CN116589585A

CN116589585A - anti-IL13Ra2 nano antibody and application thereof

Info

Publication number: CN116589585A
Application number: CN202310611114.9A
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: 2023-08-15
Anticipated expiration: 2041-12-29
Also published as: CN116444671A; CN114409782A; CN116514982B; CN116514982A; CN116589585B; CN116813773A; CN116444671B; CN114409782B

Abstract

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

Description

anti-IL13Ra2 nano antibody and application thereof

The application relates to a divisional application of a patent application with the application number of 202111681616.6 (the application date of the original application is 2021, 12 and 29 days, and the application name is an anti-IL13Ra2 nanobody and application thereof).

Technical Field

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

Background

Brain gliomas are the most common primary malignant tumors in the cranium, gliomas can be classified into grades I to IV from low to high according to WHO classification, I and II are low-grade gliomas, III and IV are high-grade gliomas, wherein glioblastoma multiforme (Glioblastoma multiforme, GBM) has the highest malignant degree and accounts for more than 80% of malignant gliomas. Although diagnostic and therapeutic methods have been rapidly developed over the last decade, the prognosis is still not ideal due to the characteristics of high grade gliomas (grade iii-iv), strong aggressiveness, high difficulty in treatment, poor prognosis, high recurrence rate, etc., with a median survival of only about 14 months ^[1] Survival rate of 2 years is less than 25 percent ^[2,3] . Although low grade gliomas (grade I-II) have a higher prognosis and are still associated with neurological symptoms such as epilepsy and cognitive dysfunction。

Traditional treatment schemes of gliomas are that surgical excision is combined with auxiliary temozolomide chemotherapy and synchronous radiotherapy, but treatment measures for malignant gliomas which are difficult to carry out surgical treatment at multiple and growing positions are limited. Such non-specific treatment regimens do not provide complete relief of glioblastoma and can cause damage to healthy brain tissue, which is not beneficial to improving patient quality of life. In addition, individual glioma cells can metastasize through white matter tracts, callus or cerebrospinal fluid, leading to recurrence, with more than half of patients finding metastases in the contralateral hemispheres ^[4] . In view of the poor effect of traditional therapies, the development of chimeric antigen receptor T-cells (CAR-T) has been fast in recent years, which brings new eosin to the treatment of GBM.

A number of in vitro pre-clinical results have demonstrated that the 5 tumor-associated antigens IL13Rα2 (Interleukin-13 receptor αchain variable 2, interleukin 13receptor α2), EGFR VIII (epidermal growth factor receptor-VIII, EGFR variant VIII), erbB2 (epidermal growth factor receptor, EGFR 2), ephA2 (ephrinA 2receptor, tyrosine protein kinase receptor A2) and B7-H3 are likely targets for the treatment of GBM, have achieved good results in preclinical animal models and in part of the clinical trials, and have been developed in clinical trials.

Interleukin 13receptor alpha 2 (IL 13Rα2), a membrane-bound protein, is closely related to IL13Rα1. The IL13Rα2 expression of adult brain tumor and meningioma in children is rich, on the contrary, it can not be detected in normal brain tissue, its expression quantity on GBM cell surface is 3 ten thousand times of that of normal tissue cell, 58% of the gene expression of patient is up-regulated ^[5] Flow cytometry analysis of the average expression levels in U373 cell lines and 3 GBM patient cells was as high as 73% ^[6] Is an effective target for immunotherapy GBM. It plays an important role in immune response and tumor microenvironment, and can block apoptosis pathways, resulting in immune escape. The first generation and second generation CAR-T targeting IL13 Ralpha 2 show obvious anti-tumor effect ^[7-9] . First generation CAR-T cells expressing IL13E13Y muteins have higher affinity for IL13Rα2Competes for binding to IL-13a1/IL4Ra expressed in the central nervous system, as reported ^[10] 3 cases of recurrent GBM were injected directly into CD8 (+) CTL cells of IL13 (E13Y) -zetakine multiple times through the cranial cavity after surgery, 2 cases had a survival period exceeding 14 months, and adverse reactions were not obvious. However, the survival time of first generation CAR-T cells in glioma environments is limited to within 15 days. Recent research results indicate that ^[8] The survival time of the CAR-T cells fused with the co-stimulatory structure is prolonged, and stronger anti-glioma activity can be exerted. In vitro experiments ^[9] Second generation CAR-T expressing E13K and/or E13Y can kill IL13rα2 ⁺ Without causing damage to normal tissue cells. In-situ glioma experiment of mice proves ^[11] The second generation E13KCAR-T cells fused with CD28 can kill tumor cells, and prolong the survival time of experimental mice. Brown et al ^[12] IL13-zetakine modified targeted T cells were found to kill IL13Rα2 simultaneously ⁺ Glioma cells and IL13Rα2 ⁺ Stem cell-like tumor cell subpopulations of (2), perhaps for complete eradication of IL13Rα2 ⁺ Is helpful for glioma.

In summary, the anti-IL13Rα2 antibody with high specificity and affinity can be effectively applied to preparing the CAR-T taking IL13Rα2 as a target, and has important significance in the field of glioma treatment.

Reference to the literature

1. Xu Pengfei, yang Jian, yang Xue, yuan Fanen Chen Qianxue. Research progress on childhood gliomas and adult gliomas. 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, zhong Xiaosong Li Wen recent advances in chimeric antigen receptor T cell therapy for glioblastoma multiforme Chinese tumor clinic (2017) 44 (6): 794-799.

5.Bart Thaci,Christine E.Brown,Emanuela Binello,Katherine Werbaneth,Prakash SampathSengupta S.Significance ofinterleukin-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 ofadoptively 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-13RA2 expressed 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,ShamiAF,Martinez C,D'apuzzo M,Barish M E,Forman SJJensen M C.Stem-like tumor-initiating cells isolated from IL13Ralpha2 expressing 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 actual demands of the prior art, the application provides an anti-IL13Ra2 nano antibody and application thereof. The nanometer antibody of the anti-IL13Ra2 has high affinity, can be used as an antigen binding domain of a chimeric antigen receptor molecule to prepare the CAR-T cell, and has good application prospect in the aspect of tumor treatment.

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

in a first aspect, the application provides an anti-IL13Ra2 nanobody, the amino acid sequence of CDR1 of which 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 which 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 which comprises the sequence shown in SEQ ID No.8, SEQ ID No.9 or SEQ ID No. 10.

According to the application, an unimmunized alpaca is immunized by IL13Ra2 recombinant protein, a phage display nanobody library is constructed, and screening is carried out on anti-IL13Ra2 antibodies according to the phage display nanobody library, so that monoclonal antibodies capable of specifically binding IL13Ra2 antigens are obtained.

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 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, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID NO. 8.

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

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

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

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

Preferably, the heavy chain variable region of the nanobody further comprises: a framework region 1 (FR 1) shown by SEQ ID No.11, SEQ ID No.12 or SEQ ID No.13, a framework region 2 (FR 2) shown by SEQ ID No.14, SEQ ID No.15 or SEQ ID No.16, a framework region 3 (FR 3) shown by SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 or SEQ ID No.20, a framework region 4 (FR 4) 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 as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.26 or SEQ ID NO. 27.

SEQ ID NO.23：

QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIAWFRQAPGKRREGVSCISSSEGSTN YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSTGRCWARPLYEYDYRGQGT QVTVSS。

SEQ ID NO.24：

QVQLVESGGGLAHPGGRLRVTCTASGANLKNYAVGWFRQRPGKQREEVACLRVRYAN TNKAPSVRERVHVFREENNNLVYMLMSDLTPEDTGIYYCAARPQQPSADCSLLANDYDNW GQGIQVTVSE。

SEQ ID NO.25：

QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIAWFRQAPGKRREGVSCISSSGGST NYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSTGRCWARPLYEYDYRGQ GTQVTVSS。

SEQ ID NO.26：

QVQLVESGGGSVQAGGSLRLSCTSSRFTLDYYAIAWFRQAPGKRREGVSCISSSEGSTN YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADSTGRCWARPLYEYDYRGQGT QVTVSS。

SEQ ID NO.27：

QVQLVESGGGLAHPGGRLRVTCTASGANLKNYAVGWFRRRPGKQREEVACLRVRYAN SNKAPSVRERVHVFREEKNNLVYMLMSDLTPEDTGIYYCAARPQQPSADCSLSANDYDNW GQGIQVTVSE。

In the application, the phage display technology is utilized to screen an 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 of targeted IL13Ra 2.

In a second aspect, the application provides a nucleic acid molecule comprising a gene encoding the nanobody of anti-IL13Ra2 of the first aspect.

In a third aspect, the application provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain comprising a nanobody of anti-IL13Ra2 of the first aspect, a hinge region, a transmembrane region, and a signal transduction 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 or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region or a DAP10 transmembrane region.

Preferably, the signal transduction domain comprises an immunoreceptor tyrosine activation motif (cd3ζ).

Preferably, the signal transduction domain further comprises a co-stimulatory molecule comprising any one or a combination of at least two of 4-1BB, the intracellular domain of CD28, OX40, ICOS or the intracellular domain of DAP 10.

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

In a fourth aspect, the present application 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 comprising a gene encoding the chimeric antigen receptor according to the third aspect, preferably a lentiviral vector.

In a fifth aspect, the present application 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 application 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 the substrate aspect of the fourth aspect.

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

In a seventh aspect, the application 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 carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.

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

Preferably, the tumor comprises a tumor expressing IL13Ra 2.

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

(1) The application uses IL13Ra2 recombinant protein to immunizeConstructing phage display nano antibody library by using the non-immunized alpaca, screening anti-IL13Ra2 antibody according to the phage display nano antibody library, and obtaining nano antibodies which can specifically bind IL13Ra2 antigen and have good affinity, wherein KD (M) is 1.61 multiplied by 10 respectively through determination of antibody affinity ^-8 、3.24×10 ^-8 、1.07×10 ^-9 、1.66×10 ^-8 And 2.57×10 ^-9 ；

(2) The nanometer antibody of the anti-IL13Ra2 has better affinity, is used as an antigen binding domain to construct a chimeric antigen receptor, and is used for preparing T cells, wherein the CAR-T cells have killing activity on IL13Ra2 positive tumor cells, and secrete cytokines IFN-gamma and TNF-alpha with high efficiency after being co-cultured with IL13Ra2 positive cells.

Drawings

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

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

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

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

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

FIG. 2 is a graph showing the results of FACS detection of IL-13 Ra2 antigen recognized by anti-IL-13 Ra2 nanobody in example 3;

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

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

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

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

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

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

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

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

FIG. 10 is a bar graph of TNF alpha secretion by CAR-T cells in example 8;

FIG. 11 is a bar graph of IFNγ secretion by CAR-T cells in example 8.

Detailed Description

The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present application and do not represent or limit the scope of the application, which is defined by the claims.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors, with no manufacturer noted.

Example 1

In this example, phage nanobody libraries were constructed and panning were performed and primary screening was performed using ELISA, as follows:

(1) Construction of phage nanobody library

Immunizing alpaca by adopting IL13Ra2 extracellular recombinant protein, and extracting peripheral blood after ELISA detection of serum titer; separating lymphocytes, extracting total RNA, performing reverse transcription to obtain cDNA, and amplifying VHH genes by nested PCR; VHH gene was inserted into pShort phagemid and electrotransformedSeparating and purifying the phage by PEG8000/NaCI precipitation method after the competent cells are amplified to obtain an antibody library; adjusting concentration, packaging, and freezing in refrigerator at-80deg.C;

(2) Screening of phage nanobody libraries

Firstly, taking 293T cells and an antibody library for co-incubation to carry out negative screening, and then taking supernatant, and respectively incubating with IL13Ra2 positive cells 293T-IL13Ra2 cells and 293T cells; washing for 4 times by adopting a pre-cooled PT buffer solution at 4 ℃; 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 the phage by PEG8000/NaCI precipitation method, and then carrying out the next round of screening; after enrichment, the VHH region was amplified using the obtained phage as a template, and subjected to second generation sequencing to obtain 5 anti-IL13Ra2 nanobodies, the amino acid sequences of which were respectively designated VHH-1, VHH-7, VHH-8, VHH-13 and VHH-16 as shown in SE1 ID No.22, SE1 ID No.23, SE1 ID No.24, SE1 ID No.25 and SE1 ID No. 26.

Example 2

This example expresses and purifies the anti-IL13Ra2 nanobody (VHH-mIgG 2a Fc nanobody) screened in example 1 and determines the affinity of the antibody. In order to further identify the antibodies obtained by screening, it was necessary to express the antibodies by mammalian cells, and therefore, a plasmid vector carrying a mouse Fc tag-expressing VHH, designated C-4pCP. Stuffer-mCg a-FC, was constructed first, comprising the following steps:

(1) The VHH fragment was amplified using PCR, the reaction system of which is shown in Table 1, and the amplification procedure is shown in Table 2;

TABLE 1

TABLE 2

(2) EnzymesThe cleavage system is shown in Table 3, the cleavage temperature is 37 ℃ and the time is 6 hours, and the carrier after cleavage is usedPurifying 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 Table 3

Reagent(s)	Usage amount
		C-4pCP.Stuffer-mCg2a-FC	5μg
10 Xenzyme cutting Buffer (10 Xreaction Buffer)	5μL
		FspA I	2μL
PfI 23II	2μL
		ddH ₂ O	Make up to 50 mu L

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

TABLE 4 Table 4

Reagent(s)	Volume (mu L)
		Exnase II	1
2×Exnase II buffer	2
		Linearization carrier (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 (temperature)	Time
			Ice bath	0℃	5min
Heat shock	42℃	1min
			Ice bath	0℃	3min
Adding 500 μl of LB medium, and shaking culturing at 220rpm	37℃	1.5h
			mu.L was pipetted and spread on LB/Amp plates	37℃	Overnight

(5) The transformation plate is selected to be pre-identified by monoclonal PCR, and the conditions of a PCR identification system are shown in Table 6; the conditions are that the pre-denaturation is carried out for 3min at 95 ℃; denaturation at 95℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72 ℃ for 5min, preserving at 4 ℃, and sending to a sequencing company for sequencing identification, wherein the sequencing result meets the expectations, and successfully constructing a plasmid vector with the mouse Fc tag for expressing VHH in the examples of the specification.

TABLE 6

293E cells were passaged approximately 24h prior to plasmid transfection to a cell density of approximately 0.6X10 ⁶ cells/mL; when the cell density is 1.2X10 ⁶ cell/mL, activity>At 95%, 293E cells were transfected with PEI at a rate of 150. Mu.g DNA per 100mL 293E, wherein the plasmidThe ratio of DNA to PEI is 1:2;

37℃、130rpm、8％CO ₂ shaking culture for 6 days, collecting cell culture supernatant at 3000rpm for 30min, filtering the collected supernatant containing target antibody with 0.45 μm filter, and adding sample to purification column MabSelect ^TM SuRe ^TM The column was washed with 5 times PBS, the protein eluted with 0.1M Gly-HCl (pH 3.0) and neutralized with 1/10 volume of Tris-HCl at pH 8.5, followed by dialysis overnight at 4℃and then determination of A with Nanodrop 2000 ₂₈₀ The antibody purity was determined by SEC-HPLC.

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

Biacore is a bioanalytical sensing technology developed based on surface plasmon resonance (surface Plasmon resonance, SPR) and can detect and track the whole change process of binding and dissociating molecules in a solution and molecules fixed on the surface of a chip, record the whole change process in the form of a sensor map and provide dynamics and affinity data.

In the measurement, the antibodies were immobilized on the chip surface, the mobile phase was a solution containing the antigen (IL 13Ra 2), and the measurement results are shown in Table 7 and FIGS. 1A to 1E, wherein the affinities of the 5 antibodies all reached sub-nanomolar levels of 1.61E-8, 3.24-8, 1.07E-9, 1.66E-8 and 2.57E-9, respectively.

TABLE 7

Nanobody	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 describes a flow assay for the nanobody of example anti-IL13Ra 2.

293T (IL 13Ra2 negative, IL13Ra 2) ^- ) 293T-Mesothelin cells were mixed with purified anti-IL13Ra2 nanobodies, incubated for 30min in an ice bath, then incubated with APC-labeled goat anti-mouse IgG antibody for 30min, and examined using a flow cytometer, as shown in FIG. 2, indicating that the anti-IL13Ra2 nanobodies of the present application recognize the cell surface IL13Ra2 antigen.

Example 4

This example prepares lentiviral vectors expressing a chimeric antigen receptor targeting IL13Ra2 (IL 13Ra2 CAR).

First, a lentiviral vector pSIN03 IL13Ra2CAR carrying an IL13Ra2CAR chimeric antigen receptor was constructed, the vector profile of which is shown in FIG. 3, and the schematic of which is shown in FIG. 4, comprising a CD8 alpha signal peptide, a nanobody against IL13Ra2 (anti-IL 13Ra2 VHH), a CD8 alpha hinge region, a transmembrane region, and an immunoreceptor tyrosine activation motif (CD 3 zeta).

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

MALPVTALLLPLALLLHAARP。

the amino acid sequence of the anti-IL13Ra2 VHH is shown as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 or SEQ ID NO. 27.

The amino acid sequences of the CD 8. Alpha. Hinge region and the transmembrane region (SEQ ID NO. 29) are:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYC。

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

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

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

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

the preparation method comprises the following steps:

(1) A PCR reaction system was prepared according to Table 8 (reagents in Table were derived from TOYOBO Inc.), each anti-IL13Ra2 nanobody fragment was amplified, and the reaction was performed according to the PCR procedure shown in Table 9, with 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(s)	Volume (mu L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10μM MF1-F	1
		10 mu 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 the resulting amplified product was preceded by a CD 8. Alpha. Signal peptide, and reacted according to the PCR procedure shown in Table 9, using the primers:

BamH-CD8αsig-F(SEQ ID NO.35)：

GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC；

table 10

After the reaction is finished, the PCR product is subjected to 1% agarose gel electrophoresis, the fragment of about 500bp is recovered, and the quantity is fixed by an ultraviolet absorption method;

(3) A PCR reaction system was prepared according to Table 11, and after completion of the preparation, a PCR reaction was performed according to the PCR procedure shown in Table 9 to amplify the CD 8. Alpha. Range-TM-41 BB-CD3Z fragment, using the primers as follows:

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

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

TABLE 11

Reagent(s)	Volume (mu L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 mu M upstream primer CD8 alpha H-F	1
		10 mu 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 about 780bp, and quantifying by an ultraviolet absorption method;

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

the 3 fragments recovered above and the vector backbone were linked by recombinase, the recombination reaction system is shown in Table 12, and after completion of the preparation, the mixture was subjected to a water bath reaction at 37℃for 0.5h, and transformed into E.coli stbl3 competent cells by a conventional method.

Table 12

Reagent(s)	Usage amount
		HD CD19 CAR	184.54ng
CD8αsingal IL13Ra2 VHH	31.32ng
		CD8αhinge-TM-41BB-CD3Z	29.72ng
5×CE buffer	2μL
		Exnase ^TM II	1μL
Sterile deionized water (PCR grade water)	Make up to 10 mu L

And selecting a monoclonal from the solid culture medium, culturing overnight, carrying out PCR identification, preparing a PCR reaction system as shown in table 13, carrying out a PCR procedure as shown in table 14, selecting a positive clone after the PCR is finished, and further carrying out sequencing identification, wherein the sequencing result meets the expectations.

TABLE 13

Reagent(s)	Volume (mu 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 packaging, concentration and titer detection were performed on the lentiviral vector HD SIN03-IL13Ra2CAR prepared in example 4, comprising the steps of:

(1) Lentivirus package

At 1.6X10 ⁷ Cell count 293T cells were seeded in 15cm dishes at 37℃with 5% CO ₂ The virus is prepared for packaging after overnight culture, and the culture medium is DMEM containing 10% of fetal calf serum; the lentiviral vector 14.5. Mu.g HD SIN03-IL13Ra2CAR, 16.7. Mu.g helper plasmid pMDLg-RRE, 16.7. Mu.g helper plasmid pRSV-REV and 6.5. Mu.g envelope plasmid VSVg were dissolved in 2mL serum-free DMEM medium and mixed well;

163.2. Mu.g PEI (1. Mu.g/. Mu.L) was dissolved in 2mL serum-free DMEM medium, vortexed at 1000rpm for 5 seconds and incubated at 25℃for 5min; adding PEI mixed solution into the DNA mixed solution, immediately vortex mixing or gently mixing after adding, and incubating for 20min at 25 ℃ to form a transfection complex; then, 4mL of the transfection complex is added into 25mL of DMEM medium containing 293T cells in a dropwise manner, and after 4 hours, the fresh medium is replaced; after 48 hours, collecting the virus liquid supernatant;

(2) Lentivirus concentration

Filtering the virus supernatant with a 0.45 μm filter membrane, collecting the filtered virus supernatant into a 50mL centrifuge tube, adding 1/4 PEG-NaCl virus concentrate, mixing the mixture upside down, and standing the mixture at 4 ℃ overnight; centrifuging at 4 ℃ at 3500rpm for 30min; removing supernatant, adding appropriate amount of RPMI 1640 medium (containing 10% FBS), and dissolving the resuspended virus precipitate; split charging 50 μl of the concentrated lentiviral suspension into each portion, storing in a finished tube, and storing at-80deg.C;

(3) Lentivirus titer detection

500 mu L K562 cells (1X 10) ⁵ Individual cells) were inoculated into 24-well plates, and the concentrated lentiviruses were 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℃at 5% CO ₂ After overnight incubation, fresh medium was changed;

after 72h infection, 400 Xg was centrifuged for 5min, the supernatant was discarded to collect cells, 100. Mu.L PBS+2% FBS was added to resuspend the cells, PE coat anti-alpaca VHH antibody was added and incubated on ice for 30min; after 2 times of washing with the flow buffer, 300 mu L of the flow buffer is added to resuspend cells, and a flow cytometer is adopted to detect the infection efficiency; the titer was calculated as follows: titer (TU/mL) =cell number (10 ⁵ ) X positive rate/viral volume (mL).

Example 6

This example uses lentivirus transduced T lymphocytes prepared in example 5, comprising the steps of:

(1) Diluting the anti-human CD3 antibody and the anti-human CD28 antibody with PBS (phosphate buffered saline) to obtain final concentrations of 1 mug/mL and 0.5 mug/mL respectively, coating an orifice plate, and standing at 4 ℃ in a refrigerator overnight; discarding the antibody coating liquid in the pore plate, and washing the plate with 1mL PBS;

(2) Human PBMC were conditioned to a density of 1X 10 with T cell culture medium (X-VIVO+10% FBS+300U/mL IL-2) ⁶ Per mL, inoculation to CD3 and CD28 anti-Activating for 24 hours in a pore plate coated with the body; the activated T cells were collected and the cell density was adjusted to 1X 10 ⁶ Lentivirus was added per mL at a multiplicity of infection (multiplicity of infection, MOI) of 10, polybrene was added to a final concentration of 5 μg/mL; at 37℃with 5% CO ₂ Replacing fresh culture medium after overnight culture in the environment, and carrying out passage every 3 days;

(3) 5 days after T cell infection, 3X 10 cells were taken ⁵ Centrifuging at 4 ℃ for 5min at 400 Xg, discarding the supernatant, and washing once with streaming buffer; adding 100 mu L of buffer solution to resuspend cells, adding PE coat anti-alpaca VHH antibody, and incubating on ice for 30min; after 2 washes with buffer, 300 μl buffer was added to resuspend cells;

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

In addition, the flow cytometer is used to detect lymphocyte phenotypes in this example, including the steps of:

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

(2) Cells were resuspended in 50. Mu.L of buffer, 1. Mu.L of FITC-labeled Anti-CD3 Ab, percp-Cy5.5 labeled Anti-CD4 Ab and PE-Cy7 labeled Anti-CD8 Ab were added and incubated on ice for 30min; after washing twice with buffer, cells were resuspended in 300. Mu.L of buffer and examined for cell phenotype using flow cytometry, as shown in FIGS. 6A and 6B, CD3 ⁺ CD4 ⁺ Cell population ratio is 33-37%, CD3 ⁺ CD8 ⁺ The cell population was 61-66%.

Example 7

In this example, an in vitro toxicity test of CAR-T cells was performed, comprising the steps of:

(1) Target cell inoculation

293-GPF-luci (IL 13Ra2 negative, IL13Ra 2) ^- )、U251-GFP-luci(IL13Ra2 ⁺ )、U87-GFP-luci(IL13Ra2 ⁺ ) As target cells, modulationTarget cell concentration was 2X 10 ⁵ 50. Mu.L of the solution was inoculated into a white 96-well plate;

(2) Effector cell seeding

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 the effective target ratio of 0.3:1 and 1:1;

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

experimental group: each target cell + CAR-T;

control group: inoculating only target cells;

(4) The detection method comprises the following steps:

after 18h of co-culture of effector cells with target cells, 100. Mu.L of Steady-Reagents (Promega, cat#E2520), after 5min of reaction, the bioluminescence signal was detected using a multifunctional microplate reader;

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

killing efficiency% = (control-experimental)/control x 100%.

The results are shown in figures 7-9, the CAR-T cells constructed by the application have no killing effect on IL13Ra2 negative 293T cells and have killing activity on IL13Ra2 positive tumor cells, and the CAR-T cells constructed by the application have high-efficiency tumor killing activity and high specificity.

Example 8

In this example, the secretion of the CAR-T cytokines TNF- α and IFN- γ was detected using the kits Human TNF- α ELISAKIT (co-products, cat# EK 182-96) and Human IFN- γ ELISAKIT (co-products, cat# EK 180-96), respectively.

1. Cell culture supernatant

Cell cultures with an effective target ratio of 1:1 were centrifuged at 400 Xg for 10min to remove sediment, and the supernatant was stored at-80℃for examination.

2. Reagent preparation

All reagents, samples were returned to 25℃before testing, and if crystallization occurred in the concentrated reagents, the incubation was performed at 37℃until the crystals were completely dissolved, and 1 Xof wash solution and 1 Xof assay buffer were prepared according to the instructions of use.

3. Standard substance and sample preparation

Standard substance: standard stock was 2-fold diluted with 5%1640 medium for a total of 8 dilution gradients, including zero concentration.

Sample: samples were diluted in ratio using 5%1640 medium.

4. Detection step

(1) Soaking the ELISA plate: adding 300 mu L of 1 Xwashing liquid, standing and soaking for 30s, discarding the washing liquid, and beating the micro-pore plate on water-absorbing paper;

(2) Adding a standard substance: standard wells were filled with 100 μl of 2-fold diluted standard and blank wells were filled with 100 μl of standard diluent (serum/plasma samples);

(3) Adding a sample: sample wells were added with 100 μl of cell culture supernatant;

(4) Adding a detection antibody: 50. Mu.L of diluted detection antibody (1:100 dilution) was added to each well;

(5) Incubation: sealing plates by using sealing plates, vibrating at 300rpm, and incubating at 25 ℃ for 2 hours;

(6) Washing: liquid was discarded, and 300. Mu.L of wash solution was added to wash the plate 6 times per well;

(7) And (3) enzyme adding and incubation: mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) was added to each well;

(8) Incubation: using a new sealing plate membrane sealing plate, oscillating at 300rpm, incubating at 25 ℃ for 45min, and washing;

(9) And (3) color development of the substrate: 100 mu L of chromogenic substrate TMB is added into each hole, and incubated for 20min at 25 ℃ in the dark;

(10) Adding a stop solution: 100 mu L of stop solution is added to each well;

(11) Detecting and reading: within 30min, performing dual-wavelength detection by using an enzyme-labeled instrument, and measuring the OD value at the maximum absorption wavelength of 450nm and the reference wavelength; the OD value after calibration was 450nm minus the measurement at the reference wavelength.

TNF- α, IFN- γ factor secretion results are shown in FIGS. 10 and 11, respectively, wherein spontaneous is a single CAR-T cell group, no cytokines were detected; no cytokines were detected in the CAR-T cell co-cultured group with 293T cells; after co-culture with target cells over-expressing MSLN, the TNF-alpha secreted by the CAR-T cells exceeds 200pg/mL, and the IFN-gamma exceeds 3000pg/mL, and the constructed CAR-T cells release cytokines to tumor cells positive for IL13Ra2, but have no obvious cytokine secretion to IL13Ra2 negative cells.

In conclusion, the nano antibody with high affinity for resisting IL13Ra2 can be screened and prepared, can be combined with IL13Ra2 in a specific way, and can be used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell, and the obtained CAR-T cell has obvious killing activity and specificity for tumor cells positive to IL13Ra2 and can secrete cytokines for killing tumors, so that the nano antibody can be effectively applied to immunotherapy, and has important significance for developing tumor therapeutic drugs.

The applicant declares that the above is only a specific embodiment of the present application, but the scope of the present application is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present application disclosed by the present application fall within the scope of the present application and the disclosure.

Claims

1. An anti-IL13Ra2 nanobody, wherein the amino acid sequence of CDR1 of said nanobody comprises the sequence shown in SEQ ID No.1, the amino acid sequence of CDR2 comprises the sequence shown in SEQ ID No.6, and the amino acid sequence of CDR3 comprises the sequence shown in SEQ ID No. 8.

2. A nucleic acid molecule comprising a gene encoding the nanobody of claim 1 against IL13Ra 2.

3. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane region, and a signal transduction domain;

the antigen binding domain comprises the nanobody of anti-IL13Ra2 of claim 1.

4. The chimeric antigen receptor according to claim 3, 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 or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region or a DAP10 transmembrane region;

preferably, the signal transduction domain comprises an immunoreceptor tyrosine activation motif;

5. The chimeric antigen receptor according to claim 4, wherein the chimeric antigen receptor comprises a CD8 a signal peptide, the nanobody of anti-IL13Ra2 of claim 1, 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 any one of claims 3-5;

preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a lentiviral vector, comprising a gene encoding the chimeric antigen receptor according to any one of claims 3 to 5.

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

8. A chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor of any one of claims 3-5;

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 the nanobody of anti-IL13Ra2 of claim 1, the nucleic acid molecule of claim 2, the chimeric antigen receptor of any one of claims 3-5, the expression vector of claim 6, the recombinant lentivirus of claim 7, the chimeric antigen receptor immune cell of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating a tumor;

preferably, the tumor comprises a tumor expressing IL13Ra 2.