CN110564694B - CAR-T cell drug secreting IL-23 antibody and targeting prostate cancer - Google Patents

Abstract

The invention discloses a prostate cancer targeted CAR-T cell drug secreting an IL-23 antibody, and relates to the technical field of immunocytology. The disclosed prostate cancer-targeted CAR-T cells contain a nucleic acid molecule having a first nucleic acid sequence encoding a chimeric antigen receptor that binds to prostate specific membrane antigen and a second nucleic acid sequence encoding an antibody or functional fragment of the antibody that specifically binds IL-23. The CAR-T cell or a medicament containing the same can target prostate specific membrane antigen of prostate cancer, and can secrete antibodies or functional fragments of the antibodies which are specifically combined with IL-23, and the secreted antibodies or the functional fragments of the antibodies act in a paracrine mode to reverse castration resistance of the prostate cancer, enhance the activity of the CAR-T cell against the prostate cancer and improve the killing capacity of the CAR-T cell against the prostate cancer.

Description

CAR-T cell drug secreting IL-23 antibody and targeting prostate cancer

Technical Field

The invention relates to the technical field of immunocytology, in particular to a CAR-T cell medicine which secretes IL-23 antibody and targets prostate cancer.

Background

Prostate cancer is one of the most common tumors of the male genitourinary system, with global morbidity accounting for the second place of malignancy in men and global mortality accounting for the fifth place of malignancy. Is the cancer with the highest incidence in the united states and is also the second most dead cancer. Data show that the prostate cancer in China has a high incidence trend in recent 10 years, large cities become 'serious disaster areas', and the incidence of the prostate cancer in China rises along with the rising of the incidence of the prostate cancer based on the characteristics of population cardinality, age structure and the like of aging day by day.

Traditional prostate cancer treatments include radical prostatectomy, radiation therapy, chemotherapy, and Androgen Deprivation Therapy (ADT), i.e., the use of drugs or surgery to reduce androgen levels in the body.

Androgen deprivation therapy is among the leading treatments for prostate cancer in patients at different stages, also known as endocrine therapy, because androgens can promote tumor growth, while interdiction therapy delays disease progression by lowering androgen levels. Although this therapy has some efficacy, it can only sustain disease remission for 1 to 4 years in most patients, in addition to side effects on cardiovascular, sexual function, metabolic, psychological and skeletal health, and patients are resistant to androgen deprivation therapy, thus allowing cancer cells to develop into more aggressive castration-resistant prostate cancer (CRPC), which is still an unmet medical need for treatment of CRPC patients because of their poor prognosis.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a prostate cancer targeted CAR-T cell drug secreting IL-23 antibodies. The CAR-T cell or the medicine containing the same can target prostate specific membrane antigen of prostate cancer, and can secrete antibody or functional fragment of antibody specifically binding IL-23, and the secreted antibody or functional fragment of antibody acts in a paracrine mode to reverse castration resistance of prostate cancer, enhance the activity of the CAR-T cell against prostate cancer and improve the killing capacity of the CAR-T cell against prostate cancer.

The invention is realized by the following steps:

IL-23 produced by Myeloid Derived Suppressor Cells (MDSCs) is a driving factor for CRPC. The specific mechanism is that IL-23 produced by MDSCs regulates the castration resistance of prostate cancer by maintaining AR signaling, so that androgen receptor pathway can be activated, and cell survival and proliferation under androgen deficiency condition can be promoted. MDSCs promote CRPC by a non-cell autonomous manner. Thus, treatment with blocking IL-23 may be resistant to MDSC-mediated castration resistance to prostate cancer.

The immunotherapy is an emerging therapy of the prostate cancer, and a programmed cell death-1 (PD-1)/programmed cell death-ligand-1 (PDL 1) inhibitor in immune checkpoint inhibitors can improve the anti-tumor immunity of the organism by activating the immune system of the organism, so as to achieve the purpose of inhibiting and killing tumor cells, and provide a new direction for the immunotherapy of the prostate cancer, but when the inhibitor is used alone, the effect in a clinical test is not optimistic, and the Objective Remission (OR) rate is low.

Adoptive T cell transfer (ACT) is currently the most promising approach to immunotherapy, and CD-19 specific CAR-T cells can show complete remission in the treatment of relapsing refractory acute lymphoblastic leukemia, with the therapeutic procedure: the patient's own T cells (or T cells from allogeneic donors) are first isolated, then activated and genetically modified to obtain chimeric antigen receptor T cells (CAR-T), and finally returned to the patient. Chimeric antigen receptors are formed by linking an extracellular antigen recognition domain (usually an antibody single chain variable fragment scFv) to an intracellular signaling domain (the CD3 zeta chain of the T cell receptor or by the concomitant introduction of one or more costimulatory signals such as CD28, 4-1BB), the extracellular portion of which confers the ability of a T cell to recognize a specific antigen. Can cross MHC restriction, can stimulate T cell proliferation through a signal transduction structural domain after being directly combined with an antigen recognized by the antigen, simultaneously activates the cytotoxicity of the T cell and promotes the secretion of cytokines, finally eliminates the cells with the antigen, and has better specificity and persistence.

The Prostate Specific Membrane Antigen (PSMA) is highly expressed in prostate cancer, is an ideal target for immunotherapy of solid tumors, and has great specificity, targeting property and fewer major histocompatibility complex limitations in tumor immunotherapy by using CAR-T cells targeting PSMA. The effectiveness and safety of CAR-T cells have been demonstrated in preclinical studies and clinical trials, but their use has been limited and further research and exploration is needed.

Based on this, in a first aspect, embodiments of the invention provide a prostate cancer-targeted CAR-T cell secreting an IL-23 antibody, said CAR-T cell comprising a nucleic acid molecule; the nucleic acid molecule has a first nucleic acid sequence encoding a chimeric antigen receptor;

the chimeric antigen receptor has an antigen binding domain, and the antigen bound by the antigen binding domain is prostate specific membrane antigen;

the nucleic acid molecule further has a second nucleic acid sequence encoding an antibody or functional fragment thereof that specifically binds IL-23;

the prostate cancer is castration-resistant prostate cancer.

The CAR-T cell provided by the invention not only has the function of targeting PSMA, but also has the characteristic of secreting and expressing an anti-IL-23 antibody or a functional fragment of the antibody, and the antibody or the functional fragment of the antibody can be specifically bound with IL-23, so that the promotion effect of IL-23 on CRPC is eliminated. And by virtue of the antigen binding domain of the CAR-T cell that binds to prostate specific membrane antigen, an anti-IL-23 antibody or functional fragment of the antibody can be secreted in the vicinity of prostate cancer cells, which in turn acts in a paracrine manner to reverse castration resistance mediated by MDSCs, reactivate the sensitivity of tumor cells to hormone therapy, and greatly enhance the activity of CAR-T cells against prostate tumors. In addition, the CAR-T cells provided by the invention can be combined with androgen deprivation therapy to reactivate tumor cell sensitivity to hormone therapy, which would greatly enhance the efficacy of castration-resistant prostate cancer. The CAR-T cell improves a new therapeutic drug selection and a treatment strategy for treating the prostatic cancer, particularly castration-resistant prostatic cancer, and also provides a certain foundation for the preclinical experiment and clinical test of the prostate cancer CAR-T cell immunotherapy.

In an alternative embodiment, the functional fragment is selected from any one of Fab, Fab ', F (ab') 2, and scFv.

In an alternative embodiment, the functional fragment is an scFv.

In an alternative embodiment, the amino acid sequence of the light chain variable region of the functional fragment is shown in SEQ ID NO. 15.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the functional fragment is shown in SEQ ID NO. 17.

In an alternative embodiment, the amino acid sequence of the hinge region between the light chain variable region and the heavy chain variable region of the functional fragment is shown in SEQ ID NO. 5.

It should be noted that one skilled in the art can select suitable hinge regions, such as, but not limited to, the hinge region shown in SEQ ID NO.5, as desired, without any creative effort, to connect the light chain variable region and the heavy chain variable region to achieve specific binding to IL-23, and whatever hinge region is selected is within the scope of the present invention. In an alternative embodiment, the antigen binding domain is selected from any one of Fab, Fab ', F (ab') 2, and scFv.

In an alternative embodiment, the antigen binding domain is an scFv.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the antigen binding domain is as set forth in SEQ ID No. 3.

In an alternative embodiment, the light chain variable region of the antigen binding domain has the amino acid sequence shown in SEQ ID No. 7.

In an alternative embodiment, the amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is set forth in SEQ ID No. 5.

In an alternative embodiment, the chimeric antigen receptor described above further has a transmembrane domain and a costimulatory signaling region.

In an alternative embodiment, the transmembrane domain is selected from the transmembrane domains of at least one of the following protein molecules: CD5, CD28, CD137, CD3 epsilon, CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134, and CD8 alpha.

In an alternative embodiment, the transmembrane domain is a CD8a transmembrane domain.

In an alternative embodiment, the aforementioned co-stimulatory signaling region comprises an intracellular domain of at least one of the following co-stimulatory molecules: OX40, CD3 γ, CD3 δ, CD134, CD5, CD79a, CD137, ICD3 ε, CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, COS, 4-1BB, and CD3 ζ.

In an alternative embodiment, the aforementioned co-stimulatory signaling region comprises the intracellular co-stimulatory element of 4-1BB and the intracellular domain of CD3 ζ.

In a second aspect, embodiments of the invention provide a nucleic acid molecule having a first nucleic acid sequence encoding a chimeric antigen receptor;

the chimeric antigen receptor has an antigen binding domain, and the antigen bound by the antigen binding domain is prostate specific membrane antigen;

the nucleic acid molecule also has a second nucleic acid sequence encoding an antibody or functional fragment of the antibody that specifically binds IL-23.

The nucleic acid molecule provided by the invention is used for preparing the CAR-T cell targeting the prostate cancer, and the CAR-T cell can be prepared by introducing the CAR-T cell into an unmodified T cell to ensure that the CAR-T cell can normally express in the cell.

In an alternative embodiment, the functional fragment is selected from any one of Fab, Fab ', F (ab') 2, and scFv.

In an alternative embodiment, the functional fragment is an scFv.

In an alternative embodiment, the amino acid sequence of the light chain variable region of the functional fragment is shown in SEQ ID NO. 15.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the functional fragment is shown in SEQ ID NO. 17.

In an alternative embodiment, the amino acid sequence of the hinge region between the light chain variable region and the heavy chain variable region of the functional fragment is shown in SEQ ID NO. 5.

In an alternative embodiment, the antigen binding domain is selected from any one of Fab, Fab ', F (ab') 2, and scFv.

In an alternative embodiment, the antigen binding domain is an scFv.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region of the antigen binding domain is as set forth in SEQ ID No. 3.

In an alternative embodiment, the light chain variable region of the antigen binding domain has the amino acid sequence shown in SEQ ID No. 7.

In an alternative embodiment, the amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is set forth in SEQ ID No. 5.

In an alternative embodiment, the chimeric antigen receptor described above further has a transmembrane domain and a costimulatory signaling region.

In an alternative embodiment, the transmembrane domain is selected from the transmembrane domains of at least one of the following protein molecules: CD5, CD28, CD137, CD3 epsilon, CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134, and CD8 alpha.

In an alternative embodiment, the transmembrane domain is a CD8a transmembrane domain.

In an alternative embodiment, the aforementioned co-stimulatory signaling region comprises an intracellular domain of at least one of the following co-stimulatory molecules: OX40, CD3 γ, CD3 δ, CD134, CD5, CD79a, CD137, ICD3 ε, CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, 4-1BB, and CD3 ζ.

In an alternative embodiment, the aforementioned co-stimulatory signaling region comprises the intracellular domains of 4-1BB and CD3 ζ.

In a third aspect, embodiments of the invention provide a CAR-T cell medicament for the treatment of prostate cancer comprising a CAR-T cell according to any preceding embodiment.

In alternative embodiments, the CAR-T cell drug further comprises a drug used in androgen deprivation therapy.

In an alternative embodiment, the drug used in the androgen deprivation therapy is selected from androgen deprivation drugs such as abiraterone and enzalutamide.

In an alternative embodiment, the prostate cancer is castration-resistant prostate cancer.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic structural diagram of an expression cassette in example 1 of the present invention. In the figure: a: a CJ-PSMA-CAR structure; b: CJ-PSMA-IL-23ab-CAR structure.

FIG. 2 is a schematic diagram of the structure of a plasmid vector in example 1 of the present invention.

FIG. 3 shows the PSMA-CAR and IL-23ab-CAR viral titer CAR positivity rates.

FIG. 4 is positive for PSMA-CART and IL-23ab-CART CAR.

FIG. 5 shows the expression of IL-23ScFv detected by WB and QPCR.

FIG. 6 is a graph of IL-23ab-CART blocking the binding of IL-23 to its receptor IL-23R, in which: a: expression of IL-23R in K562 cells; b: the IL-23ab-CART supernatant reduced the binding of IL-23 to IL-23R.

FIG. 7 shows the killing function verification of PSMA-IL-23 ab-CART.

FIG. 8 is a schematic diagram of the structure of plasmid vector pMD2. G.

FIG. 9 is a schematic diagram of the structure of the plasmid vector pSPAX 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

A plasmid vector containing an expression cassette for expressing a chimeric antigen receptor and a single-chain antibody specifically binding to IL-23 was constructed, and the structure and positional relationship of each element on the expression cassette were as shown in FIG. 1, and the plasmid vector backbone was as shown in FIG. 2.

The method comprises the following specific steps:

a first expression cassette expressing a chimeric antigen receptor targeting a prostate specific membrane antigen was synthesized by kasuga, inc (PSMA-CAR) comprising: the CD8 alpha signal peptide, the PSMA single-chain antibody heavy chain variable region, the Linker1, the PSMA single-chain antibody light chain, the CD8 hinge region, the CD8 alpha transmembrane domain, the 4-1BB intracellular costimulatory element and the CD3 zeta intracellular domain (A in figure 1) are connected in sequence, and a Kozac sequence and a corresponding enzyme cutting site are introduced into the forefront. The first expression cassette was transferred to a plasmid empty vector (lentiviral transfer vector of Carl June (CJ) team) using XbaI and SalI double digestion, and after enzymatic ligation, the chimeric antigen receptor expression vector CJ-PSMA-CAR was obtained. Adding a second expression cassette for expressing a single-chain antibody of the anti-IL-23 by taking a CJ-PSMA-CAR plasmid as a starting plasmid, wherein the first expression cassette and the second expression cassette are connected by P2A, and the second expression cassette (IL-23-ScFv) sequentially comprises: 460sp, an anti-IL-23 single-chain antibody light chain variable region, Linker2, an anti-IL-23 single-chain antibody heavy chain variable region, and HA-tag (B in FIG. 1). The resulting plasmid was designated as IL-23ab-CAR plasmid (see FIG. 2 for structure).

Wherein, the sequences of the elements of the first expression cassette for expressing the chimeric antigen receptor targeting the prostate specific membrane antigen are as follows:

the base sequence of the CD8 alpha signal peptide (CD8 alpha Leader) is shown as SEQ ID NO. 1:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG。

the base sequence of the PSMA single-chain antibody light chain variable region (PSMA-ScFv VL) is shown in SEQ ID NO. 2:

GACATCGTGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAGGATTGTGGCACCGCCGTGGACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCTCCACCAGACACACCGGCGTGCCTGACAGATTCACCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCGACTACTTCTGCCAGCAGTACAACTCCTACCCTCTGACCTTCGGCGGAGGCACCAAGCTGGAAATCAAA；

the amino acid sequence of the PSMA single-chain antibody light chain variable region is shown in SEQ ID NO. 3:

DIVMTQSPSSLSASVGDRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQPEDFADYFCQQYNSYPLTFGGGTKLEIK。

the base sequences of the Linker of the PSMA-ScFv VL and the PSMA-ScFv VH are shown in SEQ ID NO. 4:

GGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGC；

the amino acid sequence of Linker is shown in SEQ ID NO. 5:

GGGGSGGGGSGGGGS。

the base sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO. 6:

GAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGACCTCCGGCTACACCTTCACCGAGTACACCATCCACTGGGTGAAACAGGCCTCCGGCAAGGGCCTGGAATGGATCGGCAACATCAACCCTAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACCGGGCCACCCTGACCGTGGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGTCTGAGGACACCGCCGTGTACTACTGCGCCGCTGGCTGGAACTTCGACTACTGGGGCCAGGGCACCACAGTGACAGTCTCGAGC；

the amino acid sequence of the PSMA single-chain antibody heavy chain variable region (PSMA-ScFv VH) is shown in SEQ ID NO. 7:

EVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHWVKQASGKGLEWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSS。

the base sequence of the CD8 hinge region (CD8 hinge) is shown as SEQ ID NO. 8:

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT。

the base sequence of the CD8 alpha transmembrane domain (CD8a-TM) is shown as SEQ ID NO. 9:

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC。

the base sequence of the intracellular costimulatory element of 4-1BB is shown in SEQ ID NO. 10:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG。

the base sequence of the intracellular domain of CD3 ζ is shown in SEQ ID NO. 11:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC。

the base sequence of P2A is shown in SEQ ID NO. 12:

GCCACAAACTTCTCTCTGCTAAAGCAAGCAGGTGATGTTGAAGAAAACCCCGGGCCT。

the base sequence of 460sp is shown in SEQ ID NO. 13:

ATGGCCTGGATGATGCTTCTCCTCGGACTCCTTGCTTATGGATCAGGAGTCGACTCT。

the base sequence of the anti-IL-23 single-chain antibody light chain variable region (IL-23ScFv VL) is shown in SEQ ID NO. 14:

GAGGTGCAGCTGGTGCAGTCTGGCGCCGAGGTGAAGAAGCCAGGCGAGAGCCTGAAGATCTCCTGCAAGGGCTCTGGCTACTCCTTCTCTAACTATTGGATCGGATGGGTGCGGCAGATGCCAGGCAAGGGACTGGAGTGGATGGGCATCATCGACCCCAGCAATTCCTACACCAGATATTCTCCTAGCTTTCAGGGCCAGGTGACCATCAGCGCCGATAAGTCCATCTCTACAGCCTACCTGCAGTGGAGCTCCCTGAAGGCCTCCGACACAGCCATGTACTATTGTGCCCGGTGGTACTATAAGCCCTTCGACGTGTGGGGACAGGGCACCCTGGTGACAGTGTCTAGC；

the amino acid sequence of the anti-IL-23 single-chain antibody light chain variable region is shown in SEQ ID NO. 15:

EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIDPSNSYTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARWYYKPFDVWGQGTLVTVSS。

the base sequence of the Linker between the IL-23ScFv VL and the IL-23ScFv VH is shown in SEQ ID NO. 4:

GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG。

the amino acid sequence of the Linker is shown in SEQ ID NO. 5.

The base sequence of the anti-IL-23 single-chain antibody heavy chain variable region (IL-23ScFv VH) is shown in SEQ ID NO. 16:

CAGTCCGTGCTGACCCAGCCACCTAGCGTGTCCGGAGCACCAGGCCAGCGGGTGACCATCTCTTGCACAGGCAGCTCCTCTAACATCGGCAGCGGCTACGACGTGCACTGGTATCAGCAGCTGCCAGGCACAGCCCCCAAGCTGCTGATCTACGGCAATTCCAAGCGGCCTTCTGGCGTGCCAGATAGATTCTCTGGCAGCAAGTCCGGCACCTCTGCCAGCCTGGCCATCACAGGCCTGCAGTCTGAGGACGAGGCCGATTACTATTGTGCAAGCTGGACCGACGGACTGTCCCTGGTGGTGTTTGGAGGAGGCACCAAGCTGACAGTGCTG。

the amino acid sequence of the heavy chain variable region (IL-23ScFv VH) of the anti-IL-23 single-chain antibody is shown as SEQ ID number-17:

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGSGYDVHWYQQLPGTAPKLLIYGNSKRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCASWTDGLSLVVFGGGTKLTVL。

the base sequence of HA-tag is shown in SEQ ID NO. 18: TACCCATACGACGTCCCAGACTACGCT are provided.

The amino acid sequence of HA-tag is shown in SEQ ID NO. 19: YPYDVPDYA.

Example 2

(1) Construction of viruses expressing chimeric antigen receptor and anti-IL-23 scFv

The method comprises the following steps: the IL-23ab-CAR plasmid and the lentiviral packaging helper plasmids psPAX2 (shown in figure 8) and pMD2.G (shown in figure 9) are amplified by using Escherichia coli, and the correctness of the plasmids is identified by agarose gel electrophoresis and sequencing after the plasmids are extracted. 293T with good state and early passage is selected as a lentivirus packaging cell, and the three plasmids are transfected into the 293T cell by using a transfection reagent PEI. Transfection was accomplished in 10cm culture dishes totaling 10mL, and the transfection mixture for each dish of cells should be formulated in 1mL system using serum-free DMEM with the plasmid psPAX 2: plasmid pmd2. g: IL-23ab-CAR plasmid: PEI 5 μ g: 3 μ g: 5 μ g: mu.l of the transfection mixture was mixed at room temperature, left to stand for 20min and then slowly added to 293T with a cell density of 60-70% in 9mL of the existing medium, and the medium was replaced with fresh medium (DMEM + 10% FBS + 1% P/S) after 6-8 h. And respectively harvesting culture solution supernatants during 48h and 72h of culture, and performing ultrafiltration and super-separation concentration to obtain the virus expressing the chimeric antigen receptor and the anti-IL-23 scFv, wherein the obtained virus is named as IL-23ab-CAR virus.

(2) Virus titer detection

The method comprises the following steps:

selecting 293T with good growth for digestion, inoculating 500 mu l of cells with the density of 4 x 10^5/mL into a 24-well plate, adding concentrated virus solution with different gradient volumes after the cells are attached to the wall, digesting the cells after culturing for 48h, using CAR to identify the combined biotinylated PSMA protein, washing after incubating the conjugated biotinylated PSMA protein with the cells for 50min at 4 ℃, then using APC-streptavidin SA capable of being combined with biotin to stain for 30min at 4 ℃, washing after staining, loading in a tube, using a flow meter to detect the CAR positive rate, selecting the virus volume with the proper positive rate to calculate the virus titer, and using a titer calculation formula: titer (TU/mL) ═ 2 × 10^5 × CAR positive rate/virus volume.

And (2) using a virus transfected by a control plasmid PSMA-CAR plasmid (compared with the IL-23ab-CAR plasmid, the PSMA-CAR plasmid lacks a second expression cassette for expressing anti-IL-23 scFv) as a control, wherein the transfection method of the control plasmid refers to the step (1), and the virus is used for replacing the IL-23ab-CAR plasmid, namely the obtained virus is the control virus and is named as PSMA-CAR virus.

The titer detection result is shown in figure 3, after the cell connecting plate is attached to the wall according to the titer detection method, two volume gradients of 1 mul and 3 mul are respectively set for the control virus PSMA-CAR and the IL-23ab-CAR, in order to avoid false positive caused by nonspecific staining, CTRL is required to be set for CAR positive circle gate, CAR positive cells are obtained when the cells fall into the APC positive gate, and the proportion value is CAR positive rate. As shown in FIG. 3, 1. mu.l of PSMA-CAR concentrated virus infected with 20 ten thousand 293T can achieve 72.9% positive rate, and 3. mu.l of the concentrated virus has a corresponding positive rate of 92.9%; 1 mul of IL-23ab-CAR virus infected with 20 ten thousand 293T can reach 85.1% of positive rate, 3 mul corresponds to the positive rate of 92.9%, because the positive rate is too high to reflect the real titer of the virus, the titer is calculated by using the volume of 1 mul, the titer of the control virus PSMA-CAR can reach 1.5 x 10 TU/mL, and the titer of the IL-23ab-CAR virus is 1.7 x 10 TU/mL.

Example 3

(1) Construction of T cells expressing chimeric antigen receptor and anti-IL-23 scFv

The method comprises the following steps:

PBMC is separated from human blood by using lymph separation liquid, then T cells are separated by using a CD4 and CD8 magnetic bead sorting method, after the PBMC is activated by a CD3/CD28 complex for 48h, the packed PSMA-CAR and IL-23ab-CAR viruses are used for centrifugally infecting for 2h according to MOI (10), and after 24h, the packed PSMA-CAR and IL-23ab-CAR viruses are replaced by a fresh culture medium (XVIVO + 10% FBS + IL-2), and the two CART cells are named as PSMA-CART cells and IL-23ab-CART cells respectively.

And after infection and fluid replacement for 48 hours, detecting the CAR expression levels of the two CARTs according to a similar method for titer detection.

The results are shown in FIG. 4, where the CAR-positivity for IL-23ab-CART and PSMA-CART was 83% and 96%, respectively.

(2) Detection of expression of IL-23-scFv:

for IL-23ab-CART, not only the expression of CAR but also the ability to express anti-IL-23 scFv need to be tested, therefore, the supernatant and cells of the two CART cells 48h under the same culture system condition are collected, and because the IL-23-scFv HAs HA-tag at the end, the expression of IL-23-scFv can be tested from the protein level by WB method (30kDa, HA-GFP is positive control 26kDa), and the collected cells are inverted after RNA extraction, and the ability of IL-23ab-CART to secrete anti-IL-23 scFv is tested from the mRNA level by QPCR method.

The results are shown in FIG. 5, and the detection results of WB and QPCR show that the IL-23ab-CART cells have the secretory expression of anti-IL-23 scFv, while the control PSMA-CART cells do not have the expression of anti-IL-23 scFv, which indicates that the CAR-T cells secreting and expressing anti-IL-23 scFv are successfully constructed by the embodiment of the invention.

Experimental example 1

Verification of IL-23 blocking function by IL-23ab-CART

The method comprises the following steps: by utilizing the characteristic that K562 cells highly express IL-23 receptor (IL-23R), as shown in A in figure 6, a flow experiment for confirming the expression of IL-23R on the surface of K562 cells is performed, IL-23 protein with His tags at the tail ends is incubated with K562 cells for 1h at 4 ℃, IL-23 is firstly combined with IL-23R on the surface of K562 cells, then PBS is used for cleaning, then a flow antibody (PE) capable of being combined with His tags is used for incubation with IL-23-stained K562 cells at 4 ℃ for 30min, and then PBS is used for cleaning, filtering and tubing and loading on a machine. The expression level of the IL-23R can be reflected by detecting the His positive rate by a flow meter. As shown by the A result in FIG. 6, the positive rate of IL-23R on the surface of K562 cells was 95%. And thus can be used to detect binding of IL-23 to IL-23R.

Based on the method for detecting the IL-23R on the surface of K562, meanwhile, because scFv of anti-IL-23 is secreted extracellularly by CART, the CART supernatant is used for verifying the blocking function of IL-23ab-CART, under the same culture system condition, the supernatant after two kinds of CART of IL-23ab-CART and PSMA-CART infect cells for 48h is collected, the supernatant is concentrated by 5 times by using an ultrafiltration tube and then is co-incubated with IL-23 protein (100ng/mL) in a refrigerator at 4 ℃ overnight, then the co-incubation system is directly co-incubated with K562 in the refrigerator at 4 ℃ for 1h, and the change of IL-23R after the blocking of the supernatant is detected by using a flow-type antibody stained with a His label and a flow-type instrument. Experimental results As shown in B in FIG. 6, the pre-overnight incubation of the IL-23ab-CART concentrated supernatant with IL-23 allowed anti-IL-23 scFv in the supernatant to bind to IL-23, blocking the binding of IL-23 and IL-23R, resulting in a decrease in IL-23 binding to IL-23R, as shown in B flow diagram in FIG. 6, the IL-23ab-CART concentrated supernatant (right panel) was positively decreased relative to the IL-23R in the PSMA-CART supernatant (left panel). Thus, the IL-23ab-CART constructed by the method has the function of blocking IL-23.

Experimental example 2

Detecting the killing effect of IL-23ab-CART on prostate cancer cells

Using IL-23ab-CART and PSMA-CART with consistent positive rate regulation as effector cells, using PC3-PSMA (human prostate cancer cell line, using lentivirus to stably express PSMA and luciferase) as target cells, firstly adding equivalent target cells into a low adsorption pore plate, and adding the target cells according to an effective target ratio (effector cells: target cells) of 5: 1. 2.5: 1. 1.25: 1. 0.63: 1 adding a corresponding number of CART effector cells, and making different gradient wells (0.5 ten thousand, 1 ten thousand, 1.5 ten thousand, 2 ten thousand, 2.5 ten thousand, 3 ten thousand, 5 ten thousand) with only target cells as standard curves. As the target cells can express luciferase, after 24h of co-incubation (co-c μ lture), after adding a substrate, the light absorption value is in a linear relationship with the number of the target cells, a standard curve can be made, and the number of the residual target cells is calculated, so that the killing efficiency lysine (%) (initial target cell number-residual target cell number)/initial target cell number is calculated, the killing efficiency lysine (%) is taken as the ordinate, different effective target ratios (E: T) are taken as the abscissa, and the result shown in figure 7 is obtained, which shows that the common CTRL-T cells basically have no killing function on the target cells, the PSMA-CART and the IL-23ab-CART have obvious killing effect on the human prostate cancer cells, and the killing effect is gradually increased along with the increase of the effective target ratios, but the killing effect of the IL-23ab-CART on the human prostate cancer cells is obviously better than that of the PSMA-CART, and shows that the ScFv which simultaneously expresses anti-IL-23 is beneficial to improving the effect on the prostate cancer cells Killing effect. Therefore, the IL-23ab-CART can be used for treating the prostatic cancer, and can also be used together with androgen deprivation medicines such as abiraterone and enzalutamide to improve the effect of treating the prostatic cancer.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of east China, Shanghai Yao Biotechnology Co., Ltd

<120> prostate cancer targeted CAR-T cell drug secreting IL-23 antibody

<160> 19

<170> PatentIn version 3.5

<210> 1

<211> 63

<212> DNA

<213> Artificial sequence

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 2

<211> 321

<212> DNA

<213> Artificial sequence

<400> 2

gacatcgtga tgacccagtc cccctcctcc ctgtctgcct ccgtgggcga cagagtgacc 60

atcacatgca aggcctccca ggattgtggc accgccgtgg actggtatca gcagaagcct 120

ggcaaggccc ctaagctgct gatctactgg gcctccacca gacacaccgg cgtgcctgac 180

agattcaccg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct 240

gaggacttcg ccgactactt ctgccagcag tacaactcct accctctgac cttcggcgga 300

ggcaccaagc tggaaatcaa a 321

<210> 3

<211> 107

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Cys Gly Thr Ala

20 25 30

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

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 4

<211> 45

<212> DNA

<213> Artificial sequence

<400> 4

ggcggaggcg gatcaggtgg tggcggatct ggaggtggcg gaagc 45

<210> 5

<211> 15

<212> PRT

<213> Artificial sequence

<400> 5

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 6

<211> 345

<212> DNA

<213> Artificial sequence

<400> 6

gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaagatc 60

tcctgcaaga cctccggcta caccttcacc gagtacacca tccactgggt gaaacaggcc 120

tccggcaagg gcctggaatg gatcggcaac atcaacccta acaacggcgg caccacctac 180

aaccagaagt tcgaggaccg ggccaccctg accgtggaca agtccacctc caccgcctac 240

atggaactgt cctccctgcg gtctgaggac accgccgtgt actactgcgc cgctggctgg 300

aacttcgact actggggcca gggcaccaca gtgacagtct cgagc 345

<210> 7

<211> 115

<212> PRT

<213> Artificial sequence

<400> 7

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 8

<211> 135

<212> DNA

<213> Artificial sequence

<400> 8

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 9

<211> 72

<212> DNA

<213> Artificial sequence

<400> 9

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 10

<211> 126

<212> DNA

<213> Artificial sequence

<400> 10

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 11

<211> 336

<212> DNA

<213> Artificial sequence

<400> 11

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 12

<211> 57

<212> DNA

<213> Artificial sequence

<400> 12

gccacaaact tctctctgct aaagcaagca ggtgatgttg aagaaaaccc cgggcct 57

<210> 13

<211> 57

<212> DNA

<213> Artificial sequence

<400> 13

atggcctgga tgatgcttct cctcggactc cttgcttatg gatcaggagt cgactct 57

<210> 14

<211> 351

<212> DNA

<213> Artificial sequence

<400> 14

gaggtgcagc tggtgcagtc tggcgccgag gtgaagaagc caggcgagag cctgaagatc 60

tcctgcaagg gctctggcta ctccttctct aactattgga tcggatgggt gcggcagatg 120

ccaggcaagg gactggagtg gatgggcatc atcgacccca gcaattccta caccagatat 180

tctcctagct ttcagggcca ggtgaccatc agcgccgata agtccatctc tacagcctac 240

ctgcagtgga gctccctgaa ggcctccgac acagccatgt actattgtgc ccggtggtac 300

tataagccct tcgacgtgtg gggacagggc accctggtga cagtgtctag c 351

<210> 15

<211> 117

<212> PRT

<213> Artificial sequence

<400> 15

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Asn Tyr

20 25 30

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

35 40 45

Gly Ile Ile Asp Pro Ser Asn Ser Tyr Thr Arg Tyr Ser Pro Ser Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Trp Tyr Tyr Lys Pro Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 333

<212> DNA

<213> Artificial sequence

<400> 16

cagtccgtgc tgacccagcc acctagcgtg tccggagcac caggccagcg ggtgaccatc 60

tcttgcacag gcagctcctc taacatcggc agcggctacg acgtgcactg gtatcagcag 120

ctgccaggca cagcccccaa gctgctgatc tacggcaatt ccaagcggcc ttctggcgtg 180

ccagatagat tctctggcag caagtccggc acctctgcca gcctggccat cacaggcctg 240

cagtctgagg acgaggccga ttactattgt gcaagctgga ccgacggact gtccctggtg 300

gtgtttggag gaggcaccaa gctgacagtg ctg 333

<210> 17

<211> 111

<212> PRT

<213> Artificial sequence

<400> 17

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

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Thr Asp Gly

85 90 95

Leu Ser Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 18

<211> 27

<212> DNA

<213> Artificial sequence

<400> 18

tacccatacg acgtcccaga ctacgct 27

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<400> 19

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

Claims (11)

1. A CAR-T cell targeted to prostate cancer, wherein the CAR-T cell comprises a nucleic acid molecule; the nucleic acid molecule has a first nucleic acid sequence encoding a chimeric antigen receptor;

the chimeric antigen receptor has an antigen binding domain that binds an antigen that is a prostate specific membrane antigen;

the nucleic acid molecule further having a second nucleic acid sequence encoding and secreting a functional fragment of an scFv that specifically binds IL-23; the second nucleic acid sequence comprises the following elements in sequence: 460sp, an anti-IL-23 single-chain antibody light chain variable region, a Linker2, an anti-IL-23 single-chain antibody heavy chain variable region and HA-tag; wherein the base sequence of 460sp is shown as SEQ ID NO.13, the base sequence of the anti-IL-23 single-chain antibody light chain variable region is shown as SEQ ID NO.14, the base sequence of the Linker of Linker2 is shown as SEQ ID NO.4, the base sequence of the anti-IL-23 single-chain antibody heavy chain variable region is shown as SEQ ID NO.16, and the base sequence of HA-tag is shown as SEQ ID NO. 18; the antigen binding structural domain is scFv, and the amino acid sequence of the heavy chain variable region of the antigen binding structural domain is shown in SEQ ID NO. 3;

the amino acid sequence of the light chain variable region of the antigen binding domain is shown as SEQ ID NO. 7;

the amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is shown in SEQ ID NO. 5.

2. The prostate cancer-targeting CAR-T cell of claim 1, wherein said chimeric antigen receptor further comprises a transmembrane domain and a costimulatory signaling region.

3. The prostate cancer targeted CAR-T cell according to claim 2, wherein the transmembrane domain is selected from the transmembrane domains of at least one of the following protein molecules: CD5, CD28, CD137, CD3 epsilon, CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134, and CD8 alpha; the costimulatory signaling region comprises an intracellular domain of at least one of the following costimulatory molecules: OX40, CD3 γ, CD3 δ, CD134, CD79a, CD137, ICD3 ε, CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, COS, 4-1BB, and CD3 ζ.

4. The prostate cancer-targeting CAR-T cell of claim 3, wherein said transmembrane domain is a CD8a transmembrane domain.

5. The prostate cancer-targeting CAR-T cell according to claim 3, wherein said costimulatory signaling region comprises the intracellular costimulatory element of 4-1BB and the intracellular domain of CD3 ζ.

6. A nucleic acid molecule having a first nucleic acid sequence encoding a chimeric antigen receptor;

the chimeric antigen receptor has an antigen binding domain that binds an antigen that is a prostate specific membrane antigen;

the nucleic acid molecule further having a second nucleic acid sequence encoding a scFv functional fragment that specifically binds IL-23; the second nucleic acid sequence comprises the following elements in sequence: 460sp, an anti-IL-23 single-chain antibody light chain variable region, a Linker2, an anti-IL-23 single-chain antibody heavy chain variable region and HA-tag; wherein the base sequence of 460sp is shown as SEQ ID NO.13, the base sequence of the anti-IL-23 single-chain antibody light chain variable region is shown as SEQ ID NO.14, the base sequence of the Linker of Linker2 is shown as SEQ ID NO.4, the base sequence of the anti-IL-23 single-chain antibody heavy chain variable region is shown as SEQ ID NO.16, and the base sequence of HA-tag is shown as SEQ ID NO. 18; the antigen binding domain is a scFv; the amino acid sequence of the heavy chain variable region of the antigen binding domain is shown as SEQ ID NO. 3;

the amino acid sequence of the light chain variable region of the antigen binding domain is shown as SEQ ID NO. 7;

the amino acid sequence of the hinge region between the heavy chain variable region and the light chain variable region of the antigen binding domain is shown in SEQ ID NO. 5.

7. The nucleic acid molecule of claim 6, wherein said chimeric antigen receptor further comprises a transmembrane domain and a costimulatory signaling region.

8. The nucleic acid molecule according to claim 7, wherein the transmembrane domain is selected from the transmembrane domains of at least one of the following protein molecules: CD5, CD28, CD137, CD3 epsilon, CD154, CD45, CD4, CD9, CD37, CD16, CD33, CD22, CD134, and CD8 alpha; the costimulatory signaling region comprises an intracellular domain of at least one of the following costimulatory molecules: OX40, CD3 γ, CD3 δ, CD134, CD79a, CD137, ICD3 ε, CD154, CD22, CD66d, CD2, CD28, CD4, CD5, CD79b, 4-1BB, and CD3 ζ.

9. The nucleic acid molecule of claim 8, wherein the transmembrane domain is a CD8a transmembrane domain.

10. The nucleic acid molecule of claim 8, wherein the costimulatory signaling region comprises the intracellular domains of 4-1BB and CD3 ζ.

11. A CAR-T cell medicament for the treatment of prostate cancer comprising a CAR-T cell according to any of claims 1 to 5.

Images