CN114409782A

CN114409782A - anti-IL13Ra2 nano antibody and application thereof

Info

Publication number: CN114409782A
Application number: CN202111681616.6A
Authority: CN
Inventors: 狄升蒙; 冯冬歌; 陈鑫; 李嘉涛; 范艳秋; 田琳; 余学军; 刘芳
Original assignee: Huadao Shanghai Biopharma Co ltd
Current assignee: Huadao Shanghai Biopharma Co ltd
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2022-04-29
Anticipated expiration: 2041-12-29
Also published as: CN116444671A; CN116813773B; CN116514982A; CN114409782B; CN116444671B; CN116589585A; CN116813773A; CN116514982B; CN116589585B

Abstract

The invention provides a nano antibody for resisting IL13Ra2 and application thereof. The amino acid sequence of the CDR3 of the nano antibody comprises a sequence shown in SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10. The anti-IL13Ra2 nano antibody can be specifically combined with IL13Ra2 antigen, has good affinity, is used as an antigen binding domain to construct a chimeric antigen receptor and CAR-T cells, and has obvious killing activity on IL13Ra2 positive tumor cells.

Description

anti-IL13Ra2 nano antibody and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-IL13Ra2 nano antibody and application thereof.

Background

Brain glioma is the most common primary malignant tumor in the cranium, and according to WHO classification, glioma can be classified into I-IV grades from low malignancy degree to high degree, wherein I and II are low-grade gliomas, and III and IV are high-grade gliomas, wherein the malignant degree of Glioblastoma multiforme (GBM) is the highest, and accounts for more than 80% of malignant glioma. Although diagnostic and therapeutic methods have rapidly progressed over the past decade, the prognosis is still not ideal due to the characteristics of high-grade glioma (grade III-IV) of strong invasiveness, high treatment difficulty, poor prognosis and high recurrence rate, and the median survival time is only about 14 months^[1]And 2-year survival rate is less than 25 percent^[2,3]. Although the prognosis of low-grade glioma (grade I-II) is of a high grade, the glioma still can bring nervous system symptoms such as epilepsy and cognitive disorder to patients.

The traditional treatment scheme of brain glioma is surgical resection combined with auxiliary temozolomide chemotherapy and synchronous radiotherapy, and the treatment measures for malignant glioma with the characteristics of multiple occurrence and growth positions and unsuitability for surgical treatment are limited. This non-specific treatment regimen does not provide complete relief of the malignant glioma and can cause damage to healthy brain tissue and is not conducive to improving the quality of life of the patient. In addition, a single glioma cell may metastasize through the white matter tract, corpus callosum or cerebrospinal fluid, leading to relapse, with more than half of patients finding metastases in the contralateral hemisphere^[4]. In view of the poor therapeutic effect of traditional methods, the rapid development of chimeric antigen-receptor T-cells (CAR-T) in recent years has brought new eosin to the treatment of GBM.

A large number of in vitro preclinical results have demonstrated that 5 tumor-associated antigens, IL13R alpha 2(interleukin-13receptor alpha chain variant 2, interleukin 13receptor alpha 2), EGFR VIII (epidermal growth factor receptor-VIII, epidermal growth factor receptor variant VIII), ErbB2(epidermal growth factor receptor 2), EphA 2(epidermal A2 receptor, tyrosine protein kinase receptor A2) and B7-H3, are likely to become targets for the treatment of GBM, and have achieved better results in preclinical animal models and in partial clinical trials and have been developed.

Interleukin 13receptor alpha 2(IL13R alpha 2), a membrane-bound protein, is closely related to IL13R alpha 1. It has been proved that the adult, children brain tumor and meningioma IL13R alpha 2 are expressed abundantly, on the contrary, the expression quantity on the surface of GBM cell is 3 ten thousand times of that of normal tissue cell, and 58% of patients have up-regulated gene expression^[5]The average expression level of the gene in U373 cell line and 3 cases of GBM patient cells analyzed by flow cytometry is up to 73%^[6]Is an effective target for immunotherapy GBM. It plays an important role in immune response and tumor microenvironment, and can block apoptosis pathway to cause immune escape. The first generation and the second generation CAR-T targeting IL13R alpha 2 show obvious anti-tumor effect^[7-9]. The report that the generation of CAR-T cells expressing IL13E13Y mutant protein has high affinity for IL13R alpha 2 and competes for binding to IL-13a1/IL4Ra expressed in the CNS^[10]After 3 recurrent GBM surgeries, CD8(+) CTL cells from IL13(E13Y) -zetakine were injected multiple times directly through the cranial cavity, 2 survived for more than 14 months with no significant adverse effects. However, the first generation CAR-T cells have a limited survival time to within 15 days in the glioma setting. Recent research results show that^[8]And the fusion costimulation structure CAR-T cell has prolonged survival time and can exert stronger anti-glioma activity. In vitro experiments^[9]Second generation CAR-T can kill IL13R alpha 2⁺Without causing damage to normal tissue cells. Mouse in situ glioma experiment proves^[11]And the second generation E13KCAR-T cell fused with CD28 can kill tumor cells and prolong the survival time of experimental mice. Brown et al^[12]IL13-zetakine modified targeting T cells were found to kill IL13R alpha 2 simultaneously⁺And IL13R alpha 2⁺Perhaps for the complete eradication of IL13R α 2⁺The glioma of (a) is of great help.

In conclusion, the anti-IL13R alpha 2 antibody with high specificity and affinity is provided, can be effectively applied to the preparation of CAR-T taking IL13R alpha 2 as a target point, and has important significance in the field of glioma treatment.

Reference to the literature

1. Schnphei, yang geian, yang xue, yuanyen, humble, progress in the study of glioma in children and glioma in adults, journal of medical research (2019)48(2):5-11.

2.Wen P YKesari S.Malignant gliomas in adults.N Engl J Med(2008)359(5):492-507.doi:10.1056/NEJMra0708126

3.Woehrer A,Bauchet LBarnholtz-Sloan J S.Glioblastoma survival:has it improvedEvidence from population-based studies.Curr Opin Neurol(2014)27(6):666-74.doi:10.1097/WCO.0000000000000144

4. Baiyue, Zhongsong Wen Dubin, the recent progress of chimeric antigen receptor T cells in the treatment of glioblastoma multiforme, Chinese tumor clinics (2017)44(6):794-799.

5.Bart Thaci,Christine E.Brown,Emanuela Binello,Katherine Werbaneth,Prakash SampathSengupta S.Significance of interleukin-13receptor alpha 2-targeted glioblastoma therapy.Neuro Oncol.(2014)16(10):1304–1312.

6.Hegde M,Corder A,Chow K K,Mukherjee M,Ashoori A,Kew Y,Zhang J,Baskin D S,Merchant F A,Brawley V S,et al.Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in Glioblastoma.Mol Ther(2013).doi:10.1038/mt.2013.185mt2013185[pii].

7.Hegde M,Corder A,Chow K K,Mukherjee M,Ashoori A,Kew Y,Zhang Y J,Baskin D S,Merchant F A,Brawley V S,et al.Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.Mol Ther(2013)21(11):2087-101.doi:10.1038/mt.2013.185.

8.Krenciute G,Krebs S,Torres D,Wu M F,Liu H,Dotti G,Li X N,Lesniak M S,Balyasnikova I VGottschalk S.Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Ralpha2-positive Glioma.Mol Ther(2016)24(2):354-363.doi:10.1038/mt.2015.199.

9.Debinski W,Dickinson P,Rossmeisl J H,Robertson JGibo D M.New agents for targeting of IL-13RA2expressed in primary human and canine brain tumors.PLoS One(2013)8(10):e77719.doi:10.1371/journal.pone.0077719.

10.Brown Ce,Badie B,Barish Me,Weng L,Ostberg Jr,Chang Wc,Naranjo A,Starr R4,Wagner J,Wright C4,et al.Bioactivity and Safety of IL13Rα2-Redirected Chimeric Antigen Receptor CD8+T Cells in Patients with Recurrent Glioblastoma.Clin Cancer Res(2015)21(18):4062-72.

11.Kong S,Sengupta S,Tyler B,Bais A J,Ma Q,Doucette S,Zhou J,Sahin A,Carter B S,Brem H,et al.Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells.Clin Cancer Res(2012)18(21):5949-60.doi:10.1158/1078-0432.CCR-12-0319.

12.Brown C E,Starr R,Aguilar B,Shami A F,Martinez C,D'apuzzo M,Barish M E,Forman S JJensen M C.Stem-like tumor-initiating cells isolated from IL13Ralpha2expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.Clin Cancer Res(2012)18(8):2199-209.doi:10.1158/1078-0432.CCR-11-1669.

Disclosure of Invention

Aiming at the defects and practical needs of the prior art, the invention provides a nanobody resisting IL13Ra2 and application thereof. The anti-IL13Ra2 nano antibody has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to prepare CAR-T cells, and the obtained CAR-T cells have good application prospects in the aspect of tumor treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a nanobody against IL13Ra2, wherein the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3, the amino acid sequence of CDR2 of the nanobody comprises the sequence shown in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7, and the amino acid sequence of CDR3 of the nanobody comprises the sequence shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

In the invention, IL13Ra2 recombinant protein is used to immunize an unimmunized alpaca, a phage display nano antibody library is constructed, and anti-IL13Ra2 antibody is screened according to the phage display nano antibody library to obtain the monoclonal antibody which can be specifically combined with IL13Ra2 antigen.

SEQ ID NO.1：GFTLDYYA。

SEQ ID NO.2：GANLKNYA。

SEQ ID NO.3：RFTLDYYA。

SEQ ID NO.4：ISSSEGST。

SEQ ID NO.5：LRVRYANT。

SEQ ID NO.6：ISSSGGST。

SEQ ID NO.7：LRVRYANS。

SEQ ID NO.8：AADSTGRCWARPLYEYDY。

SEQ ID NO.9：AARPQQPSADCSLLANDYDN。

SEQ ID NO.10：AARPQQPSADCSLSANDYDN。

Preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.1, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.4, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 8.

Preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 9.

Preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.6, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.8, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 9.

Preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.3, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.4, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 8.

Preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.7, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 10.

Preferably, the heavy chain variable region of the nanobody further comprises: framework region 1(FR1) shown by SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO.13, framework region 2(FR2) shown by SEQ ID NO.14, SEQ ID NO.15 or SEQ ID NO.16, framework region 3(FR3) shown by SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.20, and framework region 4(FR4) shown by SEQ ID NO.21 or SEQ ID NO. 22.

SEQ ID NO.11：QVQLVESGGGLVQPGGSLRLSCAAS。

SEQ ID NO.12：QVQLVESGGGLAHPGGRLRVTCTAS。

SEQ ID NO.13：QVQLVESGGGSVQAGGSLRLSCTSS。

SEQ ID NO.14：IAWFRQAPGKRREGVSC。

SEQ ID NO.15：VGWFRQRPGKQREEVAC。

SEQ ID NO.16：VGWFRRRPGKQREEVAC。

SEQ ID NO.17：

NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC。

SEQ ID NO.18：

NKAPSVRERVHVFREENNNLVYMLMSDLTPEDTGIYY。

SEQ ID NO.19：

NYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC。

SEQ ID NO.20：

NKAPSVRERVHVFREEKNNLVYMLMSDLTPEDTGIYYC。

SEQ ID NO.21：RGQGTQVTVSS。

SEQ ID NO.22：WGQGIQVTVSE。

Preferably, the heavy chain variable region of the nanobody comprises the amino acid sequence shown in SEQ ID No.23, SEQ ID No.24, SEQ ID No.26 or SEQ ID No. 27.

SEQ ID NO.23：

QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIAWFRQAPGKRREGVSCISSSEGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSTGRCWARPLYEYDYRGQGTQVTVSS。

SEQ ID NO.24：

QVQLVESGGGLAHPGGRLRVTCTASGANLKNYAVGWFRQRPGKQREEVACLRVRYANTNKAPSVRERVHVFREENNNLVYMLMSDLTPEDTGIYYCAARPQQPSADCSLLANDYDNWGQGIQVTVSE。

SEQ ID NO.25：

QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIAWFRQAPGKRREGVSCISSSGGSTNYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSTGRCWARPLYEYDYRGQGTQVTVSS。

SEQ ID NO.26：

QVQLVESGGGSVQAGGSLRLSCTSSRFTLDYYAIAWFRQAPGKRREGVSCISSSEGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSTGRCWARPLYEYDYRGQGTQVTVSS。

SEQ ID NO.27：

QVQLVESGGGLAHPGGRLRVTCTASGANLKNYAVGWFRRRPGKQREEVACLRVRYANSNKAPSVRERVHVFREEKNNLVYMLMSDLTPEDTGIYYCAARPQQPSADCSLSANDYDNWGQGIQVTVSE。

In the invention, the phage display technology is utilized to screen the IL13Ra2 immune camel VHH library, and the obtained nano antibody has high affinity and has important application prospect in the aspect of constructing a chimeric antigen receptor targeting IL13Ra 2.

In a second aspect, the present invention provides a nucleic acid molecule comprising a gene encoding the nanobody against IL13Ra2 of the first aspect.

In a third aspect, the present invention provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain comprising the nanobody of the first aspect against IL13Ra2, a hinge region, a transmembrane region, and a signaling domain.

Preferably, the signal peptide comprises a CD8 a signal peptide.

Preferably, the hinge region comprises a CD8 a hinge region.

Preferably, the transmembrane region comprises any one of, or a combination of at least two of, a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region.

Preferably, the signaling domain comprises an immunoreceptor tyrosine activation motif (CD3 ζ).

Preferably, the signaling domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular domain, OX40, ICOS or DAP10 intracellular domain, or a combination of at least two thereof.

Preferably, the chimeric antigen receptor comprises a CD8 a signal peptide, the nanobody against IL13Ra2 of the first aspect, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine activation motif.

In a fourth aspect, the present invention provides an expression vector comprising a gene encoding the chimeric antigen receptor of the third aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to the third aspect, preferably a lentiviral vector.

In a fifth aspect, the present invention provides a recombinant lentivirus comprising the expression vector of the fourth aspect.

Preferably, the recombinant lentivirus is prepared from mammalian cells transfected with the expression vector and helper plasmid of the fourth aspect.

In a sixth aspect, the present invention provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the third aspect.

Preferably, the chimeric antigen receptor immune cell comprises the recombinant lentivirus of the expression vector and/or substrate aspects of the fourth aspect.

Preferably, the chimeric antigen receptor immune cells include any one of T cells, B cells, NK cells, mast cells or macrophages or a combination of at least two thereof.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the sixth aspect.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.

In an eighth aspect, the present invention provides a nanobody against IL13Ra2 of the first aspect, a nucleic acid molecule of the second aspect, a chimeric antigen receptor of the third aspect, an expression vector of the fourth aspect, a recombinant lentivirus of the fifth aspect, a chimeric antigen receptor immune cell of the sixth aspect, or a pharmaceutical composition of the seventh aspect, for use in the preparation of a medicament for the treatment of tumors.

Preferably, the tumor comprises a tumor expressing IL13Ra 2.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) according to the invention, IL13Ra2 recombinant protein is used to immunize unimmunized alpaca, a phage display nano antibody library is constructed, anti-IL13Ra2 antibody is screened according to the phage display nano antibody library, the obtained nano antibody can be specifically combined with IL13Ra2 antigen, the affinity is good, and the KD (M) is 1.61 multiplied by 10 respectively according to the determination of the antibody affinity^-8、3.24×10^-8、1.07×10^-9、1.66×10^-8And 2.57X 10^-9；

(2) The anti-IL13Ra2 nano antibody provided by the invention has better affinity, is used as an antigen binding structural domain to construct a chimeric antigen receptor, and utilizes the chimeric antigen receptor to prepare a T cell, the CAR-T cell has killing activity on IL13Ra2 positive tumor cells, and efficiently secretes cell factors IFN-gamma and TNF-alpha after being co-cultured with IL13Ra2 positive cells, so that the nano antibody can be effectively applied to immunotherapy, and has important significance for developing tumor treatment medicines.

Drawings

FIG. 1A is a graph showing the affinity of VHH-1 detected by Biacore as an anti-IL13Ra2 nanobody in example 2;

FIG. 1B is a graph showing the affinity of VHH-7 detected by Biacore as an anti-IL13Ra2 nanobody in example 2;

FIG. 1C is a graph showing the affinity of VHH-8, an anti-IL13Ra2 nanobody detected using Biacore in example 2;

FIG. 1D is a graph showing the affinity of VHH-13 detected by Biacore as an anti-IL13Ra2 nanobody in example 2;

FIG. 1E is a graph showing the affinity of VHH-16, an anti-IL13Ra2 nanobody detected using Biacore in example 2;

FIG. 2 is a graph showing the results of FACS detection of IL13Ra2 antigen recognized by anti-IL13Ra2 nanobody in example 3;

FIG. 3 is a plasmid map of a chimeric antigen receptor lentiviral vector targeting IL13Ra2 in example 4;

FIG. 4 is a schematic diagram of the structure of a chimeric antigen receptor expressing IL13Ra2 in example 4;

FIG. 5 is a graph showing the results of flow measurement of the expression rate of the chimeric antigen receptor of T lymphocytes in example 6;

FIG. 6A shows FACS detection of T-cell and CAR-T cell phenotypes in example 6 (CD 3)⁺CD4⁺) The obtained result graph;

FIG. 6B FACS detection of T-cell and CAR-T cell phenotypes in example 6 (CD 3)⁺CD8⁺) The obtained result graph;

FIG. 7 is a graph of the killing effect of CAR-T cells on 293T cells in example 7;

FIG. 8 is a graph of the killing effect of CAR-T cells on glioma cells U87 in example 7;

FIG. 9 is a graph showing the killing effect of CAR-T cells on glioma cells U251 in example 7;

FIG. 10 is a histogram of TNF α secretion by CAR-T cells in example 8;

FIG. 11 is a histogram of the secretion of IFN γ by CAR-T cells in example 8.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

In this example, phage nanobody library was constructed and panning was performed, and preliminary screening was performed using ELISA, which specifically comprises the following steps:

(1) construction of phage Nanobody libraries

Adopting IL13Ra2 extracellular recombinant protein to immunize alpaca, and extracting peripheral blood after ELISA (enzyme-linked immunosorbent assay) is used for detecting the serum titer; separating lymphocytes, extracting total RNA, performing reverse transcription to obtain cDNA, and amplifying VHH gene by using nested PCR; the VHH gene was inserted into the pShort phagemid and electrotransformed

After the competent cells are amplified, the phage clones are separated and purified by a PEG8000/NaCI precipitation method to obtain an antibody library; adjusting concentration, subpackaging and freezing at-80 deg.C in refrigerator for use;

(2) screening of phage Nanobody libraries

Firstly, incubating 293T cells and an antibody library for negative screening, then taking supernatant, and respectively incubating with 293T-IL13Ra2 cells and 293T cells which are IL13Ra2 positive cells; washing with pre-cooled PT buffer solution at 4 deg.C for 4 times; infecting NEB alpha 5F' cells, adding helper phage, and culturing overnight; coating a plate by a Drop method, and counting the enrichment degree the next day; separating and purifying phage by PEG8000/NaCI precipitation method, and performing the next round of screening; after enrichment, the obtained phage is used as a template, a VHH region is amplified, and secondary sequencing is carried out to obtain 5 kinds of nano antibodies of anti-IL13Ra2, wherein the amino acid sequences are respectively shown as SE1ID NO.22, SE1ID NO.23, SE1ID NO.24, SE1ID NO.25 and SE1ID NO.26 and are respectively named as VHH-1, VHH-7, VHH-8, VHH-13 and VHH-16.

Example 2

This example expresses and purifies the nanobody against IL13Ra2 (VHH-mIgG2a Fc nanobody) screened in example 1, and performs the determination of the affinity of the antibody. In order to further identify the screened antibodies, the antibodies need to be expressed by mammalian cells, therefore, a plasmid vector for expressing VHH with a mouse Fc tag is firstly constructed, which is marked as C-4pCP.Stuffer-mCg2a-FC, and the specific steps are as follows:

(1) the VHH fragments were amplified using PCR, the reaction system is shown in Table 1, and the amplification procedure is shown in Table 2 below;

TABLE 1

TABLE 2

(2) The enzyme digestion system is shown in Table 3, the enzyme digestion temperature is 37 ℃, the time is 6h, the carrier after enzyme digestion is used

Purifying by using a PCR purification kit, dissolving the recovered DNA in 45 mu L of water, and detecting the concentration of the DNA;

TABLE 3

Reagent	Amount of the composition used
		C-4pCP.Stuffer-mCg2a-FC	5μg
10 Xdigestion Buffer (10 Xreaction Buffer)	5μL
		FspA I	2μL
PfI 23II	2μL
		ddH₂O	Make up to 50. mu.L

(3) Connecting the PCR amplification product into the enzyme-digested linearized vector by adopting a homologous recombination mode, wherein the system is shown in table 4, and the conditions are that water bath is carried out at 37 ℃ for 30 min;

TABLE 4

Reagent	Volume (μ L)
		Exnase II	1
2×Exnase II buffer	2
		Linearized vector	4
Insert (Insert fragment)	3

(4) Adding all homologous recombination reaction systems into DH5 alpha competent cells, and transforming DH5 alpha competent cells under the transformation conditions shown in Table 5;

TABLE 5

Procedure	Temperature of	Time
			Ice bath
	0℃	5min
			Heat shock	42℃	1min
Ice bath	0℃	3min
			Adding 500. mu.L LB medium, shaking at 220rpm	37℃	1.5h
Pipette
	200. mu.L of the solution and spread on LB/Amp plates	37℃	Overnight

(5) Selecting monoclonal PCR for pre-identification by the transformation plate, wherein the conditions of a PCR identification system are shown in Table 6; the condition is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 5min at 72 ℃, storing at 4 ℃, sending to a sequencing company for sequencing and identifying, wherein the sequencing result is in accordance with expectation, and the plasmid vector with the mouse Fc tag expression VHH is successfully constructed in the embodiment of the specification.

TABLE 6

293E cells were passaged to a cell density of about 0.6X 10 approximately 24h prior to plasmid transfection⁶cells/mL; when the cell density is 1.2X 10⁶cells/mL, viability>At 95%, transfecting 293E cells by a PEI method at a ratio of 150 mu g DNA/100mL 293E, wherein the ratio of plasmid DNA to PEI is 1: 2;

37℃、130rpm、8％CO₂shaking culturing for 6 days at 3000rpm for 30min, collecting cell culture supernatant, filtering the collected supernatant containing target antibody with 0.45 μm filter, and loading onto purification column MabSelect^TM SuRe^TMThe cartridge was washed with 5-fold PBS, and the protein eluted with 0.1M Gly-HCl (pH 3.0) and neutralized with 1/10 volumes of Tris-HCl pH 8.5, followed by dialysis at 4 deg.CAfter overnight, A was measured with NanoDrop 2000₂₈₀The method of (3) is quantitative, and the SEC-HPLC is used for determining the purity of the antibody.

In addition, the affinity of the purified VHH antibody was also determined by Biacore in this example.

Biacore is a bioanalytical sensing technology developed based on Surface Plasmon Resonance (SPR), and can detect the whole process of change of binding and dissociation of molecules in a tracking solution and molecules fixed on the surface of a chip, record the change in the form of a sensorgram, and provide kinetic and affinity data.

In the measurement process, the antibody was immobilized on the chip surface, and the mobile phase was a solution containing the antigen (IL13Ra2), and the results of the measurement are shown in Table 7 and FIGS. 1A to 1E, where the affinity of each of the 5 antibodies was in the sub-nanomolar range of 1.61E-8, 3.24-8, 1.07E-9, 1.66E-8, and 2.57E-9.

TABLE 7

Nanobodies	Ka(1/Ms)	Kd(1/s)	KD(M)
				VHH-1(SEQ ID NO.23)	5.53E+04	1.56E-03	1.61E-08
VHH-7(SEQ ID NO.24)	6.54E+04	6.6E-04	3.24E-08
				VHH-8(SEQ ID NO.25)	4.95E+04	6.98E-03	1.07E-09
VHH-13(SEQ ID NO.26)	6.83E+04	4.33E-03	1.66E-08
				VHH-16(SEQ ID NO.27)	6.25E+04	3.64E-03	2.57E-09

Example 3

This example was performed by flow assay of nanobodies against IL13Ra 2.

293T (negative for IL13Ra2, IL13Ra2)^-) 293T-Mesothelin cells and purified nano-antibody against IL13Ra2 were mixed, incubated for 30min in ice bath, then incubated for 30min with APC-labeled goat anti-mouse IgG antibody, and detected by flow cytometry, the results are shown in FIG. 2, which indicates that the nano-antibody against IL13Ra2 of the present invention can recognize IL13Ra2 antigen on the cell surface.

Example 4

This example prepared a lentiviral vector expressing a chimeric antigen receptor targeting IL13Ra 2(IL13 Ra2 CAR).

First, a lentiviral vector pSIN03IL13Ra2CAR carrying a chimeric antigen receptor for IL13Ra2CAR was constructed, the vector map is shown in FIG. 3, the schematic diagram of the chimeric antigen receptor is shown in FIG. 4, and the lentiviral vector comprises a CD8 α signal peptide, a nanobody against IL13Ra2 (anti-IL13Ra2VHH), a CD8 α hinge region, a transmembrane region, and an immunoreceptor tyrosine activation motif (CD3 ζ).

Wherein the amino acid sequence of the signal peptide (SEQ ID NO.28) is:

MALPVTALLLPLALLLHAARP。

the amino acid sequence of anti-IL13Ra2VHH is shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 or SEQ ID NO. 27.

The amino acid sequence of the hinge and transmembrane regions of CD8 α (SEQ ID NO.29) is:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC。

the amino acid sequence of the 4-1BB intracellular domain (SEQ ID NO.30) is:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

the amino acid sequence of CD3 ζ (SEQ ID NO.31) is:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

the preparation method comprises the following steps:

(1) PCR reaction systems were prepared according to table 8 (in table, reagents from TOYOBO Inc.), and nanobody fragments against IL13Ra2 were amplified and reacted according to the PCR procedure shown in table 9, with the primer sequences:

MS1-F(SEQ ID NO.32)：

tgccgctggccttgctgctccacgccgccaggccgcaggtgcagctggtggag；

MS1-R(SEQ ID NO.33)：cgctggcgtcgtggtgctagacactgtcacctg；

MS9-R(SEQ ID NO.34)：cgctggcgtcgtggtagagctcactgtcacctg。

TABLE 8

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10μM MF1-F	1
		10 μ M MS1-R or MS9-R	1
Template DNA (cDNA clone)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high fidelity PCR enzyme	1

TABLE 9

(2) A PCR reaction system was prepared according to table 10, and CD8 α signal peptide was added before the resulting amplification product, and the reaction was performed according to the PCR procedure shown in table 9 using the primers:

BamH-CD8αsig-F(SEQ ID NO.35)：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC；

watch 10

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 μ M upstream primer (BamH-CD8 α sig-F)	1
		10 μ M downstream primer (MS1-R or MS9-R)	1
Template DNA (VHH fragment PCR reaction solution)	4
		Sterile deionized water (PCR grade water)	30
KOD-Plus-Neo high fidelity PCR enzyme	1

After the reaction is finished, carrying out 1% agarose gel electrophoresis on the PCR product, recovering fragments of about 500bp, and quantifying by an ultraviolet absorption method;

(3) a PCR reaction system was prepared according to Table 11, and after the preparation, a PCR reaction was carried out according to the PCR procedure shown in Table 9 to amplify a CD 8. alpha. hinge-TM-41BB-CD3Z fragment using the following primers:

CD8αH-F(SEQ ID NO.36)：ACCACGACGCCAGCGCCGCGAC；

Vector-R(SEQ ID NO.37)：TCGATAAGCTTGATATCG；

TABLE 11

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 μ M of the forward primer CD8 α H-F	1
		10 μ M downstream primer Vector-R	1
Template DNA (HD CD19 CAR)	1
		Sterile deionized water (PCR grade water)	33
KOD-Plus-Neo high fidelity PCR enzyme	1

After the PCR is finished, carrying out 1% agarose gel electrophoresis, recovering fragments of about 780bp, and quantifying by an ultraviolet absorption method;

(4) carrying out BamHI and EcoRI double enzyme digestion on 5 mu g of HD SIN03 CD19 CAR plasmid constructed in a laboratory, carrying out water bath reaction at 37 ℃ for 2h, and then recovering the vector;

the 3 fragments recovered above were ligated with the vector backbone by recombinant enzyme, and the recombinant reaction system was as shown in Table 12, followed by preparation, reaction in water bath at 37 ℃ for 0.5h, and transformation into E.coli stbl3 competent cells by a conventional method.

TABLE 12

Reagent	Amount of the composition used
		HD CD19 CAR	184.54ng
CD8α singal IL13Ra2 VHH	31.32ng
		CD8α hinge-TM-41BB-CD3Z	29.72ng
5×CE buffer	2μL
		Exnase^TMII	1μL
Sterile deionizationSeed water (PCR grade water)	Make up to 10. mu.L

Selecting a single clone from a solid culture medium, culturing overnight, performing PCR identification, preparing a PCR reaction system as shown in table 13, performing PCR program as shown in table 14, selecting a positive clone after PCR is finished, further sequencing identification, and ensuring that the sequencing result is in line with expectation.

Watch 13

Reagent	Volume (μ L)
		Taq PCR Master Mix	10
10μM F Seq-trEF1a-F	1
		10μM R Vector-R	1
Template DNA bacterial liquid	1
		Sterile deionized water (PCR grade water)	7

TABLE 14

Example 5

In this example, lentiviral vector HD SIN03-IL13 Ra2CAR prepared in example 4 was subjected to lentiviral packaging, concentration and titer detection, comprising the following steps:

(1) lentiviral packaging

At 1.6X 10⁷ Cell number 293T cells were plated in 15cm dishes at 37 ℃ with 5% CO₂Culturing overnight to prepare packaged virus, wherein the culture medium is DMEM containing 10% fetal calf serum; dissolving 14.5 mu g of HD SIN03-IL13 Ra2CAR, 16.7 mu g of helper plasmid pMDLg-RRE, 16.7 mu g of helper plasmid pRSV-REV and 6.5 mu g of envelope plasmid VSVg in 2mL of serum-free DMEM culture solution, and mixing uniformly;

dissolving 163.2. mu.g PEI (1. mu.g/. mu.L) in 2mL serum-free DMEM medium, vortexing at 1000rpm for 5 seconds, and incubating at 25 ℃ for 5 min; adding the PEI mixed solution into the DNA mixed solution, immediately mixing by vortex or mixing lightly, and incubating for 20min at 25 ℃ to form a transfection compound; dripping 4mL of the transfection compound into 25mL of DMEM medium containing 293T cells, and replacing the fresh medium after 4 hours; after 48h, collecting the virus liquid supernatant;

(2) lentiviral concentration

Filtering the virus supernatant with a 0.45-micron filter membrane, collecting the filtrate in a 50mL centrifuge tube, adding 1/4 PEG-NaCl virus concentrated solution, turning upside down, mixing uniformly, and standing at 4 ℃ overnight; centrifuging at 4 deg.C and 3500rpm for 30 min; removing supernatant, adding appropriate amount of RPMI 1640 culture medium (containing 10% FBS), and dissolving and resuspending virus precipitate; subpackaging the concentrated lentivirus suspension into 50 μ L portions, storing in a finished product tube, and storing at-80 deg.C;

(3) lentiviral titer detection

500 μ L K562 cells (1X 10)⁵Individual cells) were inoculated into 24-well culture plates, and the concentrated lentivirus was added to the cell suspension in volumes of 1. mu.L, 0.2. mu.L, and 0.04. mu.L, respectively,and polybrene was added to a final concentration of 5. mu.g/mL, 37 ℃, 5% CO₂After overnight culture, the fresh medium was replaced;

after infection for 72h, centrifuging at 400 Xg for 5min, discarding cells collected from the supernatant, adding 100. mu.L PBS + 2% FBS to resuspend the cells, adding PE coat anti-alpaca VHH antibody, and incubating on ice for 30 min; after washing for 2 times, adding 300 mu L of flow buffer solution to resuspend cells, and detecting infection efficiency by adopting a flow cytometer; the titer was calculated as follows: titer (TU/mL) is cell number (10)⁵) X positive rate/virus volume (mL).

Example 6

This example uses the lentiviruses prepared in example 5 to transduce T lymphocytes, comprising the following steps:

(1) diluting anti-human CD3 antibody and anti-human CD28 antibody with PBS to final concentrations of 1 μ g/mL and 0.5 μ g/mL respectively, coating the well plate, and standing overnight in a refrigerator at 4 deg.C; discarding the antibody coating solution in the pore plate, and washing the plate by 1 mLPBS;

(2) human PBMC were adjusted to a density of 1X 10 with T cell culture medium (X-VIVO + 10% FBS +300U/mL IL-2)⁶mL, inoculated into CD3 and CD28 antibody coated well plates for 24h activation; collecting activated T cells, adjusting cell density to 1 × 10⁶(iv)/mL, lentivirus was added at a multiplicity of infection (MOI) of 10, polybrene was added to a final concentration of 5. mu.g/mL; at 37 ℃ with 5% CO₂After overnight culture in the environment, replacing the fresh culture medium, and carrying out passage every 3 days;

(3) after 5 days of T cell infection, 3X 10 cells were taken⁵Centrifuging the T cells at the temperature of 4 ℃ and the speed of 400 Xg for 5min, removing supernatant, and washing the cells once by using a flow buffer; adding 100 μ L buffer solution to resuspend cells, adding PE coat anti-alpaca VHH antibody, and incubating on ice for 30 min; adding a buffer solution for washing for 2 times, and then adding 300 mu L of the buffer solution for resuspending the cells;

the chimeric antigen receptor expression rate of the T lymphocytes was measured by flow cytometry, and the results are shown in fig. 5, where the infection efficiency of each set of CAR-T cells is: 24.8%, 25.8%, 14.8%, 26.2% and 23.9%, indicating successful construction of CAR-T cells.

In addition, in this example, flow cytometry was also used to detect lymphocyte phenotype, including the following steps:

(1) after 5 days of T cell infection, 3X 10 cells were taken⁵Centrifuging the T cells at 4 ℃ for 5min at 400g, discarding the supernatant, and washing the cells once with PBS (containing FBS with the mass fraction of 2%);

(2) add 50 u L buffer heavy suspension cells, add 1 u L FITC labeled Anti-CD3 Ab, Percp-Cy5.5 labeled Anti-CD4 Ab and PE-Cy7 labeled Anti-CD8 Ab, ice incubation for 30 min; after washing twice with buffer, 300. mu.L of buffer was added to resuspend the cells, and the cell phenotype was examined by flow cytometry, as shown in FIGS. 6A and 6B, CD3⁺CD4⁺The percentage of the cell population is 33-37%, CD3⁺CD8⁺The proportion of the cell population is 61-66%.

Example 7

In this example, CAR-T cell in vitro toxicity experiments were performed, comprising the following steps:

(1) target cell seeding

293-GPF-luci (IL13Ra2 negative, IL13Ra2)^-)、U251-GFP-luci(IL13Ra2⁺)、U87-GFP-luci(IL13Ra2⁺) As the target cells, the concentration of the target cells was adjusted to 2X 10⁵mL, inoculating 50 μ L to a white 96-well plate;

(2) seeding of effector cells

IL13Ra2 CAR-T and control T cells are effector cells, and CAR-T cells and control T cells are added into a 96-well plate according to an effective target ratio of 0.3:1 and 1: 1;

(3) each group was set with 3 replicate wells, the average of the 3 replicate wells was taken, with each experimental and each control group as follows:

experimental groups: each target cell + CAR-T;

control group: seeding only target cells;

(4) the detection method comprises the following steps:

after 18h of co-culture of effector cells and target cells, 100. mu.L of each well was added

A reagent (Promega, Cat # E2520) reacts for 5min, and a bioluminescent signal is detected by using a multifunctional microplate reader;

(5) the CAR-T killing efficiency calculation formula is as follows:

the killing efficiency%.

The results are shown in fig. 7-fig. 9, the CAR-T cells constructed by the invention have no killing effect on 293T cells negative to IL13Ra2, and have killing activity on tumor cells positive to IL13Ra2, which indicates that the CAR-T cells constructed by the invention not only have high-efficiency tumor killing activity, but also have high specificity.

Example 8

In this example, the secretion of the CAR-T cytokines TNF-alpha and IFN-gamma was detected using Human TNF-alpha ELISA Kit (Council organism, cat # EK182-96) and Human IFN-gamma ELISA Kit (Council organism, cat # EK 180-96).

1. Cell culture supernatant

Centrifuging the cell culture with the effective target ratio of 1:1 at 400 Xg for 10min to remove precipitate, taking supernatant, and storing at-80 deg.C for detection.

2. Reagent preparation

Before the detection, all reagents and samples were returned to 25 ℃, and if the concentrated reagents appeared to crystallize, the samples were incubated at 37 ℃ until all crystals were dissolved, and 1 × washing solution, 1 × detection buffer, was prepared according to the instructions.

3. Standard and sample preparation

And (3) standard substance: the standard stock was diluted 2-fold using 5% 1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample preparation: samples were diluted proportionally using 5% 1640 medium.

4. Detection step

(1) Soaking the enzyme label plate: adding 300 μ L of 1 × lotion, standing, soaking for 30s, discarding the lotion, and patting the microporous plate on absorbent paper;

(2) adding a standard substance: adding 100 μ L of 2-fold diluted standard into the standard well, and adding 100 μ L of standard diluent (serum/plasma sample) into the blank well;

(3) adding a sample: adding 100 mu L of cell culture supernatant into the sample hole;

(4) adding a detection antibody: add 50. mu.L of diluted detection antibody per well (1:100 dilution);

(5) and (3) incubation: incubating for 2h at 25 ℃ by using a sealing plate and a membrane sealing plate and oscillating at 300 rpm;

(6) washing: discarding the liquid, adding 300 mu L of washing liquid into each hole, washing the plate for 6 times;

(7) adding enzyme for incubation: add 100. mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) per well;

(8) and (3) incubation: sealing the plate with a new sealing plate membrane, oscillating at 300rpm, incubating at 25 ℃ for 45min, and washing;

(9) adding a substrate for color development: adding 100 μ L chromogenic substrate TMB into each well, keeping out of the sun, and incubating for 20min at 25 ℃;

(10) adding a stop solution: adding 100 mu L of stop solution into each hole;

(11) and (3) detection reading: within 30min, performing dual-wavelength detection by using an enzyme-labeling instrument, and determining OD values under the maximum absorption wavelength of 450nm and the reference wavelength; the OD value after calibration was the value measured at 450nm minus the value measured at the reference wavelength.

The TNF-alpha and IFN-gamma factor secretion results are shown in FIG. 10 and FIG. 11, respectively, wherein no cytokine was detected in the independent CAR-T cell group; CAR-T cells were co-cultured with 293T cells, and no cytokine was detected; after being co-cultured with target cells over-expressing MSLN, the secreted TNF-alpha of the CAR-T cells exceeds 200pg/mL, and IFN-gamma exceeds 3000pg/mL, the CAR-T cells constructed by the invention release cytokines on IL13Ra2 positive tumor cells, and have no obvious cytokine secretion on IL13Ra2 negative cells.

In conclusion, the nano antibody with high affinity and anti-IL13Ra2 can be efficiently and specifically combined with IL13Ra2, and can be used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell, so that the obtained CAR-T cell has obvious killing activity and specificity on IL13Ra2 positive tumor cells, and can secrete tumor killing cytokines, and the nano antibody provided by the invention can be effectively applied to immunotherapy and has important significance for developing tumor treatment medicines.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Sequence listing

<110> Huadao (Shanghai) biopharmaceutical Co., Ltd

<120> nano antibody for resisting IL13Ra2 and application thereof

<130> 2021-12-28

<160> 37

<170> PatentIn version 3.3

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Gly Phe Thr Leu Asp Tyr Tyr Ala

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Gly Ala Asn Leu Lys Asn Tyr Ala

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Arg Phe Thr Leu Asp Tyr Tyr Ala

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Ile Ser Ser Ser Glu Gly Ser Thr

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Leu Arg Val Arg Tyr Ala Asn Thr

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Ile Ser Ser Ser Gly Gly Ser Thr

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Leu Arg Val Arg Tyr Ala Asn Ser

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Ala Ala Asp Ser Thr Gly Arg Cys Trp Ala Arg Pro Leu Tyr Glu Tyr

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Asp Tyr

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Ala Ala Arg Pro Gln Gln Pro Ser Ala Asp Cys Ser Leu Leu Ala Asn

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Asp Tyr Asp Asn

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Ala Ala Arg Pro Gln Gln Pro Ser Ala Asp Cys Ser Leu Ser Ala Asn

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Asp Tyr Asp Asn

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser

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

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Arg Leu Arg Val Thr Cys Thr Ala Ser

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Thr Ser Ser

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Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Arg Glu Gly Val Ser

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Cys

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Val Gly Trp Phe Arg Gln Arg Pro Gly Lys Gln Arg Glu Glu Val Ala

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Cys

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Val Gly Trp Phe Arg Arg Arg Pro Gly Lys Gln Arg Glu Glu Val Ala

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Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

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Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

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Thr Ala Val Tyr Tyr Cys

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Asn Lys Ala Pro Ser Val Arg Glu Arg Val His Val Phe Arg Glu Glu

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Asn Asn Asn Leu Val Tyr Met Leu Met Ser Asp Leu Thr Pro Glu Asp

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Thr Gly Ile Tyr Tyr

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Asn Tyr Ala Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

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Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

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Thr Ala Val Tyr Tyr Cys

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Asn Lys Ala Pro Ser Val Arg Glu Arg Val His Val Phe Arg Glu Glu

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

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Thr Gly Ile Tyr Tyr Cys

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Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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Trp Gly Gln Gly Ile Gln Val Thr Val Ser Glu

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

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Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Arg Glu Gly Val

35 40 45

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

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

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

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Ala Ala Asp Ser Thr Gly Arg Cys Trp Ala Arg Pro Leu Tyr Glu Tyr

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

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

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

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Ala Val Gly Trp Phe Arg Gln Arg Pro Gly Lys Gln Arg Glu Glu Val

35 40 45

Ala Cys Leu Arg Val Arg Tyr Ala Asn Thr Asn Lys Ala Pro Ser Val

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Arg Glu Arg Val His Val Phe Arg Glu Glu Asn Asn Asn Leu Val Tyr

65 70 75 80

Met Leu Met Ser Asp Leu Thr Pro Glu Asp Thr Gly Ile Tyr Tyr Cys

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Ala Ala Arg Pro Gln Gln Pro Ser Ala Asp Cys Ser Leu Leu Ala Asn

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Asp Tyr Asp Asn Trp Gly Gln Gly Ile Gln Val Thr Val Ser Glu

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

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Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Arg Glu Gly Val

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Ser Cys Ile Ser Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asn Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

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

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Ala Ala Asp Ser Thr Gly Arg Cys Trp Ala Arg Pro Leu Tyr Glu Tyr

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

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Thr Ser Ser Arg Phe Thr Leu Asp Tyr Tyr

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Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Arg Arg Glu Gly Val

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

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

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

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Ala Ala Asp Ser Thr Gly Arg Cys Trp Ala Arg Pro Leu Tyr Glu Tyr

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

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

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

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Ala Val Gly Trp Phe Arg Arg Arg Pro Gly Lys Gln Arg Glu Glu Val

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Ala Cys Leu Arg Val Arg Tyr Ala Asn Ser Asn Lys Ala Pro Ser Val

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Arg Glu Arg Val His Val Phe Arg Glu Glu Lys Asn Asn Leu Val Tyr

65 70 75 80

Met Leu Met Ser Asp Leu Thr Pro Glu Asp Thr Gly Ile Tyr Tyr Cys

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Ala Ala Arg Pro Gln Gln Pro Ser Ala Asp Cys Ser Leu Ser Ala Asn

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Asp Tyr Asp Asn Trp Gly Gln Gly Ile Gln Val Thr Val Ser Glu

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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His Ala Ala Arg Pro

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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

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Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

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Ile Thr Leu Tyr Cys

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Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

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Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

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Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

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Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

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Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

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Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

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Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

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<212> DNA

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tgccgctggc cttgctgctc cacgccgcca ggccgcaggt gcagctggtg gag 53

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cgctggcgtc gtggtgctag acactgtcac ctg 33

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cgctggcgtc gtggtagagc tcactgtcac ctg 33

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gctgcaggtc gactctagag gatcccgcca ccatggcctt accagtgacc gccttgctcc 60

tgccgctggc cttgc 75

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accacgacgc cagcgccgcg ac 22

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tcgataagct tgatatcg 18

Claims

1. A nanobody against IL13Ra2, characterized in that the amino acid sequence of CDR1 of said nanobody comprises the sequence shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3;

the amino acid sequence of the CDR2 of the nano antibody comprises a sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7;

the amino acid sequence of the CDR3 of the nano antibody comprises a sequence shown in SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10.

2. The nanobody against IL13Ra2 according to claim 1, wherein the amino acid sequence of CDR1 of the nanobody comprises the sequence shown in SEQ ID No.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.4, the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 8;

preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.2, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 9;

preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.1, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.6, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 8;

preferably, the amino acid sequence of the CDR1 of the nanobody comprises the sequence shown in SEQ ID NO.3, the amino acid sequence of the CDR2 comprises the sequence shown in SEQ ID NO.4, and the amino acid sequence of the CDR3 comprises the sequence shown in SEQ ID NO. 8;

3. A nucleic acid molecule comprising a gene encoding the nanobody against IL13Ra2 of claim 1 or 2.

4. A chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signaling domain;

the antigen binding domain comprises the nanobody of claim 1 or 2 against IL13Ra 2.

5. The chimeric antigen receptor according to claim 4, wherein the signal peptide comprises a CD8 a signal peptide;

preferably, the hinge region comprises a CD8 a hinge region;

preferably, the transmembrane region comprises any one of, or a combination of at least two of, a CD8 a transmembrane region, a CD28 transmembrane region, or a DAP10 transmembrane region;

preferably, the signaling domain comprises an immunoreceptor tyrosine activation motif;

preferably, the signaling domain further comprises a co-stimulatory molecule comprising any one of the 4-1BB, CD28 intracellular domain, OX40, ICOS or DAP10 intracellular domain or a combination of at least two thereof;

preferably, the chimeric antigen receptor comprises a CD8 a signal peptide, the nanobody of claim 1 or 2 against IL13Ra2, a CD8 a hinge region, a CD8 a transmembrane region, and an immunoreceptor tyrosine activation motif.

6. An expression vector comprising a gene encoding the chimeric antigen receptor of claim 4 or 5;

preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to claim 4 or 5, preferably a lentiviral vector.

7. A recombinant lentivirus comprising the expression vector of claim 6.

8. A chimeric antigen receptor immune cell, wherein said chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 4 or 5;

preferably, the chimeric antigen receptor immune cell comprises the expression vector of claim 6 and/or the recombinant lentivirus of claim 7;

9. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of claim 8;

preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients;

10. Use of a nanobody against IL13Ra2 of claim 1 or 2, a nucleic acid molecule of claim 3, a chimeric antigen receptor of claim 4 or 5, an expression vector of claim 6, a recombinant lentivirus of claim 7, a chimeric antigen receptor immune cell of claim 8 or a pharmaceutical composition of claim 9 for the preparation of a medicament for the treatment of tumors;

preferably, the tumor comprises a tumor expressing IL13Ra 2.