CN114763387B - Method for preparing trispecific antibody based on structure-optimized protein activity - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a method for preparing a trispecific antibody based on structure optimization protein activity. The construction method of the invention takes Fab of a monoclonal antibody of a first structural domain as a structural basis, and adds antibody fragments targeting 2 tumor specific or related antigens, namely a second structural domain and a third structural domain, wherein the second structural domain and the third structural domain can be connected to the N end of a heavy chain variable region of the Fab, between 184S-187L of a heavy chain constant region (CH 1) structural domain and the C end of the heavy chain constant region through connecting peptides (Linker); or linked to the N-terminus of the Fab light chain variable region, between the light chain constant region (CL) domains 171S-173D, the C-terminus of the light chain constant region. By adopting the method, the prepared structure-optimized trispecific antibody can effectively mediate the targeting recognition and the killing activity inhibition of T cells on blood tumors and solid tumors, prevent the problems of tumor antigen immune escape, drug resistance and the like, and has the functions of synergizing targeting treatment and enhancing the immune treatment effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing a trispecific antibody based on structure optimization protein activity.
Background
In recent years, the progress of tumor immunotherapy greatly expands the treatment means of malignant tumors. Common immunotherapies include immune checkpoint blocking therapies or engineering T cells using genetic engineering means such that the T cells have a higher and sustained killing capacity against tumor cells. Although immunotherapy has good effects on some malignant tumors, most malignant tumors still do not respond to immunotherapy, development of new and more effective immunotherapeutic 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.). is urgently needed, one of which is a bispecific antibody which can specifically recognize antigen on the surface of target cells and CD3 molecules on the surface of T cells, respectively, guide T cells to specific malignant tumor cell targets to exert cell killing effect, and 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.). that 2 monoclonal antibodies are chemically coupled to construct a first bispecific antibody according to (Brennan M,et al.Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments.Science.1985 Jul 5;229(4708)：81-3.), different targets after the first bispecific antibody is constructed by chemical coupling in the manner of Brennan et al in 1985, Bispecific antibodies of different forms are widely studied, but due to the problems of heavy chain mismatch, antibody inactivation, non-human related immunogenicity and the like, the wider clinical application (Thakur A,et al.Bispecific antibody based therapeutics：Strengths and challenges.Blood Rev.2018 Jul;32(4)：339-347.). of the bispecific antibodies is limited along with the rapid development of genetic engineering technology, people can control the size, affinity, half-life, stability, solubility and other characteristics of the bispecific antibodies through means of recombinant DNA and the like, chimeric or humanized Abs can be constructed to meet different requirements (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.). to target Blinatumomab of CD3/CD19 and Catumaxomab to target CD3/EpCAM to be the only bispecific antibodies approved to be marketed for tumor immunotherapy at present, In order to overcome the immune escape of tumors and the activation limit of T cells, a trispecific antibody SAR441236 which pays importance to the report of (Runcie K,et al.Bi-specifc and tri-specific antibodies-the next big thing in solid tumor therapeutics.Mol Med.2018 Sep 24;24(1)：50.).2017 in recent years is designed and developed to be capable of independently combining 3 different epitopes of HIV envelope and has a certain antiviral capability on animal models (Mullard A. Trispecific antibodies take to the clinical Nat Rev Drug discovery.2020 Oct; 19 (10): 657-658.). The research and development team envisages applying a multi-specific antibody strategy to the field of tumor immunotherapy, and by targeting more molecules on the surface of lymphocytes, more activation models are transferred, so that activated T cells can eliminate tumor cells with better specificity.
T cell binding trispecific antibodies (T cell-ENGAGING TRISPECIFIC antibodies, tsAbs) represent a very effective 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 tumor therapeutics.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.).CD3 as part of the T cell receptor, expressed on mature T cells, capable of transducing activation signals generated by TCR recognition antigens. TsAbs can be combined with surface tumor antigen and CD3 epsilon subunit of T cell receptor at the same time, and can provide a physical connection between T cell and tumor cell so as to effectively activate static T cell to kill tumor cell and attain the effect of curing tumor. Because of the co-stimulatory requirements of T cell bispecific bypass 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. Wu and other scientific researchers develop a novel trispecific antibody which can improve and enhance the targeting ability and signal transduction ability of T cells through T cell receptor co-stimulation and continuously activate the T cells. Compared with the double antibody, the trispecific antibody is additionally provided with a targeted T cell surface CD28 protein, so that the expression of the kinesin Bcl-xL can prevent T cell apoptosis in the presence of Bcl-xL, and in a multiple myeloma model of a mouse, the trispecific antibody can also enhance the capability of resisting a myeloma cell line (Wu L,et al.Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.Nature Cancer 1,86-98.).
TsAbs has important application prospect for solving the limitation of clinical use of the existing bispecific antibody, however, aiming at different antibodies related to TsAbs, the positions of the cell surfaces of the binding epitopes of the antibodies are different, which means that the structural design of the antibodies needs to be adjusted in a personalized way. Generally, tsAbs mediates the binding of T cells to tumor cells involving the formation of an immune synapse, a highly complex, ordered supramolecular domain formed between T cells and tumor cells or antigen presenting cells during T cell activation, a special structure (Wu L,et al.Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor 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.). that binds to antigen complexes by a central cluster of TCRs surrounded by a loop of adhesion molecules of the integrin family, during immune synapse formation, the distance between T cells and target cells being about 15nm due to the structure of the TCRs and antigen complexes. Phosphorylases CD45 and CD148 are displaced out of the synapse by having an outer functional region well longer than 15nm, thus facilitating TCR-induced tyrosine phosphorylation without negative regulatory factors. Studies have shown that mutant CD45 proteins with shorter external functional regions attenuate T cell-related signals, which also verifies the importance of molecular extrusion in T cell signals (Basu R,et al.Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.Cell.2016 Mar 24;165(1)：100-110.;Basu R,et al.Mechanical Communication at the Immunological Synapse.Trends Cell Biol.2017 Apr;27(4)：241-254.).TsAbs described above and the spatial distance between different antigens may also be important for corresponding T cell immune synapse formation and T cell signal activation, except that this spatial distance depends on entirely different structural factors, including the location of the epitope and the junction region between antibody and T cell membrane.
In summary, in order to achieve the optimal killing effect of TsAbs mediated T cells on antigen expressed tumor cells, different antibody connection modes and different connection positions need to be examined, and an optimal TsAbs design scheme based on mediated T cell targeted tumor killing is established, so that the TsAbs molecular design of the mediated T cell targeted tumor cells with optimal performance can be achieved through optimization and screening of structural design for any 3 antigen targeted antibody molecules.
Disclosure of Invention
The multi-target antibody drug becomes the key to solve the similar problems, so the invention provides a novel method for constructing the trispecific antibody in order to find a more effective treatment means, which provides more drug selection for patients.
The invention adopts the site-specific biological chimeric technology to fusion express different functional antibodies at different positions of the framework of the other antibody, and further establishes a design and construction strategy of the trispecific antibody with optimal efficient screening function through comparison of different structural designs and efficacy relations.
In a first aspect of the invention, a method of constructing a trispecific antibody molecule is provided. The method for constructing the trispecific antibody takes a first structural domain, namely Fab of a monoclonal antibody, as a structural basis, and simultaneously adds antibody fragments targeting 2 tumor specific or related antigens, namely a second structural domain and a third structural domain, wherein the second structural domain and the third structural domain can be connected to the Fab fragments through connecting peptides (Linker).
Specifically, the construction method comprises the following steps:
(1) Ligating the second domain to the heavy chain domain of the first domain Fab;
(2) Linking the third domain to the light chain domain of the first domain Fab;
(3) The heavy and light chains bind via disulfide bonds in CH1 and CL to form a heterodimeric, trispecific antibody.
The connection mode in the step (1) comprises that the second structural domain can be connected with the N end of a Fab heavy chain variable region (VH) structural domain through rigid connection peptides respectively, the N end can be marked as HNT, and the rigid connection peptides can be HE Linker and PD Linker; or the second domain is linked between 184S-187L of the Fab heavy chain constant region (CH 1) domain by a coiled-coil linker peptide (Coiled Coil Linker), and deletion 185S 186g, which can be labeled H184-187; or the second domain is linked to the C-terminal end of the Fab heavy chain constant region (CH 1) domain by a flexible linker peptide, which can be labeled as HCT.
The connection mode in the step (2) comprises that the third domain is connected to the N end of the variable region (VL) domain of the Fab light chain through rigid connection peptide, which can be marked as LNT, and the rigid connection peptide can be HE Linker and PD Linker; or the third domain is linked between Fab light chain constant region (CL) domains 171S-173D by a coiled coil linker peptide (Coiled Coil Linker) and deleted 172K, which may be labeled L171-173; or linked to the C-terminal end of the Fab light chain constant region (CL) domain via a flexible linker peptide third domain, can be labeled as LCT.
The rigid connecting peptide HE linker disclosed by the invention has a sequence shown in SEQ ID NO:20, and the rigidly connected peptide PD linker has an amino acid sequence as set forth in SEQ ID NO:21, said coiled coil connecting peptide (Coiled Coil Linker) having the amino acid sequence set forth in SEQ ID NO: 22. 23, and the flexible connecting peptide is (G4S) 3.
Step (1), step (2) the first domain, second domain, third domain each independently have binding specificity for a relevant target antigen selected for 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 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、ICOS、GITR、CD28、4-1BB、OX40、CD27、CD24、CD47、CXCR4、DLL3、Integrin, and the like.
The second domain and the third domain in the step (1) and the step (2) may be in the form of Adnectin (human fibronectin), an affibody, an anticalin (anti-carrier protein), a bicyclic peptide, DARPin (natural ankyrin repeat), fynomer, kunitz-type domain, E7 immune protein, lymphocyte receptor variable region, single domain antibody, whole antibody, antibody fragment, single chain antibody, aptamer, etc.
The first domain Fab fragment may be derived from sources including, but not limited to, anti-CD 3 monoclonal antibodies.
In a second aspect of the invention, a method of constructing a dimeric trispecific antibody molecule targeting the immune effector T cell antigen CD3, which mediates T cell relocation to tumor cells, is disclosed. The method for constructing the trispecific antibody takes Fab of an anti-CD 3 monoclonal antibody (clone SP 34) as a structural basis, the second structural domain and the third structural domain are respectively nanobodies (VHH) aiming at HER2 and VEGFR2 targets, and the second structural domain and the third structural domain are respectively connected to the SP34 Fab fragments through a Linker. The construction method comprises the following steps:
(1) The anti-HER 2-VHH is linked to the heavy chain domain of the SP34 Fab structure.
(2) The anti-VEGFR 2-VHH is linked to the light chain domain of the SP34 Fab structure.
(3) In the invention, the recombinant heavy chain and the recombinant light chain are combined through disulfide bonds in CH1 and CL in the SP34 Fab to form a heterodimer, namely an anti-HER 2/VEGFR2/CD3 trispecific antibody.
Preferably, step (1) can attach an anti-HER 2-VHH to the heavy chain domain of the SP34 Fab structure by:
(a) Ligating an anti-HER 2-VHH to the SP34 Fab heavy chain variable region (VH) domain N-terminal 1E by a rigid ligating peptide HE Linker;
(b) Ligating an anti-HER 2-VHH to the SP34 Fab heavy chain variable region (VH) domain N-terminal 1E by a rigid ligating peptide PD Linker;
(c) Chimeric anti-HER 2-VHH between SP34 Fab heavy chain constant region (CH 1) domains 184S-187L by a spirally wound linker peptide Coiled Coil Linker, and deleted 185S, 186G;
(d) The anti-HER 2-VHH was linked to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain via a flexible linker peptide (G4S) 3 Linker.
The HER2/CD3 bispecific antibodies prepared in the manner described in (a) - (d) above can be labeled HER2/CD3 HNT (HE Linker), HER2/CD3 HNT (PD Linker), HER2/CD 3H 184-187, HER2/CD3 HCT, respectively.
Further preferably, step (1) may connect the anti-HER 2-VHH to the heavy chain domain of the SP34 Fab structure by:
(a) Ligating an anti-HER 2-VHH to the SP34 Fab heavy chain variable region (VH) domain N-terminal 1E by a rigid ligating peptide HE Linker;
(b) Ligating an anti-HER 2-VHH to the SP34 Fab heavy chain variable region (VH) domain N-terminal 1E by a rigid ligating peptide PD Linker;
(c) The anti-HER 2-VHH was linked to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain via a flexible linker peptide (G4S) 3 Linker.
Still further preferably, step (1) is performed by attaching an anti-HER 2-VHH to the heavy chain domain of the SP34 Fab structure by:
(a) The anti-HER 2-VHH was linked to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain via (G4S) 3 Linker.
Preferably, step (2) may connect the anti-VEGFR 2-VHH to the light chain domain of the SP34 Fab structure by:
(a) The anti-VEGFR 2-VHH was linked to the SP34 Fab light chain variable region (VL) domain N-terminal 1Q by a rigid linking peptide HE Linker;
(b) Connecting the anti-VEGFR 2-VHH to the N-terminal 1Q of the SP34 Fab light chain variable region (VL) domain by a rigid connecting peptide PD Linker;
(c) Chimeric anti-VEGFR 2-VHH between SP34 Fab light chain constant region (CL) domains 171S-173D by coiled-coil linker peptide Coiled Coil Linker and deleted 172K;
(d) The anti-VEGFR 2-VHH was linked to the SP34 Fab light chain constant region (CL) domain C-terminal 217C via a flexible linker peptide (G4S) 3 Linker.
The VEGFR2/CD3 bispecific antibodies prepared in the above (a) - (d) can be labeled as VEGFR2/CD3 LNT (HE Linker), VEGFR2/CD3 LNT (PD Linker), VEGFR2/CD 3L 171-173, VEGFR2/CD3 LCT, respectively.
Further preferably, step (2) may connect the anti-VEGFR 2-VHH to the light chain domain of the SP34 Fab structure by:
(a) Connecting the anti-VEGFR 2-VHH to the N-terminal 1Q of the SP34 Fab light chain variable region (VL) domain by a rigid connecting peptide PD Linker;
(b) Chimeric anti-VEGFR 2-VHH between SP34 Fab light chain constant region (CL) domains 171S-173D by coiled-coil linker peptide Coiled Coil Linker and deleted 172K;
(c) VEGFR2-VHH was linked to the SP34 Fab light chain constant region (CL) domain C-terminus 217C via flexible linker peptide (G4S) 3 Linker.
Still further preferably, step (2) may connect the anti-VEGFR 2-VHH to the light chain domain of the SP34 Fab structure by:
(a) anti-VEGFR 2-VHH was chimeric between SP34 Fab light chain constant region (CL) domains 171S-173D by coiled-coil linker peptide Coiled Coil Linker and deleted 172K.
In a preferred embodiment, the HER2/VEGFR2/CD3 (Structural Optimization, SO) trispecific antibody is based on an anti-CD 3 Fab structure, constructed by the following method:
(1) Ligating an anti-HER 2-VHH to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain via a flexible ligating peptide (G4S) 3 Linker;
(2) Chimeric anti-VEGFR 2-VHH between SP34 Fab light chain constant region (CL) domains 171S-173D via a spirally wound connecting peptide Coiled Coil Linker, and deleted 172K;
(3) The Heavy (HC) and Light (LC) chains combine via disulfide bonds in CH1 and CL to form a heterodimeric anti-CD 3 Fab (clone SP 34), the HER2/VEGFR2/CD3 trispecific antibody HER2/VEGFR2/CD3 (SO) after structure optimization.
In one embodiment, using the method, a HER2/VEGFR2/CD3 trispecific antibody is constructed based on a structural optimization design, the HER2/VEGFR2/CD3 trispecific antibody comprising the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, and a recombinant heavy chain amino acid sequence having binding specificity to VEGFR2/CD3 as set forth in SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO: 7.
Preferably, the HER2/VEGFR2/CD3 (SO) trispecific antibody has a binding specificity for HER2/CD3 as set forth in SEQ ID NO:4, and a recombinant heavy chain amino acid sequence as set forth in SEQ ID NO:6, and a recombinant light chain amino acid sequence shown in the specification.
In a third aspect of the invention, a method of constructing a dimeric trispecific antibody molecule targeting the immune effector T cell antigen CD3, which mediates T cell relocation to tumor cells, is disclosed. The method for constructing the trispecific antibody is to take Fab of an anti-CD 3 monoclonal antibody (clone SP 34) as a structural basis, the second structural domain is an anti-CD 19 single chain antibody (SINGLE CHAIN variable fragment, scFv), the third structural domain is an anti-CD 22 monovalent nanobody (VHH), and the Fab are respectively connected to an SP34 Fab fragment through a Linker. The construction method comprises the following steps:
(1) The anti-CD 19-scFv was linked to the heavy chain domain of the SP34 Fab structure.
(2) The anti-CD 22-VHH is linked to the light chain domain of the SP34 Fab structure.
(3) In the present invention, the heavy and light chains are combined by disulfide bonds in CH1 and CL to form a heterodimeric anti-CD 3 Fab (SP 34), i.e., an anti-CD 19/CD22/CD3 trispecific antibody.
Preferably, step (1) can link the anti-CD 19-scFv to the heavy chain domain of the SP34 Fab structure by:
(a) The anti-CD 19-scFv was linked to the SP34 Fab heavy chain variable region (VH) domain N-terminal 1E by a rigid linking peptide PD Linker;
(b) The anti-CD 19-scFv was chimeric between the SP34 Fab heavy chain constant region (CH 1) domains 184S-187L by a spirally wound linker peptide Coiled Coil Linker, and deleted 185S, 186G;
(c) The anti-CD 19-scFv was linked to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain by a flexible linker peptide (G4S) 3 Linker.
The CD19/CD3 bispecific antibodies prepared in the above manner (a) - (c) may be labeled as CD19/CD3 HNT, CD19/CD 3H 184-187, CD19/CD3 HCT, respectively.
Further preferably, step (1) can link the anti-CD 19-scFv to the heavy chain domain of the SP34 Fab structure by:
(a) The anti-CD 19-scFv was chimeric between the SP34 Fab heavy chain constant region (CH 1) domains 184S-187L by a spirally wound linker peptide Coiled Coil Linker, and deleted 185S, 186G;
(b) The anti-CD 19-scFv was linked to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain by a flexible linker peptide (G4S) 3 Linker.
Still further preferably, step (1) may link the anti-CD 19-scFv to the heavy chain domain of the SP34 Fab structure by:
(a) The anti-CD 19-scFv was linked to the C-terminal 228C of the SP34 Fab heavy chain constant region (CH 1) domain by a flexible linker peptide (G4S) 3 Linker.
Preferably, step (2) can attach the anti-CD 22-VHH to the light chain domain of the SP34 Fab structure by:
(a) Ligating an anti-CD 22-VHH to the SP34 Fab light chain variable region (VL) domain N-terminal 1Q by a rigid ligating peptide PD Linker;
(b) The anti-CD 22-VHH was chimeric between SP34 Fab light chain constant region (CL) domains 171S-173D by a spirally coiled-connecting peptide Coiled Coil Linker and deleted 172K;
(c) The anti-CD 22-VHH was linked to the SP34 Fab light chain constant region (CL) domain C-terminal 217C by a flexible linking peptide (G4S) 3 Linker.
The CD22/CD3 bispecific antibodies prepared in the manner described in (a) - (c) above can be labeled as CD22/CD3 LNT, CD22/CD 3L 171-173, CD22/CD3 LCT, respectively.
Further preferably, step (2) can link the anti-CD 22-VHH to the light chain domain of the SP34 Fab structure by:
(a) The anti-CD 22-VHH was chimeric between SP34 Fab light chain constant region (CL) domains 171S-173D by a spirally coiled-connecting peptide Coiled Coil Linker and deleted 172K;
(b) The anti-CD 22-VHH was linked to the SP34 Fab light chain constant region (CL) domain C-terminal 217C by a flexible linking peptide (G4S) 3 Linker.
Still further preferably, step (2) is performed by linking the anti-CD 22-VHH to the light chain domain of the SP34 Fab structure by:
(a) The anti-CD 22-VHH was linked to the SP34 Fab light chain constant region (CL) domain C-terminal 217C by a flexible linking peptide (G4S) 3 Linker.
In a preferred embodiment, the CD19/CD22/CD3 (SO) trispecific antibody is based on an anti-CD 3 Fab structure, constructed by the following method:
(1) The anti-CD 19-scFv was linked to the C-terminus 228C of the SP34 Fab heavy chain constant region (CH 1) domain via a flexible linking peptide (G4S) 3 Linker;
(2) Ligating the anti-CD 22-VHH to the SP34 Fab light chain constant region (CL) domain C-terminus 217C via a flexible ligating peptide (G4S) 3 Linker;
(3) The Heavy (HC) and Light (LC) chains combine via disulfide bonds in CH1 and CL to form heterodimeric anti-CD 3 Fab (SP 34), an anti-CD 19/CD22/CD3 (SO) trispecific antibody.
In one embodiment, using the method, a CD19/CD22/CD3 trispecific antibody is constructed based on a structural optimization design, said CD19/CD22/CD3 trispecific antibody comprising the amino acid sequence as set forth in SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10, and a recombinant heavy chain amino acid sequence having binding specificity for CD22/CD3 as set forth in SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID NO:13, and a recombinant light chain amino acid sequence shown in seq id no.
Preferably, the CD19/CD22/CD3 (SO) trispecific antibody comprises the amino acid sequence as set forth in SEQ ID NO:10, and a recombinant heavy chain amino acid sequence having binding specificity for CD22/CD3 as set forth in SEQ ID NO:13, and a recombinant light chain amino acid sequence shown in seq id no.
In a fourth aspect of the invention, there is also provided a method of preparing the trispecific antibody of the invention. It comprises the following steps:
(1) Obtaining fusion genes of the trispecific antibody and constructing an expression vector of the trispecific antibody;
(2) Transfecting the expression vector into a host cell by a genetic engineering method;
(3) Culturing the above-described host cell under conditions that allow production of the trispecific antibody;
(4) And separating and purifying the produced antibody protein.
The expression vector in the step (1) may be eukaryotic expression vector pFuse, pSeqtag, pCMV, pcDNA, pFastBac, pPIC9K, etc. and prokaryotic expression vector pET, pGEX, pMAL, pQE, pTrc, pBV, pTXB, preferably eukaryotic expression vector.
The host cell in the step (2) can be escherichia coli, bacillus thuringiensis, pichia pastoris, insect cells, 293 suspension cells and Chinese insect ovary cells, preferably 293 suspension cells.
In a fifth aspect of the invention there is provided the use of said antibody in the manufacture of a medicament for the treatment of cancer. Such cancers include, but are not limited to, breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gall bladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urinary tract cancer, lung cancer, non-small cell lung cancer, brain tumor, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, renal cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma, or lymphoma. The include tumor cells that express CD19, CD22, HER2, VEGFR2, and the like.
In one embodiment, the use for treating tumors that concurrently express HER2/VEGFR2 positive. The HER2/VEGFR2/CD3 trispecific antibody molecule can remarkably kill HER2+ tumor cells in vitro, has better killing effect on tumor cells simultaneously expressing VEGFR2, but has no nonspecific killing effect on HER2/VEGFR2 double-negative cells. And simultaneously, the tumor killing effect in vitro and in vivo is obviously higher than that of the bispecific antibody of the same type and the combination thereof.
In one embodiment, the method can be used for treating tumors simultaneously expressing CD19/CD22, and the target CD19/CD22/CD3 trispecific antibody can obviously kill CD19/CD22 double-positive tumor cells or CD19 and CD22 single-positive tumor cells in vitro, and has no nonspecific killing effect on CD19/CD22 double-negative cells. Meanwhile, the in-vitro tumor killing effect is also obviously higher than that of a clinically obtained CD19/CD3 bispecific antibody Bonauzumab (Blinatumomab).
In a sixth aspect of the application, there is provided a method for treating a tumor comprising administering to a subject in need thereof a trispecific antibody of the application in an amount effective to treat the tumor.
The technical scheme of the invention has the beneficial technical effects that:
1. The trispecific antibody medicament disclosed by the invention realizes multi-target treatment of simultaneously targeting T cells, tumor cells and tumor blood vessels for the first time, is simpler and more convenient to express and purify compared with antibodies with Fc regions, has stronger penetrating power in solid tumor microenvironments, and has longer half-life in vivo compared with common single-chain antibody tandem structures, and is more beneficial to reducing the administration times and dosage.
2. The linking of VHH or scFv antibody molecules to different positions on Fab via different Linker peptides has different effects: in constructing the trispecific antibodies of the present invention, the linkage and functional distinction between domains is considered in combination. When the structural domain is added to the N end of the Fab, the targeting of the Fab structural domain is important, and in order to effectively isolate 2 different structural domains, the invention uses a rigid connecting peptide Linker, so that the targeting of the 2-end antibody is ensured; when the structural domain is added at the C end of the Fab, the added structural domain needs to be paid attention to the fact that the added structural domain can be expressed normally, and meanwhile, the independence of the structural domain is ensured, so that the invention uses the flexible connecting peptide Linker; the invention also provides a method for embedding the domains into the light/heavy chain constant region through the spirally-curled connecting peptide (Coiled Coil Linker), so that the independence and targeting among 3 domains can be more effectively ensured. By introducing VHH or scFv at different positions of Fab, the distance between the targeted tumor cells and T cells can be effectively adjusted, so that effective immune synapses are formed, and the influence of immunosuppressive signals on the activation and killing effects of the T cells is avoided. Based on this, we found that the optimal site for introducing HER2, CD19 and CD22 targeting antibodies at the CD3 targeting Fab is the C-terminus of the Fab, while the optimal site for introducing the VEGFR2 targeting antibody is the constant region of the Fab.
3. Through literature summary and early test results, antibody clones targeting HER2, VEGFR2, CD19, CD22 and CD3 are successfully selected, and are respectively expressed in a eukaryotic expression system through a molecular cloning technology, and through in vitro verification, the selected antibodies can be confirmed to have good binding specificity and affinity with target antigens.
4. The bispecific antibodies of different structures are successfully expressed by selecting a proper expression system, taking anti-CD 3 antibody fragment Fab as a basic bracket, respectively constructing bispecific antibodies of HER2/CD3 and VEGFR2/CD3 and bispecific antibodies of CD19/CD3 and CD22/CD3, and confirming that the bispecific antibodies can show good binding stability and mediate T cell immune killing target cell effect through structure optimization and activity comparison.
5. Through structural optimization and activity verification, the optimized molecular structure of the HER2/VEGFR2/CD3 (SO) trispecific antibody is determined, the protein with stable structure is further obtained through a eukaryotic expression system, the stable HER2, VEGFR2 and CD3 target binding specificity and affinity are shown in vitro, and meanwhile, the killing effect of T cells on target cells can be effectively mediated. Compared with the existing curative bispecific antibody, the HER2/VEGFR2/CD3 (SO) trispecific antibody not only can effectively kill HER2/VEGFR2 double-positive and single-positive tumors, but also can effectively inhibit the formation of tumor microenvironment blood vessels, has the effects of effectively preventing tumor antigens from immune escape, and simultaneously enhances the synergistic immunotherapy effect on the regulation and control of the microenvironment of solid tumors.
6. Through structural optimization and activity verification, the optimized molecular structure of the CD19/CD22/CD3 (SO) trispecific antibody is determined, the protein with stable structure is further obtained through a eukaryotic expression system, the stable target binding specificity and affinity are shown in vitro, and meanwhile, the killing effect of T cells on blood tumor cells can be effectively mediated. Compared with the currently curative bispecific antibody Bonaku monoclonal antibody (Blinatumomab), the CD19/CD22/CD3 (SO) trispecific antibody can target CD19/CD22 double-positive and single-positive tumors at the same time, SO that immune escape caused by the fact that the bispecific antibody aims at single-target recognition is effectively prevented, and the functions of synergizing targeted therapy and enhancing the immune therapeutic effect are achieved.
Drawings
The structure design of bispecific antibodies of different targets in fig. 1 and the design of trispecific antibodies based on structure-optimized activity are schematically shown.
Figure 2 binding functions of HER2/CD3 bispecific antibodies of different structures to HER2 antigen were verified.
Figure 3 in vitro killing activity of different HER2/CD3 bispecific antibodies against HER2 positive MDA-MB-231 tumor cells.
Figure 4 in vitro killing activity of different HER2/CD3 bispecific antibodies against HER2 positive MDA-MB-435 tumor cells.
Figure 5 in vitro killing activity of different HER2/CD3 bispecific antibodies against HER2 positive PC3 tumor cells.
Figure 6 in vitro killing activity of different HER2/CD3 bispecific antibodies against HER2 negative MDA-MB-468 tumor cells.
FIG. 7 shows the binding function of VEGFR2/CD3 bispecific antibodies with different structures to VEGFR2 antigen.
FIG. 8 validation of in vitro killing activity of different VEGFR2/CD3 bispecific antibodies against VEGFR2 positive PC3 tumor cells.
FIG. 9 validation of in vitro killing activity of different VEGFR2/CD3 bispecific antibodies against VEGFR2 positive HUVEC cells.
FIG. 10 in vitro killing activity of different VEGFR2/CD3 bispecific antibodies against VEGFR2 positive MDA-MB-468/VEGFR2 tumor cells was verified.
FIG. 11 in vitro killing activity of different VEGFR2/CD3 bispecific antibodies against VEGFR2 negative MDA-MB-468 tumor cells was verified.
Figure 12 binding function verification of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure to HER2 antigen.
Fig. 13 binding function verification of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure to VEGFR2 antigen.
Figure 14 in vitro killing activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure against HER2/VEGFR2 double positive PC3 tumor cells with HER2/CD3 bispecific antibodies of different structures.
Figure 15 confirmation of in vitro killing activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure with HER2/CD3 bispecific antibodies of different structures on HER2/VEGFR2 biscationic HUVEC cells.
FIG. 16 in vitro killing activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure against HER2/VEGFR2 double negative MDA-MB-468 cells with different structure HER2/CD3 bispecific antibodies.
FIG. 17 in vitro killing activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure against HER2/VEGFR2 double positive PC3 tumor cells with different structures of VEGFR2/CD3 bispecific antibodies.
FIG. 18 in vitro killing activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure against HER2/VEGFR2 double positive HUVEC cells with bispecific antibodies of different structures.
FIG. 19 in vitro killing activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure against HER2/VEGFR2 double negative MDA-MB-468 tumor cells with different structures of VEGFR2/CD3 bispecific antibodies.
Figure 20 HER2/VEGFR2/CD3 (SO) trispecific antibodies of optimized structure compared to the anti-tumor activity of different bispecific antibodies and combinations thereof in PC3 prostate cancer loaded mice.
Figure 21 effect of HER2/VEGFR2/CD3 (SO) trispecific antibodies with different bispecific antibodies and combinations thereof on mouse body weight with optimized structure.
FIG. 22 shows a comparison of binding capacity of different CD19/CD3 bispecific antibodies to CD19 positive K562-CD19 tumor cells.
FIG. 23 shows a comparison of binding capacity of different CD19/CD3 bispecific antibodies to CD19 negative K562 tumor cells.
FIG. 24 shows in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19/CD22 biscationic Nalm6 tumor cells.
FIG. 25 in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19 positive CD22 negative Nalm6-KO22 tumor cells.
FIG. 26 in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19 negative CD22 positive Nalm6-KO19 tumor cells.
FIG. 27 in vitro killing activity of different CD19/CD3 bispecific antibodies against CD19/CD22 double negative K562 tumor cells.
FIG. 28 shows a comparison of binding capacity of different CD22/CD3 bispecific antibodies to CD22 positive K562-CD22 tumor cells.
FIG. 29 shows a comparison of binding capacity of different CD22/CD3 bispecific antibodies to CD22 negative K562 tumor cells.
FIG. 30 shows in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19/CD22 biscationic Nalm6 tumor cells.
FIG. 31 in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19 positive CD22 negative Nalm6-KO22 tumor cells.
FIG. 32 in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19 negative CD22 positive Nalm6-KO19 tumor cells.
FIG. 33 in vitro killing activity of different CD22/CD3 bispecific antibodies against CD19/CD22 double negative K562 tumor cells.
FIG. 34 shows functional verification of binding of CD19/CD22/CD3 (SO) trispecific antibodies of optimized structure to CD19/CD3, CD22/CD3 bispecific antibodies of optimized structure to CD19 positive K562-CD19 tumor cells.
FIG. 35 binding function of CD19/CD22/CD3 (SO) trispecific antibody of optimized structure to CD19/CD3, CD22/CD3 bispecific antibody of optimized structure to CD22 positive K562-CD22 tumor cells was verified.
FIG. 36 in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies of optimized structure against CD19/CD3, CD22/CD3 bispecific antibodies of optimized structure against CD19/CD22 biscationic Nalm6 tumor cells.
FIG. 37 in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies with optimized structure and CD19/CD3, CD22/CD3 bispecific antibodies against CD19 positive CD22 negative Nalm6-KO22 tumor cells.
FIG. 38 in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies of optimized structure against CD19/CD3, CD22/CD3 bispecific antibodies of optimized structure against CD19 negative CD22 positive Nalm6-KO19 tumor cells.
FIG. 39 in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies of optimized structure against CD19/CD3, CD22/CD3 bispecific antibodies of optimized structure against CD19/CD22 double negative K562 tumor cells.
FIG. 40 shows in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies of optimized structure against CD19 positive CD22 negative K562-CD19 tumor cells compared to commercial CD19/CD3 bispecific antibodies Blinatumomab.
FIG. 41 compares the in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies of the optimized structure against CD19 negative CD22 positive K562-CD22 tumor cells with commercial CD19/CD3 bispecific antibodies Blinatumomab.
FIG. 42 compares the in vitro killing activity of CD19/CD22/CD3 (SO) trispecific antibodies of the optimized structure with commercial CD19/CD3 bispecific antibodies Blinatumomab against CD19/CD22 double negative K562 tumor cells.
Detailed Description
The present invention is further illustrated and described below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.
In the following examples, materials used in the experiments are all available and can be prepared by referring to the prior disclosed technology; both source and gauge are not indicated as commercially available; various processes and methods not described in detail are conventional methods well known in the art.
EXAMPLE 1 construction and eukaryotic expression of bispecific/trispecific antibodies
1.1 Vector construction
Construction of CD3-HC-HER2-VHH and CD3-LC expression vector:
The heavy chains of the HER2/CD3 bispecific antibody were linked by CD3-HC (SP 34) and HER2-VHH, respectively, in the following manner: ligating a HER2-VHH to the N-terminal 1E of the CD3-HC Variable (VH) domain via a rigid ligating peptide HE Linker; or by ligating HER2-VHH to the N-terminal 1E of the CD3-HC Variable (VH) domain via a rigid ligating peptide PD Linker; or the HER2-VHH is chimeric between the CD3-HC constant region (CH 1) domain 184S-187L by a spirally-coiled-coil connecting peptide Coiled Coil Linker, and 185S and 186G are deleted; or HER2-VHH is linked to the C-terminal 228C of the CD3-HC constant region (CH 1) domain by a flexible linking peptide (G4S) 3 Linker; the light chain CD3-LC was not modified at all. The coding genes of the CD3-HC-HER2-VHH and the CD3-LC are respectively synthesized according to a conventional molecular biology method, and the obtained synthetic genes are inserted into a pFUSE eukaryotic expression vector with Zeocin resistance through a homologous recombination method. The post-expressed bispecific antibodies were labeled HER2/CD3 HNT (HE Linker), HER2/CD3 HNT (PD Linker), HER2/CD 3H 184-187, HER2/CD3 HCT, respectively, depending on the mode of attachment. In addition to the HE Linker, PD Linker, coiled Coil Linker and (G4S) 3 Linker, which were selected by the present experiment, the anti-HER 2-VHH can be linked to the CD3-HC, and alternative links can be made using Linker peptide linkers known to those skilled in the art. The relevant sequences are shown in table 1:
TABLE 1
| Name of the name | Amino acid sequence numbering | Nucleotide sequence numbering |
| CD3-HC | SEQ ID NO：14 | SEQ ID NO：38 |
| HER2-VHH | SEQ ID NO：16 | SEQ ID NO：40 |
| HE Linker | SEQ ID NO：20 | SEQ ID NO：44 |
| PD Linker | SEQ ID NO：21 | SEQ ID NO：45 |
| Coiled Coil Linker | SEQ ID NO：22、23 | SEQ ID NO：46、47 |
| (G4S)3 Linker | SEQ ID NO：24 | SEQ ID NO：48 |
| HER2/CD3 HNT(HE Linker) | SEQ ID NO：1 | SEQ ID NO：25 |
| HER2/CD3 HNT(PD Linker) | SEQ ID NO：2 | SEQ ID NO：26 |
| HER2/CD3 H184-187 | SEQ ID NO：3 | SEQ ID NO：27 |
| HER2/CD3 HCT | SEQ ID NO：4 | SEQ ID NO：28 |
| CD3-LC | SEQ ID NO：15 | SEQ ID NO：39 |
Construction of CD3-LC-VEGFR2-VHH and CD3-HC expression vector:
The light chain of the VEGFR2/CD3 bispecific antibody was linked by CD3-LC (SP 34) and VEGFR2-VHH, respectively, as follows: connecting VEGFR2-VHH to the N-terminal 1Q of the CD3-LC Variable (VL) domain via a rigid connecting peptide PD Linker; or by chimeric VEGFR2-VHH between CD3-LC constant region (CL) domains 171S-173D via a spirally-coiled-connecting peptide Coiled Coil Linker, and deleted 172K; or by linking VEGFR2-VHH to the C-terminus 217C of the CD3-LC constant region (CL) domain via a flexible linking peptide (G4S) 3 Linker; the heavy chain CD3-HC did not undergo any modification. The coding genes of the CD3-LC-VEGFR2-VHH and the CD3-HC are respectively synthesized according to a conventional molecular biology method, and the obtained synthetic genes are inserted into a pFUSE eukaryotic expression vector with Zeocin resistance through a homologous recombination method. The post-expressed bispecific antibodies were labeled VEGFR2/CD3 LNT, VEGFR2/CD 3L 171-173, VEGFR2/CD3 LCT, respectively, depending on the mode of ligation. Wherein the anti-VEGFR 2-VHH is linked to CD3-LC by the PD Linker, coiled Coil Linker and (G4S) 3 Linker selected in this experiment, alternative links can also be made using a Linker peptide Linker known to those skilled in the art. The relevant sequences are shown in table 2:
TABLE 2
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Construction of CD3-HC-CD19-scFv and CD3-LC expression vector:
The heavy chain of the CD19/CD3 bispecific antibody was linked by CD3-HC (SP 34) and CD19-scFv, respectively, in the following manner: ligating the CD19-scFv to the N-terminal 1E of the CD3-HC variable region (VH) domain via a rigid ligating peptide PD Linker; or chimeric CD19-scFv between CD3-HC constant region (CH 1) domain 184S-187L via spirally-coiled-coil linker peptide Coiled Coil Linker, with deletions of 185S, 186G; or the CD19-scFv is linked to the C-terminal 228C of the CD3-HC constant region (CH 1) domain by a flexible linking peptide (G4S) 3 Linker; the light chain CD3-LC was not modified at all. The coding genes of the CD3-HC-CD19-scFv and the CD3-LC are respectively synthesized according to a conventional molecular biology method, and the obtained synthetic genes are inserted into a pFUSE eukaryotic expression vector with Zeocin resistance through a homologous recombination method. Depending on the mode of ligation, the post-expressed bispecific antibodies were labeled CD19/CD3 HNT, CD19/CD 3H 184-187, CD19/CD3 HCT, respectively. In addition to the linking of anti-CD 19-scFv to CD3-HC by PD Linker, coiled Coil Linker and (G4S) 3 Linker selected in this experiment, alternative links can be made using Linker peptides Linker well known to those skilled in the art. The relevant sequences are shown in table 3:
TABLE 3 Table 3
| Name of the name | Amino acid sequence numbering | Nucleotide sequence numbering |
| CD3-HC | SEQ ID NO：14 | SEQ ID NO：38 |
| CD19-scfv | SEQ ID NO：19 | SEQ ID NO：43 |
| PD Linker | SEQ ID NO：21 | SEQ ID NO：45 |
| Coiled Coil Linker | SEQ ID NO：22、23 | SEQ ID NO：46、47 |
| (G4S)3 Linker | SEQ ID NO：24 | SEQ ID NO：48 |
| CD19/CD3 HNT | SEQ ID NO：8 | SEQ ID NO：32 |
| CD19/CD3 H184-187 | SEQ ID NO：9 | SEQ ID NO：33 |
| CD19/CD3 HCT | SEQ ID NO：10 | SEQ ID NO：34 |
| CD3-LC | SEQ ID NO：15 | SEQ ID NO：39 |
Construction of CD3-LC-CD22-VHH and CD3-HC expression vector:
The light chain of the CD22/CD3 bispecific antibody was linked by CD3-LC (SP 34) and CD22-VHH, respectively, as follows: ligating the CD22-VHH to the N-terminal 1Q of the CD3-LC variable region (VL) domain by means of a rigid ligating peptide PD Linker; or CD22-VHH is chimeric between CD3-LC constant region (CL) domains 171S-173D by a spirally-coiled-coil linker peptide Coiled Coil Linker, and 172K is deleted; or the CD22-VHH is linked to the C-terminal 217C of the CD3-LC constant region (CL) domain by a flexible linking peptide (G4S) 3 Linker; the heavy chain CD3-HC did not undergo any modification. The coding genes of the CD3-LC-CD22-VHH and the CD3-HC are respectively synthesized according to a conventional molecular biology method, and the obtained synthetic genes are inserted into a pFUSE eukaryotic expression vector with Zeocin resistance through a homologous recombination method. Depending on the mode of ligation, the post-expressed bispecific antibodies were labeled CD22/CD3 LNT, CD22/CD 3L 171-173, CD22/CD3 LCT, respectively. In addition to the linking of anti-CD 22-VHH to CD3-LC by PD Linker, coiled Coil Linker and (G4S) 3 Linker selected in this experiment, alternative links can be made using Linker peptides Linker well known to those skilled in the art. The relevant sequences are shown in table 4:
TABLE 4 Table 4
| Name of the name | Amino acid sequence numbering | Nucleotide sequence numbering |
| CD3-LC | SEQ ID NO：15 | SEQ ID NO：39 |
| CD22-VHH | SEQ ID NO：18 | SEQ ID NO：42 |
| PD Linker | SEQ ID NO：21 | SEQ ID NO：45 |
| Coiled Coil Linker | SEQ ID NO：22、23 | SEQ ID NO：46、47 |
| (G4S)3 Linker | SEQ ID NO：24 | SEQ ID NO：48 |
| CD22/CD3 LNT | SEQ ID NO：11 | SEQ ID NO：35 |
| CD22/CD3 L171-173 | SEQ ID NO：12 | SEQ ID NO：36 |
| CD22/CD3 LCT | SEQ ID NO：13 | SEQ ID NO：37 |
| CD3-HC | SEQ ID NO：14 | SEQ ID NO：38 |
Construction of HER2/VEGFR2/CD3 (SO) expression vector:
The heavy chain of the HER2/VEGFR2/CD3 trispecific antibody was linked by CD3-HC (SP 34) and HER2-VHH as follows: attaching HER2-VHH to the C-terminal 228C of the CD3-HC constant region (CH 1) domain via a flexible linker peptide (G4S) 3 Linker; the light chain of the HER2/VEGFR2/CD3 (SO) trispecific antibody was linked by CD3-LC (SP 34) and VEGFR2-VHH as follows: VEGFR2-VHH was chimeric between CD3-LC constant region (CL) domains 171S-173D by a spirally-coiled-coil linker peptide Coiled Coil Linker and 172K was deleted. And inserting the obtained gene into a pFDSE eukaryotic expression vector with Zeocin resistance by a homologous recombination method. Depending on the mode of ligation, the post-expressed trispecific antibody was labeled HER2/VEGFR2/CD3 (SO). Wherein the HER2-VHH, VEGFR2-VHH can be alternatively linked using Linker peptide Linker well known to those skilled in the art, in addition to the linking to CD3-HC or CD3-LC by (G4S) 3 Linker and Coiled Coil Linker selected in this experiment. The relevant sequences are shown in table 5:
TABLE 5
| Name of the name | Amino acid sequence numbering | Nucleotide sequence numbering |
| CD3-HC | SEQ ID NO：14 | SEQ ID NO：38 |
| CD3-LC | SEQ ID NO：15 | SEQ ID NO：39 |
| HER2-VHH | SEQ ID NO：16 | SEQ ID NO：40 |
| VEGFR2-VHH | SEQ ID NO：17 | SEQ ID NO：41 |
| Coiled Coil Linker | SEQ ID NO：22、23 | SEQ ID NO：46、47 |
| (G4S)3 Linker | SEQ ID NO：24 | SEQ ID NO：48 |
| HER2/CD3 HCT | SEQ ID NO：4 | SEQ ID NO：28 |
| VEGFR2/CD3 L171-173 | SEQ ID NO：6 | SEQ ID NO：30 |
Construction of a CD19/CD22/CD3 (SO) expression vector:
The heavy chain of the CD19/CD22/CD3 (SO) trispecific antibody was linked by CD3-HC (SP 34) and CD19-scFv as follows: ligating the CD19-scFv to the C-terminal 228C at the CD3-HC constant region (CH 1) domain via a flexible ligating peptide (G4S) 3 Linker; the light chain of the CD19/CD22/CD3 (SO) trispecific antibody was linked by CD3-LC (SP 34) and CD22-VHH as follows: CD22-VHH was linked to the C-terminal 217C of the CD3-LC constant region (CL) domain by a flexible linking peptide (G4S) 3 Linker. And inserting the obtained gene into a pFDSE eukaryotic expression vector with Zeocin resistance by a homologous recombination method. Depending on the mode of ligation, the post-expressed trispecific antibody was labeled CD19/CD22/CD3 (SO). In addition to the attachment of CD19-scFv, CD22-VHH to CD3-HC or CD3-LC by the (G4S) 3 Linker selected in this experiment, alternative attachments may be made using Linker peptides Linker well known to those skilled in the art. The relevant sequences are shown in table 6:
TABLE 6
The structural design of the bispecific antibody of different targets and the optimized structure of the trispecific antibody are shown in figure 1.
1.2 Eukaryotic expression of proteins
Inoculating 1.5X10 6/mL 293 suspension cells, shaking and culturing in 500mL shaking flask 200mL culture medium at 37 ℃ at 165rpm in a shaking incubator with 5% CO 2 concentration for 24 hours, counting cells, adjusting the cell density to 3X 10 6/mL, uniformly mixing the light chain and heavy chain plasmids constructed in the step 1.1 in 10mL Opti-MEM, standing at room temperature for 5min, adding 500 mu L (1:2.5) PEI 40000 in 10mL Opti-MEM, gently mixing, standing at room temperature for 5min, mixing the plasmids and PEI mixed solution, gently mixing, and standing at room temperature for 20min. The mixed solution is added into 200mL 293 suspension cell culture solution drop by drop, the culture flask is gently shaken while the mixed solution is added drop by drop, and the mixed solution is put into a shaking table for transfection for 72h. After transfection, 100g is centrifuged for 5min, supernatant is taken, 100mL of culture medium is added into a culture flask for re-suspension culture for 48h, and then centrifugation is carried out at 3000rpm for 5min, supernatant is taken, and the supernatant of 293 suspension cells collected twice is mixed and frozen at-20 ℃.
EXAMPLE 2 purification of trispecific antibodies
2.1 Protein G affinity purification
400ML of the cell supernatant of 1.2 in example 1 above was centrifuged at 15000rpm at 4℃for 30min, and the supernatant was collected, filtered through a 0.45 μm filter membrane, and placed on ice for use. 4mL of Protein G (20% ethanol/Protein G1:1) was placed in the column, washed 3 times with Binding buffer, and the pad was used to hold down the resin surface. Protein G column was equilibrated with 20mL Binding buffer. Samples were passed through the Protein G column at constant speed (about 0.5 mL/min) every 10 mL. 40mL Binding buffer at constant speed (about 1 mL/min) the Protein G column was washed. First, 10% of the volume of the eluent was added to the collection tube of the Elution tube, and the column was filled with the solution buffer and 5mL of the solution was eluted once until the protein concentration could not be quantified. The collected protein samples were concentrated using an Amicon Ultra-15 centrifuge filter and centrifuged at 3000rpm at 4℃for 20 minutes to quantify the protein concentration.
EXAMPLE 3 evaluation of biological Functions of HER2/VEGFR2/CD3 trispecific antibodies
3.1 Antigen binding validation of HER2/CD3 bispecific antibodies of different structures
HER2 ECD-Fc antigen protein was diluted to 10 μg/mL with PBS and coated in 96-well ELISA plates, 100 μl/well, overnight at 4 ℃. The plate was discarded, and after 2 PBST washes, 200 μl/well of 5% skim milk in PBST solution was added and blocked at room temperature for 2 hours, and the blocked solution in the wells was drained. Bispecific antibodies to be tested HER2/CD3 HNT (HE Linker), HER2/CD3 HNT (PD Linker), HER2/CD 3H 184-187, HER2/CD3 HCT were 10-fold diluted with blocking solution for 8 gradients, starting antibody concentration 100nM, 3 multiplex wells per concentration gradient, 100 μl/well added to the well plate and incubated for 2H at room temperature. Plates were washed 3 times with PBST and drained, HRP-labeled anti-kappa chain antibodies were diluted 1:5000 with 5% skimmed milk PBST and added to the well plates, 100. Mu.L/well, and incubated for 1h at room temperature. After washing the plate 3 times with PBST, draining, adding 100. Mu.L of TMB color development liquid into each well, developing at room temperature in dark for 10min, adding 50. Mu.L of 2M H 2SO4 into each well, stopping the color development reaction, and detecting the absorbance value of OD450nm by using a microplate reader. And (3) taking the sample concentration value as an abscissa and the absorbance value as an ordinate, performing a four-parameter nonlinear regression graph, and calculating an EC50 value.
As shown in table 7 and fig. 2, bispecific antibodies of different structures were able to bind HER2 ECD-Fc antigen protein effectively compared to control CD3-Fab, and the EC50 differences between HER2/CD3 HNT (HE Linker) and HER2/CD3 HNT (PD Linker), HER2/CD3 HCT were smaller, and the antigen binding capacity was essentially the same, except that the antigen binding effect of HER2/CD 3H 184-187 was poor.
TABLE 7
| Constructs | EC50(pM) |
| CD3-Fab | Not determined |
| HER2/CD3 HNT(HE Linker) | 215.6 |
| HER2/CD3 HNT(PD Linker) | 218.6 |
| HER2/CD3 H184-187 | 2764 |
| HER2/CD3 HCT | 384.3 |
3.2 In vitro tumor killing Activity of different Structure HER2/CD3 bispecific antibodies
Activated human T cells (PBMC from healthy volunteers) were cultured with RPMI-1640 complete medium containing IL2 at a concentration of 300IU/mL, and breast cancer cells MDA-MB-231 (HER2+), MDA-MB-435 (HER2+), MDA-MB-468 (HER2-) and prostate cancer cells PC3 (HER2+), were cultured with DMEM medium containing 10% fetal bovine serum. T cells were cultured 24h prior to the experiment with IL 2-depleted RPMI-1640 medium. T cell density was adjusted to 2X 10 6, tumor cell density was adjusted to 2X 10 5, 2 cells were mixed at an effective target ratio of 10:1, and the cell mixture was added to a 96-well cell plate at 100. Mu.L/well. Bispecific antibodies to be tested HER2/CD3 HNT (HE Linker), HER2/CD3 HNT (PD Linker), HER2/CD 3H 184-187, HER2/CD3 HCT were 10-fold diluted with medium for 8 gradients, starting antibody concentration 10nM, 2 multiplex wells per concentration gradient, 10. Mu.L/well were added to the well plate. The experiment was run with both positive control (tumor cells directly into cell lysate) and negative control (tumor cells and T cells) at 37 ℃ in a 5% co 2 incubator for 24h. And detecting the target cell lysis degree in the 96-well plate by using a lactate dehydrogenase detection kit, detecting the OD490 reading in the well by using a multifunctional enzyme-labeled instrument, performing data analysis by using GRAPHPAD PRISM software, performing a four-parameter nonlinear regression graph by using an antibody concentration value as an abscissa and an absorbance value as an ordinate, and calculating an IC50 value.
The results are shown in FIG. 3, FIG. 4, FIG. 5 and FIG. 6, where the 4 structural HER2/CD3 bispecific antibodies were significantly cytotoxic to HER2 positive tumor cells MDA-MB-231 (HER2+), MDA-MB-435 (HER2+), and PC3 (HER2+), but were not significantly cytotoxic to tumor cells MDA-MB-468 (HER2-). From the IC50 values, HER2/CD3 HCT showed significantly enhanced tumor cell killing compared to other 3 structural bispecific antibodies; thus, by comparison of HER2 antigen binding activity and HER2 positive tumor cell killing activity, it was determined that HCT with HER2-VHH fused to CD3-Fab was the optimal recombinant antibody structure. In addition, no significant antigen binding differences and tumor cell killing differences were shown for HER2/CD3 HNT using either HE Linker or PD Linker, so the next bispecific antibodies used PD Linker for N-terminal fusion ligation of CD3-HC or CD 3-LC.
3.3 Antigen binding validation of different Structure VEGFR2/CD3 bispecific antibodies
VEGFR2 ECD-Fc antigen protein was diluted to 10. Mu.g/mL with PBS and coated in 96-well ELISA plates, 100. Mu.L/well, overnight at 4 ℃. The plate was discarded, and after 2 PBST washes, 200 μl/well of 5% skim milk in PBST solution was added and blocked at room temperature for 2 hours, and the blocked solution in the wells was drained. The bispecific antibodies to be tested, VEGFR2/CD3 LNT, VEGFR2/CD 3L 171-173, VEGFR2/CD3 LCT were 10-fold diluted with blocking solution for 8 gradients, starting antibody concentration was 100nM, 3 multiplex wells per concentration gradient, 100. Mu.L/well were added to the well plate and incubated for 2h at room temperature. Plates were washed 3 times with PBST and drained, HRP-labeled anti-kappa chain antibodies were diluted 1:5000 with 5% skimmed milk PBST and added to the well plates, 100. Mu.L/well, and incubated for 1h at room temperature. After washing the plate 3 times with PBST, draining, adding 100. Mu.L of TMB color development liquid into each well, developing at room temperature in dark for 10min, adding 50. Mu.L of 2M H 2SO4 into each well, stopping the color development reaction, and detecting the absorbance value of OD450nm by using a microplate reader. And (3) taking the sample concentration value as an abscissa and the absorbance value as an ordinate, performing a four-parameter nonlinear regression graph, and calculating an EC50 value.
The results are shown in Table 8 and FIG. 7, and compared with the control CD3-Fab, the bispecific antibodies with different structures can effectively bind to VEGFR2 ECD-Fc antigen protein; further analysis of the EC50 values found that the antigen binding capacity of VEGFR2/CD3 LNT and VEGFR2/CD 3L 171-173 was significantly stronger than that of VEGFR2/CD3 LCT; further comparison shows that VEGFR2/CD 3L 171-173 has good antigen binding capacity compared with bispecific antibodies of 2 other structures.
TABLE 8
| Constructs | EC50(pM) |
| CD3-Fab | Not determined |
| VEGFR2/CD3 LNT | 1856 |
| VEGFR2/CD3 L171-173 | 1492 |
| VEGFR2/CD3 LCT | 10206 |
3.4 In vitro tumor killing Activity of bispecific antibodies to VEGFR2/CD3 of different Structure
Activated human T cells (PBMC from healthy volunteers), prostate cancer cells PC3 (VEGFR2+), human umbilical vein endothelial cells HUVEC (VEGFR2+), breast cancer cells MDA-MB-468 (VEGFR 2-) and engineered MDA-MB-468/VEGFR2 (VEGFR2+), were cultured in complete medium of RPMI-1640 with IL2 concentration of 300IU/mL, and in medium of DMEM with 10% fetal bovine serum. T cells were cultured 24h prior to the experiment with IL 2-depleted RPMI-1640 medium. T cell density was adjusted to 2X 10 6, tumor cell density was adjusted to 2X 10 5, and the mixture of 2 cells was mixed according to an effective target ratio of 10:1 and added to a 96-well cell plate at 100. Mu.L/well. The bispecific antibodies to be tested, VEGFR2/CD3 LNT, VEGFR2/CD 3L 171-173, VEGFR2/CD3 LCT were 10-fold diluted with medium for 8 gradients, starting antibody concentration 10nM, 2 multiplex wells per concentration gradient, 10. Mu.L/well were added to the well plate. The experiment was run with both positive control (tumor cells directly into cell lysate) and negative control (tumor cells and T cells) at 37 ℃ in a 5% co 2 incubator for 24h. And detecting the target cell lysis degree in the 96-well plate by using a lactate dehydrogenase detection kit, detecting the OD490 reading in the well by using a multifunctional enzyme-labeled instrument, performing data analysis by using GRAPHPAD PRISM software, performing a four-parameter nonlinear regression graph by using an antibody concentration value as an abscissa and an absorbance value as an ordinate, and calculating an IC50 value.
The results are shown in FIG. 8, FIG. 9, FIG. 10 and FIG. 11, the 3 structural VEGFR2/CD3 bispecific antibodies were antigen specific cytotoxic to all of VEGFR2 positive tumor cells PC3 (VEGFR2+), HUVEC (VEGFR2+), MDA-MB-468/VEGFR2 (VEGFR2+), but not to tumor cells MDA-MB-468 (VEGFR 2-), which did not express VEGFR 2. Comparison of IC50 shows that VEGFR2/CD 3L 171-173 shows significantly enhanced tumor cell specific killing effect on different VEGFR2 positive tumor cells compared with other bispecific antibodies with 2 structures. Thus, by comparing the VEGFR2 antigen binding activity with the VEGFR2 positive tumor cell killing activity, L171-173 chimeric to CD3-Fab by VEGFR2-VHH was determined as the optimal recombinant antibody structure.
3.5 Antigen binding validation of HER2/VEGFR2/CD3 (SO) trispecific antibodies with optimized structure
The specific implementation procedure is the same as 3.1, and the results are shown in table 9 and fig. 12, and the comparison of EC50 shows that the optimized structure HER2/VEGFR2/CD3 (SO) has no obvious difference in binding capacity to HER2 antigen compared with the HER2/CD3 bispecific antibody with different structure, indicating that HER2/VEGFR2/CD3 (SO) retains its binding affinity and specificity to HER2 antigen; furthermore, the procedure was performed as 3.3, and the results are shown in table 10 and fig. 13, and comparing EC50 results show that the optimized structure HER2/VEGFR2/CD3 (SO) has no significant difference in binding capacity to VEGFR2 antigen compared to VEGFR2/CD3 bispecific antibodies of different structures, indicating that HER2/VEGFR2/CD3 (SO) retains its binding affinity and specificity to VEGFR2 antigen. Wherein the HER2/VEGFR2/CD3 (SO) trispecific antibody has binding specificity for HER2/CD3 as set forth in SEQ ID NO:4, and a recombinant heavy chain amino acid sequence as set forth in SEQ ID NO:6, and a recombinant light chain amino acid sequence shown in the specification.
TABLE 9
| Constructs | EC50(pM) |
| VEGFR2/CD3 L171-173 | Not determined |
| HER2/CD3 HNT(HE Linker) | 133.5 |
| HER2/CD3 HNT(PD Linker) | 254.5 |
| HER2/CD3 H184-187 | 3499 |
| HER2/CD3 HCT | 339.4 |
| HER2/VEGFR2/CD3(SO) | 216.8 |
Table 10
| Constructs | EC50(pM) |
| HER2/CD3 HNT(PD Linker) | Not determined |
| VEGFR2/CD3 LNT | 300.7 |
| VEGFR2/CD3 L171-173 | 1140 |
| VEGFR2/CD3 LCT | 2513 |
| HER2/VEGFR2/CD3(SO) | 276.4 |
3.6 In vitro tumor cell killing Activity comparison of optimized Structure HER2/VEGFR2/CD3 (SO) trispecific antibodies with different Structure HER2/CD3 bispecific antibodies
The specific implementation steps are 3.2, and the results are shown in fig. 14, fig. 15 and fig. 16, and comparing the IC50 values show that compared with HER2/CD3 bispecific antibodies with different structures, the trispecific antibody HER2/VEGFR2/CD3 (SO) after structure optimization has better killing effect on HER2 and VEGFR2 double positive cell lines PC-3 (her2+/vegfr2+) and HUVEC (her2+/vegfr2+), and has no nonspecific killing effect on HER2 and VEGFR2 double negative cell lines MDA-MB-468.
3.7 Comparison of in vitro tumor cell killing Activity of optimized Structure HER2/VEGFR2/CD3 (SO) trispecific antibodies with different Structure VEGFR2/CD3 bispecific antibodies
The specific implementation steps are 3.4, and the results are shown in fig. 17, 18 and 19, and the comparison of the IC50 values shows that compared with the VEGFR2/CD3 bispecific antibodies with different structures, the trispecific antibody HER2/VEGFR2/CD3 (SO) after structure optimization has a significantly improved killing effect on HER2 and VEGFR2 double positive cell lines PC-3 (her2+/vegfr2+) and HUVEC (her2+/vegfr2+), and has no nonspecific killing effect on HER2 and VEGFR2 double negative cell lines MDA-MB-468.
3.8 In vivo anti-tumor Activity of HER2/VEGFR2/CD3 (SO) trispecific antibodies with optimized Structure
25 NCG male mice, 6 weeks old, were inoculated subcutaneously with PC3 tumor cells. Specifically, matrigel and PC-3 tumor cells were mixed at a volume ratio of 1:1, and each mouse was inoculated with 2X 10 6 cells at a dose of 100. Mu.L. The vernier caliper detects the tumor volume, and when the tumor volume reaches 200mm 3, the abdominal cavity is inoculated with activated T cells each 2.5X10 7, and the dosage is 200 mu L. Treatment with antibody was started after one day, specifically 25 mice were divided into 5 groups, and physiological saline, HER2/CD3 HCT, VEGFR2/CD 3L 171-172, a double antibody cocktail (HER 2/CD3 HCT+VEGFR2/CD 3L 171-172), HER2/VEGFR2/CD3 (SO) were injected into the tail vein, the dose was 15nmol/kg, the dosing interval was two days, and the total dose was 7 times, and tumor volumes and mouse weights were measured once every 2 days.
As shown in fig. 20, the results demonstrate that both bispecific and trispecific antibodies inhibited tumor growth, whereas HER2/VEGFR2/CD3 (SO) trispecific antibodies inhibited more significantly, indicating that the trispecific antibodies mediate more pronounced killing effects of T cells on tumor cells and micro-environmental vessels than the bispecific antibodies. As shown in fig. 21, the results show that the body weight of 5 groups of mice is not obviously changed while killing the tumor, which indicates that the trispecific antibody does not cause obvious increase of toxicity in vivo after increasing the target resistance, and the safety is ensured. In conclusion, through the comparison of the antigen binding activity and the tumor cell killing activity, the HER2/VEGFR2/CD3 trispecific antibody with optimized structural design has obvious synergistic inhibition and killing functions on solid tumors.
Example 4 evaluation of in vitro biological Functions of CD19/CD22/CD3 trispecific antibodies
4.1 Cell binding Capacity of different Structure CD19/CD3 bispecific antibodies
Cell counts were performed using CD19 engineered K562 cell lines K562-CD19 (CD19+CD22-) and K562 (CD 19-CD 22-) to adjust cell densities to 3X 10 6/mL 100. Mu.L per well. Bispecific antibodies to be tested, CD19/CD3 HNT, CD19/CD 3H 184-187, CD19/CD3 HCT were 5-fold diluted 5-fold with FACS diluent at an initial antibody concentration of 50nM, 2 multiplex wells per concentration, 100. Mu.L/well were added to the well plate and incubated on ice for 2H. Preparing a secondary antibody: APC labeled anti-human Kappa chain, antibody concentration 1 μg/mL. Cells were resuspended at 100 μl per well and ice for 1h. Centrifuging to remove supernatant, and cleaning once. 200 μL FACS resuspended cells. And (3) carrying out flow detection, namely plotting with the protein concentration as an abscissa and the average fluorescence intensity as an ordinate, making a four-parameter nonlinear regression graph, and calculating an EC50 value.
As shown in Table 11, FIG. 22 and FIG. 23, the CD19/CD3 bispecific antibodies of different structures were each able to bind effectively to K562-CD19 (CD19+CD22-), while not showing any nonspecific binding to K562 (CD 19-CD 22-), and showed better antigen binding affinity compared to CD19/CD3 HNT and CD19/CD 3H 184-187, CD19/CD3 HCT, as compared to EC50 value comparison.
TABLE 11
4.2 In vitro tumor killing Activity of bispecific antibodies to CD19/CD3 of different Structure
Activated human T cells (PBMC from healthy volunteers), human B cell leukemia cells Nalm6 (CD19+CD22+), and Nalm6 cells knocked out of CD19 or knocked out of CD22, nalm6-KO19 (CD 19-CD22+), nalm6-KO22 (CD19+CD22-), K562 (CD 19-CD 22-), respectively, were cultured with RPMI-1640 complete medium containing IL2 at a concentration of 300 IU/mL. T cells were cultured 24h prior to the experiment with IL 2-depleted RPMI-1640 medium. Tumor cells were stained with fluorescent dye CFSE, T cell density was adjusted to 2X 10 6, tumor cell density was adjusted to 2X 10 5, and the mixture of 2 cells was mixed at an effective target ratio of 10:1 and added to a 96 well cell plate at 100. Mu.L/well. The bispecific antibodies to be tested CD19/CD3 HNT, CD19/CD 3H 184-187, CD19/CD3 HCT were diluted 10-fold with medium for 3 gradients, starting antibody concentrations of 100pM, 2 multiplex wells per concentration gradient, and 10. Mu.L/well were added to the well plate. Experiments were run with controls (tumor cells and T cells) at 37 ℃ in a 5% co 2 incubator for 24h. Flow cytometry was performed after adding 7-AAD staining for 30min per well, and CFSE positive 7-AAD negative tumor cells were counted using the formula: % lysis= (number of control-number of experimental-group live tumor cells)/number of control-group live tumor cells×100. And carrying out data analysis by adopting GRAPHPAD PRISM software, carrying out a four-parameter nonlinear regression graph by taking the value of the antibody concentration as an abscissa and taking% Lysis as an ordinate, and calculating an IC50 value.
As a result, as shown in FIG. 24, FIG. 25, FIG. 26 and FIG. 27, the 3-structure CD19/CD3 bispecific antibody had CD19 antigen-specific cytotoxicity to both Nalm6 (CD19+CD22+) and Nalm6-KO22 (CD19+CD22-) and had no killing activity to CD 19-negative Nalm6-KO19 (CD 19-CD22+) and K562 (CD 19-CD 22-). Comparison of IC50 shows that CD19/CD3 HCT shows significantly improved tumor cell specific killing effect compared with CD19/CD3 HNT and CD19/CD 3H 184-187 bispecific antibody. Thus, by comparison of the binding activity of CD19 antigen and the killing activity of CD19 positive tumor cells, it was determined that HCT of CD19-scFv fused to CD3-Fab was the optimal recombinant antibody structure.
4.3 Cell binding Capacity of different Structure CD22/CD3 bispecific antibodies
Cell counts were performed using CD22 engineered K562 cell lines K562-CD22 (CD 19-CD 22+) and K562 (CD 19-CD 22-) to adjust cell density to 3X 10 6/mL 100. Mu.L per well. The bispecific antibodies to be tested, CD22/CD3 LNT, CD22/CD 3L 171-173, CD22/CD3 LCT were diluted 5-fold with FACS diluent for 7 gradients at an initial antibody concentration of 200nM, 2 multiplex wells per concentration, 100. Mu.L/well were added to the well plate and incubated on ice for 2h. Preparing a secondary antibody: APC labeled anti-human Kappa chain, antibody concentration 1 μg/mL. Cells were resuspended at 100 μl per well and ice for 1h. Centrifuging to remove supernatant, and cleaning once. 200 μL FACS resuspended cells. And (3) carrying out flow detection, namely plotting with the protein concentration as an abscissa and the average fluorescence intensity as an ordinate, making a four-parameter nonlinear regression graph, and calculating an EC50 value.
Results Table 12, as shown in FIGS. 28 and 29, the CD22/CD3 bispecific antibodies of different structures were each able to bind effectively to K562-CD22 (CD 19-CD22+), while not showing any nonspecific binding to K562 (CD 19-CD 22-), and the CD22/CD3 LCT had good antigen binding capacity compared to the CD22/CD3 LNT and CD22/CD 3L 171-173 bispecific antibodies in comparison of EC50 values.
Table 12
4.4 In vitro tumor killing Activity of CD22/CD3 bispecific antibodies of different Structure
Activated human T cells (PBMC from healthy volunteers), human B cell leukemia cells Nalm6 (CD19+CD22+), and Nalm6 cells knocked out of CD19 or knocked out of CD22, nalm6-KO19 (CD 19-CD22+), nalm6-KO22 (CD19+CD22-), K562 (CD 19-CD 22-), respectively, were cultured with RPMI-1640 complete medium containing IL2 at a concentration of 300 IU/mL. T cells were cultured 24h prior to the experiment with IL 2-depleted RPMI-1640 medium. Tumor cells were stained with fluorescent dye CFSE, T cell density was adjusted to 2X 10 6, tumor cell density was adjusted to 2X 10 5, and the mixture of 2 cells was mixed at an effective target ratio of 10:1 and added to a 96 well cell plate at 100. Mu.L/well. The bispecific antibodies to be tested, CD22/CD3 LNT, CD22/CD 3L 171-173, CD22/CD3 LCT, were diluted 10-fold with medium for 5 gradients, starting antibody concentration 1nM, 2 multiplex wells per concentration gradient, and 10. Mu.L/well were added to the well plate. Experiments were run with controls (tumor cells and T cells) at 37 ℃ in a 5% co 2 incubator for 24h. Flow cytometry was performed after adding 7-AAD staining for 30min per well, and CFSE positive 7-AAD negative tumor cells were counted using the formula: % lysis= (number of control-number of experimental-group live tumor cells)/number of control-group live tumor cells×100. And carrying out data analysis by adopting GRAPHPAD PRISM software, carrying out a four-parameter nonlinear regression graph by taking the value of the antibody concentration as an abscissa and taking% Lysis as an ordinate, and calculating an IC50 value.
As a result, as shown in FIG. 30, FIG. 31, FIG. 32 and FIG. 33, the 3-structure CD22/CD3 bispecific antibody had CD22 antigen-specific cytotoxicity to Nalm6 (CD19+CD22+) and Nalm6-KO19 (CD 19-CD22+) and had no killing activity to CD 19-negative Nalm6-KO22 (CD19+CD22-) and K562 (CD 19-CD 22-). Comparison of IC50 shows that the CD22/CD3 LCT shows significantly improved tumor cell specific killing effect compared with the CD22/CD3 LNT and the CD22/CD 3L 171-173 bispecific antibody; in addition, CD22/CD3 LNT showed nonspecific cytotoxicity to CD22 negative Nalm6-KO22 (CD19+CD22-) at high concentrations. Thus, by comparison of the binding activity of CD22 antigen and the killing activity of CD22 positive tumor cells, the LCT of CD22-VHH fused to CD3-Fab was determined as the optimal recombinant antibody structure.
4.5 Antigen binding validation of CD19/CD22/CD3 (SO) trispecific antibodies with optimized Structure
The specific procedures are carried out in the same manner as in 4.1 and 4.3, and the results are shown in Table 13, FIG. 34 and FIG. 35, and by comparing the EC50 values, it is found that the optimized structure CD19/CD22/CD3 (SO) trispecific antibody retains the specificity and affinity of the CD19-scFv and CD22-VHH antibodies, and does not significantly differ from the binding capacity of the target cells K562-CD19 (CD19+CD22-) or K562-CD22 (CD 19-CD22+), compared to the best active bispecific antibodies CD19/CD3 HCT and CD22/CD3 LCT. Wherein the CD19/CD22/CD3 (SO) trispecific antibody has binding specificity for CD19/CD3 as set forth in SEQ ID NO:10, and a recombinant heavy chain amino acid sequence as set forth in SEQ ID NO:13, and a recombinant light chain amino acid sequence shown in seq id no.
TABLE 13
4.6 Comparison of tumor cell killing Activity of CD19/CD22/CD3 (SO) trispecific antibodies with bispecific antibodies
The specific implementation steps are the same as those of 4.2 and 4.4, and the results are shown in fig. 36, 37, 38 and 39, and comparing the IC50 values, the result shows that compared with the bispecific antibodies CD19/CD3 HCT and CD22/CD3 LCT with optimal activity, the trispecific antibody CD19/CD22/CD3 (SO) has significantly improved killing effect on CD19 and CD22 double positive cell line Nalm6 (CD 19+CD22+), CD19 single positive cell line Nalm6-KO22 (CD 19+CD22+), and CD22 single positive cell line Nalm6-KO19 (CD 19-CD22+), and has no nonspecific killing effect on CD19 and CD22 double negative cell line K562 (CD 19-CD 22+).
4.7 Comparison of the Activity of CD19/CD22/CD3 (SO) trispecific antibodies with Blinatumomab killer tumor cells
Activated human T cells (PBMC from healthy volunteers), CD19 engineered K562 cell lines K562-CD19 (CD19+CD22-), CD22 engineered K562 cell lines K562-CD22 (CD 19-CD22+) and K562 (CD 19-CD 22-) were cultured with RPMI-1640 complete medium containing IL2 at a concentration of 300 IU/mL. T cells were cultured 24h prior to the experiment with IL 2-depleted RPMI-1640 medium. Tumor cells were stained with fluorescent dye CFSE, T cell density was adjusted to 2X 10 6, tumor cell density was adjusted to 2X 10 5, and the mixture of 2 cells was mixed at an effective target ratio of 10:1 and added to a 96 well cell plate at 100. Mu.L/well. The trispecific antibodies to be tested CD19/CD22/CD3 (SO) and Blinatumomab were diluted 10-fold with medium in 4 gradients with an initial antibody concentration of 1nM and 2 multiplex wells per concentration gradient, 10. Mu.L/well were added to the well plate. Experiments were run with controls (tumor cells and T cells) at 37 ℃ in a 5% co 2 incubator for 24h. Flow cytometry was performed after adding 7-AAD staining for 30min per well, and CFSE positive 7-AAD negative tumor cells were counted using the formula: % lysis= (number of control-number of experimental-group live tumor cells)/number of control-group live tumor cells×100. And carrying out data analysis by adopting GRAPHPAD PRISM software, carrying out a four-parameter nonlinear regression graph by taking the value of the antibody concentration as an abscissa and taking% Lysis as an ordinate, and calculating an IC50 value.
As a result, as shown in FIG. 40, FIG. 41 and FIG. 42, the CD19/CD22/CD3 (SO) trispecific antibody had antigen-specific killing activity against both K562-CD19 (CD19+CD22-) and K562-CD22 (CD 19-CD22+), but had no killing activity against negative cells K562 (CD 19-CD 22-). Whereas Blinatumomab had killing activity only on K562-CD19 (CD19+CD22-) cells, no killing could be recognized on K562-CD22 (CD 19-CD22+) cells that mimic the immune escape of the CD19 antigen. Therefore, the CD19/CD22/CD3 (SO) trispecific antibody has wider application of multi-target recognition of tumor cells, and particularly has killing effect on the tumor cells which have drug resistance of Blinatumomab and positive CD 22. In conclusion, through the comparison of the antigen binding activity and the tumor cell killing activity, the CD19/CD22/CD3 trispecific antibody with optimized structural design has remarkable blood tumor immune killing function, and can prevent the problems of immune escape caused by single target recognition, drug resistance caused by Blinatumomab treatment and the like.
Claims (2)
1. The method for constructing the trispecific antibody based on the structure-optimized protein activity is characterized in that the trispecific antibody is a HER2/VEGFR2/CD3 trispecific antibody, and the specific steps when constructing the HER2/VEGFR2/CD3 trispecific antibody are as follows:
S1) taking a CD3 monoclonal antibody Fab as a structural basis;
S2) attaching the anti-HER 2-VHH to the heavy chain domain of the CD3 Fab structure, including attaching the HER2-VHH to the C-terminal 228C of the CH1 domain of the CD3 Fab heavy chain constant region via a flexible linker peptide;
S3) the anti-VEGFR 2-VHH is linked to the light chain domain of the CD3 Fab structure, comprising grafting the anti-VEGFR 2-VHH between CD3 Fab light chain constant region domains 171S-173D by Coiled Coil Linker and deleting 172K;
S4) links the heavy and light chains by disulfide bonds in CH1 and CL.
2. The method for constructing a trispecific antibody according to claim 1,
Said Coiled Coil Linker has the sequence as set forth in SEQ ID NO: 22. 23, and the flexible connecting peptide is (G4S) 3 Linker.
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