CN110950957A

CN110950957A - Polypeptide capable of binding CTLA4 and application thereof

Info

Publication number: CN110950957A
Application number: CN201911280508.0A
Authority: CN
Inventors: 吴稚伟; 吴喜林; 施海霞; 赵振民
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2020-04-03
Anticipated expiration: 2039-12-13
Also published as: CN110950957B

Abstract

The invention relates to a polypeptide capable of binding CTLA4, which comprises 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NO. 1-8, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 9-16, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 17-25. The invention develops a nano antibody medicament aiming at a tumor patient, screens a nano antibody VHH specifically combined with CTLA4 by preparing CTLA4 protein, immune alpaca, a platform technology for displaying a nano monoclonal antibody by utilizing a phage library and the like, and identifies a CDR sequence of the nano antibody VHH. The invention provides a potential nano-antibody new drug for clinical treatment of patients with tumors and infectious diseases.

Description

Polypeptide capable of binding CTLA4 and application thereof

Technical Field

The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding CTLA4, and the application of said polypeptide in preparing CTLA4 detection agent or medicine for curing tumor patient.

Background

Tumor refers to a new organism formed by abnormal proliferation of cells in local tissues of the body, and is often represented as a local tumor mass. Tumors are generally classified into benign tumors and malignant tumors according to their properties. Malignant tumor grows fast, is easy to spread to surrounding tissues, is easy to metastasize and endangers life. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a co-stimulatory molecule expressed on the surface of T cells, is highly homologous to CD28, and has the common ligands B7-1(CD80) and B7-2(CD 86). A small amount of CTLA-4 is effective in competing with CD28 for ligand binding, weakening the structure of CD28 and ligand, and inhibiting T cell activation signaling. .

In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH2 and CH3), a hinge region and a heavy chain variable region (VHH, i.e., antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of conventional antibodies, and with a molecular height and diameter at the nanometer level, is the smallest functional antibody fragment currently available, and thus is also referred to as Nanobody (Nb). Because the nano monoclonal antibody has the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80 percent with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is widely applied to the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.

Some anti-CTLA 4 antibodies have been used to treat melanoma, but they often have serious side effects, limiting the use of anti-CTLA-4. Therefore, there is a need to prepare new anti-CTLA-4 antibodies, which are reserved for the development of new anti-tumor and anti-infectious diseases drugs with less side effects.

Disclosure of Invention

The camel source nanometer monoclonal antibody and the VHH thereof are obtained by immunizing the alpaca with the antigen and are used for diagnosing and treating patients with high expression of CTLA 4. Based on these studies, the present invention provides a polypeptide that binds to CTLA4, comprising 3 complementarity determining regions CDR1-3, CDR1 sequence is or comprises one of the sequences shown in SEQ ID NOs 1-8, CDR2 sequence is or comprises one of the sequences shown in SEQ ID NOs 9-16, and CDR3 sequence is or comprises one of the sequences shown in SEQ ID NOs 17-25.

In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being staggered with respect to said CDR 1-3. The sequence of the framework region can be designed according to the species. For example, the FR1-4 sequence can be designed as shown in SEQ ID NOS: 26-29, but the scope of the present invention is not limited thereto, and the framework region FR1-4 sequence can also be humanized as shown in SEQ ID NOS: 30-33. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences are not so much affected and can be designed according to species, which is well known in the art. For example, FR region sequences of human, murine or alpaca origin can be designed to link the above CDRs, resulting in a polypeptide or domain that binds human PD 1.

In a preferred embodiment, the polypeptide is a monoclonal antibody.

In a preferred embodiment, the polypeptide is VHH.

In a preferred embodiment, the polypeptide is a VHH of camelid origin or a humanized VHH.

In one embodiment, the CDR sequences of the polypeptides are as follows:

I) the sequence of CDR2 is SEQ ID NO: 34, wherein X at the second position represents serine or threonine; and is

II) the sequence of CDR1 is SEQ ID NO 1 or 2; and the sequence of CDR3 is selected from the group consisting of SEQ ID NOs 17-19.

Preferably, the sequence of CDR1 is SEQ ID NO 1, the sequence of CDR2 is SEQ ID NO 9 and the sequence of CDR3 is SEQ ID NO 17; or

The sequence of CDR1 is SEQ ID NO. 1, the sequence of CDR2 is SEQ ID NO. 9 and the sequence of CDR3 is SEQ ID NO. 18; or

The sequence of CDR1 is SEQ ID NO. 2, the sequence of CDR2 is SEQ ID NO. 10 and the sequence of CDR3 is SEQ ID NO. 19.

In a specific embodiment, the sequence of CDR1 is SEQ ID NO 3, the sequence of CDR2 is SEQ ID NO 11 and the sequence of CDR3 is SEQ ID NO 20.

In a specific embodiment, the sequence of CDR1 is SEQ ID NO. 4, the sequence of CDR2 is SEQ ID NO. 12 and the sequence of CDR3 is SEQ ID NO. 21.

In a specific embodiment, the sequence of CDR1 is SEQ ID NO 5, the sequence of CDR2 is SEQ ID NO 13 and the sequence of CDR3 is SEQ ID NO 22.

In a specific embodiment, the sequence of CDR1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 14 and the sequence of CDR3 is SEQ ID NO 23.

In a specific embodiment, the sequence of CDR1 is SEQ ID NO. 7, the sequence of CDR2 is SEQ ID NO. 15 and the sequence of CDR3 is SEQ ID NO. 24.

In a specific embodiment, the sequence of CDR1 is SEQ ID NO 8, the sequence of CDR2 is SEQ ID NO 16 and the sequence of CDR3 is SEQ ID NO 25.

The invention also provides the application of the polypeptide in medicaments for treating tumors or infectious diseases.

The present invention also provides the nucleic acid encoding sequence of the polypeptide.

In one embodiment, the nucleic acid sequence is a DNA sequence or an RNA sequence.

In a specific embodiment, the nucleic acid sequence is present in a gene expression cassette.

The invention also provides the application of the polypeptide in tumor treatment medicines.

The invention aims at tumor and infectious disease patients who escape from immune mechanism through CTLA-4 way to develop nano antibody drug, and the nano antibody VHH specifically combined with CTLA-4 protein is screened by preparing CTLA-4 protein, immune alpaca, platform technology utilizing phage library to display nano monoclonal antibody and the like, and the CDR sequence is identified. The invention provides a potential nano antibody new drug for clinical treatment of tumor and infectious disease patients who escape from an immune mechanism through a CTLA-4 way.

Drawings

Figure 1 is an antiserum titer test curve one week after the 4 th and 5 th immunization of camels with sCTLA 4;

figure 2 is a graph of binding of antiserum to cell-expressed CTLA4 at various dilutions one week after the 5 th immunization camel, with preimmune serum as a control;

FIG. 3 is an electrophoretogram of PCR products amplified using sCTLA4-VHH phage antibody library as a template;

FIG. 4 shows the panning identification of sCTLA4-VHH phage antibody library, in which A is the ELISA detection statistical chart after phage library panning against sCTLA4 protein; b is the second wheel (2)^nd) And a third wheel (3)^rd) Respectively selecting 39 and 47 clones from the panned phage antibody library to carry out phage ELISA detection statistical chart;

FIG. 5 is a statistical chart of ELISA detection of prokaryotically expressed VHH antibodies, each dot representing one clone, with OD450 for sGN/OD 450 of blank on the ordinate, and a positive ratio greater than 5.0 being defined.

Detailed Description

1. Preparation of immunogens

According to CTLA4 protein sequence and gene sequence information on NCBI website, polypeptide sCTLA4 capable of effectively inducing alpaca to generate specific antibody against CTLA4 protein is analyzed and designed, and His-tag (sCTLA4-His) or rabbit Fc (sCTLA4-rFc) is connected at C terminal for subsequent purification and detection.

2. Camel immunization and antiserum procurement

Priming the alpaca with an emulsified mixture of 250 mu g of sCTLA4-rFc protein and 250 mu l of Freund's complete adjuvant, boosting the sCTLA4-rFc protein and 250 mu l of Freund's incomplete adjuvant 3 times on days 14, 28, 42 and 56, and collecting blood to detect the antiserum titer 1 week after 2, 3 and 4 weeks of immunization; after 1 week of the 5 th immunization, 200ml of blood was collected for the construction of phage antibody library.

Antiserum titers were determined by ELISA, plates were coated with 0.5. mu.g/ml CTLA4 protein, and 100. mu.l of either antiserum or purified antibody was added to each well in a gradient (control pre-immunization)Llama serum), incubated at 37 ℃ for 1.5h, washed 2 times, 1: 10000 diluted second antibody of horse radish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 mu L of TMB substrate is added, incubation is carried out for 10min at 37 ℃, and 50 mu L of 0.2M H is added₂SO₄The reaction was stopped and the OD450 nm was measured. ELISA assay serum titers were specified as the highest dilution at OD450 that was more than 2.1-fold over the empty control and greater than 0.2.

As shown in FIG. 1, the antiserum titers of 4-and 5-wells were 3.65X 10⁵And 1.09X 10⁶. Therefore, the antigen can induce the alpaca to generate high-titer antiserum specifically aiming at the CTLA4 protein.

To further verify whether the high titer llama antiserum was able to effectively bind to cell surface CTLA4 protein, serum cell binding experiments were performed. Antisera and preimmune sera at various dilutions were incubated with CTLA4 plasmid-transfected cells for 90min at 4 ℃, followed by 2 washes with 2% FBS in PBS, followed by addition of fluorescent secondary antibodies, and the proportion of bound cells was determined by flow cytometry after incubation for 1h at 4 ℃. The results of cell binding experiments showed that CTLA 4-induced antisera were highly specific for binding to cells transfected with CTLA4 plasmid (figure 2). In summary, CTLA4 induced high titers of antisera with high specific binding ability to CTLA 4.

Construction and panning of VHH phage library

Collecting 200ml of immunized alpaca peripheral blood, separating by using lymphocyte separation liquid (GE Ficoll-Paqueplus) to obtain camel PBMC, extracting RNA according to a TRIzol operation manual, inverting by using oligo (dT) into cDNA, cloning the VHH gene of the alpaca to phagemid plasmid through technologies such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. In order to further identify whether the sCTLA4-VHH phage library is successfully constructed, the VHH target gene of immune sCTLA4 small alpaca is amplified by PCR, and the target band is 500bp and the size is in line with the expectation (figure 3), which indicates that the sCTLA4-VHH phage antibody library contains the VHH gene. 50 clones were selected for sequencing and the sequencing results were shownThus, the sequenced columns do not have perfectly identical repeats; the alignment results show that the difference sequence is mostly in the CDR binding region. Through detection, the library volume of the constructed sCTLA4-VHH phage antibody library is 1.37 multiplied by 10⁹The positive rate was 100%, the sequence Diversity (Diversity) was 100%, and the effective insertion rate (In frame rate) was 100%.

The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The phage antibody library was enriched three times with 50. mu.g/ml of sCTLA4-His protein. And (3) carrying out elution, transformation, plate coating and monoclonal picking on the enriched phage, carrying out binding identification on the phage and sCTLA4 protein ELISA, and sequencing the clone with the binding reading value of more than 1.0.

The elutriated library was tested for binding to CTLA4 protein. The phage ELISA results showed that the binding reading values of the sCTLA4-VHH phage library and sCTLA4 protein before enrichment were-0.41, and the reading values of the phage library after one, two and three rounds of enrichment were 0.30, 0.65 and 1.63 respectively (FIG. 4, left). To further verify the positive phage rate of binding sCTLA4-VHH proteins in the enriched library, 39 and 47 clones were selected from the enriched libraries in round 2 and round 3, respectively, for single phage ELISA detection. The results showed that 10% of the individual phage clones were positive in the library round 2, 13% of the phage clones were positive in the library round 3, and the mean reading of binding was around 3.0 (right in FIG. 4), and the high binding sGN-VHH phage library was successfully enriched by sCTLA4 protein panning.

Construction of VHH prokaryotic expression library and VHH expression

PCR amplification is carried out on the enriched 2nd-sCTLA4-VHH and 3rd-sCTLA4-VHH phage antibody libraries after the two and three rounds of panning; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; coating a plate with a prokaryotic expression antibody library, culturing overnight, randomly picking 1000 monoclonal colonies the next day, inducing and expressing an antibody supernatant by IPTG, and carrying out ELISA combined detection on the antibody supernatant and sCTLA4 protein.

The results showed that 182 bacterial supernatants bound sCTLA4 protein while not binding to the blank, sCTLA4 binding/blank reading was greater than 5.0 (figure 5 and table 1). And (3) sequencing and aligning 182 sequences, and removing repeated sequences to finally obtain 38 VHH antibody sequences. Further experiments demonstrated that both these 38 VHH antibodies and CDRs derived from the VHH antibodies can specifically bind CTLA4 protein.

TABLE 138 binding values of VHH antibodies to sGN protein and their CDR sequences

5. Detection of binding of VHH antibodies to CTLA-4 positive T cells by flow cytometry

VHH antibody was incubated with H9 cells (T lymphocyte cell line) mixed at 100. mu.l/sample for 1H at 4 ℃; washing twice with 0.5% PBSF, adding secondary Alexa Fluor 488 coat anti human IgG, and keeping the temperature at 4 ℃ for 30 min; after washing twice with 0.5% PBSF, the machine is used for detection. MOCK is PBS control; neg group is negative control, namely prokaryotic expression supernatant control without antibody; positive control is positive control, namely positive antibody control capable of binding CTLA-4. The results show that all VHHs described above exhibit binding to H9 cells. Similar results were obtained using humanized VHH antibodies. Therefore, the VHH antibody has the capacity of binding CTLA-4 on the surface of a T lymphocyte, and simultaneously, the binding chance of common ligands B7-1(CD80) and B7-2(CD86) of CD28 and CTLA-4 can be blocked by shielding CTLA-4 molecules on the surface of the cell, so that the CTLA-4 is prevented from inhibiting the activation of the T cell through a CD28 channel, the tumor is prevented from escaping from an immune mechanism, and the effect of treating or inhibiting the growth of the tumor is achieved, therefore, the VHH antibodies are potential to become novel antibody drugs for treating the tumor. The VHH antibodies have the capacity of CTLA-4 on the surface of T lymphocytes, and simultaneously can prevent the activation of the T cells from being inhibited by shielding CTLA-4 molecules on the surface of the cells, so that the aim of preventing and controlling infectious diseases is finally fulfilled by enhancing the capacity of the T lymphocytes to kill viruses or bacteria, and the VHH antibodies have the potential to become novel antibody medicaments for treating the infectious diseases.

6. In vivo experiments using humanized VHH loaded AAV viral vectors

Adeno-associated virus (AAV) is derived from non-pathogenic wild adeno-associated virus, and is considered as one of the most promising gene transfer vectors due to its high safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long time for expressing foreign genes in vivo, and is widely used in gene therapy and vaccine research worldwide.

AAV Helper-Free viral packaging system was purchased from Cell Biolabs, San Diego USA. Inserting the DNA coding sequence of the VHH into the pAAV-MCS plasmid by a molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab, pHelper and pAAV-DJ plasmids are cotransfected with AAV-293T cells by using a PEI transfection reagent according to the mass ratio of 1:1: 1. Supernatants were collected at 48, 72, 96 and 120 hours post transfection and concentrated with 5xPEG8000(sigma) and finally purified with 1.37g/ml cesium chloride. Purified AAV was dissolved in PBS, identified and stored at-80 ℃ after packaging.

Multiple melanoma model mice received AAV-VVH (1X 10)¹¹gc/100. mu.l) were injected intramuscularly, and AAV-GFP was used as a control group. The results show that AAV-VVH has therapeutic effect on melanoma.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the subject matter of the invention is to be construed as broadly as the appended claims.

Sequence listing

<110> Source daolong (Suzhou) medical science and technology, Inc

<120> CTLA 4-binding polypeptides and uses thereof

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<210>8

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His Lys His Asp Pro Trp Gly Arg Asp Tyr

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<213> Artificial Sequence (Artificial Sequence)

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Ser Leu Arg Leu Ser Cys Val Val Ser

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Ala

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35

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Gly Gln Gly Thr Arg Val Thr Val Ser Ser

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Claims

1. A polypeptide that binds CTLA4, comprising 3 complementarity determining regions CDR1-3, CDR1 sequence is or comprises one of the sequences shown in SEQ ID NOs 1-8, CDR2 sequence is or comprises one of the sequences shown in SEQ ID NOs 9-16, and CDR3 sequence is or comprises one of the sequences shown in SEQ ID NOs 17-25.

2. The polypeptide of claim 1, wherein said polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being sequentially staggered from said CDR 1-3.

3. The polypeptide of claim 2, wherein the polypeptide is a monoclonal antibody.

4. The polypeptide of claim 2, wherein the polypeptide is a VHH.

5. The polypeptide of claim 4, wherein the polypeptide is a VHH of camelid or humanized VHH.

6. Use of the polypeptide of any one of claims 1 to 5 for detecting cell-surface CTLA 4.

7. Use of the polypeptide of any one of claims 1-5 for the manufacture of a medicament for the treatment of a tumor or an infectious disease.

8. A nucleic acid sequence encoding the polypeptide of any one of claims 1 to 5.

9. Use of the nucleic acid sequence of claim 8 for the preparation of a medicament for gene therapy.

10. A reagent for detecting CTLA4 on a cell surface comprising the polypeptide of any one of claims 1 to 5.