CN112175084A - Targeting Frizzled7 humanized antibody and preparation method and application thereof - Google Patents

Abstract

The invention relates to a targeted Frizzled7 humanized antibody and a preparation method and application thereof. The humanized antibody of the targeted Frizzled7 has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.11, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 12. The humanized antibody SHH002-hu1 targeting the Frizzled7 of the invention reserves the high affinity (KD values are all less than 1 × 10) of the murine monoclonal antibody SHH002 and the recombinant human Fzd7 protein^‑12M), specifically binds to Fzd7 and does not cross-react with other receptors of the Fzd family, reducing the risk of off-target of the antibody in potential clinical applications. SHH002-hu1 can bind with Fzd7 on the surface of tumor cells to block Wnt signal channel; and can be effectively combined with Fzd7 in the tumor tissue of a patient, has potential tumor targeting property, and is a new candidate drug for future antitumor treatment.

Description

Targeting Frizzled7 humanized antibody and preparation method and application thereof

The application is a divisional application, the application date of a parent application of the divisional application is 2019, 11 and 6, the application number is 201911078254.4, and the invention name is 'a targeted Frizzled7 humanized antibody and a preparation method and application thereof'.

Technical Field

The invention relates to the technical field of biology, in particular to a targeted Frizzled7 humanized antibody and a preparation method and application thereof.

Background

The Wnt signaling pathway is a highly conserved signaling pathway in the process of species evolution, and plays a crucial role in the early development of animal embryos, organogenesis and the development of tumors. The family of Wnt proteins bind to the N-terminal extracellular domain CRD (cysteine-rich domain) of the receptor frizzled (Fzd), interact with Dvl (secreted) proteins and activate downstream signaling pathways. Fzd proteins are a family of Wnt receptors with seven transmembrane molecular structures, 10 member proteins of which Frizzled-7(Fzd7) is one member. Studies have shown that the Wnt receptor Fzd7 is aberrantly expressed in different kinds of cancers (including Triple Negative Breast Cancer (TNBC), non-small Cell lung cancer (NSCLC), liver cancer (HCC), etc.) (Polakis P,2012, EMBO J,31: 2737-46; King TD et al,2012, J Cell Biochem,113: 13-8); fzd7 induces tumor development and metastasis by activating the canonical Wnt pathway (Wnt/β -catenin).

The Wnt pathway is involved in invasion and metastasis of tumors, has a regulating effect on tumor angiogenesis, and can remarkably inhibit tumor cell proliferation, transplanted tumor growth and angiogenesis by gene knockout of Fzd 7. Wnt signaling is also closely linked to tumor stem cell behavior, Fzd7 is one of the tumor stem cell markers, and upregulation of Fzd7 in these tumor stem cells is significantly associated with tumor chemotherapy tolerance and low survival in patients (Barker N et al, 2009, Nature,457: 608-11). In addition, "context-specific functions" of Fzd7 suggest that selective targeting of Fzd7 can treat cancer without affecting normal tissue homeostasis. In summary, Fzd7 is a potential therapeutic target for a variety of tumors (Kahn M,2014, Nat Rev Drug Discov,13: 513-32).

At present, the targeted Fzd7 method mainly comprises RNA interference, small molecule inhibitors, small interfering peptides, soluble recombinant Fzd7 protein and anti-Fzd 7 monoclonal antibodies. The Fzd7shRNA can effectively inhibit the proliferation and invasion of TNBC cells and the growth of tumors; small interfering peptides RHPDs or small molecule inhibitors FJ9 interfere with the binding of Fzd7 and dispersin (Dvl) to block Wnt signaling and effectively inhibit the growth of mouse transplantable tumors (Nambotin SB et al, 2011, J Hepatol,54: 288-99; Fujii N et al, 2007, Cancer Res,67: 573-9); the extracellular domain recombinant protein of Fzd7 (sFzd7) was used to competitively bind Wnt3 ligand with cell surface Fzd7 to block Wnt/β -catenin signaling and also inhibit tumor development (Wei W et al, 2011, Mol Cancer,10: 16); the above methods are still in preclinical research phase. In contrast, a more favorable approach to clinical transformation is to use antibodies to specifically target Fzd7, and Oncomed has developed a Fzd7CRD domain targeting monoclonal antibody OMP-18R5(vantictumab) currently in phase I clinical trials of advanced non-small cell lung Cancer (NSCLC), advanced HER2 (human epidermal growth factor receptor 2) negative breast Cancer, and advanced pancreatic Cancer (Flanagan DJ et al, 2019, Cancer Res,79: 970-81; Gurney A et al,2012, Proc Natl Acad Sci USA,109: 11717-22). OMP-18R5 also cross-reacts with Fzd1, Fzd2, Fzd5 and Fzd8 in the Fzd receptor family, suggesting that OMP-18R5 may be used in the treatment of various cancers, but lack of specificity for target proteins may lead to off-target effects that affect antibody efficacy.

The Wnt protein family specifically binds to the N-terminal extracellular domain CRD (cysteine-rich domain) of Fzd, interacts with Dvl proteins and activates downstream signaling pathways. CRD of Fzd plays a crucial role in binding to ligands, and the sequence of this region of the Fzd receptor family is highly conserved across species as well as species. The CRD region sequence of Fzd7 has more than 40% homology to other members of the Fzd family, particularly with Fzd1 and Fzd2 to nearly 80% (Janda, CY et al,2012, Science,337: 59-63). The Fzd7CRD segment is selected as an antibody obtained by antigen screening, which can block the key site of the binding of Fzd and its ligand, but it also has the probability of binding with other members of the Fzd family, especially Fzd1/2, so that the antibody can have off-target risk in the course of treatment (OMP-18R5 is an antibody obtained by screening using Fzd7CRD segment protein as antigen). When an antibody specifically targeting only one Fzd receptor is selected, it is possible to not only reduce off-target effects and enhance antitumor activity, but also study "context-specific roles" of each Fzd receptor as a powerful tool, thereby elucidating Wnt signaling pathway and its mechanism of action.

Compared with antibody Fab fragments, single chain antibodies (scFv) and Nanobody antibodies (Nanobody), IgG-like Full-length antibodies (Full-length antibodies) have a complete antibody structure, are stable in properties, have a long half-life in vivo, and can exert immune effects such as Fc fragment-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) in addition to blocking a target signal pathway. Only 1 humanized Fab fragment and 1 nano antibody in 43 antibody drugs on the market of 2014-2018 are almost full-length antibodies except a few Fc fusion proteins and antibody coupling drugs (ADC), and the excellent clinical and pharmaceutical values of the full-length antibodies are shown.

Among the commonly used antibody screening technologies, the murine full-length antibody obtained by screening through the hybridoma technology has higher affinity. However, murine antibodies in clinical use can cause patients to develop a human anti-mouse antibody response (HAMA); splicing the variable region (V region) gene of the murine antibody and the constant region (C region) gene of the human antibody into a chimeric antibody gene, wherein the expressed chimeric antibody reduces the risk of the patient to develop HAMA; the human antibody developed by further grafting the complementarity determining regions (CDR regions) of the murine antibody into the human variable domain further reduces the risk of an anti-drug antibody (ADA) response against the mouse variable region gene in the chimeric antibody. In antibody humanization design, antibodies of two subtypes, IgG1 and IgG4, are usually selected as templates, and the Fc end of the antibodies can further enhance or weaken cellular immune activities such as ADCC and CDC by site mutation, deglycosylation, and the like according to physiological mechanisms.

In the antibody V region, the framework regions (FR regions) that function as scaffolds not only provide the steric conformational environment for the CDRs, but also participate in the formation of the correct conformation of the antibody binding site. Thus, simple CDR grafting tends to lose or reduce the affinity of the original antibody (Jones PT et Al, 1986, Nature,321: 522-5; Al-Lazikani B et Al, 1997, J Mol Biol,273: 927-48). The bioinformatics software discovery and analysis is applied to design non-key amino acid humanized mutation of the murine FR region, so that the risk of loss of antibody affinity can be effectively reduced. The advantages of humanization design using antibody databases and bioinformatics software are mainly: 1. searching a Human Germline database according to sequences of a heavy chain variable region (VH) and a light chain variable region (VL), and respectively selecting sequences with the highest homology with VH and VL as humanized design templates; 2. structural simulation is carried out on the murine antibody through discovery software, a simulation result with the highest reliability is selected to guide humanized design, murine amino acids playing a key role in structural stability of the antibody are reserved, and unimportant sites are mutated into humanized amino acids according to a template. The method can obtain the antibody with the highest humanization degree, and ensure the activity of the antibody to the maximum degree.

Disclosure of Invention

The invention aims to provide a targeted Frizzled7 humanized antibody, and a preparation method and application thereof.

The invention uses hybridoma technology to screen anti-Frizzled 7(Fzd7) antibodies and humanize them. The obtained humanized antibody targeting the Frizzled7 retains the high affinity of the murine antibody and the recombinant human Frizzled7 protein (rhFzd7), can be specifically combined with Fzd7 on the surface of tumor cells and tumor tissues, and effectively blocks the Wnt/beta-catenin signal pathway; is a brand-new high-specificity genetic engineering antibody with the functions of targeting Fzd7 and potential anti-tumor activity.

The purpose of the invention can be realized by the following technical scheme:

the invention provides a targeted Frizzled7 monoclonal antibody, marked as SHH002, the targeted Frizzled7 monoclonal antibody SHH002 can also be expressed as anti-Fzd 7 murine antibody SHH002, the targeted Frizzled7 monoclonal antibody has a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the following three Complementarity Determining Regions (CDRs): CDR1 shown in SEQ ID No.5, CDR2 shown in SEQ ID No.6, and CDR3 shown in SEQ ID No. 7;

the light chain variable region comprises the following three complementarity determining regions CDRs: CDR1 ' shown in SEQ ID No.8, CDR2 ' shown in SEQ ID No.9, and CDR3 ' shown in SEQ ID No. 10.

The heavy chain variable region nucleotide sequence of the targeted Frizzled7 monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2.

The nucleotide sequence of the light chain variable region of the targeted Frizzled7 monoclonal antibody is shown as SEQ ID No.3, and the amino acid sequence is shown as SEQ ID No. 4.

The murine monoclonal antibody SHH002 of the invention can be specifically combined with Fzd7, and shows high affinity with recombinant human Fzd7 protein, and affinity constant KD is less than 1 x 10 detected by biofilm interference technique (BLI)^-12M。

The invention provides a targeting Frizzled7 humanized antibody, which is marked as SHH002-hu1, wherein the targeting Frizzled7 humanized antibody has a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.11, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 12. SHH002-hu1, and compared with murine antibody SHH002, its humanization degree is as high as 96.5%.

The base sequence of the heavy chain of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.21, wherein the constant region is from 346 th to 1335 th of the base sequence shown as SEQ ID No. 21.

The light chain base sequence of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.22, wherein the constant region is from 334 th to 654 th of the base sequence shown as SEQ ID No. 22.

The heavy chain amino acid sequence of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.23, wherein the constant region is from 116 th to 445 th positions of the base sequence shown as SEQ ID No. 23.

The light chain amino acid sequence of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.24, wherein the constant region is from 112 th to 218 th of the base sequence shown as SEQ ID No. 24.

The invention provides another humanized antibody targeting Frizzled7, which is marked as SHH002-hu2, and the humanized antibody targeting Frizzled7 has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 14.

The invention provides another humanized antibody targeting Frizzled7, which is marked as SHH002-hu3, and the humanized antibody targeting Frizzled7 has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 16.

The invention provides another humanized antibody targeting Frizzled7, which is marked as SHH002-hu4, and the humanized antibody targeting Frizzled7 has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.17, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 18.

The invention provides another humanized antibody targeting Frizzled7, which is marked as SHH002-hu5, and the humanized antibody targeting Frizzled7 has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.19, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 20.

The present invention provides a recombinant protein having:

(i) any one of the Frizzled7 targeting monoclonal antibody SHH002, the Frizzled7 targeting humanized antibody SHH002-hu1, the Frizzled7 targeting humanized antibody SHH002-hu2, the Frizzled7 targeting humanized antibody SHH002-hu3, the Frizzled7 targeting humanized antibody SHH002-hu4, and the Frizzled7 targeting humanized antibody SHH002-hu 5; and (ii) optionally a tag sequence to facilitate expression and/or purification.

The present invention provides a nucleotide encoding a polypeptide selected from the group consisting of:

(1) any one of the Frizzled7 targeting monoclonal antibody SHH002, the Frizzled7 targeting humanized antibody SHH002-hu1, the Frizzled7 targeting humanized antibody SHH002-hu2, the Frizzled7 targeting humanized antibody SHH002-hu3, the Frizzled7 targeting humanized antibody SHH002-hu4, and the Frizzled7 targeting humanized antibody SHH002-hu 5; the recombinant protein according to (1) or (2).

The present invention provides an expression vector containing the nucleic acid.

The present invention provides a recombinant host cell comprising the expression vector.

The invention provides a recombinant cell strain which can stably express any one of the humanized antibody SHH002-hu1 targeting the Frizzled7, the humanized antibody SHH002-hu2 targeting the Frizzled7, the humanized antibody SHH002-hu3 targeting the Frizzled7, the humanized antibody SHH002-hu4 targeting the Frizzled7 and the humanized antibody SHH002-hu5 targeting the Frizzled 7.

The invention provides a kit, which is characterized by comprising: the targeting Frizzled7 monoclonal antibody SHH002, targeting Frizzled7 humanized antibody SHH002-hu1, targeting Frizzled7 humanized antibody SHH002-hu2, targeting Frizzled7 humanized antibody SHH002-hu3, targeting Frizzled7 humanized antibody SHH002-hu4, targeting Frizzled7 humanized antibody SHH002-hu 5.

The invention provides an immunoconjugate, comprising:

(a) any one of the Frizzled7 targeting monoclonal antibody SHH002, Frizzled7 targeting humanized antibody SHH002-hu1, Frizzled7 targeting humanized antibody SHH002-hu2, Frizzled7 targeting humanized antibody SHH002-hu3, Frizzled7 targeting humanized antibody SHH002-hu4, Frizzled7 targeting humanized antibody SHH002-hu5 or the recombinant protein; and (b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

The invention provides a pharmaceutical composition, which is characterized in that the composition comprises any one of the Frizzled7 targeting monoclonal antibody SHH002, the Frizzled7 targeting humanized antibody SHH002-hu1, the Frizzled7 targeting humanized antibody SHH002-hu2, the Frizzled7 targeting humanized antibody SHH002-hu3, the Frizzled7 targeting humanized antibody SHH002-hu4, the Frizzled7 targeting humanized antibody SHH002-hu5, the recombinant protein, or the immunoconjugate; and a pharmaceutically acceptable carrier.

The invention provides a preparation method of the targeted Frizzled7 monoclonal antibody SHH002, which comprises the following steps:

a BALB/c mouse is immunized by using Fzd7 extracellular CRD region protein, spleen cells of the immunized mouse are taken and fused with myeloma cells to obtain a hybridoma cell strain capable of expressing a mouse monoclonal antibody SHH002, and the cell strain is used for preparing the antibody.

The invention provides a preparation method of any one of the humanized antibody SHH002-hu1 targeting Frizzled7, the humanized antibody SHH002-hu2 targeting Frizzled7, the humanized antibody SHH002-hu3 targeting Frizzled7, the humanized antibody SHH002-hu4 targeting Frizzled7 and the humanized antibody SHH002-hu5 targeting Frizzled7, which comprises the following steps:

immunizing BALB/c mouse with Fzd7 extracellular CRD region protein, taking spleen cell of the immunized mouse and fusing it with myeloma cell to obtain hybridoma cell strain capable of expressing target Frizzled7 monoclonal antibody, and preparing target Frizzled7 monoclonal antibody by using said cell strain; the obtained hybridoma cell strain is classified and named as a hybridoma cell strain FL253-1 for expressing an anti-Fzd 7 murine antibody SHH002, the cell strain is preserved for 10 and 30 months in 2019, and the preservation organization is as follows: china Center for Type Culture Collection (CCTCC) with the preservation number: CCTCC NO of C2019279, and the preservation address is Wuhan university in Wuhan, China;

the discovery systestydio software is used for designing non-key amino acid mutation of the FR region of the targeted Frizzled7 monoclonal antibody, and finally the targeted Frizzled7 humanized antibody with the humanization degree of more than 96 percent is obtained.

Specifically, the V region gene of the targeted Frizzled7 monoclonal antibody is spliced with the C region gene of the human IgG1 antibody to construct an expression chimeric antibody SHH002-C, and the affinity constant detected by a BLI method is 8.02 multiplied by 10^-11M, reserving CDR regions of a heavy chain and a light chain of the chimeric antibody SHH 002-C; performing structure simulation on the murine antibody by using discovery software, reserving murine amino acids which play a key role in the structural stability of the antibody, mutating unimportant sites into human amino acids according to a template, and finally obtaining the high affinity (KD value is less than 1 × 10)^-12M) is greater than 96% of the humanized antibody targeted to Frizzled 7. Compared with the murine antibody SHH002, the humanized degree is up to 96.5%.

The invention also verifies the affinity, specificity, targeting property and preliminary biological activity of a monoclonal antibody SHH002 targeting Frizzled7, a humanized antibody SHH002-hu1 targeting Frizzled7, a humanized antibody SHH002-hu2 targeting Frizzled7, a humanized antibody SHH002-hu3 targeting Frizzled7, a humanized antibody SHH002-hu4 targeting Frizzled7 and a humanized antibody SHH002-hu5 targeting Frizzled 7.

The invention also provides application of the Frizzled7 targeting monoclonal antibody SHH002, the Frizzled7 targeting humanized antibody SHH002-hu1, the Frizzled7 targeting humanized antibody SHH002-hu2, the Frizzled7 targeting humanized antibody SHH002-hu3, the Frizzled7 targeting humanized antibody SHH002-hu4 and the Frizzled7 targeting humanized antibody SHH002-hu5 in preparation of a tumor treatment drug.

The action mechanisms of the Frizzled7 targeting monoclonal antibody, the Frizzled7 targeting humanized antibody SHH002-hu1, the Frizzled7 targeting humanized antibody SHH002-hu2, the Frizzled7 targeting humanized antibody SHH002-hu3, the Frizzled7 targeting humanized antibody SHH002-hu4 and the Frizzled7 targeting humanized antibody SHH002-hu5 are as follows: the derivative specifically binds to human Fzd7, inhibits the binding of Wnt ligand to Fzd7 receptor, and blocks the transduction of Wnt signal channel.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the anti-Fzd 7 targeting Frizzled7 humanized antibody SHH002-hu1 reserves the high affinity (KD values are less than 1 × 10) of the mouse monoclonal antibody SHH002 and the recombinant human Fzd7 protein^-12M), specifically binds to Fzd7 and does not cross-react with other receptors of the Fzd family, reducing the risk of off-target of the antibody in potential clinical applications. SHH002-hu1 can bind with Fzd7 on the surface of tumor cells to block Wnt signal channel; and can be effectively combined with Fzd7 in the tumor tissue of a patient, has potential tumor targeting property, and is a new candidate drug for future antitumor treatment.

Drawings

FIG. 1 is a graph showing the results of Western blot for verifying the binding of murine antibody SHH002 to the immunogen rhFzd 7. FIG. 1A: the immunogen recombinant protein rhFzd7 is transferred to an NC membrane after SDS-PAGE electrophoresis, and is subjected to early spring red staining, and lanes 1-5 are rhFzd7 protein. FIG. 1B: the different lanes of the NC membrane were incubated with antibodies, lanes 1-4 with # 1, # 2, # 3, and # 11 ascites, respectively, lane 5 with mouse serum (as a positive control), followed by goat anti-mouse-HRP-labeled secondary antibody, and finally DAB light-shielded color development. Target bands are detected in the four ascites plants, wherein the ascites 1#, 2# and 3# have better effects. The band indicated by the arrow is the position of the target band, and the lower band is suspected to be a protein degradation binding band (the picture has a distinct band at the position after the protein is stained with Lichun red).

FIG. 2 shows the results of SDS-PAGE electrophoresis on purified antibodies # 1, # 2 and # 3. Lanes 1-7 are non-reducing samples and lanes 8-10 are reducing samples. Lanes 1-3: antibodies # 1, # 2, and # 3; lane 4: an IPI positive control antibody; lane 5: blank; lane 6: N/A quality control; lane 7: control BSA; lanes 8-10: antibodies # 1, # 2, and # 3; lane 11: an IPI positive control antibody; lane 12: blank; lane 13: control BSA.

FIG. 3 is a graph showing the reaction of indirect ELISA to determine the affinity of murine anti-Fzd 7 antibody. FIG. 3A: reaction profile of antibody # 1 with rhFzd 7; FIG. 3B: graph of the reaction of antibody # 2 with rhFzd 7; graph of the reaction of antibody # 3 with rhFzd 7. The measured antibody affinities were respectively for # 1 antibody: 4.06X 10^-11M; antibody # 2: 9.74X 10^-11M; 3# antibody: 7.59X 10^-11M。

FIG. 4 is a graph fitted with the dynamic process of binding and dissociation of antigen (rhFzd7) antibody (SHH002) measured by Fortebio instrument. 600s to 1000s indicate binding and 1000s to 1580s indicate dissociation, and 2 curves in the figure represent, from top to bottom, the binding of the antibody to rhFzd7 at a concentration of 750nM, 300nM, respectively. Two curves were obtained for each rhFzd7 concentration, the green curve was fit by a computer, and the other curve was the actual measurement curve of the instrument.

FIG. 5 is a nucleic acid electrophoretic map of PCR amplification of the SHH002 heavy-light chain V region gene. The PCR products were electrophoresed through 1.5% agarose, lanes 1-5 correspond to different primer PCR results, respectively; lane 1: SHH002 heavy chain variable region PCR results, lanes 2-5: as a result of PCR of the SHH002 light chain variable region, the gene of interest was found to be at the 330bp position.

FIG. 6 shows the alignment of the SHH002 VH and VL sequences to the Human Germline database sequence. FIG. 6A: as a result of the alignment of the SHH002 VL sequence with the Human Germline database sequence, the VL has the highest homology with the Human Germline IGKV1 large-class sequence. FIG. 6B: as a result of alignment of the SHH002 VH sequence with the Human Germline database sequence, the VH and Human Germline IGHV1 have the highest homology. In the figure, red marked sequences in the VH and VL sequences are CDR1, CDR2 and CDR3 in sequence.

FIG. 7 shows the results of the SHH002 potential modification site analysis and immunogenicity analysis. FIG. 7A: as a result of analysis of potential modification sites of SHH002, potential modification sites of VH/VL are predicted (double-dashed marker sequences, and single-dashed marker sequences indicate CDR1, CDR2, and CDR3 in this order). FIG. 7B: as a result of the SHH002 immunogenicity analysis, VH/VL is predicted to have partial strong immunogenicity sites (in-frame marked sequences, the underlined marked sequences of CDR1, CDR2 and CDR3 are indicated by the underlined marked sequences, and the underlined double-lined sequences are indicated by the Hot Spot).

Fig. 8 shows the results of structural simulation of SHH002 using the bioinformatics software discovery. FIG. 8A: the variable region structure is simulated, the reliability is 98 percent, the left side is a heavy chain variable region, and the right side is a light chain variable region; FIG. 8B: variable region CDR structure simulation, CDR region (grey marker) position in structural model; FIG. 8C: surface charge simulation diagram of the variable region, the surface charge diagram describes: red is negative charge, blue is positive charge, arrows point to the CDR regions (left), white oval boxes are labeled CDR regions (right).

FIG. 9 shows the result of SDS-PAGE electrophoresis of chimeric antibody SHH 002-C. Lanes 1-4 are non-reducing samples and lanes 5-7 are reducing samples. Lane 1: N/A quality control; lane 2: SHH 002-C; lanes 3, 4: an IPI positive control antibody; lane 5: N/A quality control; lane 6: SHH 002-C; lane 7: IPI positive control antibody.

FIG. 10 is a graph fitted with the kinetics of dissociation of binding of antigen (rhFzd7) antibody (SHH002-C) measured by Fortebio apparatus. 600s to 1000s indicate binding and 1000s to 1580s indicate dissociation, and 3 curves in the figure represent, from top to bottom, the binding of the antibody to rhFzd7 at a concentration of 300nM, 100nM, and 33.33nM, respectively. Two curves were obtained for each rhFzd7 concentration, the blue curve was fit by a computer, and the other curve was the actual measurement curve of the instrument.

FIG. 11 shows the result of SDS-PAGE of the humanized antibody SHH002-hu targeted Frizzled 7. Lanes 1-7 are non-reducing samples and lanes 8-14 are reducing samples. Lane 1: N/A quality control; lane 2: an IPI positive control antibody; lanes 3-7: SHH002-hu 1-SHH 002-hu 5; lane 8: N/A quality control; lane 9: an IPI positive control antibody; lanes 10-14: SHH002-hu 1-SHH 002-hu 5.

FIG. 12 shows the results of SHH002-hu SEC-HPLC (size exclusion chromatography) detection of the humanized antibody against Frizzled7, and FIG. 12 includes FIGS. 12-1 and 12-2. In FIG. 12-1A: an SEC-HPLC chromatogram of the IPI positive control antibody has the reference product peak time of 11.2min, symmetrical peak pattern, stable base line and system adaptability; b in FIG. 12-1: an SEC-HPLC chromatogram of a murine antibody SHH002 shows that the peak time of a reference product is 9.5min, the peak patterns are symmetrical, the base line is stable, and the system adaptability is passed; C-G in FIGS. 12-1 and 12-2: the SEC-HPLC chromatograms of humanized antibodies SHH002-hu 1-SHH 002-hu5 targeting Frizzled7 respectively have peak output time of 9.7min, 10.8min, 9.5min, 9.6min and 9.5min respectively, the peak patterns are symmetrical, the base line is stable, and the system adaptability is passed.

FIG. 13 is a graph showing the results of ELISA detection of the binding of SHH002-hu to rhFzd 7. Coating rhFzd7 on enzyme label plate, adding primary antibodies (SHH002 and SHH002-hu 1-SHH 002-hu5) with different concentrations (8 concentration gradients), diluting with primary 10 μ g/ml by 3 times gradient, incubating at room temperature for 1h, adding corresponding secondary antibody, developing with TMB, and performing OD (optical Density) color development₄₅₀The absorbance was read. Obtaining a fitting curve graph of a light absorption value along with the change of the antibody concentration through Excel software, and calculating to obtain the EC of the antibody combined with the antigen₅₀The value is obtained. The results showed that SHH002 binds to antigen-bound EC₅₀The value was 0.53. mu.g/ml; EC of SHH002-hu 1-SHH 002-hu3₅₀The values were 0.30. mu.g/ml, 0.23. mu.g/ml, 0.19. mu.g/ml, respectively, and EC compared with SHH002₅₀The value is not reduced; SHH002-hu4 and SHH002-hu5 failed to bind antigen effectively, indicating a loss of affinity.

FIG. 14 is a graph fitted with the dynamic process of binding and dissociation of an antigen (rhFzd7) antibody (SHH002-hu 1-SHH 002-hu3) measured by a Fortebio instrument. FIGS. 14A-C are the dissociation curves for binding of SHH002-hu1, SHH002-hu2, SHH002-hu3 to rhFzd7, respectively, with binding indicated by 0s to 300s and dissociation indicated by 300s to 600s, and the 4 curves in the figure, from top to bottom, show binding of the antibody to rhFzd7 at concentrations of 400nM, 100nM, 25nM and 6.25nM, respectively. Two curves were obtained for each rhFzd7 concentration, the red curve was obtained by computer fitting, and the other curve was the actual curve measured by the instrument.

FIG. 15 is a graph of the binding dissociation kinetics process fit of SHH002-hu1 and protein rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc measured by Fortebio instrument. FIGS. 15A-D are the dissociation curves for binding of the SHH002-hu1 antibody to rhFzd1-Fc, rhFzd2-Fc, rhFzd5-Fc, and rhFzd8-Fc proteins, respectively, with binding at 0s to 300s and dissociation at 300s to 600s, and the 4 curves in the figure from top to bottom represent binding of the antibody to rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc at concentrations of 400nM, 100nM, 25nM and 6.25nM, respectively. Each protein concentration has two curves, the fluctuation curve is the actual measurement curve of the instrument, and the stable curve is obtained by computer fitting. The results in the figure show that no significant binding of SHH002-hu1 to rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc protein was observed.

FIG. 16 is a graph showing the results of detecting the binding of SHH002-hu1 to cell surface Fzd7 by immunofluorescence assay, and FIG. 16 includes FIGS. 16-1 and 16-2. In FIG. 16-1A: western blot detection of the expression of Fzd7 in tumor cells (triple negative breast cancer cell lines: MDA-MB-231, MDA-MB-468; non-small cell lung cancer cell lines: H1299, H1975, A549). The detection Antibody was Rabbit Anti-Human Fzd7 Antibody (purchased from Abcam), and HEK293T cells were used as normal cell controls. B in FIG. 16-1: results of cellular immunofluorescence assay for SHH002-hu1 binding to cells overexpressing Fzd7 HEK293T (Fzd7 OE) are shown, with HEK293T untransfected and HEK293T transfected with empty vector (GFP tag) as controls at bar 50 μm. C in FIG. 16-2: the results of the cytoimmunofluorescence method for detecting the binding of SHH002-hu1 with MDA-MB-231, MDA-MB-468, H1299, H1975 and A549 cells show that bar is 50 μm.

FIG. 17 is a graph showing the results of immunohistochemical methods to detect the binding of SHH002-hu1 to Fzd7 in tumor tissue. The two right panels show the results of the examination of tumor tissues in patients with non-small cell lung cancer, and the two left panels show the results of the examination of tissues beside the cancer. The upper two panels show the results of detection of the detection Antibody (Rabbit Anti-Human Fzd7 Antibody, purchased from Abcam), and the lower two panels show the results of detection of SHH002-hu 1. Bar 20 μm.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1: preparation of hybridoma cell of anti-Fzd 7 monoclonal antibody

1. Immunizing animals: the immune animal used in the invention is a BALB/c mouse, which is provided by the animal experiment center of the first human civil hospital (Songjiang) in Shanghai, the immune adjuvant is purchased from Sigma company, and the immunogen is recombinant human Fzd7 extracellular CRD region protein (rhFzd 7). BALB/c mice were immunized using conventional immunization protocols. Immunization was performed once every four weeks after priming, three times in total. Detecting the antibody titer of the immune mouse serum by using an indirect ELISA method, selecting a mouse with the highest immune titer detection value for additional immunization once, and taking the spleen of the mouse for cell fusion three days later.

2. Cell fusion:

1) preparation of myeloma cells (Sp 2/0): two weeks before fusion Sp2/0 was recovered and placed in 5% CO₂And subculturing in a constant temperature incubator at 37 ℃. In the culture process, 8-nitrogen guanine is added into the culture solution and is continuously treated for 3 times, so that Sp2/0 cells are more sensitive to HAT culture solution. The myeloma cells were passaged once a day before the fusion so that they were in the logarithmic growth phase on the day of the fusion.

2) Preparation of feeder cells: one day before cell fusion, taking abdominal cavity macrophages of healthy BALB/c mice of 3-4 weeks old as feeder cells, and adjusting the cell concentration to l-5 × 10⁵cells/mL were transferred to 96-well cell culture plates at 100. mu.L/well.

3) Preparation of splenocytes: taking a mouse which is boosted for immunization three days before, taking the spleen aseptically, collecting spleen cell suspension, and counting the spleen cells for later use.

4) Fusion of cells: sp2/0 cells and immune spleen cell suspension were mixed well, cells were fused using polyethylene glycol, and then transferred to 96-well cell culture plates in 5% CO₂And culturing in a constant temperature incubator at 37 ℃.

5) Screening of hybridoma cells: after the fusion, the growth of the hybridoma cells is observed, and the supernatant is aspirated when the cells grow to 1/4-1/3 of the bottom of the well for indirect ELISA detection. P/N is more than or equal to 2.5 as a positive judgment standard, the serum of the non-immune mouse is used as a negative control, and the antibody diluent is used as a blank control. The cells that were detected as positive were subjected to amplification culture and simultaneously to cloning culture.

6) Cloning and culturing hybridoma cells: cloning culture was performed by limiting dilution and indirect ELISA was performed on cell supernatants until cells grew to l/4-1/3 of the bottom of the wells. Positive wells with a low number of clones and a high OD450nm value were selected and recloned. And (3) cloning for 3-4 times until the positive rate of all cloned cell holes reaches 100%, determining to obtain the hybridoma cell strain secreting the specific monoclonal antibody, and performing amplification culture and cryopreservation in time.

Example 2: preparation and identification of murine anti-Fzd 7 antibody

1. Ascites collection: after the hybridoma cells are subjected to amplification culture, the abdominal cavity of the mouse is injected, the abdominal cavity of the mouse is obviously swollen 7 to 10 days later, and ascites is collected at the moment. Centrifuging at 4 deg.C for 20min at 5000g, removing cell debris and oil in ascites (Western blot for detecting the combination of four ascites strains and antigen, FIG. 1 shows that 1#, 2#, 3# ascites are combined well with antigen rhFzd7), adding equal volume of glycerol, and storing at-20 deg.C.

2. Purification of murine anti-Fzd 7 antibody: the antibody was purified using a Protein G affinity column.

The method comprises the following specific steps:

1) the Protein G affinity chromatography column was washed with 10 times the column volume.

2) The Protein G affinity chromatography column was washed with 10 column volumes of 20mM sodium phosphate buffer (pH 7.0).

3) The desired purified sample is pumped into the chromatography column.

4) Eluting with 100mM glycine buffer solution (pH 3.5, pH 2.7), and collecting the elution peak; the collection was neutralized with 1M Tris buffer (pH 9.0).

5) Antibodies were initially identified by SDS-PAGE electrophoresis (FIG. 2). The results show that: a target band appears at 150KD under the non-reduction condition, and the main band is obvious; the bands (heavy and light chains) appeared at 50kD and 25kD, respectively, under reducing conditions; the 1#, 2#, 3# antibodies heavy and light chains were expressed correctly and assembled completely.

3. Characterization of murine anti-Fzd 7 antibody

1) Immunoglobulin subtype identification:

the mouse monoclonal antibody subtype identification kit of Wuhan Sanying biotechnology limited is adopted to identify the 1#, 2#, and 3# cell strains secreting antibody subtypes. The results were: the heavy chain subtype of the 1# antibody is IgG2b, and the light chain subtype is Kappa; the heavy chain subtype of the 2# antibody is IgG1, and the light chain subtype is Kappa; the 3# antibody heavy chain subtype is IgG2a and the light chain subtype is Kappa.

2) The indirect ELISA method is used for detecting the affinity of the murine anti-Fzd 7 antibody:

A. coating the enzyme label plate with rhFzd7 at the concentration of 1, 0.5 and 0.25 mu g/mL; B. measuring the antibody titer of different antigen coating concentrations by using an indirect ELISA method; C. taking the logarithmic value of the monoclonal antibody concentration as an abscissa and the OD450 as an ordinate, and drawing a reaction curve; D. the OD value of the upper flattening section of each curve is taken as OD-100, the molar concentration (mol/L) of the monoclonal antibody corresponding to OD-50 is found, Ab1, Ab0.5 and Ab0.25 are found, and according to the formula:

K1＝1/(2×[Ab0.5]－[Ab1])

K2＝1/(2×[Ab0.25]－[Ab0.5])

K3＝1/(2×[Ab0.25]－[Ab1])

and calculating to obtain three K values, and taking the average number as the antibody affinity constant. The antibody affinities measured by indirect ELISA were 1# antibody: 4.06X 10^-11M; antibody # 2: 9.74X 10^-11M; 3# antibody: 7.59X 10^-11And M. The reaction curve measured by ELISA method is shown in FIG. 3.

3) The 1# antibody with the highest affinity (namely SHH002) is selected, and the affinity is further verified by a BLI method:

A. using an instrument: the protein interactor Fortebio Octet Red 96.

B. Solution preparation: KB buffer: 0.1% BSA, 0.05% Tween 20 dissolved in PBS pH 7.2; antibody working solution: the Biotin marked SHH002 antibody is prepared into 10 mu g/mL by KB buffer; antigen working solution: rhFzd7 was formulated with KB buffer at 300nM and 750 nM.

C. The operation process comprises the following steps: and opening a Fortebio instrument and related software, and selecting an advanced Kientics experiment mode. Firstly, an Anti-Biotin Sensor is used for capturing an antibody SHH002 coupled with Biotin, then the captured antibody is used for combining antigens rhFzd7 with different concentrations, then dissociation is carried out in a buffer solution, and finally the affinity kinetic constant KD of antigen-antibody combination is calculated through an instrument algorithm.

The experimental results showed that the binding constant ka (1/Ms) of SHH002 to rhFzd7 was 2.85 × 10⁴Dissociation constant kd (1/s)<1×10^-7Affinity constant KD<1×10^-12M, binding dissociation kinetics process the fitted graph is shown in fig. 4. Compared with the indirect ELISA result, the affinity constant of SHH002 to antigen is at least one order of magnitude smaller, which shows that the affinity of SHH002 measured by BLI method is higher; the reason for this analysis may be that in the BLI method, the result isThe antibody is coupled on the chip to capture the antigen in the mobile phase, and compared with the indirect ELISA method in which the antigen is coated on an ELISA plate to capture the antibody, the steric hindrance caused by the combination of the antibody and the antigen is avoided more effectively.

The hybridoma cell line expressing the 1# antibody (SHH002) in the example is classified and named as hybridoma cell line FL253-1 expressing anti-Fzd 7 murine antibody SHH002, and the cell line is preserved for 10, 30 and 2019, and the preservation organization is as follows: china Center for Type Culture Collection (CCTCC) with the preservation number: CCTCC NO is C2019279, and the preservation address is Wuhan university in Wuhan, China.

Example 3: cloning and sequencing of heavy-light chain V region gene of 1# hybridoma cell strain

1. Extracting, amplifying and initially identifying Fzd7 monoclonal antibody SHH002 heavy-light chain V region gene

1) Extracting total RNA: collecting hybridoma cells in logarithmic growth phase, extracting total RNA with RNA extraction kit (Shanghai bio-organism), dissolving in 20-50 μ L RNase-free water, and storing at-70 deg.C.

2) And (3) cDNA synthesis: the first strand of cDNA was synthesized by reverse transcription using total RNA as a template (reverse transcription kit was purchased from Shanghai Probiotics).

3) PCR amplification of antibody heavy and light chain V region genes:

A. polymerase: primerstar high fidelity polymerase

B. Primer: the degenerate primer is designed according to the upstream and downstream gene sequences of the heavy and light chain V region of the hybridoma cell strain antibody, and the sequences of two upstream primers VH F1 and VH F2 and a downstream primer VH R in the heavy chain V region and an upstream primer VL F and a downstream primer VL R in the light chain V region are respectively as follows:

VH F1：ctt ccg gaa ttc sar gtn mag ctg sag tc

VH F2：ctt ccg gaa ttc sar gtn mag ctg sag tcw gg

VH R：gga aga tct ata gac aga tgg ggg tgt cgt ttt ggc

VL F：gg gag ctc gay att gtg mts acm car wct mca

VL R：ggt gca tgc gga tac agt tgg tgc agc atc

(degenerate codon specification: r ═ a, g; y ═ c, t; m ═ a, c; s ═ c, g; w ═ a, t)

C. PCR reaction systems were configured to amplify antibody heavy and light chain V region genes (30 Total System, 27 reaction systems PCR) according to Table 1

TABLE 1 PCR reaction System and reaction conditions

And D, carrying out 1.5% agarose electrophoresis on the PCR products, wherein the target bands are all bands with the size of about 330bp (shown in a figure 5).

E. Adding adenine (A) at 3' end of gel recovered DNA product

2. Sequencing and analysis of anti-Fzd 7 monoclonal antibody heavy and light chain V region gene

1) The PCR product obtained in 1 was ligated to the pMD18-T vector and the ligated product was transformed into competent DH 5. alpha. cells, and positive clones were selected for sequencing.

2) The sequencing result shows that the 1# hybridoma cell strain antibody V region gene is successfully obtained. The anti-SHH 002 antibody heavy and light chain V region genes were analyzed using the IMGT-VQUEST database.

The heavy chain variable region of SHH002 of the monoclonal antibody targeted to Frizzled7 includes the following three CDRs: CDR1 shown in SEQ ID No.5, CDR2 shown in SEQ ID No.6, and CDR3 shown in SEQ ID No. 7; the light chain variable region comprises the following three complementarity determining regions CDRs: CDR1 ' shown in SEQ ID No.8, CDR2 ' shown in SEQ ID No.9, and CDR3 ' shown in SEQ ID No. 10. The heavy chain variable region nucleotide sequence of the targeted Frizzled7 monoclonal antibody is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2. The nucleotide sequence of the light chain variable region of the targeted Frizzled7 monoclonal antibody is shown as SEQ ID No.3, and the amino acid sequence is shown as SEQ ID No. 4.

Example 4: humanized design of SHH002 antibody

1. The VH and VL of SHH002 were aligned to the Human Germline sequence and the results are shown in FIG. 6.

2. SHH002 potential modification site analysis predicted VH/VL potential modification sites, and the results are shown in fig. 7A.

3. SHH002 immunogenicity analysis predicted VH/VL to have partially strong immunogenic sites (B cell immunogenicity: weak, T cell immunogenicity: medium), with the results shown in FIG. 7B.

4. SHH002 sequence analysis results:

1) VH has the highest sequence homology with the human Germline IGHV1 large family: FR 1-3 contains 26 murine sites (V gene) and FR 4 contains 0 murine sites (J gene).

2) VL shares the highest sequence homology with the human Germline IGKV1 major class: FR 1-3 contains 24 murine sites (V gene) and FR 4 contains 0 murine sites (J gene).

3) IGHV1/IGKV1 with the highest homology is selected as a humanized design template, IGHV1/IGKV1 is a common pairing form, and the drug occupancy rate on the market is high.

5. SHH002 was structurally simulated using the bioinformatics software discovery:

1) simulation of variable region Structure (FIG. 8A)

2) Simulation of variable CDR Structure (FIG. 8B)

3) Variable zone surface charge simulation diagram (FIG. 8C)

4) SHH002 analogue structure results analysis:

shh002 sequence homology (Identity) to antibody structure database was close to 90%, and simulated structure Confidence (Confidence) was over 95%.

Shh002 structural simulation results showed common antibody structures without specific secondary-tertiary structure.

Shh002 murine antibody has a uniform surface charge distribution and a low probability of forming aggregates or aggregates.

D. And selecting two structure models with the highest credibility to guide the humanized design.

6. Based on the structural model selected in 5 as guidance, murine amino acids that are critical to the structural stability of the antibody were retained, and unimportant sites were mutated to humanized amino acids according to the template IGHV1/IGKV1, to obtain the following 5 humanized design schemes (SHH002-hu1, SHH002-hu2, SHH002-hu3, SHH002-hu4, SHH002-hu 5).

SHH002-hu1, the heavy chain variable region amino acid sequence is shown as SEQ ID No.11, and the variable region amino acid sequence is shown as SEQ ID No. 12. SHH002-hu1, and compared with murine antibody SHH002, its humanization degree is as high as 96.5%.

The base sequence of the heavy chain of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.21, wherein the constant region is from 346 th to 1335 th of the base sequence shown as SEQ ID No. 21. The light chain base sequence of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.22, wherein the constant region is from 334 th to 654 th of the base sequence shown as SEQ ID No. 22.

The heavy chain amino acid sequence of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.23, wherein the constant region is from 116 th to 445 th positions of the base sequence shown as SEQ ID No. 23. The light chain amino acid sequence of the humanized antibody SHH002-hu1 targeting Frizzled7 is shown as SEQ ID No.24, wherein the constant region is from 112 th to 218 th of the base sequence shown as SEQ ID No. 24.

SHH002-hu2, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID No.13, and the light chain variable region amino acid sequence is shown as SEQ ID No. 14.

SHH002-hu3, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID No.15, and the light chain variable region amino acid sequence is shown as SEQ ID No. 16.

SHH002-hu4, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID No.17, and the light chain variable region amino acid sequence is shown as SEQ ID No. 18.

SHH002-hu5, wherein the heavy chain variable region amino acid sequence is shown as SEQ ID No.19, and the light chain variable region amino acid sequence is shown as SEQ ID No. 20.

Example 5: expression and identification of anti-Fzd 7 chimeric antibody SHH002-C

1. The key first step in antibody humanization is the construction of chimeric antibodies: the heavy chain constant region of SHH002 was substituted for the heavy chain constant region of human IgG1, and the light chain constant region of SHH002 was substituted for the light chain constant region of human IgG1 Kappa. The recombinant heavy and light chain genes were linked to pCDNA3.4 vectors, respectively, co-transfected with EXPICHO-S cells (purchased from Invitrogen), and cell culture supernatants were collected and purified by separation on Protein G columns. The protein of interest was detected by SDS-PAGE (FIG. 9), and the results showed that: a target band appears at 150KD under the non-reduction condition, and the main band is obvious; the bands (heavy and light chains) appeared at 50kD and 25kD, respectively, under reducing conditions; indicating that the SHH002-C heavy and light chain is correctly expressed and completely assembled.

2. Determination of affinity constant of SHH002-C and rhFzd7 by BLI method

1) Using an instrument: same as example 2

2) Solution preparation: KB buffer: 0.1% BSA, 0.05% Tween 20 dissolved in PBS pH 7.2; antibody working solution: the SHH002-C antibody is prepared into 10 mu g/mL by using KB buffer; antigen working solution: rhFzd7 was formulated with KB buffer at 300nM, 100nM and 33.33 nM.

3) The operation process comprises the following steps: and opening a Fortebio instrument and related software, and selecting an advanced Kientics experiment mode. Firstly, using Anti-Human Fab-CH 12 nd Generation Sensor to capture SHH002-C, then using the captured antibody to bind antigen rhFzd7 with different concentrations, then dissociating in buffer solution, and finally calculating the affinity kinetic constant KD of antigen-antibody binding by an instrument algorithm.

The results of the experiment showed that the binding constant ka (1/Ms) of SHH002-C to rhFzd7 was 8.24 × 10⁵Dissociation constant kd (1/s) 2.01X 10^-5Affinity constant KD of 2.44X 10^-11M (binding dissociation kinetics process fitted graph shown in fig. 10), the affinity change was within the acceptable range compared to the murine antibody SHH 002.

The successful construction and expression of the chimeric antibody SHH002-C are identified as further humanization of SHH002, and a solid foundation is laid.

Example 6: expression and identification of anti-Fzd 7 targeting Frizzled7 humanized antibody SHH002-hu

1. Connecting 5 humanized sequences of the SHH002 heavy chain variable region in the example 4 with a human IgG1 heavy chain constant region, and connecting 5 humanized sequences of the SHH002 light chain variable region with a human IgG1 Kappa light chain constant region to obtain 5 pairs of recombinant humanized heavy and light chain genes; the 5 heavy and light chain genes are respectively linked to pCDNA3.4 vectors, and then are co-transfected with EXPICHO-S cells, cell culture supernatant is collected, and 5 kinds of target Frizzled7 humanized antibodies SHH002-hu1, SHH002-hu2, SHH002-hu3, SHH002-hu4 and SHH002-hu5 are obtained by separation and purification through a Protein G column. The protein of interest was detected by SDS-PAGE (FIG. 11), and the results showed that: a target band appears at 150KD under the non-reduction condition, and the main band is obvious; the bands (heavy and light chains) appeared at 50kD and 25kD, respectively, under reducing conditions; indicating that the SHH002-hu heavy and light chains are correctly expressed and completely assembled.

2. The purity of the humanized antibody targeting Frizzled7 was further verified using SEC-HPLC (size exclusion chromatography) method (using IPI positive protein and murine antibody SHH002 as control). High molecular polymer substances, monomer substances and low molecular weight substances in the antibody purification sample have different retention times in a molecular exclusion chromatographic column, and the content of each substance is determined according to the ratio of peak areas by integrating each substance which generates peaks at different times in a chromatogram. The experimental procedure was as follows:

1) using an instrument: agilent HPLC 1100 (from Agilent Japan)

2) Size exclusion chromatography column: TSKgelG3000SWXL (7.8X 300, available from Tosoh Co., Japan)

3) Column temperature: 20 deg.C

4) Sample introduction amount: 5 μ L

5) Detector parameters: the detection wavelength is 280nm, the bandwidth is 16nm, the reference wavelength is 360nm, the bandwidth is 100nm, and the peak width (response time) >0.1min (2 s); a slit is 4 nm; the negative absorbance baseline was 100 mAU.

6) System adaptability criteria: and the reference product protein peak time is 11.2min, the peak patterns are symmetrical, and the base line is stable, so that the system adaptability is passed.

The results show that the monomer peak content of the murine antibody SHH002 is greater than 90% in both the murine antibody SHH002 and the 5-strain humanized antibody SHH002-hu targeting the Frizzled7, as shown in FIG. 12.

3. Determination of EC binding of SHH002-hu to rhFzd7 by ELISA method₅₀Value (SHH002 as control)

1) The rhFzd7 protein was coated on an ELISA plate overnight at 4 ℃.

2) The plates were washed with PBST and blocked for 1h at room temperature.

3) PBST plates were washed and incubated with primary antibody for 1h at room temperature (SHH002, SHH002-hu 1-SHH 002-hu 56 antibodies were each set to 8 concentration gradients, 10. mu.g/ml in the first well, followed by 3-fold gradient dilution).

4) PBST plates were washed and incubated with Anti-Mouse-IgG-Fc-HRP/Anti-Huamn-IgG-Fc-HRP.

5) PBST plate washing, TMB color development, OD₄₅₀And reading the data.

The results showed that SHH002 binds to antigen-bound EC₅₀The value was 0.53. mu.g/ml; EC of SHH002-hu 1-SHH 002-hu3₅₀The values were 0.30. mu.g/ml, 0.23. mu.g/ml, 0.19. mu.g/ml, respectively, and EC compared with SHH002₅₀The value is not reduced; SHH002-hu4 and SHH002-hu5 failed to bind antigen effectively, indicating a loss of affinity. The results of the experiment are shown in FIG. 13.

4. Determination of affinity constant of SHH002-hu and rhFzd7 by BLI method

1) Using an instrument: same as example 5

2) Solution preparation: KB buffer: 0.1% BSA, 0.05% Tween 20 dissolved in PBS pH 7.2; antibody working solution: SHH002-hu1, SHH002-hu2 and SHH002-hu3 antibodies were prepared to 10. mu.g/mL with KB buffer, respectively; antigen working solution: rhFzd7 was formulated with KB buffer at 400nM, 100nM, 25nM and 6.25 nM.

3) The operation process comprises the following steps: and opening a Fortebio instrument and related software, and selecting an advanced Kientics experiment mode. Firstly, an Anti-Human Fab-CH 12 nd Generation Sensor is used for capturing an SHH002-hu antibody, then the captured antibody is used for binding antigens rhFzd7 with different concentrations, then dissociation is carried out in a buffer solution, and finally the affinity kinetic constant KD of antigen-antibody binding is calculated through an instrument algorithm.

The results of the experiments are shown in table 2, and the binding dissociation kinetics process fitted graph is shown in fig. 14. KD represents the value of affinity, kon represents the value of binding, kdis represents the value of dissociation, R²Representing the correlation coefficient between the fitted curve and the binding dissociation curve, a closer value to 1 indicates that the result calculated by fitting is closer to the true result. When the affinity of the antigen and the antibody is high, the dissociation time needs to be long, and the longer the dissociation time is, the larger the baseline fluctuation is due to the limitation of a Fortebio instrument, so that the specific dissociation constants of SHH002-hu1, SHH002-hu2 and SHH002-hu3 are not calculated due to the higher affinity ratioNumerical values, and therefore affinity constants KD, are shown to be less than 1.0X 10^-12And M. No loss of affinity compared to murine antibody.

TABLE 2 SHH002-hu affinity kinetics assay results

Example 7: affinity detection of SHH002-hu1 with other members of Fzd receptor family

Combining the results of the ELISA and BLI experiments, SHH002-hu1 showed the greatest reaction value (absorbance) for binding to rhFzd7 in the ELISA assay and the greatest Kon value in the BLI assay, and SHH002-hu1 was selected for further study.

The CRD region sequence of Fzd7 has over 40% homology with other members of Fzd family, especially with Fzd1 and Fzd2, and its homology is up to 80%. OMP-18R5 is the antibody screened by using Fzd7CRD fragment protein as antigen, and research shows that the antibody has cross reaction with Fzd1/2/5/8, which results in off-target risk of the antibody in the treatment process. Therefore, the present invention focuses on whether SHH002-hu1 could cross-react with Fzd1/2/5/8 (4 of Fzd receptor family with the highest homology to Fzd7CRD region), and uses BLI method to determine the affinity of SHH002-hu with rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc (purchased from R & D Systems) as follows:

1) using an instrument: same as example 5

2) Solution preparation: KB buffer: 0.1% BSA, 0.05% Tween 20 dissolved in PBS pH 7.2; antibody working solution: SHH002-hu1 was made up to 10. mu.g/mL with KB buffer; mobile phase working solution: rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc was formulated with KB buffer at 400nM, 100nM, 25nM and 6.25nM, respectively.

3) The operation process comprises the following steps: and opening a Fortebio instrument and related software, and selecting an advanced Kientics experiment mode. Firstly, an Anti-Human Fab-CH 12 nd Generation Sensor is used for capturing an SHH002-hu1 antibody, then the captured antibody is used for combining rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc protein with different concentrations, then dissociation is carried out in a buffer solution, and finally a combined dissociation simulation curve is obtained.

The results showed no significant binding of SHH002-hu1 to either rhFzd1-Fc/rhFzd2-Fc/rhFzd5-Fc/rhFzd8-Fc protein, as shown in FIG. 15.

Example 8: detection of binding ability of SHH002-hu1 to cell surface Fzd7

Example 6 demonstrates that the humanized antibody SHH002-hu1 targeting Frizzled7 has high affinity with recombinant human Fzd7 protein, and then the invention uses cell immunofluorescence to demonstrate whether SHH002-hu1 can bind to Fzd7 naturally expressed on the cell surface, the specific steps are as follows:

1. constructing a HEK293T cell model for over-expressing human Fzd7 protein (transmembrane and extracellular region): the transmembrane region and extracellular region gene (shown as SEQ ID No. 25) of the human Fzd7 protein and a lentiviral vector

pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO (purchased from Hanbio Biotechnology) was ligated to obtain recombinant plasmid HBLV-h-FZD7-3 FLAG-GFP-PURO, which was lentivirally packaged with HBLV-GFP-PURO (GFP control), and HEK293T cells were infected.

2. The Western blot method was used to screen 5 tumor cells (triple negative breast cancer cell lines: MDA-MB-231, MDA-MB-468; non-small cell lung cancer cell lines: H1299, H1975, A549) highly expressing Fzd7, and the results are shown in FIG. 16A.

3. HEK293T (blank cell group, GFP control group, Fzd7 overexpression group), MDA-MB-231, MDA-MB-468, H1299, H1975, A549 cells were plated on six-well plates, the confluency of the cells reached 80-90%, and immunofluorescence assay was performed using SHH002-hu1 as primary antibody and Goat Anti-Human IgG H & L (Alexa Fluor 647, purchased from Abcam) as secondary antibody.

The experimental result shows that SHH002-hu1 has no obvious combination with blank HEK293T cells and GFP control HEK293 cells, and has obvious combination with HEK293T cells over expressing Fzd 7; SHH002-hu1 also effectively bound tumor cells (MDA-MB-231, MDA-MB-468, H1299, H1975, A549) with high surface expression of Fzd7, as shown in FIG. 16B, C. In conclusion, SHH002-hu1 can specifically bind to cell surface Fzd 7.

Example 9: detection of binding of SHH002-hu1 to Fzd7 in tumor tissue

Example 8 demonstrates specific binding of SHH002-hu1 to native Fzd7 on the surface of tumor cells, and then the present invention uses Immunohistochemistry (IHC) method to detect the expression of Fzd7 in tumor tissues of non-small cell lung cancer patients (rabbitti-Human Fzd7 Antibody, purchased from Abcam, using detection Antibody), and para-carcinoma tissues as control; and SHH002-hu1 was tested for binding to Fzd7 in tumor tissue using the same method. The detection antibody immunohistochemistry result shows that the non-small cell lung cancer tumor tissue has high expression of Fzd7, but has almost no expression in the paracarcinoma tissues; the histochemical results of SHH002-hu1 showed that it could bind effectively to lung cancer tissues with high expression of Fzd7 and hardly to paracarcinoma tissues, as shown in FIG. 17. The results suggest that SHH002-hu1 can effectively detect Fzd7 in tumor tissues and has potential tumor tissue specific targeting.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> Shanghai health medical college

<120> targeting Frizzled7 humanized antibody and preparation method and application thereof

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

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<213> Mus musculus

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tcctgcaagg cttctggcta cacctttact acctacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gaagtggtta taccaattac 180

aatcagaaat tcaaggacaa ggccacagtg actgcagaca aatcctccag cacagcctac 240

atgcaactga acagcctgac atctgaggac tctgcagtct attattgtgc aagaaggggc 300

tattttgact actggggcca agggaccacg gtcaccgtct cctca 345

<210> 2

<211> 115

<212> PRT

<213> Mus musculus

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Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Val Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 3

<211> 307

<212> DNA

<213> Mus musculus

<400> 3

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atatcctgca gagccagtga aagtgttgat gattatggca atacttttat gcactggtac 120

caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattgat 240

cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatccctac 300

acgttcg 307

<210> 4

<211> 111

<212> PRT

<213> Mus musculus

<400> 4

Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp

65 70 75 80

Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 5

<211> 8

<212> PRT

<213> Mus musculus

<400> 5

Gly Tyr Thr Phe Thr Thr Tyr Thr

1 5

<210> 6

<211> 10

<212> PRT

<213> Mus musculus

<400> 6

Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr

1 5 10

<210> 7

<211> 6

<212> PRT

<213> Mus musculus

<400> 7

Arg Arg Gly Tyr Phe Asp

1 5

<210> 8

<211> 12

<212> PRT

<213> Mus musculus

<400> 8

Glu Ser Val Asp Asp Tyr Gly Asn Thr Phe Met His

1 5 10

<210> 9

<211> 4

<212> PRT

<213> Mus musculus

<400> 9

Leu Ala Ser Asn

1

<210> 10

<211> 7

<212> PRT

<213> Mus musculus

<400> 10

Gln Gln Asn Asn Glu Asp Pro

1 5

<210> 11

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 12

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly Asp Arg

1 5 10 15

Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr Gly Asn

20 25 30

Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser Ser Val

65 70 75 80

Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Glu Asp

85 90 95

Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Val Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 14

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 15

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 16

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

1 5 10 15

Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr Gly Asn

20 25 30

Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val

65 70 75 80

Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn Glu Asp

85 90 95

Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Val Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 18

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 20

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 21

<211> 1335

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

caggtgcagc tggtgcagtc cggcgctgag gtgaagaagc ccggcgctag cgtgaagatg 60

agctgtaagg cctccggcta caccttcacc acctacacca tgcactgggt gaggcaggcc 120

cctggccagg gactggagtg gatcggctat atcaaccctc ggagcggcta caccaactat 180

aatcagaagt tccagggccg ggccaccgtg accgccgata agtccaccag caccgcttac 240

atggagctga gctccctgag gtccgaggac accgccgtgt attattgcgc taggcggggc 300

tacttcgatt attggggcca gggcaccacc gtgaccgtgt ccagcgcttc caccaagggc 360

ccctccgtgt tccccctggc tccctcttcc aagagcacca gcggcggcac cgctgctctg 420

ggatgtctgg tgaaggacta cttccctgag cctgtgaccg tgtcctggaa ttccggcgcc 480

ctgacctccg gcgtgcacac attccctgct gtgctgcagt cctccggcct gtatagcctg 540

tcctccgtgg tgacagtgcc tagctccagc ctgggcaccc agacctatat ctgcaacgtg 600

aaccacaagc ctagcaatac caaggtggac aagaaggtgg agcctaagag ctgcgacaag 660

acccacacct gtcctccatg tcctgctcca gaactgctcg gcggaccttc cgtgttcctg 720

tttcctccaa agcctaagga caccctgatg atcagcagaa cccctgaagt gacctgcgtg 780

gtggtggatg tgtcccacga ggatcccgaa gtgaagttca attggtacgt ggacggcgtg 840

gaagtgcaca acgccaagac caagcctaga gaggaacagt acaacagcac ctacagagtg 900

gtgtccgtgc tgaccgtgct gcaccaggat tggctgaacg gcaaagagta caagtgcaag 960

gtgtccaaca aggccctgcc tgctcctatc gagaaaacca tcagcaaggc caagggccag 1020

cctagggaac cccaggttta cacactgcct ccaagcaggg acgagctgac caagaatcag 1080

gtgtccctga cctgcctggt caagggcttc tacccttccg atatcgccgt ggaatgggag 1140

agcaatggcc agcctgagaa caactacaag acaacccctc ctgtgctgga cagcgacggc 1200

tcattcttcc tgtacagcaa gctgacagtg gacaagagca gatggcagca gggcaacgtg 1260

ttcagctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa gtccctgagc 1320

ctgtctcctg gcaaa 1335

<210> 22

<211> 654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gatatccagc tgacccagag ccctagctcc ctgtccgtgt ccgtgggcga tcgggccacc 60

atcacctgtc gggcctccga gtccgtggac gactatggca ataccttcat gcactggtac 120

cagcagaagc ctggcaaggc tcccaagctg ctgatctacc tggcctccaa cctgcagagc 180

ggcgtgcctt ccaggttctc cggcagcggc agccggaccg acttcaccct gaccatcagc 240

agcgtgcagg ctgaggacgc cgccacctat tactgccagc agaataatga ggatccttat 300

accttcggcc agggcaccaa gctggagatc aagaggaccg tggctgcccc cagcgtgttc 360

atcttccctc ctagcgacga gcagctgaag agcggcaccg ctagcgtggt gtgtctgctg 420

aataacttct atcccaggga ggccaaggtg cagtggaagg tggataacgc cctgcagagc 480

ggcaactccc aggagtccgt gaccgagcag gactccaagg acagcaccta ctccctgagc 540

tccaccctga ccctgtccaa ggctgattat gagaagcaca aggtgtatgc ttgcgaggtg 600

acacaccagg gcctgtccag ccctgtgacc aagagcttca accggggcga gtgc 654

<210> 23

<211> 445

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Ala Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 24

<211> 218

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Asp Tyr

20 25 30

Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Val Gln Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 25

<211> 1725

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

atgcgggacc ccggcgcggc cgctccgctt tcgtccctgg gcctctgtgc cctggtgctg 60

gcgctgctgg gcgcactgtc cgcgggcgcc ggggcgcagc cgtaccacgg agagaagggc 120

atctccgtgc cggaccacgg cttctgccag cccatctcca tcccgctgtg cacggacatc 180

gcctacaacc agaccatcct gcccaacctg ctgggccaca cgaaccaaga ggacgcgggc 240

ctcgaggtgc accagttcta cccgctggtg aaggtgcagt gttctcccga actccgcttt 300

ttcttatgct ccatgtatgc gcccgtgtgc accgtgctcg atcaggccat cccgccgtgt 360

cgttctctgt gcgagcgcgc ccgccagggc tgcgaggcgc tcatgaacaa gttcggcttc 420

cagtggcccg agcggctgcg ctgcgagaac ttcccggtgc acggtgcggg cgagatctgc 480

gtgggccaga acacgtcgga cggctccggg ggcccaggcg gcggccccac tgcctaccct 540

accgcgccct acctgccgga cctgcccttc accgcgctgc ccccgggggc ctcagatggc 600

agggggcgtc ccgccttccc cttctcatgc ccccgtcagc tcaaggtgcc cccgtacctg 660

ggctaccgct tcctgggtga gcgcgattgt ggcgccccgt gcgaaccggg ccgtgccaac 720

ggcctgatgt actttaagga ggaggagagg cgcttcgccc gcctctgggt gggcgtgtgg 780

tccgtgctgt gctgcgcctc gacgctcttt accgttctca cctacctggt ggacatgcgg 840

cgcttcagct acccagagcg gcccatcatc ttcctgtcgg gctgctactt catggtggcc 900

gtggcgcacg tggccggctt ccttctagag gaccgcgccg tgtgcgtgga gcgcttctcg 960

gacgatggct accgcacggt ggcgcagggc accaagaagg agggctgcac catcctcttc 1020

atggtgctct acttcttcgg catggccagc tccatctggt gggtcattct gtctctcact 1080

tggttcctgg cggccggcat gaagtggggc cacgaggcca tcgaggccaa ctcgcagtac 1140

ttccacctgg ccgcgtgggc cgtgcccgcc gtcaagacca tcactatcct ggccatgggc 1200

caggtagacg gggacctgct gagcggggtg tgctacgttg gcctctccag tgtggacgcg 1260

ctgcggggct tcgtgctggc gcctctgttc gtctacctct tcataggcac gtccttcttg 1320

ctggccggct tcgtgtccct cttccgtatc cgcaccatca tgaaacacga cggcaccaag 1380

accgagaagc tggagaagct catggtgcgc atcggcgtct tcagcgtgct ctacacagtg 1440

cccgccacca tcgtcctggc ctgctacttc tacgagcagg ccttccgcga gcactgggag 1500

cgcacctggc tcctgcagac gtgcaagagc tatgccgtgc cctgcccgcc cggccacttc 1560

ccgcccatga gccccgactt caccgtcttc atgatcaagt acctgatgac catgatcgtc 1620

ggcatcacca ctggcttctg gatctggtcg ggcaagaccc tgcagtcgtg gcgccgcttc 1680

taccacagac ttagccacag cagcaagggg gagactgcgg tatga 1725

Claims (9)

1. A targeted Frizzled7 humanized antibody, which is characterized in that the targeted Frizzled7 humanized antibody has a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.19, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 20.

2. A recombinant protein, said recombinant protein having:

(i) the antibody of claim 1; and (ii) optionally a tag sequence to facilitate expression and/or purification.

3. A nucleotide, wherein the nucleotide encodes a polypeptide selected from the group consisting of:

(1) the antibody of claim 1; or (2) the recombinant protein of claim 2.

4. An expression vector comprising the nucleotide of claim 3.

5. A recombinant host cell comprising the expression vector of claim 4.

6. A kit, comprising: the antibody of claim 1.

7. An immunoconjugate, comprising:

(a) the antibody or antibody fragment of claim 1, or the recombinant protein of claim 2; and (b) a conjugate moiety of a drug selected from the group consisting of a detectable label, a toxin, a cytokine, a radionuclide, or an enzyme.

8. A pharmaceutical composition comprising the antibody or antibody fragment of claim 1, the recombinant protein of claim 2, or the immunoconjugate of claim 7; and a pharmaceutically acceptable carrier.

9. Use of the antibody of claim 1 for the preparation of a medicament for the treatment of a tumor.

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