Patent Application
20240018481
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The sequence listing submitted herewith is contained in the text file created Apr. 27, 2023, entitled “23-0690-WO-US_Sequence-Listing_ST25.txt” and 20,480 bytes in size.
The present application belongs to the field of immune effect detection, and specifically relates to a recombinant CHO cell, a method of constructing the recombinant cell, and a detection system and method for detecting an ADCC effect and a CDC effect of an antibody using the cell.
ADCC, i.e., antibody-dependent cell-mediated cytotoxicity, refers to that after an antibody specifically binds to antigenic determinants on a surface of a target cell through Fab segment, a Fc segment thereof binds to an effector cell with FcyR, triggering killing activity of the effector cell to directly kill the target cell. An ADCC effect is an important immune mechanism for anti-infection and anti-tumor of the body. The effector cells involved in ADCC mainly include natural killed (NK) cell, phagocyte, eosinophil, basophil and mast cell, and the like.
CDC i.e., complement dependent cytotoxicity, refers to a cytotoxic effect in which a complement is involved, that is, a complex is formed to activate a complement classical pathway through the binding of a specific antibody to the corresponding antigen on the surface of cell membrane. Complement protein C1q initiates the complement classical pathway firstly, and then complement proteins C2-C9 are activated to form a membrane attack complex (MAC), which exerts a lysis effect on the target cell.
In view of the important role of the ADCC effect and CDC effect in tumor immunity, in the current development of anti-tumor antibodies, many researchers expect to obtain the improved ADCC effect and CDC effect through a specific design of antibody structures. However, in the development of other types of antibodies, the antibody-mediated ADCC effect and CDC effect are not always desired.
Nerve growth factor (NGF), the earliest found one among neurotrophic factors, is produced in the positions on neocortex and hippocampus, and consists of two a subunits, one β subunit and two y subunits, which can promote the growth, development, differentiation, and maturation of central and peripheral neurons, maintain the normal functions of the nervous system, and accelerate the repair to the nervous system after damage. At present, the recombinant NGF is mainly used in treating various neuropathy and nerve damage.
Chinese hamster ovarian cell (CHO cell) is one of the most widely used expression systems of mammalian cells, which has good post-translational processing ability, so that the expression product can maintain its natural structure and activity. Currently, CHO has been used to express NGF and secrete the recombinant NGF outside the cell, and therefore NGF can be produced and obtained in a large scale (for example, Xu L et al., Expression, purification, and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells. Protein Expr Purif, 2014, 104: 41-49; Wang X Y et al., Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells. Genet Mol Res, 2015, 14(3): 9191-9199). However, studies on the stable expression of NGF on the surface of a recombinant CHO cell have not been reported.
In order to be able to accurately detect the ADCC effect and CDC effect of an anti-NGF antibody, the inventors have developed a recombinant CHO cell stably expressing NGF on its surface through research, which is used as a target cell capable of being effectively used to evaluate the ADCC effect and CDC effect of the anti-NGF antibody.
In one aspect, the present application relates to a recombinant CHO cell, wherein NGF is stably expressed on the surface of the cell.
In another aspect, the present application relates to a method of constructing the above-mentioned recombinant CHO cell, comprising:
In another aspect, the present application relates to a system for detecting an ADCC effect of an antibody, comprising:
In another aspect, the present application relates to a system for detecting a CDC effect of an antibody, comprising:
In yet another aspect, the present application relates to use of the above-mentioned recombinant CHO cell in detecting an ADCC effect or a CDC effect of an antibody.
In yet another aspect, the present application relates to a method of detecting an ADCC effect of an antibody, comprising co-incubating the above-mentioned recombinant CHO cell as a target cell with an antibody to be detected and an effector cell.
In yet another aspect, the present application relates to a method of detecting a CDC effect of an antibody, comprising co-incubating the above-mentioned recombinant CHO cell as a target cell with an antibody to be detected and a complement.
In conventional recombinant NGF expression studies, a mammalian cell such as a CHO cell is usually used as an expression system to express and secrete NGF out of the cell, so that it is present in the supernatant in a free form similar to its natural form. In addition, some researchers have also tried to use prokaryotic systems (e.g., E. coli) to express NGF, and the expression product obtained is present as an insoluble inclusion body (Jiang Jing et al., Comparison of Recombinant Human β-NGF from E. coli and CHO in Biochemical Characters, China Biotechnology, 2006, 26(2): 8-12). There is considerable uncertainty on whether NGF, as a free protein rather than a transmembrane protein, can be expressed on the cell surface. Therefore, regarding the stable expression of NGF on the surface of the CHO cell, there have been no relevant reports in the art so far. In the present application, by concatenating the nucleic acid sequence of NGF with the nucleic acid sequence of an appropriate hinge region and transmembrane region and then introducing the same into a CHO cell, stable expression of NGF on the surface of the recombinant CHO cell is achieved, thereby making NGF present on the cell surface rather than being secreted into the culture fluid, and enabling the proper post-translational processing (including correct folding, glycosylation, etc.) of the expressed NGF and thus maintaining natural conformation and activity thereof. The recombinant CHO cell thus obtained has NGF on the cell surface and can be effectively used as a target cell for detecting the ADCC effect and CDC effect of an anti-NGF antibody, and thus can be used to detect the ADCC effect and CDC effect of the anti-NGF antibody.
Hereinafter, the present application is further described in combination with particular embodiments, and the advantages and features of the present application will become clearer with description. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present application. It should be understood by the person skilled in the art that although modifications or substitutions may be made to the details and form of the technical solutions of the present application without departing from its spirit and scope, such modifications and substitutions fall within the protection scope of the present application.
Unless defined otherwise, the technical and scientific terms used herein have the same meanings as commonly understood by the person of ordinary skill in the art to which this disclosure belongs, which can be seen in, for example, Singleton eta!, Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (2012); Abbas et al., Cellular and Molecular Immunology, Elsevier Science Health Science div (2009); He Wei et al., Medical Immunology (2nd Edition), People's Medical Publishing House Co., Ltd., 2010.
Unless stated otherwise, the terms “comprise”, “comprises” and “comprising” or equivalents thereof (“contain”, “contains”, “containing”, “include”, “includes” and “including”) herein are open-ended mode expressions which mean that other unspecified elements, components and steps may be included in addition to those already listed.
Unless stated otherwise, all the numbers used herein to express amounts of ingredients, measured values, or reaction conditions should be understood as being modified in all cases by the term “about”. When being attached to a percentage, the term “about” can represent, e.g., ±1%, preferably ±0.5%, and more preferably ±0.1%.
Unless explicitly indicated otherwise in the context, a singular term encompasses the plural indicating objects, and vice versa. Similarly, unless explicitly indicated otherwise in the context, the word “or” intends to include “and”.
The percentage of identity (degree of homology) between sequences herein can be determined by aligning two sequences using, for example, freely accessible computer programs in the World Wide Web (e.g., www.ncbi.nlm.nih.gov) that are commonly used for this purpose (e.g., BLASTp or BLASTn with default settings).
Unless stated otherwise, the term “codon optimization” herein refers to a process of modifying nucleic acid sequences by replacing at least one codon of a natural sequence with a codon that is used more or most frequently in the host cell to improve expression in the host cell while maintaining the natural amino acid sequence. Various species have specific preference for specific codons of specific amino acids, and this codon preference (difference in codon use among different organisms) is typically related to the translation efficiency of mRNA.
In the present invention, “expression vector” refers to a vector comprising a desired coding sequence and an appropriate nucleic acid sequence essential to express the coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more marker genes. In order to express the target sequence of the present invention, for example, any one of the following several expression control sequences can be used in the vector: replication origin, polyadenylation signal, promoter, enhancer, terminator, etc.
Since the first monoclonal antibody drug OKT3 (muromonab-CD3) was approved by the U.S. FDA in 1986, antibody drugs have been increasingly studied as an option for disease treatment. So far, FDA has approved hundreds of antibody drugs (https://www.nature.com/articles/d41573-021-00079-7). The mechanism by which antibodies activate the human immune system is very complex, mainly including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
In general, since NGF per se is a free protein, the recombinant cell obtained by the construction capable of expressing NGF will secrete the expressed NGF out of the cell, so that it is present in the culture fluid in a free form substantially equivalent to the natural form, thereby making the NGF-expressing recombinant cell obtained by the common construction unsuitable as a target cell used in ADCC and CDC assays. There is considerable uncertainty on expressing NGF on the cell surface, and even if NGF is expressed on the surface of a recombinant cell, it is also unknown whether the expressed NGF can maintain the same natural conformation and activity as those of the free NGF. Therefore, recombinant CHO cells or other recombinant cells that can stably express NGF on their surfaces have not been reported.
In some embodiments, the present application relates to a recombinant CHO cell, wherein NGF is stably expressed on the surface of the cell.
Any NGF of known sequences can be used herein, such as the sequence available in NCBI, ENSMBL, or UniProt database, or a sequence obtained by performing codon optimization to a known sequence according to the codon preference of the host cell (existing software such as codon W can be used) can be employed. In some preferred embodiments, the NGF can be, but is not limited to, a mammalian NGF, such as a human NGF, a mouse NGF, a rat NGF, a bovine NGF, a horse NGF, a pig NGF, etc. The exemplary amino acid sequences and nucleic acid sequences of NGFs can be found in the following records: Katherine A. Fitzgerald et al., The Cytokine FactsBook and Webfacts (Second Edition), Academic Press, 2001; Wei Ma et al., Cloning and Sequencing of Matured Fragment of Human Never Growth Factor Gene, ACAD XI TU, 15(1), 2003: 62-65; V A Barde et al., The nerve growth factor family, Progress in Growth Factor Research, 2, 1990: 237-248; Wiesmann, C. et al., Nerve growth factor: structure and function. CMLS, Cell. Mol Life Sci, 58, 2001: 748-759; Eric Adriaenssens et al, Nerve Growth Factor Is a Potential Therapeutic Target in Breast Cancer, Cancer Res., 68(2), 2008: Jan. 15, 2008: 346-351; WO2021120900A1, WO2021093134A1, WO2021091363A1, WO2019201133A1, US2021079053A1, WO2010128398A1, US2014170137A1, etc., the contents of each of which are incorporated herein by reference in their entireties.
In some preferred embodiments, the amino acid sequence of NGF comprises an amino acid sequence set forth in any one of SEQ ID NO: 1-SEQ ID NO: 3 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the genome of the cell is integrated with a nucleic acid sequence of NGF.
In some preferred embodiments, the nucleic acid sequence of NGF comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 4-SEQ ID NO: 6 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the genome of the cell is integrated with a nucleic acid sequence of a concatenated NGF, hinge region and transmembrane region (also known as “transmembrane domain”).
In further preferred embodiments, the amino acid sequence of the hinge region has 3 or more amino acids, 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, or 100 or less amino acids, 90 or less amino acids, 80 or less amino acids, 70 or less amino acids, 60 or less amino acids, or amino acids within the range of the above any two numerical values, such as 3-100 amino acids, 10-90 amino acids, 10-80 amino acids, 10-70 amino acids, 10-60 amino acids, 10-50 amino acids, and 12-45 amino acids.
In further preferred embodiments, the amino acid sequence of the transmembrane region has 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, or 60 or less amino acids, 50 or less amino acids, 40 or less amino acids, or amino acids within the range of the above any two numerical values, such as 10-60 amino acids, 10-50 amino acids, 10-40 amino acids, 10-30 amino acids, 15-30 amino acids, 20-30 amino acids, and 20-25 amino acids.
In further preferred embodiments, the hinge region may be a CD8 hinge region (e.g., CD8α hinge region) or a human IgG1 hinge region.
In further preferred embodiments, the transmembrane region may be a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region.
In some preferred embodiments, the amino acid sequence of the CD8 hinge region comprises an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 7, amino acid sequence of the CD8 hinge region
In some preferred embodiments, the amino acid sequence of the human IgG1 hinge region comprises an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 8, amino acid sequence of the human IgG1 hinge region EPKSCDKTHICP
In some preferred embodiments, the nucleic acid sequence encoding the CD8 hinge region comprises a nucleic acid sequence set forth in SEQ ID NO: 9 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 9, nucleic acid sequence of the CD8 hinge region
In some preferred embodiments, the amino acid sequence of the CD8 transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 10, amino acid sequence of the CD8 transmembrane region
In some preferred embodiments, the amino acid sequence of the PGFRA transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 11, amino acid sequence of the PGFRA transmembrane region
In some preferred embodiments, the amino acid sequence of the CD80 transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 12, amino acid sequence of the CD80 transmembrane region
In some preferred embodiments, the nucleic acid sequence encoding the CD8 transmembrane region comprises a nucleic acid sequence set forth in SEQ ID NO: 13 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
SEQ ID NO: 13, nucleic acid sequence of the CD8 transmembrane region
In some preferred embodiments, the hinge region is a CD8 hinge region, and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some instances, the hinge region is a CD8 hinge region, and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a CD8 hinge region, and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a CD8 hinge region, and the transmembrane region is a CD80 transmembrane region.
In some preferred embodiments, the hinge region is a human IgG1 hinge region, and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some instances, the hinge region is a human IgG1 hinge region, and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a human IgG1 hinge region, and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a human IgG1 hinge region, and the transmembrane region is a CD80 transmembrane region.
In some preferred embodiments, the nucleic acid sequence of the concatenated NGF, hinge region and transmembrane region is a nucleic acid sequence of concatenated NGF, CD8 hinge region and CD8 transmembrane region.
In some embodiments, the present application relates to a method of constructing the above-mentioned recombinant CHO cell, comprising:
In some preferred embodiments, the NGF can be, but is not limited to, a mammalian NGF, such as a human NGF, a mouse NGF, a rat NGF, a bovine NGF, a horse NGF, a pig NGF, etc.
In some preferred embodiments, the amino acid sequence of NGF comprises an amino acid sequence set forth in any one of SEQ ID NO: 1-SEQ ID NO: 3 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the nucleic acid sequence of NGF comprises a nucleic acid sequence set forth in any one of SEQ ID NO: 4-SEQ ID NO: 6 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In further preferred embodiments, the amino acid sequence of the hinge region has 3 or more amino acids, 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, or 100 or less amino acids, 90 or less amino acids, 80 or less amino acids, 70 or less amino acids, 60 or less amino acids, or amino acids within the range of the above any two numerical values, such as 3-100 amino acids, 10-100 amino acids,10-90 amino acids, 10-80 amino acids, 10-70 amino acids, 10-60 amino acids, 10-50 amino acids, and 12-45 amino acids.
In a further preferred embodiment, the amino acid sequence of the transmembrane region has 10 or more amino acids in length, 20 or more amino acids, 30 or more amino acids, or 60 or less amino acids, 50 or less amino acids, 40 or less amino acids, or amino acids within the range of the above any two numerical values, such as 10-60 amino acids, amino acids, 10-40 amino acids, 10-30 amino acids, 15-30 amino acids, 20-30 amino acids, and 20-25 amino acids.
In some preferred embodiments, the hinge region may be a CD8 hinge region (e.g., CD8α hinge region) or a human IgG1 hinge region.
In some preferred embodiments, the transmembrane region may be a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region.
In some preferred embodiments, the amino acid sequence of the CD8 hinge region comprises an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto. In some preferred embodiments, the amino acid sequence of the human IgG1 hinge region comprises an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the nucleic acid sequence encoding the CD8 hinge region comprises a nucleic acid sequence set forth in SEQ ID NO: 9 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the amino acid sequence of the CD8 transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto. In some preferred embodiments, the amino acid sequence of the PGFRA transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto. In some preferred embodiments, the amino acid sequence of the CD80 transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the nucleic acid sequence encoding the CD8 transmembrane region comprises a nucleic acid sequence set forth in SEQ ID NO: 13 or a nucleic acid sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher) sequence identity thereto.
In some preferred embodiments, the hinge region is a CD8 hinge region, and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some instances, the hinge region is a CD8 hinge region, and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a CD8 hinge region, and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a CD8 hinge region, and the transmembrane region is a CD80 transmembrane region.
In some preferred embodiments, the hinge region is a human IgG1 hinge region, and the transmembrane region is a CD8 transmembrane region, a PGFRA transmembrane region, or a CD80 transmembrane region. For example, in some instances, the hinge region is a human IgG1 hinge region, and the transmembrane region is a CD8 transmembrane region. Or for example, the hinge region is a human IgG1 hinge region, and the transmembrane region is a PGFRA transmembrane region. Or for example, the hinge region is a human IgG1 hinge region, and the transmembrane region is a CD80 transmembrane region.
In some preferred embodiments, the nucleic acid sequence of the concatenated NGF, hinge region and transmembrane region is a nucleic acid sequence of a concatenated NGF, CD8 hinge region and CD8 transmembrane region.
In some preferred embodiments, the expression vector can be, but is not limited to, a lentiviral expression vector, an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a poxvirus vector or a herpes simplex virus vector. As an example of the lentiviral expression vector, the lentiviral expression vector can be, for example, a Lenti-CMV-puro lentiviral expression vector, a lentiviral expression vector of pCDH series, or a lentiviral expression vector of pLenti series, but not limited thereto.
In some preferred embodiments, in step (2), the recombinant expression vector and a virus packaging plasmid are co-transfected into a packaging cell. After culturing, a virus comprising the nucleic acid sequence is prepared, and then the virus is used to transfect a CHO cell to obtain the recombinant CHO cell.
In further preferred embodiments, the virus packaging plasmid can be pMD2.G, psPAX2, Lenti-packaging Mix, pCMV-dR8.91, pCMV-dR8.74, pLP1, pLP2, pVSV-G, etc., but not limited thereto.
In further preferred embodiments, the packaging cell may be a 293T cell, a 293FT cell, etc., but not limited thereto.
Unless stated otherwise, cell culture herein is conducted using common culture conditions and common cell culture media (such as 1640 culture medium, DMEM culture medium, MEM culture medium, F12 culture medium, etc.) in the art, which can be found in, for example, Lan Rong et al., Cell Culture Technology, Chemical Industry Press Co., Ltd., 2007; R. I. Fershney, Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (Sixth Edition), John Wiley & Sons, Inc; 2010; Liu Bin et al., Cell Culture (Third Edition), World Publishing Corporation, 2018; the contents of each of which are incorporated herein by reference in their entireties.
Transfection is one of the commonly used means known in the art for introducing a target exogenous DNA (extracted from a donor of interest or directly synthesized) into host cells. Virus-mediated transfection is one of the commonly used transfection methods in the art, which uses a method of infecting cells with a virus packaging a desired exogenous gene enabling the exogenous gene of interest entering the cell. The method has the advantages such as the highest transfection efficiency and low cytotoxicity.
In some embodiments, the present application relates to a system for detecting an ADCC effect of an antibody, comprising:
In the present disclosure, the effector cell can be any common effector cell known in the art. As an example, in some preferred embodiments, the effector cell can include, but are not limited to, effector T cell, NK cell, phagocyte, eosinophil, basophil and mast cell, such as Jurkat-NFAT-Luciferase-CD16 cell, primary NK cell, etc.
In some preferred embodiments, the effector-target ratio (E:T) of the effector cell to the recombinant CHO cell by number can be 1:1 to 1:20, e.g., 1:5.
In some preferred embodiments, the system further includes a luciferase substrate. As a typical luciferase, firefly luciferase is a protein with a molecular weight of about 61 kDa, which can catalyze the oxidation of luciferin to oxyluciferin in the presence of ATP, magnesium ions and oxygen, and emits biofluorescence during the oxidation process.
In some embodiments, the present application relates to a system for detecting a CDC effect of an antibody, comprising:
Complement, also known as complement system, is a protein present in human and animal serum or tissue fluid that can mediate immune response and inflammation, including more than 30 soluble proteins and membrane binding proteins. In some preferred embodiments, the complement is a complement serum comprising complement proteins C1-C9.
In some preferred embodiments, the system further comprises a luciferase substrate.
In some embodiments, the present application relates to use of the above-mentioned recombinant CHO cell in detecting an ADCC effect or a CDC effect of an antibody.
In some embodiments, the present application relates to a method of detecting an ADCC effect of an antibody, comprising co-incubating the above-mentioned recombinant CHO cell as a target cell with an antibody to be detected and an effector cell. In further preferred embodiments, the effector-target ratio of the effector cell to the target cell by number can be 1:1 to 1:20, e.g., 1:5. In some preferred embodiments, the method further comprises adding a luciferase substrate to the system after co-incubation.
In some embodiments, the present application relates to a method of detecting a CDC effect of an antibody, comprising co-incubating the above-mentioned recombinant CHO cell as a target cell with an antibody to be detected and a complement. In further preferred embodiments, the complement is a complement serum comprising complement proteins C1-C9. In some preferred embodiments, the method further comprises adding a luciferase substrate to the system after co-incubation.
Hereinafter, the present application will be described in details through examples, but it is not intended to limit the protection scope of the present application thereto. For the skilled in the art, without departing from the spirit and scope of the present application, various changes, modifications and improvements can be made based on the descriptions of the following examples, and the resulting solutions still falls within the protection scope of the present application.
It should be illustrated that the operational steps involved in the present application, such as the design, synthesis, and cloning of genes, the linkage of gene fragments, the construction of expression vectors, analysis and identification of sequences, the construction of a recombinant cell, and the isolation and purification of expression products, can be performed in accordance with techniques known in the art (e.g., see the records in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY). Unless specified otherwise, the technical means used in the examples are common means well known by the skilled in the art. Unless stated otherwise, the reagents, materials, and devices used in the following examples are commercially available.
The main reagents, consumable materials and devices used in the following examples and sources thereof are shown in the table below. The skilled in the art can use any other reagents, consumable materials and devices with equivalent effects.
The nucleic acid sequence of NGF could be concatenated with the nucleic acid 5 sequences encoding the hinge region and the transmembrane region and inserted into an appropriate expression vector, thereby achieving the heterologous expression of the above nucleic acid sequences. Regarding NGF, a NGF from mammals could be selected, such as a human NGF, a mouse NGF, a rat NGF, a bovine NGF, a horse NGF, a pig NGF, etc., and a NGF and nucleic acid sequence thereof could be synthesized based on the known sequences disclosed by commonly used databases of nucleic acid sequences and protein sequences, such as NCBI, ENSMBL or UniProt.
Here, a human NGF was taken as an example, and a CD8 hinge region and a CD8 transmembrane region were used to be concatenated with the NGF. Specifically, the full-length sequence of the human NGF was codon optimized according to the codon preference of the host cell CHO-S cell to obtain NGF with an amino acid sequence set forth in SEQ NO ID: 1 (the corresponding nucleic acid sequence was set forth in SEQ ID NO: 4), which was concatenated with the CD8 hinge region (the amino acid sequence thereof was set forth in SEQ ID NO: 7; the nucleic acid sequence thereof was set forth in SEQ ID NO: 9) and the CD8 transmembrane region (the amino acid sequence thereof was set forth in SEQ ID NO: 10; the nucleic acid sequence thereof was set forth in SEQ ID NO: 13). Gene synthesis was conducted after concatenation and the synthesized gene fragments were subcloned to Lenti-CMV-puro lentiviral expression vector (iCarTab Biotechnology (Suzhou) co.ltd). The target gene overexpression vector Lenti-CMV-NGF-Full (CD8-TM) vector was prepared; and after being sequenced to verify that the sequence was correct, the endotoxin-removing plasmid was prepared for future use.
Next, lentivirus was prepared by the steps as follows:
Using the Lenti-CMV-NGF-Full (CD8-TM) overexpression lentivirus prepared in Example 1, recombinant CHO cells (also known as “CHO-NGF recombinant cells” or “CHO-NGF cells”) that can stably express NGF on the cell surface were prepared by the following process:
Amount of virus (mL)=(number of cells×MOI)/virus titer
Using a positive control antibody, the recombinant CHO cells constructed in Example 2 were identified by the flow cytometry. Wherein, the positive control antibody HAB20-6-2 was prepared according to the following process:
The detection results were shown in
Based on the positive control antibody HAB20-6-2 prepared above, the recombinant CHO cells could be identified by the flow cytometry. For example, the recombinant CHO cells (CHO-NGF cells) constructed in Example 2 could be identified by the following process:
The flow cytometry identification results were shown in
In this example, the CHO-NGF recombinant cell that stably expressed human NGF on the cell surface constructed in the above Example 2 was used as a target cell, and the Jurkat-NFAT-Luciferase-CD16 cell line that stably transfected with luciferase was used as an effector cell. Upon the Fab segment of the sample to be detected or the positive control antibody bound to the antigen on the target cell, the Fc segment of the sample to be detected or the positive control antibody bound to CD16 on the surface of the effector cell Jurkat-NFAT-Luciferase-CD16, and the intracellular NFAT-related signaling pathway was activated, which in turn led to an increase in the luciferase expression level. By detecting changes in luciferase activity, the ADCC effects of the sample to be detected and the positive control antibody were evaluated.
Tanezumab, the sample to be detected, was a recombinant anti-NGF humanized monoclonal antibody developed by Pfizer, which targeted human nerve growth factor and was synthesized by a CHO cell expression system. The relevant information on Tanezumab and the positive control antibody HAB20-6-2 used in this example was shown in the table below.
The ADCC effect assay experiment was conducted using ADCC Reporter Bioassay, and the experimental steps were as follows.
In this example, the CHO-NGF recombinant cell that stably expressed NGF on the cell surface constructed in Example 2 was used as a target cell, and the abilities of the positive control antibody HAB20-6-2 and Tanezumab to mediate the ADCC effect were evaluated by a reporter gene method using the existing effector cell. The detection results of the ADCC effect were shown in
It can be seen that under the above experimental conditions, the positive control antibody produced a significant ADCC effect, but Tanezumab, which had the same variable region (from which it could be expected that the antibody Tanezumab could recognize and specifically bind to the same antigen, i.e., the NGF expressed by the CHO-NGF recombinant cell), did not produce a significant ADCC effect. Therefore, the CHO-NGF recombinant cell of the present invention could be effectively used as a target cell for detecting the anti-NGF antibody-mediated ADCC effect in vitro, thereby accurately detecting whether the antibody to be detected could cause the effector cell to produce the ADCC effect.
In this example, the CHO-NGF recombinant cell that stably expressed human NGF on the cell surface constructed in Example 2 was used as a target cell, and the target cell, human complement serum, and the sample to be detected or the positive control antibody were incubated together. Upon the Fab segment of the sample to be detected or the positive control antibody bound to the antigen on the target cell, the Fc segment of the sample to be detected or the positive control antibody bound to the complement in human serum and resulted in a CDC effect, causing the target cell to be killed. ATP was an important indicator of the metabolism of living cells, and the content thereof directly reflected the number of living cells. Cell Counting-Lite 3D Luminescent reagent was added to the reaction system after completing the incubation. The reagent comprised a thermostable luciferase and the substrate luciferin thereof, and could promote the lysis of living cells in the reaction system to release ATP. ATP promoted the effect of luciferase on the substrate, leading to luminescence of the substrate. By detecting the luminous intensity, the number of living cells in the detection system could be reflected thereby, and the lysis effect of the sample to be detected or the positive control antibody on the target cell via the CDC effect could be further reflected.
The relevant information on Tanezumab, which was the sample to be detected, and the positive control antibody HAB20-6-2 were the same as that in Example 4. The CDC effect detection experiment was conducted according to the following steps:
In this example, the abilities of the positive control antibody HAB20-6-2 and Tanezumab to mediate the CDC effect were evaluated using the CHO-NGF recombinant cell that stably expressed NGF on the cell surface constructed in Example 2 as a target cell. According to the detection results in
It can be seen that under the above experimental conditions, the positive control antibody HAB20-6-2 produced a significant CDC effect, but Tanezumab (which had the same variable region as that of the positive control antibody, and it could be expected that the antibody Tanezumab could also recognize and specifically bind to the NGF expressed on the surface of the CHO-NGF recombinant cell) did not produce a significant CDC effect. Therefore, the CHO-NGF recombinant cell of the present invention could be effectively used as a target cell for detecting the anti-NGF antibody-mediated CDC effect in vitro, thereby accurately detecting whether the antibody to be detected could produce the CDC effect on the target cell.
All patents, patent applications and other publications are hereby expressly incorporated herein by reference for the purpose of describing and disclosing. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements regarding the date or representations regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission of the correctness of the dates or contents of these documents. Moreover, in any country, any reference to these publications herein does not constitute recognition that the publications become a part of the common knowledge in the art.
The skilled in the art will realize that the scope of the present application is not limited to various specific embodiments and examples described above, but can be modified, replaced, or recombined without departing from the spirit of the present application, which all fall within the protection scope of the present application.
This application is a section 371 U.S. national phase of PCT/CN2021/109763, filed Jul. 30, 2021, which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/109763 | 7/30/2021 | WO |
