CN116082510B - Monoclonal antibody or antigen binding fragment thereof and application of monoclonal antibody or antigen binding fragment thereof in resisting tumor - Google Patents
Monoclonal antibody or antigen binding fragment thereof and application of monoclonal antibody or antigen binding fragment thereof in resisting tumor Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3023—Lung
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Abstract
A monoclonal antibody or antigen binding fragment thereof is disclosed, which antibody or fragment comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity thereto: the CDR sequence of the heavy chain variable region is 1-11; the light chain variable region CDR sequence SEQ ID NO. 12-26, and the monoclonal antibody or antigen binding fragment thereof is selected from scFv amino acid sequence SEQ ID NO. 27-33. The monoclonal antibody or the antigen binding fragment thereof specifically recognizes LL/2-Luc2 or other tumor cell related antigens, has anti-tumor effect and application, and has good application prospect.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a monoclonal antibody or an antigen binding fragment thereof and an anti-tumor application thereof.
Background
Lung cancer currently represents the first cause of cancer death in the world, and is one of the malignant tumors with higher morbidity and mortality in the world. The treatment of lung cancer is numerous, but the prognosis of lung cancer is still poor, and the overall treatment effect is still not ideal. Because early diagnosis of lung cancer is difficult and an effective treatment method is lacking, the selection of a lung cancer animal model which is simple and easy to implement, short in molding time and high in molding rate is necessary for deeply discussing the cancerogenic factors and pathogenesis of lung cancer and formulating effective treatment means and prevention measures.
Lewis lung carcinoma has been widely used in a number of important studies. Lewis lung carcinoma cell lines (LLC) were at the earliest adaptive cultured cell clone lines isolated from mouse Lewis lung carcinoma. The C57BL/6 mouse has normal immune function, low price, strong tolerance and mature model, and is commonly used for lung cancer metastasis modeling type construction of Lewis cells. LLC cells maintain high tumorigenicity and pulmonary metastasis in C57BL/6mice, and the LLC cells can express luciferase or green fluorescent protein after being modified by adopting a genetic engineering technology, so that the tumor cells can realize the in-vivo visualization effect (Bertram JS, jan ik P.Estab l i shment of a C loned l ine of Lewi s l ung carcinoma ce l l s adapted to ce l l cu lture [ J ]. Cancer Lett,1980,11 (1): 63-73;Zhu H,et a l.A s imp le bio l uminescence imaging method for studying Cancer ce l l growth and metastas i s after subcutaneous inject ion of Lewi s l ung carcinoma ce l l s in syngeneic C57BL/6mice [ J ]. React Oxyg Species (Apex), 2018,5 (14): 118-125).
With the advent of phage display technology and the development of genetically engineered antibody technology, phage antibody library technology has become a new method for preparing monoclonal antibodies. The majority of phage antibody libraries are immune phage libraries at present, the antibody level in animals is improved after immunization, so that the antibodies aiming at specific antigens can be more conveniently screened, and the subsequent diagnosis and treatment effects are more advantageous (Xu Jing, zhang Lei and the like, construction and expression of B3HM cell immune mouse spleen cell scFv phage display libraries, journal of Chinese bioengineering, 2007, 27 (7): 12-16).
No report on screening specific monoclonal antibodies from LL/2-Luc2 cell immunized animals is currently available. The invention screens out the monoclonal antibody of the specific targeting LL/2-Luc2 cells by utilizing phage display technology, and provides a basis for obtaining the antibody for specifically recognizing tumor cells and further preparing antitumor drugs.
Disclosure of Invention
The invention provides a monoclonal antibody or antigen binding fragment thereof, which specifically targets LL/2-Luc2 lung cancer cells. By using a genetic engineering method, monoclonal antibodies or antigen binding fragments thereof which can specifically bind to LL/2-Luc2 cells are elutriated and identified in a murine immune phage antibody library, so that the aim of effectively and rapidly identifying LL/2-Luc2 lung cancer cells is fulfilled, and ideas and methods are provided for obtaining monoclonal antibodies which specifically target tumor cells.
The following embodiments are provided for the purpose of the present invention.
A monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region CDR having an amino acid sequence selected from the group consisting of SEQ id NOs 1 to 11 or an amino acid sequence having at least 95% identity thereto and a light chain variable region CDR having an amino acid sequence selected from the group consisting of SEQ id NOs 12 to 26 or an amino acid sequence having at least 95% identity thereto.
In a specific embodiment, the heavy chain variable region CDRs are CDR-H1, CDR-H2 and CDR-H3 and the light chain variable region CDRs are CDR-L1, CDR-L2 and CDR-L3 of a monoclonal antibody or antigen binding fragment thereof of the present invention, said monoclonal antibody or antigen binding fragment having an amino acid sequence selected from the group consisting of:
1) CDR-H1 has an amino acid sequence as shown in SEQ ID NO. 1, CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 4 and CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 8 and CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 12, CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 16 and CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 21.
2) CDR-H1 has an amino acid sequence as shown in SEQ ID NO. 1, CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 4 and CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 8 and CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 13, CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 17 and CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 22.
3) CDR-H1 has an amino acid sequence as shown in SEQ ID NO.2, CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 5 and CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 9 and CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 14, CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 18 and CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 23;
4) CDR-H1 has the amino acid sequence shown as SEQ ID NO. 1, CDR-H2 has the amino acid sequence shown as SEQ ID NO. 6 and CDR-H3 has the amino acid sequence shown as SEQ ID NO. 9 and CDR-L1 has the amino acid sequence shown as SEQ ID NO. 14, CDR-L2 has the amino acid sequence shown as SEQ ID NO. 18 and CDR-L3 has the amino acid sequence shown as SEQ ID NO. 24;
5) CDR-H1 has an amino acid sequence as shown in SEQ ID NO. 1, CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 4 and CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 8 and CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 15, CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 19 and CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 25.
6) CDR-H1 has an amino acid sequence as shown in SEQ ID NO. 1, CDR-H2 has an amino acid sequence as shown in SEQ ID NO. 4 and CDR-H3 has an amino acid sequence as shown in SEQ ID NO. 11 and CDR-L1 has an amino acid sequence as shown in SEQ ID NO. 14, CDR-L2 has an amino acid sequence as shown in SEQ ID NO. 20 and CDR-L3 has an amino acid sequence as shown in SEQ ID NO. 26;
7) CDR-H1 has the amino acid sequence shown as SEQ ID NO.3, CDR-H2 has the amino acid sequence shown as SEQ ID NO. 7 and CDR-H3 has the amino acid sequence shown as SEQ ID NO. 10 and CDR-L1 has the amino acid sequence shown as SEQ ID NO. 12, CDR-L2 has the amino acid sequence shown as SEQ ID NO. 16 and CDR-L3 has the amino acid sequence shown as SEQ ID NO. 21.
Preferably, the monoclonal antibody or antigen-binding fragment thereof of the present invention has an amino acid sequence selected from the group consisting of those shown in SEQ ID NOS.27 to 33.
More preferably, the monoclonal antibody or antigen-binding fragment thereof of the present invention has a sequence selected from the group consisting of antibody numbers 501, Y003, Y005, Y008, Y009, Y010, and RA001 in table 1.
The present invention also provides a DNA molecule encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention described above.
The invention also provides an expression vector, which contains the recombinant vector of the monoclonal antibody or the antigen binding fragment thereof, and the expression sequence of the I L-10 signal peptide-monoclonal antibody is cloned to the Not1/Xba1 enzyme multiple cloning site of the pcDNA3.4 vector.
The invention also provides a eukaryotic host cell, which is inserted into the expression vector and transformed.
The eukaryotic host cell described above, which is a CHO cell.
Terminology: CDR-H1, CDR-H2 and CDR-H3 represent heavy chain complementarity determining region 1, heavy chain complementarity determining region 2, heavy chain complementarity determining region 3, respectively; CDR-L1, CDR-L2 and CDR-L3 represent light chain complementarity determining region 1, light chain complementarity determining region 2 and light chain complementarity determining region 3, respectively.
In another embodiment, the invention provides a monoclonal antibody or antigen binding fragment thereof that targets LL/2-Luc2 cells, comprising heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), and light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), light chain complementarity determining region 3 (CDR-L3). The amino acid sequence of the CDR of the heavy chain variable region is shown as SEQ ID NO. 1-11; the amino acid sequence of the CDR of the light chain variable region is shown in SEQ ID NO. 12-26.
Preferably, in another specific embodiment described above, the monoclonal antibody of the invention that targets LL/2-Luc2 cells or antigen binding fragment thereof, the amino acid sequence of the CDRs is selected from the group consisting of:
(1) The amino acid sequence of CDR-H1 is SEQ ID NO 1, the amino acid sequence of CDR-H2 is SEQ ID NO 4, and the amino acid sequence of CDR-H3 is SEQ ID NO 8; the amino acid sequence of CDR-L1 is SEQ ID NO 12, the amino acid sequence of CDR-L2 is SEQ ID NO 16, and the amino acid sequence of CDR-L3 is SEQ ID NO 21;
(2) The amino acid sequence of CDR-H1 is SEQ ID NO 1, the amino acid sequence of CDR-H2 is SEQ ID NO 4, and the amino acid sequence of CDR-H3 is SEQ ID NO 8; the amino acid sequence of CDR-L1 is SEQ ID NO 13, the amino acid sequence of CDR-L2 is SEQ ID NO 17, and the amino acid sequence of CDR-L3 is SEQ ID NO 22;
(3) The amino acid sequence of CDR-H1 is SEQ ID NO 2, the amino acid sequence of CDR-H2 is SEQ ID NO 5, and the amino acid sequence of CDR-H3 is SEQ ID NO 9; the amino acid sequence of CDR-L1 is SEQ ID NO 14, the amino acid sequence of CDR-L2 is SEQ ID NO 18, and the amino acid sequence of CDR-L3 is SEQ ID NO 23;
(4) The amino acid sequence of CDR-H1 is SEQ ID NO 1, the amino acid sequence of CDR-H2 is SEQ ID NO 6, and the amino acid sequence of CDR-H3 is SEQ ID NO 9; the amino acid sequence of CDR-L1 is SEQ ID NO 14, the amino acid sequence of CDR-L2 is SEQ ID NO 18, and the amino acid sequence of CDR-L3 is SEQ ID NO 24;
(5) The amino acid sequence of CDR-H1 is SEQ ID NO 1, the amino acid sequence of CDR-H2 is SEQ ID NO 4, and the amino acid sequence of CDR-H3 is SEQ ID NO 8; the amino acid sequence of CDR-L1 is SEQ ID NO 15, the amino acid sequence of CDR-L2 is SEQ ID NO 19, and the amino acid sequence of CDR-L3 is SEQ ID NO 25;
(6) The amino acid sequence of CDR-H1 is SEQ ID NO 1, the amino acid sequence of CDR-H2 is SEQ ID NO 4, and the amino acid sequence of CDR-H3 is SEQ ID NO 11; the amino acid sequence of CDR-L1 is SEQ ID NO 14, the amino acid sequence of CDR-L2 is SEQ ID NO 20, and the amino acid sequence of CDR-L3 is SEQ ID NO 26;
(7) The amino acid sequence of CDR-H1 is SEQ ID NO 3, the amino acid sequence of CDR-H2 is SEQ ID NO 7, and the amino acid sequence of CDR-H3 is SEQ ID NO 10; the amino acid sequence of CDR-L1 is SEQ ID NO. 12, the amino acid sequence of CDR-L2 is SEQ ID NO. 16, and the amino acid sequence of CDR-L3 is SEQ ID NO. 21.
Further, the monoclonal antibody or antigen binding fragment thereof of the present invention is selected from scFv amino acid sequences as set forth in SEQ id NO:27 to 33.
The present invention provides a method for screening scFv sequences of said monoclonal antibodies or antigen binding fragments thereof, said method comprising the steps of:
firstly, constructing a phage antibody library;
step two, panning of phage antibody library;
thirdly, after the enrichment of the positive cell LL/2-Luc2 is successful, coating the enriched phage bacterial liquid, and selecting positive monoclonal phage to send to a sequencing company for sequencing to obtain heavy chain and light chain variable region sequences of monoclonal antibodies or antigen binding fragments thereof targeting the LL/2-Luc2 cell;
fourth, the heavy chain and light chain variable region sequences obtained are assembled into scFv sequences to express the corresponding antibodies.
Preferably, the constructing of the phage antibody library in the first step comprises:
(1) By using Lewis lung cancer LL/2-Luc2 cells in exponential growth phase, 6C 57 mice with the age of 6-8 weeks are immunized by intraperitoneal injection, and each half of male and female is injected with 1x10 5 Cells were injected once every week, mice were sacrificed after 3 immunizations;
(2) Taking spleens of 6 immunized mice to extract RNA;
(3) Reverse transcription of RNA into cDNA, PCR amplification of VH (heavy chain variable region) and VL (light chain variable region) with the cDNA as template, and splicing to form scFv fragment;
(4) Electrotransformation of scFv fragments into phage and assessment of library quality;
(5) Collecting antibody library phage, and establishing an scFv phage antibody library original library.
The invention also provides a DNA molecule encoding the monoclonal antibody or antigen binding fragment thereof.
The invention provides a recombinant vector containing the monoclonal antibody targeting LL/2-Luc2 cells, wherein the vector comprises the DNA molecule; the expression sequence of the I L-10 signal peptide-monoclonal antibody was cloned into the pcDNA3.4 vector at the Not1/Xba1 enzyme multiple cloning site.
The present invention provides a host cell transformed with the above expression vector. In a preferred embodiment, the host cell is a CHO cell.
The present invention provides a method for preparing the monoclonal antibody of the present invention, comprising:
(1) Providing an expression vector comprising a nucleic acid sequence encoding the monoclonal antibody of the invention described above, and an expression control sequence operably linked to the sequence;
(2) Transforming a host cell with the expression vector of step (1);
(3) Culturing the host cell of step (2) under conditions suitable for expression of said monoclonal antibody;
(4) And separating and purifying to obtain the monoclonal antibody.
The monoclonal antibody is an idiotype antibody and can be specifically combined with LL/2-Luc2 cells. The detection result of the flow cytometry shows that the binding rate of the chimeric antibody containing 501 antibody sequences which are expressed in vitro in a recombination way and LL/2-Luc2 positive cells is up to 35.84 percent, and the binding rate of the chimeric antibody and negative cells is about 3.36 percent; the results of immunocytochemistry experiments show that the chimeric antibody containing the 501 antibody sequence has strong positive binding with LL/2-Luc2 positive cells and almost no binding with negative cells, and all the chimeric antibody has the characteristic of targeting LL/2-Luc2 cells.
By combining the technical scheme, the invention has the advantages and positive effects that: the monoclonal antibody targeting LL/2-Luc2 cells or the antigen binding fragment thereof provides support for constructing the genetic engineering antibody specifically targeting tumor cells. The monoclonal antibody or the antigen binding fragment thereof specifically targets LL/2-Luc2 lung cancer cells and inhibits growth thereof, and thus has antitumor activity.
Drawings
FIG. 1 is a pcDNA3.4 vector and shows the elements and cleavage sites therein;
FIG. 2 shows the expression vector constructed in the present invention, which contains the heavy chain variable region and light chain variable region genes (light region is the target gene) of a monoclonal antibody specifically recognizing LL/2-Luc 2;
FIG. 3 is a diagram of the electrophoresis of protein purification of a chimeric antibody comprising the 501 antibody sequence of the present invention;
FIG. 4 is a high performance liquid chromatogram of a chimeric antibody of the invention comprising a 501 antibody sequence;
FIG. 5 is a graph showing the results of flow cytometry detection of specific binding of a chimeric antibody of the present invention comprising a 501 antibody sequence to LL/2-Luc 2;
FIG. 6 is a graph showing the results of immunocytochemistry detection of specific binding of a chimeric antibody comprising a 501 antibody sequence of the present invention to LL/2-Luc2 (dark color is positive expression);
FIG. 7 shows the growth inhibitory activity of the chimeric antibodies of the invention on LL/2-Luc2 cells.
Detailed Description
The following examples are provided to further illustrate and understand the nature of the present invention, but are not intended to limit the scope of the invention in any way, and any simple modifications and variations that come within the spirit of the invention are within the scope of the invention.
Positive cells: LL/2-Luc2 mouse Lewis lung carcinoma cells, purchased from ATCC,
negative cells: normal lung epithelial cells of mice were purchased from Shanghai Fuxiang biosciences, inc.
EXAMPLE 1 screening of antibody libraries for antibody Gene variable regions targeting LL/2-Luc2 cells
1) Construction of murine antibody libraries
By using Lewis lung cancer LL/2-Luc2 cells in exponential growth phase, 6C 57 mice with the age of 6-8 weeks are immunized by intraperitoneal injection, and each of male and female mice is injected with 1X10 5 Cells were injected once every week, immunized 3 times, tail vein blood was taken on day 3 after last immunization and sacrificed.
Mouse blood was taken to isolate serum and antibody titers were detected by conventional ELISA. Coating of exponential growth phase LL/2-Luc2 cells in 96 well ELISA plates, 1×10 5 Cell/well, PBS buffer as negative control. The primary antibody is serum of mice after immunization, diluted according to corresponding proportion, and the serum of the mice is injected by PBS buffer solution as negative control. The secondary antibody is HRP marked goat anti-mouse IgG antibody, tetramethyl benzidine (TMB) is developed, stop solution is added, and an enzyme-labeled instrument is used for detecting the light absorption value at the wavelength of 450 nm. OD (optical density) 450 The minus blank value > the negative control is positive by 2 times, and the result shows that the average value of the titers of the 6 mouse serum samples is more than 1:10.
Spleen of 6 immunized mice were mixed and spleen lymphocytes were isolated. Taking 2X 10 7 Spleen lymphocytes extract total RNA from the mouse spleen according to the total RNA extraction kit instructions.
Reverse transcription was performed immediately after total RNA extraction using a reverse transcription kit, the PCR reaction procedure was: pre-denaturation at 95℃for 2min followed by denaturation at 94℃for 30s, annealing at 55℃for 45s and extension at 72℃for 1min for 30 cycles. The obtained cDNA can be temporarily stored at 4deg.C, and the cDNA which needs long-term storage can be stored at-20deg.C, so as to avoid repeated freezing and thawing.
Using cDNA as a template, and obtaining a VH/VL fragment through one round of PCR amplification by using high-fidelity enzyme; then taking VL as a template, and obtaining a Linker-VL fragment through a second round of PCR amplification; scFv fragments were obtained by over ap PCR with VH and Linker-VL templates.
And (3) carrying out enzyme digestion and recovery on the purified scFv gene product, connecting the scFv gene product with a carrier fragment subjected to the same enzyme digestion and purification, carrying out electric shock conversion on the connecting product, activating the electric conversion product and collecting antibody library phage.
And counting the stock capacity, randomly picking 10 monoclone from the LB/Carb50 plate for PCR identification, and counting that the GA insertion efficiency is more than 90%.
2) Panning of phage antibody libraries
Antibody phages that specifically bind to LL/2-Luc2 were screened by subtractive panning (negative selection followed by positive selection). First using normal or control cells (3X 10) 6 Individual) and library phage supernatant (titre 2X 10) 10 ) Suspending, incubating at 4deg.C for 2 hr, adsorbing nonspecific antibody, centrifuging to obtain supernatant, and collecting target cell LL/2-Luc2 (5×10) 6 And personal) are combined with phage supernatant, and incubated for 2h at 4 ℃ for affinity screening. Washing with PT buffer solution pre-cooled at 4deg.C for 6-8 times, and infectingalpha5F, helper phage was added overnight for amplification culture. Phage were isolated and purified by PEG8000/NaCl precipitation. 3-4 rounds of phage panning were performed and the final round of phage library was stored in a-80 ℃ refrigerator with 50% glycerol added.
3) Selecting positive monoclonal phage antibody and sequencing to obtain scFv sequence
And (3) coating a plate of the phase bacterial liquid enriched by the LL/2-Luc2 cells with positive monoclonal phage antibodies, and sending the positive monoclonal phage antibodies to a sequencing company for sequencing to obtain heavy chain variable region and light chain variable region sequences of the monoclonal antibodies targeting the LL/2-Luc2 cells, and assembling the heavy chain variable region and the light chain variable region sequences into single-chain antibodies 501, Y003, Y005, Y008, Y009, Y010 and RA001. The scFv amino acid sequence is shown in Table 1.
TABLE 1 scFv amino acid sequence targeting LL/2-Luc2 cells
The amino acid sequences of the CDRs, which are the key fragments of the variable region amino acid sequences of the antibodies, are shown in Table 2.
TABLE 2 CDR amino acid sequences of monoclonal antibodies targeting LL/2-Luc2 cells
The composition of Complementarity Determining Regions (CDRs) of each specific monoclonal antibody or antigen binding fragment thereof targeting LL/2-Luc2 cells is shown in table 3.
TABLE 3 amino acid sequences of complementarity determining regions of specific monoclonal antibodies
EXAMPLE 2 preparation of recombinant vector
Antibody expression was performed using the antibody sequences obtained by screening in the form of scfv+rabbit Fc protein. Finishing the antibody sequence to I L-10 signal peptide-chimeric antibody form, cloning into pcDNA3.4 vector, wherein
Nucleotide sequence of the junction region SEQ id No.34:
GGTGGTGGCGGATCAGGTGGGGGAGGCTCTGGTGGAGGCGGTAGT。
i L-10 nucleotide sequence of signal peptide SEQ ID NO.35:
GCGGCCGCAAACTACAAGACAGACTTGCAAAAGAAGGCATGCACAGCTCAGCACTGCTCTGTTGC CTGGTCCTCCTGACTGGGGTGAGGGCC。
nucleotide sequence of Fc fragment SEQ id No.36:
GCCCCCTCCACCGCTTCCAAGCCCACCTGCCCCCCCCCTGAGCTGCTGGGAGGACCTTCCGTGTTCATCTTCCCCCCCAAACCCAAAGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGACGACCCCGAGGTGCAGTTTACATGGTACATTAACAACGAGCAGGTGAGAACCGCCAGACCCCCCCTGCGCGAGCAGCAGTTCAACAGCACCATCAGAGTGGTGAGCACCCTGCCCATCGCCCACCAGGATTGGCTGAGAGGCAAGGAATTCAAGTGCAAAGTGCACAACAAGGCCCTGCCCGCCCCCATTGAGAAAACCATCAGCAAAGCCAGAGGCCAGCCCCTGGAACCCAAAGTGTACACCATGGGGCCCCCCAGAGA AGAACTGAGCAGCAGAAGCGTGAGCCTGACCTGCATGATCAACGGCTTCTACCCCAGCGACATCAGCGTGGAATGGGAAAAAAACGGCAAGGCCGAGGACAACTACAAGACCACCCCCGCCGTGCTGGACAGCGACGGCAGCTACTTCCTGTACAGCAAGCTGAGCGTGCCCACCAGCGAGTGGCAGAGAGGCGACGTGTTCACCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCATCAGCAGAAGCCCCGGCAAGTGATTCTAGA。
the integrated sequence is delivered to Shanghai Baiying Biotechnology Co Ltd for complete sequence synthesis, cloned to pcDNA3.4 plasmid (shown in figure 1), and entrusted Baiying organism is constructed by an upstream NotI cleavage site and a downstream XbaI cleavage site to obtain a recombinant vector (shown in figure 2). FIG. 2 is a schematic representation of an expression vector constructed to contain the heavy and light chain variable region genes of a monoclonal antibody specifically recognizing LL/2-Luc 2.
EXAMPLE 3 expression and purification of antibodies
The constructed expression Vector pcDNA3.4-vector+scFv+Fc transformed E.coli DH 5. Alpha. Strain with chimeric antibody gene was inoculated into 100mL of LB medium for amplification, and the plasmid DNA was purified by extraction with an ultrapure plasmid DNA purification kit (U ltrapurePl asmid DNA Pur ificat ion Kit). The above purified plasmid DNA was co-transfected into CHO cells using the Liposome kit (l ipofectamine 2000transfect ion reagent,11668-027) from Invitrogen, and the procedure was carried out according to the manufacturer's instructions.
Transformed CHO cells were selected on MTX selection medium (s igma a-6770) for 2 weeks in succession, and finally subjected to limiting dilution culture on 96-well plates, 3 times in succession, and subjected to monoclonalization.
The selected monoclonal cell line is cultured on RPMI 1640 medium (I nvitrogen), the chimeric antibody is directly separated and purified from cell culture supernatant by using a Protein A affinity chromatography column, and the purified Protein collection is taken for SDA-PAGE electrophoresis identification. As can be seen from FIG. 3, the chimeric antibody protein containing the 501 antibody sequence was screened for significant expression on CHO cells.
The affinity chromatography product is subjected to molecular sieve chromatography again, and the purity results of the chimeric antibodies after 7 expression and purification are shown in Table 4 through HPLC identification. As shown in fig. 4, chimeric antibodies containing 501 antibody sequences gave samples with purity > 98%.
TABLE 4 chimeric antibody purity targeting LL/2-Luc2 cells
EXAMPLE 4 flow cytometry detection of chimeric antibodies
The cultured positive and negative cells are digested by pancreatin and then blown fully to form single cell suspension, and the positive cells and the negative cells are respectively taken 1X10 6 And 250g of the culture medium is discarded after centrifugation for 5min, the cells are washed 3 times by precooled PBS, and 250g of the culture medium is centrifuged for 5min, so that cell precipitates are collected for standby. 100 mu L PBS was used to resuspend the cells, 5uL chimeric antibody was added and mixed, and incubated at 4℃for 30min in the absence of light; centrifugation at 350g for 5min, the supernatant was discarded. The cells were washed 3 times with pre-chilled PBS to remove unbound antibody, and positive and negative cell-antibody complexes were obtained, respectively.
And (3) uniformly mixing the cell-antibody complex with fluorescence-labeled secondary-antibody donkey anti-rabbit IgG respectively, incubating for 30min at 4 ℃ in a dark place, pre-cooling PBS (phosphate buffer solution) for washing the cells for 3 times, and removing unbound fluorescence secondary-antibody donkey anti-rabbit IgG to obtain positive and negative cell-antibody-secondary-antibody complexes respectively.
Positive and negative cells were used as blank control in combination with secondary antibody alone, respectively.
The cell-antibody-secondary antibody complex and the cell-secondary antibody complex were resuspended in 300uL PBS and detected using a flow cytometer. The streaming results were analyzed by the software cytpert.
The mouse rabbit chimeric antibody specifically recognizes LL/2-Luc2 cells, and then recognizes the chimeric antibody with a fluorescence-labeled donkey anti-rabbit secondary antibody, whereby cells with secondary antibody fluorescence development are considered positive cells capable of binding to the chimeric antibody. The flow results are shown in FIG. 5 and Table 5, and FIG. 5 shows the results of flow cytometry detection of chimeric antibodies containing 501 antibody sequences specifically binding to LL/2-Luc2 cells. The binding rate of the chimeric antibody containing the 501 antibody sequence to LL/2-Luc2 positive cells reaches 35.84%, and the binding rate to negative cells reaches about 3.36%, so that the chimeric antibody can be verified to specifically bind to LL/2-Luc2 cells.
TABLE 5 Positive binding Rate of chimeric antibodies targeting LL/2-Luc2 cells
EXAMPLE 5 immunocytochemical detection of chimeric antibodies
(1) Preparation of cell climbing tablet:
taking positive cells and negative cells in an exponential growth phase respectively, fully blowing the positive cells and the negative cells into single cell suspension after pancreatin digestion, centrifuging at 1000rpm for 5 minutes, sucking the supernatant, and adding a proper amount of complete culture medium for suspension counting. Regulating cell density to 1-2 x10 5 And each mL.
Taking out the sterile 6-hole plate, firstly preparing a position for placing the climbing plates in each hole to drop a small amount of culture medium according to the size of the slide, and then placing the slide to prevent the cell suspension Shi Bopian from floating up and causing double-layer cell patches. The adjusted cell suspensions were inoculated into 6-well plates (cell suspensions were dropped drop by drop onto a slide) at a cell density of about 2X 10 in 2 mL/well 5 Individual/holes, cappedThen placed at 37 ℃ and 5 percent CO 2 Culturing at constant temperature until the cells are completely adhered.
(2) Immunocytochemistry detection:
(1) fixing: taking out the pore plate, sucking the culture medium, gently washing the cells with PBS for 2 times, and fixing with 4% paraformaldehyde for 30min;
(2) closing: absorbing the fixing solution, washing for 2 times by PBS, and dripping goat serum sealing solution 5min each time, wherein the room temperature is 20min;
(3) an antibody: dropwise adding chimeric antibody solution at 4 ℃ overnight;
(4) and (2) secondary antibody: dripping biotin-marked goat anti-rabbit IgG at 37 ℃ for 30min; washing with PBS for 3 times, each time for 5min;
(5) dripping horseradish enzyme marked streptavidin working solution at 37 ℃ for 30min; washing with PBS for 3 times, each time for 5min;
(6) DAB color development: using DAB color development kit, mixing the reagents, dripping onto cell climbing slices, developing color at room temperature, controlling reaction time under a mirror, generally about 2min, and washing with distilled water;
(7) hematoxylin is lightly counterstained, dehydrated, transparent and neutral resin sealing piece.
(8) And acquiring images of the sections by adopting a microscopic imaging system of a BA200Digita l digital three-eye imaging microscopic imaging system manufactured by Miao industry group Co.
Positive cells and negative cells were used as blank control groups, respectively, to which only secondary antibodies were bound.
(3) Results
As shown in FIG. 6, the result of immunocytochemistry detection of the chimeric antibody containing 501 antibody sequence specifically binding to LL/2-Luc2 shows that LL/2-Luc2 cell climbing sheet has strong positive color development, while normal lung epithelial cell climbing sheet has no obvious color development, which indicates that the chimeric antibody containing 501 antibody sequence can specifically bind to LL/2-Luc2 cells.
EXAMPLE 6 growth inhibitory Activity of chimeric antibodies
The growth inhibition of the chimeric antibodies against LL/2-Luc2 cells was detected using the Ce l Titer-G lo luminescence cell viability assay kit (Promega, cat#: G7570). Digestion and collection of LL/2-Luc2 cells in exponential growth phase, 250g for 5 minutes and then resuspended in complete medium. Cell density was adjusted to 1X10 5 mu.L of the cell suspension was added to each well of a 96-well plate and incubated overnight at 37 ℃. The antibodies were diluted with medium to varying concentrations, 50 μl of antibody dilution was added to each well of a 96-well plate, and incubated for 3 days at 37 ℃. The 96-well plate was allowed to stand at room temperature for 30 minutes, and 100. Mu.L/well of room temperature Ce L Titer-Glo color development liquid was added. The samples were then incubated at room temperature for 10 minutes in the dark. The plate was read with PE Ensci re. Growth inhibitory Activity (%) = [1- (l uminescent samp le)/(l uminescent mock contro l)]X 100.Rabbit IgG I sotype Contro l (Thermo Fi sher Scient ific, cat#: 02-6102) as a negative control. As shown in FIG. 7, chimeric antibodies containing the 501, Y009, and RA001 antibody sequences were able to exert biological inhibitory activity on LL/2-Luc2 cells in a dose-dependent manner. The maximum growth inhibition of the antibodies against LL/2-Luc2 cells is shown in Table 6.
Table 6 maximum growth inhibitory Activity of chimeric antibodies against LL/2-Luc2 cells
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (7)
1. A targeting LL/2-Luc2 monoclonal antibody or antigen binding fragment thereof, the antibody or antigen binding fragment thereof comprising a heavy chain variable region CDR and a light chain variable region CDR, the heavy chain variable region CDR is CDR-H1, CDR-H2 and CDR-H3, the light chain variable region CDR is CDR-L1, CDR-L2 and CDR-L3, characterized in that,
the CDR-H1-CDR-H3 of the heavy chain variable region is: an amino acid sequence shown in SEQ ID NO. 1 of CDR-H1, an amino acid sequence shown in SEQ ID NO. 4 of CDR-H2 and an amino acid sequence shown in SEQ ID NO. 8 of CDR-H3;
the CDR-L1-CDR-L3 of the light chain variable region is:
1) An amino acid sequence shown in SEQ ID NO. 12 for CDR-L1, an amino acid sequence shown in SEQ ID NO. 16 for CDR-L2 and an amino acid sequence shown in SEQ ID NO. 21 for CDR-L3; or (b)
2) An amino acid sequence shown in SEQ ID NO. 13 for CDR-L1, an amino acid sequence shown in SEQ ID NO. 17 for CDR-L2 and an amino acid sequence shown in SEQ ID NO. 22 for CDR-L3; or (b)
3) The amino acid sequence of CDR-L1 is shown as SEQ ID NO. 15, the amino acid sequence of CDR-L2 is shown as SEQ ID NO. 19 and the amino acid sequence of CDR-L3 is shown as SEQ ID NO. 25.
2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 27. 28, 31.
3. A DNA molecule encoding the monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2.
4. An expression vector comprising the DNA molecule of claim 3, wherein the expression sequence of the IL-10 signal peptide-monoclonal antibody or antigen-binding fragment thereof is cloned into the pcdna3.4 vector at the Not1/Xba1 enzyme multiple cloning site.
5. A eukaryotic host cell containing the expression vector of claim 4.
6. The eukaryotic host cell of claim 5, which is a CHO cell.
7. Use of the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 for the manufacture of an anti-tumor medicament, wherein the tumor is lung cancer.
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CN108602888A (en) * | 2015-10-23 | 2018-09-28 | 美勒斯公司 | Inhibit the binding molecule of growth of cancers |
CN110267982A (en) * | 2016-10-19 | 2019-09-20 | 斯克利普斯研究所 | With humanization targeting moiety and/or by optimization Chimeric antigen receptor interaction domain Chimeric antigen receptor effector cell switch with and application thereof |
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In vivo visualization of GL261-luc2 mouse glioma cells by use of Alexa Fluor-labeled TRP-2 antibodies;Kathryn E Fenton等;《Neurosurgical Focus》;第36卷(第02期);E12 * |
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