DLL1 antibody and application thereof

Abstract

The invention discloses a DLL1 antibody which comprises three different scFv sequences, and each scFv sequence correspondingly comprises one of a 3P53 sequence, a 3P47 sequence or a 4P108 sequence; 3P53-IgG, 3P47-IgG and 4P108-IgG antibodies having high affinity with human and mouse DLL1 are constructed by using VH and VL sequences derived from 3P53, 3P47 and 4P108 sequences correspondingly contained in different scFv sequences, and the antibodies can recognize human and mouse DLL1 protein molecules, DLL1 expressed by cell membranes and DLL1 expressed by tissues, can be used for ELISA, WB, IHC and other detection methods, and can be used for detecting human and mouse DLL1 protein molecules, cell membrane expression DLL1 and tissue expression DLL1. Therefore, the DLL1 has a better application prospect in the aspect of detecting molecule, cell and tissue levels.

Description

Technical Field

[0001] The present invention relates to the field of antibody biology, and in particular to a DLL1 antibody and application thereof. Background Art

[0002] DLL1 is an antibody protein found in mammals and is one of the important ligands of the Notch signaling pathway. The Notch signaling pathway plays a key role in cell differentiation, proliferation and growth, and plays a role in a variety of biological processes such as embryonic development, tissue homeostasis and disease progression. Notch signaling is associated with the development of cancer and neurological diseases, so detecting the presence and expression level of DLL1 is particularly important in cancer research and neurobiology research.

[0003] Phage display technology is a technology used to screen and identify proteins, peptides, antibodies and other biological molecules. This technology uses bacteriophages as carriers to insert the genes of target molecules into the phage genome so that these molecules can be displayed on the surface of the phage. This display method has a wide range of applications in biology and biotechnology. Phage display is often used to screen antibodies or antibody fragments to develop high-affinity antibodies against specific antigens.

[0004] In biological research, antibodies are the most commonly used detection tools and are widely used in experimental techniques such as immunohistochemistry, immunoblotting and immunofluorescence. To study diseases related to Notch signaling, it is necessary to develop DLL1 antibodies to detect and label human DLL1 protein. In addition, mice are a commonly used animal model, and how to detect mouse DLL1 protein is also a problem that needs to be solved in in vivo animal experiments.

[0005] Therefore, it is necessary to develop an antibody that can specifically bind to human and mouse DLL1 protein in order to conduct relevant research in cancer and neurobiology. Summary of the invention

[0006] Based on the above problems, the problem to be solved by the present invention is to provide an antibody that can specifically bind to human and mouse DLL1 proteins and its application; by detecting the presence and expression level of DLL1 antibodies, relevant biological research can be carried out.

[0007] The technical solution of the present invention is as follows:

[0008] A DLL1 antibody comprises three different scFv sequences, each scFv sequence comprises one of 3P53, 3P47 and 4P108 sequences.

[0009] In one embodiment, in the above-mentioned DLL1 antibody, when the scFv sequence comprises a 3P53 sequence, the amino acid sequence of the heavy chain variable region of the scFv sequence is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the scFv sequence is as shown in SEQ ID NO: 12; accordingly, the 3P53 sequence comprises three heavy chain complementary determining regions and three light chain complementary determining regions; the three heavy chain complementary determining regions are HCDR1, HCDR2, and HCDR3, respectively, and the three light chain complementary determining regions are LCDR1, LCDR2, and LCDR3, respectively; wherein:

[0010] When the 3P53 sequence is chothia, the amino acid sequence of HCDR1 is GGTFSSYA, the amino acid sequence of HCDR2 is IIPILGIA, the amino acid sequence of HCDR3 is ARDDIAVAGNFDY, the amino acid sequence of LCDR1 is QSISTL, the amino acid sequence of LCDR2 is AAS, and the amino acid sequence of LCDR3 is QQSYSTHSIT; or

[0011] When the 3P53 sequence is Kabat, the amino acid sequence of HCDR1 is SYAIS, the amino acid sequence of HCDR2 is RIIPILGIANYAQEFQG, the amino acid sequence of HCDR3 is DDIAVAGNFDY, the amino acid sequence of LCDR1 is RTSQSISTLLN, the amino acid sequence of LCDR2 is AASTLQS, and the amino acid sequence of LCDR1 is QQSYSTHSIT.

[0012] In one embodiment, in the above-mentioned DLL1 antibody, when the scFv sequence comprises the 3P53 sequence, the amino acid sequence of the heavy chain variable region of the scFv sequence is as shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region of the scFv sequence is as shown in SEQ ID NO: 15; accordingly, the 3P47 sequence comprises three heavy chain complementary determining regions and three light chain complementary determining regions; the three heavy chain complementary determining regions are HCDR1, HCDR2, and HCDR3, respectively; the three light chain complementary determining regions are LCDR1, LCDR2, and LCDR3, respectively; wherein:

[0013] When the 3P47 sequence is chothia, the amino acid sequence of HCDR1 is GYTFTSY, the amino acid sequence of HCDR2 is NPSGGS, the amino acid sequence of HCDR3 is DSSSWYGDYYYGMDV, the amino acid sequence of LCDR1 is RASQGIGDHLA, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR3 is QQYNNYPLT; or

[0014] When the 3P47 sequence is Kabat, the amino acid sequence of HCDR1 is SYYMH, the amino acid sequence of HCDR2 is IINPSGGSTSYAQKFQG, the amino acid sequence of HCDR3 is DSSSWYGDYYYGMDV, the amino acid sequence of LCDR1 is RASQGIGDHLA, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR1 is QQYNNYPLT.

[0015] In one embodiment, in the above-mentioned DLL1 antibody, when the scFv sequence comprises the 3P53 sequence, the amino acid sequence of the heavy chain variable region of the scFv sequence is as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the scFv sequence is as shown in SEQ ID NO: 18; accordingly, the 4P108 sequence comprises three heavy chain complementary determining regions and three light chain complementary determining regions; the three heavy chain complementary determining regions are HCDR1, HCDR2, and HCDR3, respectively, and the three light chain complementary determining regions are LCDR1, LCDR2, and LCDR3, respectively; wherein:

[0016] When 4P108 is chothia, the amino acid sequence of HCDR1 is GDTFSSY, the amino acid sequence of HCDR2 is IPILGI, the amino acid sequence of HCDR3 is DDIAVAGSFDY, the amino acid sequence of LCDR1 is RASQSISRYLN, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR3 is QQSYSAQYT; or

[0017] When the 4P108 sequence is Kabat, the amino acid sequence of HCDR1 is SYAIS, the amino acid sequence of HCDR2 is RIIPILGIANYAQEFQG, the amino acid sequence of HCDR3 is DDIAVAGSFDY, the amino acid sequence of LCDR1 is RASQSISRYLN, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR1 is QQSYSAQYT.

[0018] The above-mentioned DLL1 antibody can be used to prepare IgG antibody or Fab antibody, and the IgG antibody or Fab antibody contains the heavy chain variable region amino acid (VH) sequence and light chain variable region amino acid (VL) sequence of DLL1; in this way, the VH and VL sequences of DLL1 become part of the IgG antibody or Fab antibody; wherein, IgG antibodies include IgG1, IgG2, IgG3 and IgG4 antibodies.

[0019] The present invention also provides a detection reagent for detecting tumor cells or cancer cells, wherein the detection reagent comprises the above-mentioned DLL1 antibody, IgG antibody or Fab antibody.

[0020] The DLL1 antibody provided by the present invention comprises three different scFv sequences, and the VH and VL sequences derived from the 3P53, 3P47 and 4P108 sequences corresponding to the different scFv sequences are used to construct 3P53-IgG, 3P47-IgG and 4P108-IgG antibodies with high affinity to human and mouse DLL1. These antibodies can recognize human and mouse DLL1 protein molecules, DLL1 expressed on cell membranes and DLL1 expressed on tissues, and can be used in detection methods such as ELISA, WB, IHC, etc., so that DLL1 has good application prospects in the detection of molecules, cells and tissue levels; at the same time, the three different scFv sequences can respectively and simultaneously bind to DLL1 antigens derived from humans and mice, and have the prospect of being used for the detection and functional evaluation of animal models of human DLL1-related diseases without species modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the SDS-PAGE electrophoresis analysis diagram corresponding to 3P53-IgG1 in Example 2;

[0022] Figure 2 This is the affinity curve of 3P53-IgG4 to human and mouse DLL1 detected by ELSIA in Example 5;

[0023] Figure 3 The WB photograph of the gel of 4P108-IgG2 detecting human and mouse DLL1 in Example 6;

[0024] Figure 4 This is a flow cytometric analysis diagram of 3P47-IgG1 detecting DLL1 overexpressing cell lines in Example 7;

[0025] Figure 5 This is a flow cytometric analysis of the expression of DLL1 on EO771 cells detected by 3P47-IgG3 in Example 8;

[0026] Figure 6 This is a microscope photograph of 3P53-IgG1 immunohistochemical detection of DLL1 expression in mouse spleen tissue in Example 9. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0028] The DLL1 antibody provided by the present invention has the following construction and screening schemes.

[0029] 1. Construction and screening of mouse scFv phage display library

[0030] 1. Construction of mouse scFv phage display library

[0031] Mice were immunized with recombinant human DLL1 protein (commercially purchased from Shanghai Jinan Technology Co., Ltd.) in multiple rounds. After each immunization, blood was collected from mice for ELISA to detect the titer of immune serum. ELISA was performed using a standard protocol.

[0032] The specific process steps for constructing the scFv phage display library are as follows:

[0033] S11, coating each well of a Corning EIA / RIA (commercially purchased from Corning) 96-well plate with 100 ng of recombinant human DLL1 protein, incubating at 4°C for 12 hours, and blocking each well with PBS (pH 7.4) containing 3% skim milk;

[0034] S12, after incubation for 12 hours, add the mouse immune serum to be tested to each well of the well plate, and the mouse immune serum sample is diluted in multiples with PBS and incubated at 37°C for 1 hour;

[0035] S13. After the incubation of the mouse immune serum sample is completed, the incubation culture waste liquid in each well is removed, and each well on the plate is washed with PBS containing 1‰ Tween-20 (PBST), and the washing waste liquid is discarded to retain the antibody bound to the well wall;

[0036] S14, the bound antibodies were cross-adsorbed and detected by Goat Anti-Mouse IgG H&L (HRP) (commercially purchased from abcam); during the detection process, the assay was developed with TMB (commercially purchased from Beijing Solebow Technology Co., Ltd.) substrate at room temperature and measured at 450nm using an ELISA reader; when the serum titer reached the immune response level against the immunogen, another impulse immunization was performed to complete the immunization step and the detection was terminated;

[0037] S15, taking blood from the mice immunized in step S11, separating PBMC, extracting mRNA using Trizol and chloroform, and reversely transcribing it into cDNA;

[0038] S16. Using cDNA as a template, specific primers were used to amplify the mouse antibody light chain VL and heavy chain VH sequences respectively; the process is as follows:

[0039] S161, VL and VH were amplified by overlapping PCR to form a scFv gene fragment VH-(G4S)3-VL connected with a (G4S)3 linker, and NcoI (purchased from Bao Ri Yi Biotechnology (Beijing) Co., Ltd.) and NheI (purchased from Bao Ri Yi Biotechnology (Beijing) Co., Ltd.) restriction sites were introduced at both ends;

[0040] S162, the scFv gene fragment was double-digested with NcoI and NheI, and ligated into the pHEN1 vector (purchased from Wuhan Huaerna Biotechnology Co., Ltd.) using T4 ligase (purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.) to construct a pHEN1-scFv vector;

[0041] S163, transforming TG1 competent cells with pHEN1-scFv to prepare a scFv library;

[0042] S164. Use helper phage M13KO7 (commercially purchased from Chengdu Gugel Biotechnology Co., Ltd.) to infect the scFv library and package the library into a scFv phage display library.

[0043] 2. Screening of mouse scFv phage display library

[0044] Recombinant human and mouse DLL1 were used as antigens, DLL1 was used to coat ELISA plates, and the mouse scFv phage display library was selected using solid phase screening.

[0045] The specific process steps for screening the scFv phage display library are as follows:

[0046] S21, recombinant human and mouse DLL1 proteins were coated on Corning EIA / RIA high-binding 96-well plates and incubated at 4°C for 12 h, after which each well was blocked alternately with PBS (pH 7.4) containing 3% skim milk and 5% BSA in different screening rounds;

[0047] S22, after incubation for 12 hours, add the mouse scFv phage display library obtained in step S16 of this embodiment to each well and incubate for 1 hour; then wash the well plate with PBST;

[0048] S23. Elute the scFv phage display library with triethylamine eluent at a volume of 100 μL / well; collect the washing solution after washing, add an equal volume of Tris-HCl, mix well, and obtain the eluted scFv phage display library.

[0049] 3. Amplification and collection of scFv phage display library

[0050] S31, after infecting TG1 bacteria in the logarithmic growth phase with the eluted scFv phage display library obtained in step S23, add M13KO7, place at 37°C for 30 minutes, and then culture on a shaker at 37°C and 180rpm for 1 hour; then transfer to a centrifuge tube, centrifuge at room temperature and 5000rpm for 10 minutes, remove the supernatant, and collect the bacterial precipitate; resuspend the bacterial precipitate with a culture medium containing ampicillin and kanamycin, and culture the resuspension on a shaker at 30°C and 220rpm for 12 hours;

[0051] S32, transfer the culture into a 50mL centrifuge tube, centrifuge at 4°C and 12000rpm; collect the supernatant and add a PEG / NaCl mixture, and the volume ratio of the supernatant to the mixture is 1:4; place the centrifuge tube in an ice bath for 4 hours; then transfer to another centrifuge tube, centrifuge at 4°C and 12000rpm for 20 minutes, discard the supernatant, collect the precipitate and resuspend it with 1mL PBS, centrifuge the resuspended solution at 4°C and 12000rpm for 10 minutes, collect the supernatant and transfer it to a new 1.5mL centrifuge tube, screen and obtain the scFv phage display library; extract the display library samples for titer determination;

[0052] S33, titer determination:

[0053] S331. Day 1: Dilute 10 μL of the scFv phage display library obtained in step S32 to 10 μL with culture medium in a 10-fold gradient. -4 ;

[0054] S332, taking each gradient dilution of the scFv phage display library and inoculating it into 200 μl of a freshly cultured TG1 bacterial solution with an OD600 of 0.4-0.5, and incubating at 37° C. for 30 minutes to obtain a scFv phage display library slurry;

[0055] S333, spreading the slurry on a plate containing ampicillin resistance to obtain colonies;

[0056] S334. On the second day, the colonies on the plate were counted and the titer of the scFv phage display library was calculated; the calculation formula was as follows:

[0057] S335, scFv phage display library titer = number of phage monoclones × dilution (pfu / 10uL).

[0058] 4. ELISA verification and sequencing of phage monoclones

[0059] S41, according to the titer determination result in step S33, determine whether to screen the amount of the scFv phage display library again, and whether to repeat the above steps 1 to 3 for 3 to 5 times until the desired amplified scFv phage display library is obtained;

[0060] S42, after the screening is completed, the amplified scFv phage display library is coated on a plate containing ampicillin resistance, and a phage monoclonal clone is taken for ELISA detection to verify the binding activity;

[0061] S43. In step S42, select the phage monoclones with the highest binding activity (preferably 20) for sequencing, and select different sequences in the sequencing results as candidate DLL1 antibody sequences, such as three different scFv sequences in the DLL1 antibody sequence, and one of the 3P53, 3P47 and 4P108 sequences contained in each scFv sequence.

[0062] The sequencing results were translated into the protein amino acid sequence as follows:

[0063] Group 1, DLL1 antibody contains 3P53 sequence, and the amino acid sequence of scFv is shown in SEQ ID NO: 10:

[0064] EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSTSLRSEDTAVYYCARDDIAVAGNFDYWGQGTTVTVSSGG GGSGGGGSGGGGSAIRMTQSPSSSLSALVGDRVTITCRTSQSISTLLNWYQQKPGRAPKLLIYAASTLQSGVPSRFSGTGSGTEFTLTISSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK;

[0065] in,

[0066] a1. The amino acid sequence of the heavy chain variable region (VH) of the scFv sequence is shown in SEQ ID NO: 11:

[0067] EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSTSLRSEDTAVYYCARDDIAVAGNFDYWGQGTTVTVSS;

[0068] a2. The amino acid sequence of the light chain variable region (VL) of the scFv sequence is shown in SEQ ID NO: 12:

[0069] AIRMTQSPSSSLSALVGDRVTITCRTSQSISTLLNWYQQKPGRAPKLLIYAASTLQSGVPSRFSGTGSGTEFTLTISSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK;

[0070] Accordingly, the three heavy chain complementary determining regions (HCDR1, HCDR2, HCDR3) and three light chain complementary determining regions (LCDR1, LCDR2, LCDR3) contained in the 3P53 sequence; wherein:

[0071] If 3P53 is chothia, then:

[0072] The amino acid sequence of HCDR1 is GGTFSSYA;

[0073] The amino acid sequence of HCDR2 is IIPILGIA;

[0074] The amino acid sequence of HCDR3 is ARDDIAVAGNFDY;

[0075] The amino acid sequence of LCDR1 is QSISTL;

[0076] The amino acid sequence of LCDR2 is AAS;

[0077] The amino acid sequence of LCDR3 is QQSYSTHSIT;

[0078] or

[0079] If 3P53 is Kabat, then:

[0080] The amino acid sequence of HCDR1 is SYAIS;

[0081] The amino acid sequence of HCDR2 is RIIPILGIANYAQEFQG;

[0082] The amino acid sequence of HCDR3 is DDIAVAGNFDY;

[0083] The amino acid sequence of LCDR1 is RTSQSISTLLN;

[0084] The amino acid sequence of LCDR2 is AASTLQS;

[0085] The amino acid sequence of LCDR1 is QQSYSTHSIT.

[0086] Group 2, DLL1 antibody contains 3P47 sequence, and the amino acid sequence of scFv is shown in SEQ ID NO: 13:

[0087] EVQLVQSGAEVKEPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSSWYGDYYYGMDVWGQGTLVTVS SGGGGSGGGGSGGGGSEIVLTQSPSSSLSASVGDRVTISCRASQGIGDHLAWFQQKPGKAPKSLISAASSLQSGVPSKFSGSGSGTDFTLTISNLQPEDSATYFCQQYNNYPLTFGGGTKLEIK;

[0088] in,

[0089] b1. The amino acid sequence of the heavy chain variable region (VH) of the scFv sequence is shown in SEQ ID NO: 14:

[0090] EVQLVQSGAEVKEPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSSWYGDYYYGMDVWGQGTLVTVSS;

[0091] b2. The amino acid sequence of the light chain variable region (VL) of the scFv sequence is shown in SEQ ID NO: 15:

[0092] EIVLTQSPSSSLSASVGDRVTISCRASQGIGDHLAWFQQKPGKAPKSLISAASSLQSGVPSKFSGSGSGTDFTLTISNLQPEDSATYFCQQYNNYPLTFGGGTKLEIK;

[0093] Accordingly, the three heavy chain complementary determining regions (HCDR1, HCDR2, HCDR3) and three light chain complementary determining regions (LCDR1, LCDR2, LCDR3) contained in the 3P47 sequence; wherein:

[0094] If 3P47 is chothia, then:

[0095] The amino acid sequence of HCDR1 is GYTFTSY;

[0096] The amino acid sequence of HCDR2 is NPSGGS;

[0097] The amino acid sequence of HCDR3 is DSSSWYGDYYYGMDV;

[0098] The amino acid sequence of LCDR1 is RASQGIGDHLA;

[0099] The amino acid sequence of LCDR2 is AASSLQS;

[0100] The amino acid sequence of LCDR3 is QQYNNYPLT;

[0101] or

[0102] If 3P47 is Kabat, then:

[0103] The amino acid sequence of HCDR1 is SYYMH;

[0104] The amino acid sequence of HCDR2 is IINPSGGSTSYAQKFQG;

[0105] The amino acid sequence of HCDR3 is DSSSWYGDYYYGMDV;

[0106] The amino acid sequence of LCDR1 is RASQGIGDHLA;

[0107] The amino acid sequence of LCDR2 is AASSLQS;

[0108] The amino acid sequence of LCDR1 is QQYNNYPLT.

[0109] Group 3, DLL1 antibody contains 4P108 sequence, and the amino acid sequence of scFv is shown in SEQ ID NO: 16:

[0110] EVQLVETGAEVKKPGSSVKVSCKASGDTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSTSLRSEDTAVYYCARDDIAVAGSFDYWGQGTLVTVSSG GGGSGGGGSGGGGSDIQLTQSPSSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLLYAASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSYSAQYTFGQGTKLEIK;

[0111] in,

[0112] The amino acid sequence of the heavy chain variable region (VH) of c1 and scFv sequence is shown in SEQ ID NO: 17:

[0113] EVQLVETGAEVKKPGSSVKVSCKASGDTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSTSLRSEDTAVYYCARDDIAVAGSFDYWGQGTLVTVSS;

[0114] The amino acid sequence of the light chain variable region (VL) of c2.scFv sequence is shown in SEQ ID NO:18:

[0115] DIQLTQSPSSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLLYAASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSYSAQYTFGQGTKLEIK;

[0116] Accordingly, the three heavy chain complementary determining regions (HCDR1, HCDR2, HCDR3) and three light chain complementary determining regions (LCDR1, LCDR2, LCDR3) contained in the 4P108 sequence; wherein:

[0117] If 4P108 is chothia, then:

[0118] The amino acid sequence of HCDR1 is GDTFSSY;

[0119] The amino acid sequence of HCDR2 is IPILGI;

[0120] The amino acid sequence of HCDR3 is DDIAVAGSFDY

[0121] The amino acid sequence of LCDR1 is RASQSISRYLN;

[0122] The amino acid sequence of LCDR2 is AASSLQS;

[0123] The amino acid sequence of LCDR3 is QQSYSAQYT;

[0124] or

[0125] If 4P108 is Kabat, then:

[0126] The amino acid sequence of HCDR1 is SYAIS;

[0127] The amino acid sequence of HCDR2 is RIIPILGIANYAQEFQG;

[0128] The amino acid sequence of HCDR3 is DDIAVAGSFDY;

[0129] The amino acid sequence of LCDR1 is RASQSISRYLN;

[0130] The amino acid sequence of LCDR2 is AASSLQS;

[0131] The amino acid sequence of LCDR1 is QQSYSAQYT.

[0132] 2. Preparation of IgG antibody-DLL1 antibody and Fab antibody-DLL1 antibody

[0133] First, take the candidate DLL1 antibody heavy chain VH and light chain VL sequences selected in the first item above;

[0134] Secondly, the VH region of the IgG antibody or Fab antibody is replaced with the VH sequence of the DLL1 antibody; the VL region of the kappa light chain of the IgG antibody or Fab antibody is replaced with the VL sequence of the DLL1 antibody;

[0135] Next, the replaced kappa light chain VL and heavy chain VH were constructed into the expression vector pcDNA3.1 to obtain DLL1-IgG antibody and DLL1-Fab antibody;

[0136] Finally, HEK293 cells were transiently transfected, and DLL1-IgG and DLL1-Fab antibodies were purified using a Protein A column.

[0137] The three different scFv sequences in the DLL1 antibody correspond to 3P53-IgG, 3P47-IgG and 4P108-IgG constructed from VH and VL sequences derived from 3P53, 3P47 and 4P108 sequences. They have high affinity with human and mouse DLL1 sequences, can recognize human and mouse DLL1 protein molecules, DLL1 expressed on cell membranes and DLL1 expressed in tissues, and can be used in ELISA, WB, IHC and other detection methods, making DLL1 have good application prospects in detecting molecular, cellular and tissue levels.

[0138] Therefore, the IgG antibody-DLL1 antibody and Fab antibody-DLL1 antibody prepared above can be used to prepare detection reagents for detecting solid tumors such as tumors or cancers, or to prepare drugs for treating tumors, cancers, etc.

[0139] Solid tumors mainly include brain gliomas, germ cell tumors, sarcomas, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumors, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignancies of the urinary system and other cancers and sarcomas.

[0140] The present invention is further described in detail below through some embodiments.

[0141] Example 1 Construction and Screening of Phages of DLL1 Antibodies

[0142] 1.1 Construction of scFv phage display library

[0143] Recombinant human DLL1 protein was coated at 100 ng per well in each well of a Corning EIA / RIA 96-well plate, incubated at 4°C for 12 hours, and each well was blocked with PBS (pH 7.4) containing 3% skim milk.

[0144] After 12 hours of incubation, the serum to be tested was added to each well, and the serum samples were diluted in multiples with PBS and incubated at 37°C for 1 hour.

[0145] After the serum sample incubation is completed, the waste incubation solution in each well is removed, each well on the plate is washed with PBST, and the waste washing solution is discarded to retain the antibody bound to the well wall.

[0146] The bound antibodies were cross-adsorbed and detected by Goat Anti-Mouse IgG H&L (HRP); during the detection process, the assay was developed with TMB substrate at room temperature and measured at 450nm using an enzyme reader; when the serum titer reached the immune response level against the immunogen, another impulse immunization was performed to complete the immunization step and the detection was terminated.

[0147] Blood was collected from immunized mice to separate PBMCs, and mRNA was extracted using Trizol and chloroform, and reverse transcribed into cDNA using SuperScriptTM IV Firt-Strand cDNA Synthesis Reaction Kit (commercially available from Thermo Fisher Scientific).

[0148] Using cDNA as a template, specific primers were used to amplify the mouse antibody light chain VL and heavy chain VH sequences respectively; the process is as follows:

[0149] VL and VH were amplified by overlapping PCR into scFv gene fragments connected by (G4S)3 linkers, and NcoI and NheI restriction sites were introduced at both ends;

[0150] The scFv gene fragment was double-digested with NcoI and NheI and ligated into the pHEN1 vector using T4 ligase to construct the pHEN1-scFv vector;

[0151] Transform TG1 competent cells with pHEN1-scFv to prepare a scFv library;

[0152] The scFv library was infected with helper phage M13KO7 and packaged into a scFv phage display library.

[0153] 1.2 Screening of scFv phage display library

[0154] Recombinant human and mouse DLL1 proteins were coated in each well of a Corning EIA / RIA 96-well plate and incubated at 4°C for 12 hours. Each well was blocked alternately with 300 μL of blocking solution containing 3% skim milk in PBS (pH 7.4) and 5% BSA.

[0155] After 12 hours of incubation, the blocking solution was discarded, and the mouse scFv phage display library obtained in step 1.1 of this example was added to each well. The phage display library was diluted to 1% with the blocking solution, and 100 μL was added to each well. The plates were incubated at 37° C. for 1 hour. The plates were then washed with PBS containing 0.05% Tween-20 (PBST).

[0156] The scFv phage display library was eluted with triethylamine elution solution at a volume of 100 μL / well; after washing, the washing solution was collected, and Tris-HCl with an equal volume to the washing solution was added and mixed evenly to obtain the eluted scFv phage display library; the titer of the eluted scFv phage display library was measured.

[0157] 1.3 Amplification and collection of scFv phage display library

[0158] After infecting TG1 bacteria in the logarithmic growth phase with the scFv phage display library eluted in step 1.2 of this example, M13KO7 was added, and the mixture was placed at 37°C for 30 minutes, and then cultured on a shaker at 37°C and 180 rpm for 1 hour; then the mixture was transferred to a centrifuge tube, centrifuged at 5000 rpm at room temperature for 10 minutes, the supernatant was removed, and the bacterial precipitate was collected; the bacterial precipitate was resuspended in a culture medium containing ampicillin and kanamycin, and the resuspension was cultured on a shaker at 30°C and 220 rpm for 12 hours to obtain a culture.

[0159] The culture was transferred to a 50 mL centrifuge tube and centrifuged at 4°C and 12,000 rpm; the supernatant was collected and added to a PEG / NaCl mixture, and the volume ratio of the supernatant to the mixture was 1:4; the centrifuge tube was placed in an ice bath for 4 hours; then transferred to another centrifuge tube, centrifuged at 4°C and 12,000 rpm for 20 minutes, the supernatant was discarded, the precipitate was collected and resuspended with 1 mL PBS, the resuspension was centrifuged at 4°C and 12,000 rpm for 10 minutes, the supernatant was collected and transferred to a new 1.5 mL centrifuge tube, and the scFv phage display library was screened; samples of the phage display library were extracted for titer determination.

[0160] 1.4 Titer determination

[0161] On the first day, 10 μL of the eluted scFv phage display library was diluted 10-fold in the culture medium to 10 -4 ;

[0162] Each gradient dilution of the scFv phage display library was inoculated into 200 μl of freshly cultured TG1 bacterial solution with an OD600 of 0.4 to 0.5, and incubated at 37° C. for 30 minutes to obtain a scFv phage display library slurry;

[0163] The slurry was spread on a plate containing ampicillin resistance to obtain colonies;

[0164] On the second day, the colonies on the plate were counted and the titer of the eluted scFv phage display library was calculated; the calculation formula is as follows:

[0165] scFv phage display library titer = number of phage clones × dilution (pfu / 10uL).

[0166] 1.5 ELISA verification and sequencing of phage monoclones

[0167] According to the titer determination result in step 1.4 of this example, it is determined whether to screen the amount of the scFv phage display library again, and whether to repeat the steps in items 1.1 to 1.3 for 3 to 5 times until the amplified scFv phage display library is obtained.

[0168] After the screening is completed, the amplified scFv phage display library is spread on a plate containing ampicillin resistance, and the phage single clone is taken for ELISA detection to verify the binding activity;

[0169] The phage monoclones with the highest binding activity (preferably 20) are selected for sequencing, and the different sequences in the sequencing results are selected as candidate DLL1 antibody sequences, such as the three different scFv sequences in the DLL1 antibody sequence, and the corresponding 3P53, 3P47 and 4P108 sequences in each scFv sequence.

[0170] The sequencing results were translated into protein amino acid sequences as shown above, such as SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0171] Example 2 Preparation of 3P53-IgG1, 3P53-IgG2, 3P53-IgG3, and 3P53-IgG4 antibodies

[0172] First, the 3P53 sequence screened in Example 1 was taken;

[0173] Secondly, the VH sequence of 3P53 sequence was used to replace the VH sequence of IgG1, IgG2, IgG3, and IgG4 antibodies respectively; the VL sequence of 3P53 sequence was used to replace the VL region of the corresponding kappa light chain in IgG1, IgG2, IgG3, and IgG4 antibodies respectively;

[0174] Next, the replaced light chain and heavy chain were constructed into the expression vector pcDNA3.1, respectively, to obtain the expression vectors of 3P53-IgG1, 3P53-IgG2, 3P53-IgG3, and 3P53-IgG4 antibodies;

[0175] Subsequently, each expression vector was transiently transfected into HEK293 cells, and 3P53-IgG1, 3P53-IgG2, 3P53-IgG3, and 3P53-IgG4 expression plasmids were transiently transfected into HEK293 cells and transiently expressed in HEK293 cells; HEK293 cells continued to grow for 3-5 days after transfection, and cells were counted every day; when the cell viability was less than 60%, the culture supernatant was collected by centrifugation;

[0176] Finally, the collected culture supernatant was filtered, and the 3P53-IgG1, 3P53-IgG2, 3P53-IgG3, and 3P53-IgG4 antibodies in the filtrate were purified using AKAT Purifier 100 (GE Healthcare) and Protein A Sepharose 4 Fast Flow column (GE Healthcare).

[0177] The expression vectors of 3P53-IgG1, 3P53-IgG2, 3P53-IgG3, and 3P53-IgG4 antibodies were sequenced, and the sequencing results were translated into protein sequences as follows:

[0178] 2.1. Amino acid sequences of light and heavy chains of 3P53-IgG1

[0179] The heavy chain sequence of 3P53-IgG1 is shown in SEQ ID NO: 19:

[0180] EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0181] The kappa light chain sequence of 3P53-IgG1 is shown in SEQ ID NO: 20:

[0182] AIRMTQSPSSSLSALVGDRVTITCRTSQSISTLLNWYQQKPGRAPKLLIYAASTLQSGVPSRFSGTGSGTEFTLTISSLQPEDSATYYCQQSYSTHSITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0183] 2.2. Amino acid sequences of light and heavy chains of 3P53-IgG2

[0184] The heavy chain sequence of 3P53-IgG2 is shown in SEQ ID NO: 21:

[0185] EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0186] The kappa light chain sequence of 3P53-IgG2 is shown in SEQ ID NO: 20;

[0187] 2.3. Amino acid sequences of light and heavy chains of 3P53-IgG3

[0188] The heavy chain sequence of 3P53-IgG3 is shown in SEQ ID NO: 22:

[0189] EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK;

[0190] The kappa light chain sequence of 3P53-IgG3 is shown in SEQ ID NO:20;

[0191] 2.4 Amino acid sequences of the light and heavy chains of 3P53-IgG1

[0192] The heavy chain sequence of 3P53-IgG4 is shown in SEQ ID NO:23:

[0193] EVHLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGNFDYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

[0194] The kappa light chain sequence of 3P53-IgG4 is shown in SEQ ID NO:20.

[0195] Figure 1 This is the analysis diagram of 3P53-IgG1 antibody after SDS-PAGE electrophoresis; Figure 1 In the SDS-PAGE, the non-reduced 3P53-IgG1 sample showed a band at about 150 kDa, and the reduced 3P53-IgG1 sample showed bands at about 50 kDa and 25 kDa, which were the heavy chain and light chain of the IgG1 antibody, respectively.

[0196] Example 3 Preparation of 4P108-IgG2 and 4P108-Fab antibodies

[0197] 3.1. Take the 4P108 sequence screened in Example 1;

[0198] 3.2. Replace the VH sequence of Fab and IgG2 antibodies with the VH sequence of 4P108; replace the VL region of the corresponding kappa light chain in Fab and IgG2 antibodies with the VL sequence of 4P108;

[0199] 3.3. The replaced light chain (VL) and heavy chain (VH) were respectively constructed into the expression vector pcDNA3.1 to obtain the expression vectors of 4P108-Fab and 4P108-IgG2 antibodies;

[0200] 3.4. Each expression vector was transiently transfected into HEK293 cells, and 4P108-Fab and 4P108-IgG2 expression plasmids were transiently transfected into HEK293 cells for transient expression in HEK293 cells; the cells continued to grow for 3 to 5 days after transfection, and the cells were counted every day; when the cell viability was lower than 60%, the culture supernatant was collected by centrifugation;

[0201] 3.5. After the collected culture supernatant was filtered, 4P108-Fab and 4P108-IgG2 antibodies in the filtrate were purified using AKAT Purifier 100 (GE Healthcare) and ProteinA Sepharose 4Fast Flow column (GE Healthcare);

[0202] 3.6. The expression vectors of 4P108-Fab and 4P108-IgG2 antibodies were sequenced, and the sequencing results were translated into protein sequences as follows:

[0203] The heavy chain sequence of 4P108-IgG2 is shown in SEQ ID NO:24:

[0204] EVQLVETGAEVKKPGSSVKVSCKASGDTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGSFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0205] The kappa light chain sequence of 4P108-IgG2 is shown in SEQ ID NO:25:

[0206] DIQLTQSPSSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLLYAASSLQSGVPSRFSASGSGTDFTLTISSLQPEDFATYYCQQSYSAQYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0207] The heavy chain sequence of 4P108-Fab is shown in SEQ ID NO:26:

[0208] EVQLVETGAEVKKPGSSVKVSCKASGDTFSSYAISWVRQAPGQGLEWVGRIIPILGIANYAQEFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDDIAVAGSFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC;

[0209] The kappa light chain sequence of 4P108-Fab is shown in SEQ ID NO:25.

[0210] Example 4 Preparation of 3P47-IgG1 and 3P47-IgG3 antibodies

[0211] 4.1. Take the 3P47 sequence screened in Example 1;

[0212] 4.2. Replace the VH sequence of IgG1 and IgG3 antibodies with the VH sequence of 3P47; replace the VL region of the corresponding kappa light chain in IgG1 and IgG3 antibodies with the VL sequence of 3P47;

[0213] 4.3. The replaced light chain (VL) and heavy chain (VH) were respectively constructed into the expression vector pcDNA3.1 to obtain the expression vectors of 3P47-IgG1 and 3P47-IgG3 antibodies;

[0214] 4.4. Each expression vector was transiently transfected into HEK293 cells, and 3P47-IgG1 and 3P47-IgG3 expression plasmids were transiently transfected into HEK293 cells for transient expression in HEK293 cells; HEK293 cells continued to grow for 3 to 5 days after transfection, and the cells were counted every day; when the cell viability was less than 60%, the culture supernatant was collected by centrifugation;

[0215] 4.5. After filtering the collected culture supernatant, 3P47-IgG1 and 3P47-IgG3 antibodies in the filtrate were purified using AKAT Purifier 100 (GE Healthcare) and Protein A Sepharose 4 Fast Flow column (GE Healthcare).

[0216] 4.6. The expression vectors of 3P47-IgG1 and 3P47-IgG3 antibodies were sequenced and the sequencing results were translated into protein sequences as follows:

[0217] The heavy chain sequence of 3P47-IgG1 is shown in SEQ ID NO: 27:

[0218] EVQLVQSGAEVKEPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSSWYGDYYYGMDV WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0219] The kappa light chain sequence of 3P47-IgG1 is shown in SEQ ID NO: 28:

[0220] EIVLTQSPSSLSASVGDRVTISCRASQGIGDHLAWFQQKPGKAPKSLISAASSLQSGVPSKFSGSGSGTDFTLTISNLQPEDSATYFCQQYNNYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0221] The heavy chain sequence of 4P47-IgG3 is shown in SEQ ID NO:29:

[0222] EVQLVQSGAEVKEPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDSSSWYGDYYYGMDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK;

[0223] The kappa light chain sequence of 4P47-IgG3 is shown in SEQ ID NO:28.

[0224] Example 5 Affinity Detection of 3P53-IgG4 Antibody

[0225] In this example, the candidate antibody 4P47-IgG3 prepared in Example 2 was selected for affinity detection; the recombinant human and mouse DLL1 proteins were used to coat the ELISA plate, and the affinity of the candidate antibody to human and mouse DLL1 was detected by ELISA.

[0226] The detection method is as follows:

[0227] 5.1. Coating: Coat the ELISA plate with 100 μL / well of recombinant human DLL1 and mouse DLL1 antigen proteins at a concentration of 1 ug / ml. Coat each well of the 96-well plate with each antigen protein. After coating, seal the plate with a sealing film and incubate at 4°C for 12 hours.

[0228] 5.2. Blocking: discard the ELISA plate coating solution, add 300 μL of PBST to each well, wash for 1 minute, and repeat this step 5 times; add 300 μL / well of PBS (PBSM) containing 5% skim milk powder to block for 1 hour; after blocking, discard the blocking solution, add 300 μL of PBST, wash for 1 minute, and repeat this step 5 times;

[0229] 5.3. Incubation with primary antibody: The starting concentration of 3P53-IgG4 antibody is 100 μg / ml. Dilute the antibody three times with PBSM in a 5-fold gradient. Add 100 μL / diluted antibody sample in the gradient and incubate at 37°C for 1 hour.

[0230] 5.4. Incubate with secondary antibody: Wash 20 times with PBST; after washing, prepare rabbit anti-human IgG H&L (HRP) (dilution: 1:130000, abcam) in PBSM, add 100 μL / well of secondary antibody, and incubate at 37°C for 1 hour;

[0231] 5.5. Color development: Wash 20 times with 1×PBST, add 100μL TMB, color develop for 10 min in dark, add 50μL stop solution / well to stop color development, detect OD450 on a microplate reader, and record the relevant values;

[0232] 5.6. Analysis: Draw the antibody IC50 curve based on the OD450 readings and draw an affinity graph.

[0233] The affinity curve of 3P53-IgG4 for human and mouse DLL1 detected by ELSIA is shown in the figure Figure 2 As shown; Figure 2 In the figure, hDLL1 represents the affinity curve of 3P53-IgG4 for humans; mDLL1 represents the affinity curve of 3P53-IgG4 for mice.

[0234] Example 6 WB detection of DLL1 protein by 4P108-IgG2 antibody

[0235] Western blotting was used to detect the specific binding of candidate antibodies to DLL1 protein. Recombinant human and mouse DLL1 proteins were boiled at 100°C for 5 min, denatured and loaded onto SDS-PAGE gel, and separated by electrophoresis before being transferred to a PVDF membrane. Blocking buffer was used for blocking, and finally the candidate antibody was diluted as the primary antibody in blocking buffer PBSM and incubated overnight at 4°C. Ms mAb to M13[CN-MM05](HRP) antibody was added and incubated in blocking buffer for 1 hour for color development.

[0236] In this example, the 4P108-IgG2 candidate antibody prepared in Example 3 was selected for WB detection.

[0237] The candidate antibody 4P108-IgG2 was used as the primary antibody, and the specificity with recombinant human and mouse DLL1 proteins was detected by western blotting. The specific detection method is as follows:

[0238] 6.1. Sample loading: quantify recombinant human and mouse DLL1 to 100 ng, add 3 μL 5×SDS loading buffer, use sample buffer to make up the total volume to 15 μL, boil the buffer at 100°C for 5 minutes and then cool to room temperature, load the boiled sample buffer and marker together, and load 15 μL for each sample;

[0239] 6.2. Electrophoresis: Use constant voltage method, electrophoresis at 100V for 2 hours;

[0240] 6.3. Transfer: Use methanol to soak the PVDF membrane in advance and use the wet transfer method for transfer; the transfer voltage is 300mA and the transfer time is 1 hour;

[0241] 6.4. Blocking: After the transfer is completed, place the membrane in the washing solution PBST and rinse for 2 minutes to wash away the transfer solution; then transfer it to a container containing the blocking solution PBSM and block it on a decolorizing shaker at room temperature for 1 hour;

[0242] 6.5. Primary antibody incubation: dilute the 4P108-IgG2 antibody with PBST; remove the membrane from the blocking solution, wash the membrane with PBST for 3 times, 10 minutes each time, and finally absorb the residual liquid with filter paper, and place the membrane protein side down on the antibody liquid surface; incubate at room temperature for 1 to 2 hours, and then wash it with PBST on a decolorizing shaker at room temperature for 3 times, 10 minutes each time;

[0243] 6.6. Secondary antibody incubation: Rabbit anti-human IgG H&L (HRP) antibody (commercially purchased from abcam) was diluted 1:10000 in advance, added to the membrane, and incubated for 2 hours with slow shaking at room temperature; the membrane was washed 3 times with PBST, each time for 10 minutes;

[0244] 6.7. Color development: Add developer (commercially purchased from Beijing Solebow Technology Co., Ltd.), react for 10 minutes in the dark, and place in a gel imager for photography.

[0245] Figure 3 The WB photograph of the gel showing 4P108-IgG2 detecting human and mouse DLL1 is presented; Figure 3 In the assay, a clear band was detected at about 57 KDa, and P108-IgG2 could sensitively detect human / mouse DLL1 protein.

[0246] Example 7 Specific detection of DLL1 by 3P47-IgG1 antibody

[0247] The 3P47-IgG1 candidate antibody prepared in Example 4 was used to detect the DLL1 overexpressing cell line by flow cytometry, that is, the specific detection of DLL1 by the 3P47-IgG1 antibody.

[0248] The cell line 293T-DLL1 overexpressing DLL1 was detected using candidate antibodies and isotype control antibody IgG1. 293T-DLL1 cells were taken and divided into aliquots (identified as group numbers 1# and 2#), and candidate antibodies and isotype control antibody IgG1 were added respectively; then APC anti-human IgG Fc Antibody (BioLegend) was used as a secondary antibody to incubate the cells, and flow cytometry was performed to determine the specificity of the candidate antibody by analyzing the flow cytometry data.

[0249] In this example, the 3P47-IgG1 antibody prepared in Example 4 was used for flow cytometry detection of DLL1 overexpressing cell lines. The cell line 293T-DLL1 overexpressing DLL1 was detected using the 3P47-IgG1 antibody and the isotype control antibody IgG1. 293T-DLL1 cells were taken and divided into two parts, and 3P47-IgG1 and the isotype control antibody IgG1 were added respectively, and then APC anti-human IgGFc Antibody (commercially available from BioLegend) was used as a secondary antibody to incubate the cells and detect by flow cytometry.

[0250] The specific detection methods are as follows:

[0251] 7.1. Take out two aliquots of cultured 293T-DLL1 cells, wash them with DPBS, digest them with trypsin, resuspend them in 10% DMEM medium, and count the cells in the resuspension respectively;

[0252] 7.2. According to the counting results, the cell suspension was taken into the cell wells of the 96-well plate and added at 100 μL / well. Each cell was added at 1.0×10 5 Each well was plated in 20 wells, with 10 wells in each group;

[0253] 7.3. Adjust the concentration of candidate 3P47-IgG1 antibody to 50 μg / ml;

[0254] 7.4. Centrifuge the 96-well plate at 500 g for 5 minutes at room temperature and discard the supernatant. Then add 3P47-IgG1 antibody to the corresponding cell wells of the 96-well plate at 100 μL / well and incubate the 96-well plate at room temperature for 20 minutes.

[0255] 7.5. After incubation, add 150 μL DPBS (containing 2% FBS) to each well for washing, then centrifuge the 96-well plate at 500 g for 5 minutes at room temperature; discard the supernatant, add 250 μL DPBS (containing 2% FBS) to each well for washing, then centrifuge the 96-well plate at 500 g for 5 minutes at room temperature and discard the supernatant;

[0256] 7.6. According to the grouping scheme (identified as group number 1#, 2#), add 100 μL to each well and incubate at room temperature for 20 minutes; the secondary antibody diluent is anti-human IgG Fc Antibody PE and the concentration is 0.5 μL / well;

[0257] 7.7 After 20 min incubation, add 150 μL DPBS (containing 2% FBS) to each well for washing twice, centrifuge the 96-well plate at 500 g for 5 min at room temperature, and discard the supernatant;

[0258] 7.8. Add 120 μL DPBS (containing 2% FBS) to each well of the 96-well plate for resuspending, and the resuspended liquid in each well is independently loaded on the flow cytometer.

[0259] Figure 4 Flow cytometric analysis of DLL1-overexpressing cell lines detected by 3P47-IgG1. Figure 4 The results showed that compared with the control group, 3P47-IgG1 had a significant peak shift in the detection of 293T-DLL1 overexpressing cell lines, while the isotype control IgG1 had no peak shift. 3P47-IgG1 can sensitively detect DLL1 in 293T-DLL1 overexpressing cell lines.

[0260] Example 8 Flow cytometry detection of DLL1 expression on tumor cell surface using 3P47-IgG3 antibody

[0261] 3P47-IgG3 detected the expression of DLL1 on the surface of tumor cells, and the isotype control antibody Fab was used to detect the cell line 293T-DLL1 that overexpressed DLL1.

[0262] 293T-DLL1 cells were taken and divided into two parts, and PE-labeled candidate and isotype control antibody Fab were added respectively, and the cells were detected by flow cytometry.

[0263] The candidate antibody was labeled with a PE labeling kit to detect the tumor cell line EO771 expressing DLL1. EO771 cells were taken and the candidate antibody was added or not, and then detected by flow cytometry.

[0264] In this example, the 3P47-IgG3 antibody prepared in Example 4 was used to detect the expression of DLL1 on the surface of tumor cells.

[0265] The 3P47-IgG3 antibody was labeled with a PE kit, and flow cytometry was used to detect the expression of DLL1 on the surface of tumor cells EO771; the detection method is as follows:

[0266] 8.1. Take 3E5-4E5 tumor cells EO771, centrifuge at 350g, 4°C for 5 minutes, and discard the supernatant;

[0267] 8.2. Add 200 μL FACS Buffer (1% FBS dissolved in PBS), resuspend the cells, centrifuge at 300g, 4°C for 5 minutes, and discard the supernatant;

[0268] 8.3. Prepare PE-labeled detection antibody 3P47-IgG3 (PE): the initial concentration is 1 μg / mL, add 100 μL of 3P47-IgG3 (PE) solution to each well, mix by pipetting, and incubate at 4°C for 1 hour; after the incubation, add 100 μL of FACS Buffer, centrifuge at 300g at 4°C for 5 minutes, and discard the supernatant;

[0269] 8.4. Add 200 μL FACS Buffer to each well, resuspend the cells, centrifuge at 300g, 4°C for 5 minutes, and discard the supernatant; repeat this step once; finally, use flow cytometer for detection.

[0270] Figure 5 Flow cytometric analysis of DLL1 expression on the surface of tumor cells detected by 3P47-IgG3 antibody; Figure 5 In the figure, the peak on the left is EO771 cells without the addition of 3P47-IgG3 (PE), and the peak on the right is EO771 cells after the addition of 3P47-IgG3 (PE). Figure 5 The results showed that 3P47-IgG3 could detect DLL1 expression on 72.6% of EO771 cells.

[0271] Example 9 Immunohistochemical detection of DLL1 expression in tissues using 3P53-IgG1 antibody.

[0272] Immunohistochemistry (IHC) was used to locate and quantify the expression of DLL1 in mouse tissues using candidate antibodies.

[0273] In this example, the 3P53-IgG1 antibody prepared in Example 1 was used to detect the expression of DLL1 on the surface of tumor cells.

[0274] In this embodiment, a mouse spleen tissue sample was fixed in advance using a neutral formaldehyde solution; after dewaxing and hydration, the tissue sample slices were placed in a repair basin, EDTA (pH 9.0) repair solution was added, and the slices were treated at 100°C for 10 minutes, then naturally cooled to room temperature, and the slices were rinsed with PBS 3 times, each time for 2 minutes; 3% hydrogen peroxide was dripped on the sliced ​​tissue, incubated at room temperature for 15 minutes, and rinsed with PBS 3 times, each time for 3 minutes; PBS was blotted with absorbent paper, 5% serum was added, and the slides were blocked at 37°C for 30 minutes, the liquid around the slides was blotted with absorbent paper, 3P53-IgG1 antibody solution was added, and the slides were incubated in a 4°C wet box overnight; the slices were rinsed with PBS 3 times, each time for 3 minutes, and the secondary antibody Rabbit Anti-Mouse IgG was added after the slices were wiped dry with absorbent paper H&L (HRP) (commercially purchased from abcam), incubated at 37°C for 30 minutes; rinsed the slices with PBS 4 times, 3 minutes each time, wiped the slices dry with absorbent paper, added DAE color developing solution, and when the color turned brown, rinsed the slices with tap water to stop color development; after rinsing the slices, they were dehydrated, fixed and sealed, and finally observed under a 20x microscope, and obtained the microscopic photos of 3P53-IgG1 immunohistochemical detection of mouse spleen tissue expressing DLL1, as shown in the figure. Figure 6 As shown, the arrow points to DLL1 stained with 3P53-IgG1.

[0275] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. A DLL1 antibody, characterized in that The DLL1 antibody comprises three different scFv sequences, each of which comprises one of the 3P53, 3P47 and 4P108 sequences.

2. The DLL1 antibody according to claim 1, characterized in that When the scFv sequence comprises a 3P53 sequence, the amino acid sequence of the heavy chain variable region of the scFv sequence is shown as SEQ ID NO:11, and the amino acid sequence of the light chain variable region of the scFv sequence is shown as SEQ ID NO:

12.

3. The DLL1 antibody according to claim 2, characterized in that The amino acid sequences of the three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 in the 3P53 sequence and the amino acid sequences of the three light chain complementary determining regions LCDR1, LCDR2, and LCDR3 in the 3P53 sequence are as follows: When the 3P53 sequence is a chothia structure, in the 3P53 sequence: the amino acid sequence of HCDR1 is GGTFSSYA, the amino acid sequence of HCDR2 is IIPILGIA, the amino acid sequence of HCDR3 is ARDDIAVAGNFDY, the amino acid sequence of LCDR1 is QSISTL, the amino acid sequence of LCDR2 is AAS, and the amino acid sequence of LCDR3 is QQSYSTHSIT; or When the 3P53 sequence is a Kabat structure, in the 3P53 sequence: the amino acid sequence of HCDR1 is SYAIS, the amino acid sequence of HCDR2 is RIIPILGIANYAQEFQG, the amino acid sequence of HCDR3 is DDIAVAGNFDY, the amino acid sequence of LCDR1 is RTSQSISTLLN, the amino acid sequence of LCDR2 is AASTLQS, and the amino acid sequence of LCDR1 is QQSYSTHSIT.

4. The DLL1 antibody according to claim 1, characterized in that When the scFv sequence comprises the 3P47 sequence, the amino acid sequence of the heavy chain variable region of the scFv sequence is shown in SEQ ID NO:14, and the amino acid sequence of the light chain variable region of the scFv sequence is shown in SEQ ID NO:

15.

5. The DLL1 antibody according to claim 4, characterized in that The amino acid sequences of the three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 in the 3P47 sequence and the amino acid sequences of the three light chain complementary determining regions LCDR1, LCDR2, and LCDR3 in the 3P47 sequence are as follows: When the 3P47 sequence is a chothia structure, in the 3P47 sequence: the amino acid sequence of HCDR1 is GYTFTSY, the amino acid sequence of HCDR2 is NPSGGS, the amino acid sequence of HCDR3 is DSSSWYGDYYYGMDV, the amino acid sequence of LCDR1 is RASQGIGDHLA, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR3 is QQYNNYPLT; or When the 3P47 sequence is a Kabat structure, in the 3P53 sequence: the amino acid sequence of HCDR1 is SYYMH, the amino acid sequence of HCDR2 is I INPSGGSTSYAQKFQG, the amino acid sequence of HCDR3 is DSSSWYGDYYYGMDV, the amino acid sequence of LCDR1 is RASQGIGDHLA, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR1 is QQYNNYPLT.

6. The DLL1 antibody according to claim 1, characterized in that When the scFv sequence comprises the 4P108 sequence, the amino acid sequence of the heavy chain variable region of the scFv sequence is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region of the scFv sequence is shown in SEQ ID NO:

18.

7. The DLL1 antibody according to claim 6, characterized in that The amino acid sequences of the three heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 in the 4P108 sequence and the amino acid sequences of the three light chain complementary determining regions LCDR1, LCDR2, and LCDR3 in the 4P108 sequence are as follows: When the 4P108 sequence is a chothia structure, in the 4P108 sequence: the amino acid sequence of HCDR1 is GDTFSSY, the amino acid sequence of HCDR2 is IPILGI, the amino acid sequence of HCDR3 is DDIAVAGSFDY, the amino acid sequence of LCDR1 is RASQSISRYLN, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR3 is QQSYSAQYT; or When the 4P108 sequence is a Kabat structure, in the 4P108 sequence: the amino acid sequence of HCDR1 is SYAIS, the amino acid sequence of HCDR2 is RIIPILGIANYAQEFQG, the amino acid sequence of HCDR3 is DDIAVAGSFDY, the amino acid sequence of LCDR1 is RASQSISRYLN, the amino acid sequence of LCDR2 is AASSLQS, and the amino acid sequence of LCDR1 is QQSYSAQYT.

8. An IgG antibody, characterized in that The IgG antibody comprises the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the DLL1 antibody according to any one of claims 1 to 7.

9. A Fab antibody, characterized in that The Fab antibody comprises the amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region of the DLL1 antibody according to any one of claims 1 to 7.

10. A detection reagent for detecting tumor cells or cancer cells, characterized in that: The detection reagent comprises the DLL1 antibody according to any one of claims 1 to 7, or comprises the IgG antibody according to claim 8, or comprises the Fab antibody according to claim 9.

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