19 tumor cells.
图7不同CD19/CD3双特异性抗体对CD19/CD22双阴性K562肿瘤细胞的体外杀伤活性验证。Figure 7 Verification of the in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19/CD22 double-negative K562 tumor cells.
图8不同结构CD22/CD3双特异性抗体对CD22阳性K562-CD22肿瘤细胞的结合能力比较。Figure 8 Comparison of the binding abilities of CD22/CD3 bispecific antibodies with different structures to CD22-positive K562-CD22 tumor cells.
图9不同结构CD22/CD3双特异性抗体对CD22阴性K562肿瘤细胞的结合能力比较。Figure 9 Comparison of the binding abilities of CD22/CD3 bispecific antibodies with different structures to CD22-negative K562 tumor cells.
图10不同CD22/CD3双特异性抗体对CD19/CD22双阳性Nalm6肿瘤细胞的体外杀伤活性验证。Figure 10 Verification of the in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19/CD22 double-positive Nalm6 tumor cells.
图11不同CD22/CD3双特异性抗体对CD19阳性CD22阴性Nalm6-KO22肿瘤细胞的体外杀伤活性验证。Figure 11 Verification of the in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19-positive CD22-negative Nalm6-KO22 tumor cells.
图12不同CD22/CD3双特异性抗体对CD19阴性CD22阳性Nalm6-KO19肿瘤细胞的体外杀伤活性验证。Figure 12 Verification of the in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19-negative CD22-positive Nalm6-KO19 tumor cells.
图13不同CD22/CD3双特异性抗体对CD19/CD22双阴性K562肿瘤细胞的体外杀伤活性验证。Figure 13 Verification of the in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19/CD22 double-negative K562 tumor cells.
图14优化结构的CD19/CD22/CD3(SO)三特异性抗体与结构最佳CD19/CD3、CD22/CD3双特异性抗体对CD19阳性K562-CD19肿瘤细胞的结合功能验证。Figure 14 Verification of the binding function of CD19/CD22/CD3 (SO) trispecific antibody with optimized structure and CD19/CD3 and CD22/CD3 bispecific antibodies with optimal structure on CD19-positive K562-CD19 tumor cells.
图15优化结构的CD19/CD22/CD3(SO)三特异性抗体与结构最佳CD19/CD3、CD22/CD3双特异性抗体对CD22阳性K562-CD22肿瘤细胞的结合功能验证。Figure 15 Verification of the binding function of CD19/CD22/CD3 (SO) trispecific antibody with optimized structure and CD19/CD3 and CD22/CD3 bispecific antibodies with optimal structure on CD22-positive K562-CD22 tumor cells.
图16优化结构的CD19/CD22/CD3(SO)三特异性抗体与结构最佳CD19/CD3、CD22/CD3双特异性抗体对CD19/CD22双阳性Nalm6肿瘤细胞的体外杀伤活性验证。Figure 16 Verification of the in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibody with optimized structure and CD19/CD3 and CD22/CD3 bispecific antibodies with optimal structure against CD19/CD22 double-positive Nalm6 tumor cells.
图17优化结构的CD19/CD22/CD3(SO)三特异性抗体与结构最佳CD19/CD3、CD22/CD3双特异性抗体对CD19阳性CD22阴性Nalm6-KO22肿瘤细胞的体外杀伤活性验证。Figure 17 Verification of the in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibody with optimized structure and CD19/CD3 and CD22/CD3 bispecific antibodies with optimal structure against CD19-positive CD22-negative Nalm6-KO22 tumor cells.
图18优化结构的CD19/CD22/CD3(SO)三特异性抗体与结构最佳CD19/CD3、CD22/CD3双特异性抗体对CD19阴性CD22阳性Nalm6-KO19肿瘤细胞的体外杀伤活性验证。Figure 18 Verification of the in vitro killing activity of CD19/CD22/CD3(SO) trispecific antibody with optimized structure and CD19/CD3 and CD22/CD3 bispecific antibodies with optimal structure against CD19-negative CD22-positive Nalm6-KO19 tumor cells.
图19优化结构的CD19/CD22/CD3(SO)三特异性抗体与结构最佳CD19/CD3、CD22/CD3双特异性抗体对CD19/CD22双阴性K562肿瘤细胞的体外杀伤活性验证。Figure 19 Verification of the in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibody with optimized structure and CD19/CD3 and CD22/CD3 bispecific antibodies with optimal structure against CD19/CD22 double-negative K562 tumor cells.
图20优化结构的CD19/CD22/CD3(SO)三特异性抗体与商品化CD19/CD3双特异性抗体Blinatumomab对CD19阳性CD22阴性K562-CD19肿瘤细胞的体外杀伤活性比较。Figure 20 Comparison of the in vitro killing activities of the optimized structure CD19/CD22/CD3 (SO) trispecific antibody and the commercial CD19/CD3 bispecific antibody Blinatumomab against CD19-positive CD22-negative K562-CD19 tumor cells.
图21优化结构的CD19/CD22/CD3(SO)三特异性抗体与商品化CD19/CD3双特异性抗体Blinatumomab对CD19阴性CD22阳性K562-CD22肿瘤细胞的体外杀伤活性比较。Figure 21 Comparison of the in vitro killing activities of the optimized structure CD19/CD22/CD3 (SO) trispecific antibody and the commercial CD19/CD3 bispecific antibody Blinatumomab against CD19-negative CD22-positive K562-CD22 tumor cells.
图22优化结构的CD19/CD22/CD3(SO)三特异性抗体与商品化CD19/CD3双特异性抗体Blinatumomab对CD19/CD22双阴性K562肿瘤细胞的体外杀伤活性比较。Figure 22 Comparison of the in vitro killing activities of the optimized structure CD19/CD22/CD3 (SO) trispecific antibody and the commercial CD19/CD3 bispecific antibody Blinatumomab against CD19/CD22 double-negative K562 tumor cells.
具体实施方式Detailed ways
下面结合实施例对本发明做进一步的解释与说明，应当理解为以下实施例仅用于说明本发明，不用来限制本发明的保护范围。The present invention will be further explained and illustrated below with reference to the examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.
以下各实施例中，实验所用材料均可购买，也可参照现有公开的技术制备；未标明来源和规格的均为市售可得；未详细描述的各种过程和方法是本领域中公知的常规方法。In the following examples, all materials used in the experiments can be purchased, or can be prepared with reference to existing disclosed technologies; those whose sources and specifications are not indicated are commercially available; various processes and methods not described in detail are well known in the art. conventional method.
实施例1 双/三特异性抗体的构建和真核表达Example 1 Construction and eukaryotic expression of bi/trispecific antibodies
CD3-HC-CD19-scFv与CD3-LC表达载体的构建：Construction of CD3-HC-CD19-scFv and CD3-LC expression vectors:
CD19/CD3双特异性抗体的重链由CD3-HC(SP34)和CD19-scFv分别按照以下方式进行连接：通过刚性连接肽PD Linker将CD19-scFv连接于CD3-HC可变区(VH)结构域N端1E；或者通过螺旋卷曲连接肽Coiled Coil Linker将CD19-scFv嵌合于CD3-HC恒定区(CH1)结构域184S-187L之间，同时缺失185S、186G；或者通过柔性连接肽(G4S)3 Linker将CD19-scFv连接于CD3-HC恒定区(CH1)结构域C端228C；轻链CD3-LC不做任何修饰。按常规分子生物学方法分别合成上述CD3-HC-CD19-scFv以及CD3-LC的编码基因，并将获得的合成基因通过同源重组方法插入到具有Zeocin抗性的pFUSE真核表达载体上。根据连接方式，后期表达的双特异性抗体分别标记为CD19/CD3 HNT、CD19/CD3 H184-187、CD19/CD3 HCT。其中抗CD19-scFv除通过本实验选择的PD Linker、Coiled Coil Linker以及(G4S)3 Linker连接于在CD3-HC，也可以采用本领域技术人员公知的连接肽Linker进行替换连接。相关序列如表1所示：The heavy chain of the CD19/CD3 bispecific antibody is connected by CD3-HC (SP34) and CD19-scFv in the following manner: CD19-scFv is connected to the CD3-HC variable region (VH) structure through the rigid linker peptide PD Linker. Domain N-terminal 1E; or CD19-scFv is chimeric between CD3-HC constant region (CH1) domain 184S-187L through a coiled coil linker peptide Coiled Coil Linker, while deleting 185S and 186G; or through a flexible linker peptide (G4S )3 Linker connects CD19-scFv to the C-terminal 228C of the CD3-HC constant region (CH1) domain; the light chain CD3-LC does not make any modifications. The above-mentioned CD3-HC-CD19-scFv and CD3-LC coding genes were synthesized according to conventional molecular biology methods, and the obtained synthetic genes were inserted into the Zeocin-resistant pFUSE eukaryotic expression vector through homologous recombination. According to the connection method, the bispecific antibodies expressed later were labeled as CD19/CD3 HNT, CD19/CD3 H184-187, and CD19/CD3 HCT. In addition to connecting the anti-CD19-scFv to CD3-HC through the PD Linker, Coiled Coil Linker and (G4S)3 Linker selected in this experiment, the anti-CD19-scFv can also be connected using a linking peptide Linker known to those skilled in the art. The relevant sequences are shown in Table 1:
表1Table 1
CD3-LC-CD22-VHH与CD3-HC表达载体的构建：Construction of CD3-LC-CD22-VHH and CD3-HC expression vectors:
CD22/CD3双特异性抗体的轻链由CD3-LC(SP34)和CD22-VHH分别按照以下方式进行连接：通过刚性连接肽PD Linker将CD22-VHH连接于CD3-LC可变区(VL)结构域N端1Q；或者通过螺旋卷曲连接肽Coiled Coil Linker将CD22-VHH嵌合于CD3-LC恒定区(CL)结构域171S-173D之间，并且缺失172K；或者通过柔性连接肽(G4S)3 Linker将CD22-VHH连接于CD3-LC恒定区(CL)结构域C端217C；重链CD3-HC不做任何修饰。按常规分子生物学方法分别合成上述CD3-LC-CD22-VHH以及CD3-HC的编码基因，并将获得的合成基因通过同源重组方法插入到具有Zeocin抗性的pFUSE真核表达载体上。根据连接方式，后期表达的双特异性抗体分别标记为CD22/CD3 LNT、CD22/CD3 L171-173、CD22/CD3 LCT。其中抗CD22-VHH除通过本实验选择的PD Linker、Coiled Coil Linker以及(G4S)3 Linker连接于在CD3-LC，也可以采用本领域技术人员公知的连接肽Linker进行替换连接。相关序列如表2所示：The light chain of the CD22/CD3 bispecific antibody is connected by CD3-LC (SP34) and CD22-VHH in the following manner: CD22-VHH is connected to the CD3-LC variable region (VL) structure through the rigid linker peptide PD Linker. Domain N-terminal 1Q; or CD22-VHH is chimeric between CD3-LC constant region (CL) domain 171S-173D through the coiled coil linker peptide Coiled Coil Linker, and 172K is deleted; or through the flexible linker peptide (G4S) 3 Linker connects CD22-VHH to the C-terminal 217C of the CD3-LC constant region (CL) domain; the heavy chain CD3-HC does not make any modifications. The above-mentioned CD3-LC-CD22-VHH and CD3-HC coding genes were synthesized according to conventional molecular biology methods, and the obtained synthetic genes were inserted into the Zeocin-resistant pFUSE eukaryotic expression vector through homologous recombination. According to the connection method, the bispecific antibodies expressed later were labeled as CD22/CD3 LNT, CD22/CD3 L171-173, and CD22/CD3 LCT. In addition to connecting the anti-CD22-VHH to CD3-LC through the PD Linker, Coiled Coil Linker and (G4S)3 Linker selected in this experiment, the anti-CD22-VHH can also be connected using a linking peptide Linker known to those skilled in the art. The relevant sequences are shown in Table 2:
表2Table 2
CD19/CD22/CD3(SO)表达载体的构建：Construction of CD19/CD22/CD3(SO) expression vector:
CD19/CD22/CD3(SO)三特异性抗体的重链由CD3-HC(SP34)和CD19-scFv按照以下方式进行连接：通过柔性连接肽(G4S)3 Linker将CD19-scFv连接于在CD3-HC恒定区(CH1)结构域C端228C；CD19/CD22/CD3(SO)三特异性抗体的轻链由CD3-LC(SP34)和CD22-VHH按照以下方式进行连接：通过柔性连接肽(G4S)3 Linker将CD22-VHH连接于在CD3-LC恒定区(CL)结构域C端217C。并将获得的基因通过同源重组方法插入到具有Zeocin抗性的pFUSE真核表达载体上。根据连接方式，后期表达的三特异性抗体标记为CD19/CD22/CD3(SO)。其中CD19-scFv、CD22-VHH除通过本实验选择的(G4S)3 Linker连接于CD3-HC或者CD3-LC外，也可以采用本领域技术人员公知的连接肽Linker进行替换连接。相关序列如表3所示：The heavy chain of the CD19/CD22/CD3 (SO) trispecific antibody is connected by CD3-HC (SP34) and CD19-scFv in the following manner: CD19-scFv is connected to the CD3-scFv through a flexible linker peptide (G4S) 3 Linker. The C-terminal 228C of the HC constant region (CH1) domain; the light chain of the CD19/CD22/CD3 (SO) trispecific antibody is connected by CD3-LC (SP34) and CD22-VHH in the following manner: through a flexible linker peptide (G4S )3 Linker connects CD22-VHH to 217C at the C terminus of the CD3-LC constant region (CL) domain. And the obtained gene was inserted into the Zeocin-resistant pFUSE eukaryotic expression vector through homologous recombination. According to the connection method, the later expressed trispecific antibody is labeled CD19/CD22/CD3(SO). In addition to connecting CD19-scFv and CD22-VHH to CD3-HC or CD3-LC through the (G4S)3 Linker selected in this experiment, they can also be connected using a linking peptide Linker known to those skilled in the art. The relevant sequences are shown in Table 3:
表3table 3
本发明双特异性抗体的结构设计以及基于结构优化活性设计的三特异性抗体结构如图1所示。The structural design of the bispecific antibody of the present invention and the structure of the trispecific antibody designed based on structure optimization and activity are shown in Figure 1.
1.2蛋白的真核表达1.2 Eukaryotic expression of protein
接种1.5×106/mL 293悬浮细胞于500mL摇瓶200mL培养基中，37℃，165rpm，5％CO2浓度的摇床培养箱中振摇培养24h，24h后进行细胞计数，将细胞密度调整至3×106/mL，将上述1.1步骤所构建的轻链与重链质粒于10mL Opti-MEM混合均匀后室温静置5min，加入500μL(1∶2.5)PEI 40000于10mL Opti-MEM轻柔混匀后室温静置5min，混合质粒和PEI混合液，轻柔混匀后室温静置20min。将上述混合液逐滴加入200mL 293悬浮细胞培养液中，滴加的同时轻柔摇晃培养瓶，混合均匀后放入摇床，转染72h。转染结束后，100g离心5min，取上清液，向培养瓶中补加100mL培养基重新悬浮培养48h后3000rpm离心5min，取上清液，将两次收集的293悬浮细胞上清液混合，于-20℃冻存。Inoculate 1.5 × 106 /mL 293 suspended cells into 200 mL culture medium in a 500 mL shake flask, and culture in a shaking incubator at 37°C, 165 rpm, and 5% CO2 for 24 hours. After 24 hours, count the cells and adjust the cell density. to 3×106 /mL, mix the light chain and heavy chain plasmids constructed in step 1.1 above in 10mL Opti-MEM and let stand at room temperature for 5 minutes. Add 500μL (1:2.5) PEI 40000 to 10mL Opti-MEM and mix gently. After mixing, let it stand at room temperature for 5 minutes, mix the plasmid and PEI mixture, mix gently and let it stand at room temperature for 20 minutes. Add the above mixture drop by drop to 200 mL of 293 suspension cell culture medium. Shake the culture bottle gently while adding dropwise. Mix evenly and put it into a shaker for 72 hours of transfection. After the transfection is completed, centrifuge at 100 g for 5 minutes, take the supernatant, add 100 mL of culture medium to the culture bottle, resuspend and culture for 48 hours, centrifuge at 3000 rpm for 5 minutes, take the supernatant, and mix the 293 suspended cell supernatants collected twice. Store frozen at -20°C.
实施例2 三特异性抗体的纯化Example 2 Purification of trispecific antibodies
2.1 Protein G亲和纯化2.1 Protein G affinity purification
取上述实施例1中1.2的细胞上清液400mL，15000rpm，4℃离心30min，收取上清液，0.45μm滤膜过滤，置于冰上备用。取4mL Protein G(20％乙醇/Protein G 1∶1)于层析柱内，用Binding buffer冲洗3次后，使用垫片压住resin表面。用20mL Binding buffer平衡Protein G柱。每10mL上样一次，使样品匀速(约0.5mL/min)通过Protein G柱。40mLBinding buffer匀速(约1mL/min)清洗Protein G柱。首先在洗脱管收集管中加入10％洗脱液体积的中和缓冲液，柱中加入Elution buffer，5mL洗脱一次，直到无法定量蛋白浓度。使用Amicon Ultra-15离心过滤器对收集的蛋白样品进行浓缩，4℃离心机3000rpm离心20分钟，定量蛋白浓度。Take 400 mL of the cell supernatant from 1.2 in the above Example 1, centrifuge at 15,000 rpm for 30 min at 4°C, collect the supernatant, filter it with a 0.45 μm filter, and place it on ice for later use. Put 4mL of Protein G (20% ethanol/Protein G 1:1) into the chromatography column, rinse it three times with Binding buffer, and use a gasket to press the resin surface. Equilibrate the Protein G column with 20mL Binding buffer. Load the sample every 10 mL and pass the sample through the Protein G column at a constant speed (about 0.5 mL/min). Clean the Protein G column with 40mL Binding buffer at a constant speed (about 1mL/min). First, add 10% of the elution volume of neutralization buffer to the elution tube collection tube, add Elution buffer to the column, and elute once with 5 mL until the protein concentration cannot be quantified. The collected protein samples were concentrated using Amicon Ultra-15 centrifugal filters, centrifuged at 3000 rpm at 4°C for 20 minutes, and the protein concentration was quantified.
实施例3 CD19/CD22/CD3三特异性抗体的体外生物学功能评价Example 3 In vitro biological function evaluation of CD19/CD22/CD3 trispecific antibodies
3.1不同结构CD19/CD3双特异性抗体的细胞结合能力3.1 Cell-binding abilities of CD19/CD3 bispecific antibodies with different structures
采用CD19工程化K562细胞系K562-CD19(CD19+CD22-)和K562(CD19-CD22-)进行细胞计数，调整细胞密度为3×106/mL每孔100μL。将待测双特异性抗体CD19/CD3 HNT、CD19/CD3 H184-187、CD19/CD3 HCT采用FACS稀释液5倍稀释5个梯度，起始抗体浓度为50nM，每个浓度2个复孔，100μL/孔加入孔板内，冰上孵育2h。配制二抗：APC标记抗人Kappa链，抗体浓度1μg/mL。每孔100μL重悬细胞，冰上1h。离心去上清，清洗一次。200μL FACS重悬细胞。流式检测，以蛋白浓度为横坐标，平均荧光强度为纵坐标作图，做四参数非线性回归图并计算EC50值。CD19-engineered K562 cell lines K562-CD19 (CD19+CD22-) and K562 (CD19-CD22-) were used for cell counting, and the cell density was adjusted to 3×106 /mL with 100 μL per well. The bispecific antibodies to be tested, CD19/CD3 HNT, CD19/CD3 H184-187, and CD19/CD3 HCT, were diluted 5 times in 5 gradients using FACS diluent. The initial antibody concentration was 50 nM, with 2 duplicate wells for each concentration, 100 μL. /well into the well plate and incubate on ice for 2 hours. Prepare secondary antibodies: APC-labeled anti-human Kappa chain, antibody concentration 1 μg/mL. Resuspend cells in 100 μL per well and place on ice for 1 hour. Centrifuge to remove the supernatant and wash once. Resuspend cells in 200 μL FACS. For flow cytometry, plot the protein concentration as the abscissa and the average fluorescence intensity as the ordinate, make a four-parameter nonlinear regression plot and calculate the EC50 value.
结果如表4、图2和图3所示，不同结构的CD19/CD3双特异性抗体均能有效结合K562-CD19(CD19+CD22-)，而对K562(CD19-CD22-)没有显示任何非特异性结合，从EC50值比较来看，相比较于CD19/CD3 HNT和CD19/CD3 H184-187，CD19/CD3 HCT显示了更好的抗原结合亲和力。The results are shown in Table 4, Figure 2 and Figure 3. CD19/CD3 bispecific antibodies with different structures can effectively bind to K562-CD19 (CD19+CD22-), but do not show any non-specific binding to K562 (CD19-CD22-). For heterosexual binding, judging from the EC50 value comparison, CD19/CD3 HCT shows better antigen binding affinity compared to CD19/CD3 HNT and CD19/CD3 H184-187.
表4Table 4
3.2不同结构CD19/CD3双特异性抗体的体外肿瘤杀伤活性3.2 In vitro tumor killing activity of CD19/CD3 bispecific antibodies with different structures
用含300IU/mL浓度IL2的RPMI-1640完全培养基培养活化的人T细胞(来自于健康志愿者的PBMC)、人B细胞白血病细胞Nalm6(CD19+CD22+)、以及敲除CD19或者敲除CD22的Nalm6细胞，分别为Nalm6-KO19(CD19-CD22+)、Nalm6-KO22(CD19+CD22-)、K562(CD19-CD22-)。实验前24h用去除IL2的RPMI-1640培养基培养T细胞。肿瘤细胞采用荧光染料CFSE染色，调整T细胞密度为2×106，肿瘤细胞密度为2×105，按照效靶比10∶1混合2种细胞的混合液加入96孔细胞板内100μL/孔。将待测双特异性抗体CD19/CD3 HNT、CD19/CD3 H184-187、CD19/CD3 HCT用培养基10倍稀释3个梯度，起始抗体浓度为100pM，每个浓度梯度2个复孔，10μL/孔加入孔板内。实验同时对照(肿瘤细胞和T细胞)，于37℃，5％CO2培养箱培养24h。每孔加入7-AAD染色30min后进行流式细胞分析，对CFSE阳性7-AAD阴性的肿瘤细胞进行计数，采用公式：％Lysis＝(对照组活肿瘤细胞数-实验组活肿瘤细胞数)/对照组活肿瘤细胞数×100。并采用Graphpad Prism6软件进行数据分析，以抗体浓度数值为横坐标，％Lysis为纵坐标做四参数非线性回归图并计算IC50值。Activated human T cells (PBMC from healthy volunteers), human B-cell leukemia cells Nalm6 (CD19+CD22+), and CD19 knockout or CD22 knockout were cultured in RPMI-1640 complete medium containing IL2 at a concentration of 300IU/mL. The Nalm6 cells are Nalm6-KO19 (CD19-CD22+), Nalm6-KO22 (CD19+CD22-), and K562 (CD19-CD22-). T cells were cultured in RPMI-1640 medium without IL2 24 h before the experiment. Tumor cells were stained with the fluorescent dye CFSE. The T cell density was adjusted to 2×106 and the tumor cell density was 2×105 . A mixture of the two types of cells was mixed according to the effect-to-target ratio of 10:1 and added to the 96-well cell plate at 100 μL/well. . Dilute the bispecific antibodies CD19/CD3 HNT, CD19/CD3 H184-187, and CD19/CD3 HCT 10 times into 3 gradients with culture medium. The initial antibody concentration is 100 pM. Each concentration gradient has 2 duplicate wells, 10 μL. /well is added to the orifice plate. The experiment was conducted simultaneously with controls (tumor cells and T cells) and cultured in a 37°C, 5% CO2 incubator for 24 hours. Flow cytometry analysis was performed after adding 7-AAD staining to each well for 30 minutes. The CFSE-positive 7-AAD-negative tumor cells were counted using the formula: % Lysis = (number of viable tumor cells in the control group - number of viable tumor cells in the experimental group)/ The number of viable tumor cells in the control group ×100. Graphpad Prism6 software was used for data analysis. The antibody concentration value was used as the abscissa and %Lysis was used as the ordinate to make a four-parameter nonlinear regression chart and calculate the IC50 value.
结果如图4，图5，图6和图7所示，3种结构的CD19/CD3双特异性抗体对Nalm6(CD19+CD22+)和Nalm6-KO22(CD19+CD22-)均具有CD19抗原特异性细胞毒性，而对CD19阴性的Nalm6-KO19(CD19-CD22+)和K562(CD19-CD22-)不具有杀伤活性。通过对IC50的比较发现，与CD19/CD3 HNT和CD19/CD3 H184-187双特异性抗体相比，CD19/CD3 HCT表现出显著提高的肿瘤细胞特异性杀伤效果。因此，通过CD19抗原结合活性和CD19阳性肿瘤细胞杀伤活性的比较，确定了CD19-scFv融合于CD3-Fab的HCT为最佳重组抗体结构。The results are shown in Figure 4, Figure 5, Figure 6 and Figure 7. The three structures of CD19/CD3 bispecific antibodies all have CD19 antigen specificity for Nalm6 (CD19+CD22+) and Nalm6-KO22 (CD19+CD22-). Cytotoxic, but has no killing activity against CD19-negative Nalm6-KO19 (CD19-CD22+) and K562 (CD19-CD22-). Through comparison of IC50, it was found that compared with CD19/CD3 HNT and CD19/CD3 H184-187 bispecific antibodies, CD19/CD3 HCT showed significantly improved tumor cell specific killing effect. Therefore, through the comparison of CD19 antigen-binding activity and CD19-positive tumor cell killing activity, it was determined that HCT in which CD19-scFv was fused to CD3-Fab was the best recombinant antibody structure.
3.3不同结构CD22/CD3双特异性抗体的细胞结合能力3.3 Cell-binding abilities of CD22/CD3 bispecific antibodies with different structures
采用CD22工程化K562细胞系K562-CD22(CD19-CD22+)和K562(CD19-CD22-)进行细胞计数，调整细胞密度为3×106/mL每孔100μL。将待测双特异性抗体CD22/CD3 LNT、CD22/CD3 L171-173、CD22/CD3 LCT采用FACS稀释液5倍稀释7个梯度，起始抗体浓度为200nM，每个浓度2个复孔，100μL/孔加入孔板内，冰上孵育2h。配制二抗：APC标记抗人Kappa链，抗体浓度1μg/mL。每孔100μL重悬细胞，冰上1h。离心去上清，清洗一次。200μL FACS重悬细胞。流式检测，以蛋白浓度为横坐标，平均荧光强度为纵坐标作图，做四参数非线性回归图并计算EC50值。CD22-engineered K562 cell lines K562-CD22 (CD19-CD22+) and K562 (CD19-CD22-) were used for cell counting, and the cell density was adjusted to 3×106 /mL with 100 μL per well. The bispecific antibodies to be tested, CD22/CD3 LNT, CD22/CD3 L171-173, and CD22/CD3 LCT, were diluted 5 times in 7 gradients using FACS diluent. The initial antibody concentration was 200 nM, with 2 duplicate wells for each concentration, 100 μL. /well into the well plate and incubate on ice for 2 hours. Prepare secondary antibodies: APC-labeled anti-human Kappa chain, antibody concentration 1 μg/mL. Resuspend cells in 100 μL per well and place on ice for 1 hour. Centrifuge to remove the supernatant and wash once. Resuspend cells in 200 μL FACS. For flow cytometry, plot the protein concentration as the abscissa and the average fluorescence intensity as the ordinate, make a four-parameter nonlinear regression plot and calculate the EC50 value.
结果表5、如图8和图9所示，不同结构的CD22/CD3双特异性抗体均能有效结合K562-CD22(CD19-CD22+)，而对K562(CD19-CD22-)没有显示任何非特异性结合，从EC50值比较来看，与CD22/CD3 LNT和CD22/CD3 L171-173双特异性抗体相比，CD22/CD3 LCT具有良好的抗原结合能力。Result Table 5, as shown in Figure 8 and Figure 9, CD22/CD3 bispecific antibodies with different structures can effectively bind to K562-CD22 (CD19-CD22+), but do not show any non-specificity to K562 (CD19-CD22-) From the comparison of EC50 values, CD22/CD3 LCT has good antigen-binding ability compared with CD22/CD3 LNT and CD22/CD3 L171-173 bispecific antibodies.
表5table 5
3.4不同结构CD22/CD3双特异性抗体的体外肿瘤杀伤活性3.4 In vitro tumor killing activity of CD22/CD3 bispecific antibodies with different structures
用含300IU/mL浓度IL2的RPMI-1640完全培养基培养活化的人T细胞(来自于健康志愿者的PBMC)、人B细胞白血病细胞Nalm6(CD19+CD22+)、以及敲除CD19或者敲除CD22的Nalm6细胞，分别为Nalm6-KO19(CD19-CD22+)、Nalm6-KO22(CD19+CD22-)、K562(CD19-CD22-)。实验前24h用去除IL2的RPMI-1640培养基培养T细胞。肿瘤细胞采用荧光染料CFSE染色，调整T细胞密度为2×106，肿瘤细胞密度为2×105，按照效靶比10∶1混合2种细胞的混合液加入96孔细胞板内100μL/孔。将待测双特异性抗体CD22/CD3 LNT、CD22/CD3 L171-173、CD22/CD3 LCT用培养基10倍稀释5个梯度，起始抗体浓度为1nM，每个浓度梯度2个复孔，10μL/孔加入孔板内。实验同时对照(肿瘤细胞和T细胞)，于37℃，5％CO2培养箱培养24h。每孔加入7-AAD染色30min后进行流式细胞分析，对CFSE阳性7-AAD阴性的肿瘤细胞进行计数，采用公式：％Lysis＝(对照组活肿瘤细胞数-实验组活肿瘤细胞数)/对照组活肿瘤细胞数×100。并采用Graphpad Prism6软件进行数据分析，以抗体浓度数值为横坐标，％Lysis为纵坐标做四参数非线性回归图并计算IC50值。Activated human T cells (PBMC from healthy volunteers), human B-cell leukemia cells Nalm6 (CD19+CD22+), and CD19 knockout or CD22 knockout were cultured in RPMI-1640 complete medium containing IL2 at a concentration of 300IU/mL. The Nalm6 cells are Nalm6-KO19 (CD19-CD22+), Nalm6-KO22 (CD19+CD22-), and K562 (CD19-CD22-). T cells were cultured in RPMI-1640 medium without IL2 24 h before the experiment. Tumor cells were stained with the fluorescent dye CFSE. The T cell density was adjusted to 2×106 and the tumor cell density was 2×105 . A mixture of the two types of cells was mixed according to the effect-to-target ratio of 10:1 and added to the 96-well cell plate at 100 μL/well. . Dilute the bispecific antibodies CD22/CD3 LNT, CD22/CD3 L171-173, and CD22/CD3 LCT 10 times into 5 gradients with culture medium. The initial antibody concentration is 1 nM. Each concentration gradient has 2 duplicate wells, 10 μL. /well is added to the orifice plate. The experiment was conducted simultaneously with controls (tumor cells and T cells) and cultured in a 37°C, 5% CO2 incubator for 24 hours. Flow cytometry analysis was performed after adding 7-AAD staining to each well for 30 minutes. The CFSE-positive 7-AAD-negative tumor cells were counted using the formula: % Lysis = (number of viable tumor cells in the control group - number of viable tumor cells in the experimental group)/ The number of viable tumor cells in the control group ×100. Graphpad Prism6 software was used for data analysis. The antibody concentration value was used as the abscissa and %Lysis was used as the ordinate to make a four-parameter nonlinear regression chart and calculate the IC50 value.
结果如图10，图11，图12和图13所示，3种结构的CD22/CD3双特异性抗体对Nalm6(CD19+CD22+)和Nalm6-KO19(CD19-CD22+)均具有CD22抗原特异性细胞毒性，而对CD19阴性的Nalm6-KO22(CD19+CD22-)和K562(CD19-CD22-)不具有杀伤活性。通过对IC50的比较发现，与CD22/CD3 LNT和CD22/CD3 L171-173双特异性抗体相比，CD22/CD3 LCT表现出显著提高的肿瘤细胞特异性杀伤效果；另外CD22/CD3 LNT在高浓度显示了对CD22阴性Nalm6-KO22(CD19+CD22-)的非特异细胞毒性。因此，通过CD22抗原结合活性和CD22阳性肿瘤细胞杀伤活性的比较，确定了CD22-VHH融合于CD3-Fab的LCT为最佳重组抗体结构。The results are shown in Figure 10, Figure 11, Figure 12 and Figure 13. The three structures of CD22/CD3 bispecific antibodies have CD22 antigen specificity for Nalm6 (CD19+CD22+) and Nalm6-KO19 (CD19-CD22+) cells. Toxicity, but has no killing activity against CD19-negative Nalm6-KO22 (CD19+CD22-) and K562 (CD19-CD22-). Through comparison of IC50, it was found that compared with CD22/CD3 LNT and CD22/CD3 L171-173 bispecific antibody, CD22/CD3 LCT showed significantly improved tumor cell specific killing effect; in addition, CD22/CD3 LNT showed a significantly improved tumor cell specific killing effect at high concentrations. Non-specific cytotoxicity towards CD22-negative Nalm6-KO22 (CD19+CD22-) was shown. Therefore, through the comparison of CD22 antigen-binding activity and CD22-positive tumor cell killing activity, it was determined that LCT with CD22-VHH fused to CD3-Fab was the best recombinant antibody structure.
3.5优化结构CD19/CD22/CD3(SO)三特异性抗体的抗原结合验证3.5 Antigen binding verification of optimized structure CD19/CD22/CD3(SO) trispecific antibody
具体实施步骤同3.1和3.3，结果如表6、图14和图15所示，通过对EC50的比较发现，优化结构CD19/CD22/CD3(SO)三特异性抗体与活性最佳的双特异性抗体CD19/CD3 HCT以及CD22/CD3 LCT相比，三特异性抗体保留了CD19-scFv和CD22-VHH抗体的特异性和亲和力，与靶细胞K562-CD19(CD19+CD22-)或者K562-CD22(CD19-CD22+)的结合能力无明显差别。其中CD19/CD22/CD3(SO)三特异性抗体具有对CD19/CD3结合特异性的如SEQ ID NO：3所示的重组重链氨基酸序列，和具有对CD22/CD3结合特异性的如SEQ ID NO：6所示的重组轻链氨基酸序列。The specific implementation steps are the same as 3.1 and 3.3. The results are shown in Table 6, Figure 14 and Figure 15. By comparing the EC50, it was found that the optimized structure CD19/CD22/CD3 (SO) trispecific antibody and the best activity bispecific Compared with antibodies CD19/CD3 HCT and CD22/CD3 LCT, trispecific antibodies retain the specificity and affinity of CD19-scFv and CD22-VHH antibodies, and bind to target cells K562-CD19 (CD19+CD22-) or K562-CD22 ( There is no significant difference in the binding ability of CD19-CD22+). The CD19/CD22/CD3 (SO) trispecific antibody has a recombinant heavy chain amino acid sequence shown in SEQ ID NO: 3 that is specific for CD19/CD3 binding, and has a recombinant heavy chain amino acid sequence that is specific for CD22/CD3 binding, such as SEQ ID The recombinant light chain amino acid sequence shown in NO: 6.
表6Table 6
3.6优化结构CD19/CD22/CD3(SO)三特异性抗体与双特异性抗体的肿瘤细胞杀伤活性比较3.6 Comparison of tumor cell killing activities of optimized structure CD19/CD22/CD3(SO) trispecific antibodies and bispecific antibodies
具体实施步骤同3.2和3.4，结果如图16，图17，图18和图19所示，通过对IC50值的比较发现，与活性最佳的双特异性抗体CD19/CD3 HCT以及CD22/CD3 LCT相比，结构优化后的三特异性抗体CD19/CD22/CD3(SO)对CD19和CD22双阳性细胞系Nalm6(CD19+CD22+)、CD19单阳性细胞系Nalm6-KO22(CD19+CD22-)以及CD22单阳性细胞系Nalm6-KO19(CD19-CD22+)具有显著提高的杀伤效果，并且对CD19和CD22双阴性细胞系K562(CD19-CD22-)没有非特异性杀伤效果。The specific implementation steps are the same as 3.2 and 3.4. The results are shown in Figure 16, Figure 17, Figure 18 and Figure 19. By comparing the IC50 values, it is found that compared with the bispecific antibodies CD19/CD3 HCT and CD22/CD3 LCT with the best activity In comparison, the structurally optimized trispecific antibody CD19/CD22/CD3(SO) was effective against the CD19 and CD22 double-positive cell line Nalm6 (CD19+CD22+), the CD19 single-positive cell line Nalm6-KO22 (CD19+CD22-), and CD22 The single-positive cell line Nalm6-KO19 (CD19-CD22+) has a significantly improved killing effect and has no non-specific killing effect on the CD19 and CD22 double-negative cell line K562 (CD19-CD22-).
3.7优化结构CD19/CD22/CD3(SO)三特异性抗体与Blinatumomab的杀伤肿瘤细胞活性比较3.7 Comparison of tumor cell killing activity between optimized structure CD19/CD22/CD3(SO) trispecific antibody and Blinatumomab
用含300IU/mL浓度IL2的RPMI-1640完全培养基培养活化的人T细胞(来自于健康志愿者的PBMC)、CD19工程化K562细胞系K562-CD19(CD19+CD22-)、CD22工程化K562细胞系K562-CD22(CD19-CD22+)和K562(CD19-CD22-)。实验前24h用去除IL2的RPMI-1640培养基培养T细胞。肿瘤细胞采用荧光染料CFSE染色，调整T细胞密度为2×106，肿瘤细胞密度为2×105，按照效靶比10∶1混合2种细胞的混合液加入96孔细胞板内100μL/孔。将待测三特异性抗体CD19/CD22/CD3(SO)和Blinatumomab用培养基10倍稀释4个梯度，起始抗体浓度为1nM，每个浓度梯度2个复孔，10μL/孔加入孔板内。实验同时对照(肿瘤细胞和T细胞)，于37℃，5％CO2培养箱培养24h。每孔加入7-AAD染色30min后进行流式细胞分析，对CFSE阳性7-AAD阴性的肿瘤细胞进行计数，采用公式：％Lysis＝(对照组活肿瘤细胞数-实验组活肿瘤细胞数)/对照组活肿瘤细胞数×100。并采用Graphpad Prism6软件进行数据分析，以抗体浓度数值为横坐标，％Lysis为纵坐标做四参数非线性回归图并计算IC50值。Activated human T cells (PBMC from healthy volunteers), CD19-engineered K562 cell line K562-CD19 (CD19+CD22-), and CD22-engineered K562 were cultured in RPMI-1640 complete medium containing IL2 at a concentration of 300IU/mL. Cell lines K562-CD22 (CD19-CD22+) and K562 (CD19-CD22-). T cells were cultured in RPMI-1640 medium without IL2 24 h before the experiment. Tumor cells were stained with the fluorescent dye CFSE, and the T cell density was adjusted to 2×106 and the tumor cell density was 2×105 . A mixture of the two types of cells was mixed according to an effect-to-target ratio of 10:1 and added to a 96-well cell plate at 100 μL/well. . Dilute the trispecific antibodies CD19/CD22/CD3(SO) and Blinatumomab 10 times into 4 gradients with culture medium. The initial antibody concentration is 1 nM. Each concentration gradient has 2 duplicate wells and 10 μL/well is added to the well plate. . The experiments were conducted simultaneously with controls (tumor cells and T cells) and cultured in a 37°C, 5% CO2 incubator for 24 hours. Flow cytometry analysis was performed after adding 7-AAD staining to each well for 30 minutes. The CFSE-positive 7-AAD-negative tumor cells were counted using the formula: % Lysis = (number of viable tumor cells in the control group - number of viable tumor cells in the experimental group)/ The number of viable tumor cells in the control group ×100. Graphpad Prism6 software was used for data analysis. The antibody concentration value was used as the abscissa and %Lysis was used as the ordinate to make a four-parameter nonlinear regression chart and calculate the IC50 value.
结果如图20，图21和图22所示，CD19/CD22/CD3(SO)三特异性抗体对K562-CD19(CD19+CD22-)和K562-CD22(CD19-CD22+)都具有抗原特异性杀伤活性，而对阴性细胞K562(CD19-CD22-)不具有杀伤活性。而Blinatumomab仅仅对K562-CD19(CD19+CD22-)细胞具有杀伤活性，对于模拟CD19抗原免疫逃逸的K562-CD22(CD19-CD22+)细胞无法识别杀伤。因此，CD19/CD22/CD3(SO)三特异性抗体具有更加广泛的肿瘤细胞多靶点识别应用性，尤其是对于Blinatumomab耐药性且CD22阳性的肿瘤细胞具有杀伤效果。综上所述，通过抗原结合活性和肿瘤细胞杀伤活性的比较，优化结构设计的CD19/CD22/CD3三特异性抗体具有显著的血液肿瘤免疫杀伤功能，可防止因单靶点识别造成的免疫逃逸以及Blinatumomab治疗引发耐药性等问题。The results are shown in Figure 20, Figure 21 and Figure 22. The CD19/CD22/CD3 (SO) trispecific antibody has antigen-specific killing effect on both K562-CD19 (CD19+CD22-) and K562-CD22 (CD19-CD22+). activity, but has no killing activity against negative cells K562 (CD19-CD22-). Blinatumomab only has killing activity against K562-CD19 (CD19+CD22-) cells, and cannot recognize and kill K562-CD22 (CD19-CD22+) cells that simulate immune escape of CD19 antigen. Therefore, the CD19/CD22/CD3(SO) trispecific antibody has a broader applicability in multi-target recognition of tumor cells, and is especially effective in killing Blinatumomab-resistant and CD22-positive tumor cells. In summary, through the comparison of antigen-binding activity and tumor cell killing activity, the optimized structurally designed CD19/CD22/CD3 trispecific antibody has significant blood tumor immune killing function and can prevent immune escape caused by single target recognition. And problems such as drug resistance caused by Blinatumomab treatment.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110429043.1ACN114853897B (en) | 2021-04-15 | 2021-04-15 | Anti-CD19/CD22/CD3 trispecific antibodies and their uses |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110429043.1ACN114853897B (en) | 2021-04-15 | 2021-04-15 | Anti-CD19/CD22/CD3 trispecific antibodies and their uses |
| Publication Number | Publication Date |
|---|---|
| CN114853897A CN114853897A (en) | 2022-08-05 |
| CN114853897Btrue CN114853897B (en) | 2024-01-26 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110429043.1AActiveCN114853897B (en) | 2021-04-15 | 2021-04-15 | Anti-CD19/CD22/CD3 trispecific antibodies and their uses |
| Country | Link |
|---|---|
| CN (1) | CN114853897B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025103425A1 (en)* | 2023-11-15 | 2025-05-22 | Concept To Medicine Biotech Co., Ltd. | Multispecific antibodies and uses thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188305A (en)* | 2015-06-01 | 2016-12-07 | 中山大学 | There is the bivalent antibody of the single domain Fab being fused to conventional Fab fragment |
| CN109069635A (en)* | 2016-02-04 | 2018-12-21 | 加州生物医学研究所 | Humanized CD 3-resisting antibody, conjugate and application thereof |
| CN110305210A (en)* | 2018-03-27 | 2019-10-08 | 信达生物制药(苏州)有限公司 | Novel antibody molecule, its preparation method and its use |
| CN111148761A (en)* | 2017-06-25 | 2020-05-12 | 西雅图免疫公司 | Multispecific antibodies and methods of making and using the same |
| CN111217908A (en)* | 2019-11-29 | 2020-06-02 | 深圳普瑞金生物药业有限公司 | CD22 single domain antibody, nucleotide sequence, kit, CAR-T viral vector and CAR-T cell |
| CN111349161A (en)* | 2018-12-20 | 2020-06-30 | 上海星湾生物技术有限公司 | Monoclonal antibody of anti-CD 19 antibody and application thereof |
| CN112119099A (en)* | 2018-03-02 | 2020-12-22 | Cdr-生物科技股份有限公司 | Trispecific antigen binding proteins |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018054484A1 (en)* | 2016-09-23 | 2018-03-29 | Biontech Ag | Bispecific trivalent antibodies binding to claudin 6 or claudin18.2 and cd3 for treatment of claudin expressing cancer diseases |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188305A (en)* | 2015-06-01 | 2016-12-07 | 中山大学 | There is the bivalent antibody of the single domain Fab being fused to conventional Fab fragment |
| CN109069635A (en)* | 2016-02-04 | 2018-12-21 | 加州生物医学研究所 | Humanized CD 3-resisting antibody, conjugate and application thereof |
| CN111148761A (en)* | 2017-06-25 | 2020-05-12 | 西雅图免疫公司 | Multispecific antibodies and methods of making and using the same |
| CN112119099A (en)* | 2018-03-02 | 2020-12-22 | Cdr-生物科技股份有限公司 | Trispecific antigen binding proteins |
| CN110305210A (en)* | 2018-03-27 | 2019-10-08 | 信达生物制药(苏州)有限公司 | Novel antibody molecule, its preparation method and its use |
| CN111349161A (en)* | 2018-12-20 | 2020-06-30 | 上海星湾生物技术有限公司 | Monoclonal antibody of anti-CD 19 antibody and application thereof |
| CN111217908A (en)* | 2019-11-29 | 2020-06-02 | 深圳普瑞金生物药业有限公司 | CD22 single domain antibody, nucleotide sequence, kit, CAR-T viral vector and CAR-T cell |
| Publication number | Publication date |
|---|---|
| CN114853897A (en) | 2022-08-05 |
| Publication | Publication Date | Title |
|---|---|---|
| JP6707496B2 (en) | APRIL variant | |
| Felices et al. | Generation of BiKEs and TriKEs to improve NK cell-mediated targeting of tumor cells | |
| US20200093861A1 (en) | Antigen binding receptor formats | |
| JP2015527070A5 (en) | ||
| WO2020155310A1 (en) | Novel scfv amino acid sequence, chimeric antigen receptor comprising same and application thereof | |
| WO2018068652A1 (en) | Anti-egfr and anti-cd3 bispecific antibody and applications thereof | |
| JP2018046811A (en) | Multispecific binding molecules targeting aberrant cells | |
| WO2021008610A1 (en) | Anti-dll3 chimeric antigen receptors and uses thereof | |
| CN108034006A (en) | The difunctional complementation of double antigen inductions | |
| CN113784980B (en) | Humanized anti-Claudin18.2 chimeric antigen receptor and its use | |
| CN103833852A (en) | Bispecific antibody aiming at phosphatidylinositols protein polysaccharide-3 and T cell antigen | |
| CN115279792B (en) | Antibodies specific for BCMA and chimeric antigen receptors | |
| Wang et al. | Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells | |
| CN104829725A (en) | Construction and application of bispecific antibody CD133*CD3 | |
| Guo et al. | Humanized CD30-targeted chimeric antigen receptor t cells exhibit potent preclinical activity against Hodgkin’s lymphoma cells | |
| CN114853897B (en) | Anti-CD19/CD22/CD3 trispecific antibodies and their uses | |
| CN114763387B (en) | A method for preparing trispecific antibodies based on structure-optimized protein activity | |
| CN115103857A (en) | Cell expressing immunoregulatory molecule and system expressing immunoregulatory molecule | |
| CN119894930A (en) | Bispecific antibody and application thereof | |
| WO2017028634A1 (en) | Antibody fusion protein and preparation method and use thereof | |
| CN115215938A (en) | Trispecific antibodies and uses | |
| CN117164712A (en) | CD 22-targeted single domain antibody, chimeric antigen receptor and application thereof | |
| CN116249715A (en) | Anti-CTLA4 monoclonal antibody and chimeric antigen receptor | |
| CN117534766A (en) | Method for constructing trispecific antibody targeting 4-1BB | |
| CN114539423B (en) | A tri-specific fusion protein and its use |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |