CN105585637B

CN105585637B - Tumor therapeutic agent based on IL-12 stable membrane expression and preparation method and application thereof

Info

Publication number: CN105585637B
Application number: CN201510979423.7A
Authority: CN
Inventors: 杨世成; 吕卫东
Original assignee: Individual
Current assignee: Yang Jing
Priority date: 2015-12-23
Filing date: 2015-12-23
Publication date: 2020-04-03
Anticipated expiration: 2035-12-23
Also published as: WO2017107353A1; CN105585637A

Abstract

The invention provides a tumor therapeutic agent based on IL-12 stable membrane expression, a preparation method and application thereof. Specifically, the invention provides an IL-12/mutant CD62L fusion protein. In the present invention, anti-tumor T cells are modified by using lentiviruses containing IL-12/mutant CD62L fusion genes. Experiments show that the fusion protein can be stably expressed on the membrane surface of T cells, and is independent of the cutting of wild type CD62L accompanied by tumor recognition of anti-tumor T cells. In addition, the T cell of the fusion protein expressed by the membrane maintains high activity and effectively kills tumor cells, and has obviously reduced toxic and side effects and obviously improved safety.

Description

Tumor therapeutic agent based on IL-12 stable membrane expression and preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a high-efficiency low-toxicity tumor therapeutic agent based on IL-12 stable membrane expression, and a preparation method and application thereof.

Background

IL-12 is a very important immune stimulator. IL-12 is a covalently linked heterogeneous dimeric cytokine, composed of p35 and p40 subunits, secreted in vivo by activated immune cells. IL-12 is an important cellular immune regulatory factor, in anti-infection immunity and malignant tumor immunity through NK cells and CTL play a role.

Although IL-12 has significant antitumoral effects, it is extremely limited because of systemic toxic side effects caused by systemic administration. Prior to the present invention, the clinical use of IL-12 was greatly limited due to its induced systemic toxic side effects and the unexpected death of two patients in early clinical trials by systemic administration of the whole body.

Clinical researchers apply the IL-12 to clinical trials of tumors by local injection, transient expression, multi-point injection and other ways in order to avoid toxic and side effects of systemic administration, but the local clinical scheme does not achieve a significant tumor inhibition effect because the therapeutic effect of the IL-12 is directly related to the dose of the reinfused IL-12.

To solve this problem, the present inventors have attempted to maximize the antitumor effect by genetically modifying antitumor T cells to continuously secrete IL-12, but T cells genetically modified by the continuous secretion of IL-12 regimen could not be efficiently expanded in vitro for unknown reasons (one possible reason is the toxic side effects of IL-12) and caused a large amount of T cell apoptosis.

In conclusion, there is no IL-12-based antitumor drug with high efficacy and low toxic and side effects in the field. Therefore, there is an urgent need in the art to develop a tumor therapeutic drug with high efficacy and low toxic and side effects.

Disclosure of Invention

The invention aims to provide a high-efficiency tumor treatment medicine with low toxic and side effects, and a preparation method and application thereof.

In a first aspect of the invention, there is provided a fusion protein comprising the following elements fused together:

(i) optionally a signal peptide and/or a leader peptide at the N-terminus;

(ii) a first protein element;

(iii) a second protein element; and

(iv) optionally a linker peptide element between the first protein element and the second protein element;

wherein the signal peptide is operably linked to the fusion element consisting of (ii), (iii) and (iv);

and the first protein element is an IL-12 protein element; the second protein element is that of mutant CD62L, which lacks the cleavage site of ADAM 17.

In another preferred embodiment, the fusion protein has a structure selected from the group consisting of:

(1) the structure of formula Ia:

D-A-B (Ia), or

(2) The structure of formula IIa:

D-A-C-B (Ⅱa)，

wherein,

a is an IL-12 protein element;

b is a mutant CD62L protein element;

c is an optional linker peptide element;

d is an optional signal peptide and/or leader peptide sequence;

each "-" independently represents a peptide bond or a peptide linker connecting the above elements.

In another preferred embodiment, the protein element of mutant CD62L lacks part or all of the KLDKSFS sequence, thereby rendering it non-cleavable by ADAM 17.

In another preferred embodiment, said "operably linked" means that said signal peptide can direct the expression or transmembrane transfer (localization) of said fusion element.

In another preferred embodiment, the linker peptide element comprises a linker peptide having the sequence shown in SEQ ID No. 7.

In another preferred embodiment, the IL-12 protein is derived from a human or non-human mammal.

In another preferred embodiment, the IL-12 protein includes wild type and mutant.

In another preferred embodiment, the IL-12 protein includes a full-length, mature form of IL-12, or an active fragment thereof.

In another preferred embodiment, the first protein element includes one or two subunits of the IL-12 protein.

In another preferred embodiment, the subunits of the IL-12 protein are selected from the group consisting of: p40 and P35 subunits.

In another preferred embodiment, the first protein element comprises subunits P40 and P35 of the IL-12 protein linked together.

In another preferred embodiment, the subunits P40 and P35 are "head-to-head", "head-to-tail" or "tail-to-tail" linked.

In another preferred embodiment, a linker (linker) is present or absent between the P40 and P35 subunits. Compared withPreferably, the linker is a flexible 4-20 amino acid linker, more preferably the linker is GGGGGGS (i.e., G)₆S)(SEQ IDNO.:8)。

In another preferred embodiment, the sequence of the IL12 protein element is shown in SEQ ID No. 4.

In another preferred embodiment, the mutant CD62L protein element lacks the K283-S284 cleavage site.

In another preferred embodiment, the mutant CD62L protein is derived from a human or non-human mammal.

In another preferred embodiment, the mutant CD62L protein comprises the full-length, mature form of CD62L, or an active fragment thereof.

In another preferred example, the sequence of the mutant CD62L protein element is shown in SEQ ID No. 6.

In another preferred embodiment, the peptide linker is 0-15 amino acids, preferably 1-10 amino acids in length.

In another preferred embodiment, the fusion protein further comprises a signal peptide element D.

In another preferred embodiment, the fusion protein comprises a connecting peptide, preferably a 218 peptide (SEQ ID NO: 7), between single-chain IL-12 (first protein element) and mutant CD62L (second protein element).

In another preferred embodiment, the fusion protein comprises a single-chain IL-12 as the first protein element, and the single-chain IL-12 has a linker peptide G provided between subunit P40 and subunit P35₆S(SEQ ID NO.:8)。

In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID No. 2.

In another preferred embodiment, the fusion protein has the following characteristics:

a) the fusion protein is not cleavable by ADAM17 protein and thus does not release IL-12;

b) the fusion protein comprises two subunits of IL-12, namely P40 and P35 subunits, and is connected by GGGGGGS (G6S).

In another preferred embodiment, the fusion protein is a monomer, or a dimer.

In a second aspect of the invention, there is provided an isolated polynucleotide encoding a fusion protein according to the first aspect of the invention.

In another preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID NO. 1.

In a third aspect of the invention, there is provided a vector comprising a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the vector comprises a plasmid or a viral vector.

In another preferred embodiment, the viral vector comprises: lentivirus vectors, adenovirus vectors, yellow fever virus vectors.

In another preferred embodiment, the vector comprises an expression vector.

In a fourth aspect of the invention, there is provided a host cell comprising a vector or genome according to the third aspect of the invention into which a polynucleotide according to the second aspect of the invention has been integrated.

In another preferred embodiment, the host cell includes prokaryotic cells and eukaryotic cells.

In another preferred embodiment, the host cell comprises a mammalian cell.

In another preferred embodiment, the host cell comprises an immune cell, preferably a T cell,

In a fifth aspect of the invention, there is provided a method of producing a protein according to the first aspect of the invention, comprising the steps of:

(1) culturing the host cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the fusion protein of the first aspect of the invention; and

(2) optionally isolating the fusion protein.

In a sixth aspect of the invention, there is provided an immune cell carrying a fusion protein according to the first aspect of the invention on a membrane surface.

In another preferred embodiment, the immune cell is at least 10³A (a)Preferably 10³-10⁹More preferably 10, respectively⁴-10⁸And (b) a cell population of the immune cells.

In another preferred embodiment, all or most (80% or more, preferably 90% or more) of the cells in the immune cell or immune cell population are viable.

In another preferred embodiment, at least a portion or all of the fusion protein is located on the cell membrane of the immune cell and the first protein element, the IL-12 protein element, is located extracellularly.

In another preferred embodiment, the immune cells comprise T cells.

In another preferred embodiment, the T cell surface carries MART-1 TCR.

In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

an immune cell according to the sixth aspect of the invention, and

a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in a liquid state.

In another preferred embodiment, the pharmaceutical composition comprises 1 × 10³-1×10⁷The immune cells are used per ml.

In an eighth aspect of the invention, there is provided a use of the fusion protein according to the first aspect of the invention and/or the immune cell according to the sixth aspect of the invention for the preparation of a medicament for the treatment of a tumor.

In another preferred embodiment, the tumor comprises: brain tumor, carcinoma of large intestine tumor, lung cancer tumor, hepatocarcinoma tumor, breast cancer tumor, gastric cancer tumor, and pancreatic cancer tumor.

In a ninth aspect of the present invention, there is provided a method for treating a tumor, comprising the steps of: administering to a subject in need thereof a fusion protein according to the first aspect of the invention and/or an immune cell according to the sixth aspect of the invention.

In another preferred embodiment, the fusion protein is administered in monomeric and/or dimeric form.

In another preferred embodiment, the subject is a human.

In a tenth aspect of the present invention, there is provided a non-therapeutic or therapeutic in vitro method for killing tumor cells, comprising the steps of: contacting the immune cell of the sixth aspect with a tumor cell, thereby killing the tumor cell.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the amino acid composition of the transmembrane region of CD62L and the cleavage site of ADAM 17. FIGS. 1A and 1B show that the dKSCD62L mutant (lacking KLDKSFS) is unable to undergo cleavage and release by a cleavage enzyme (such as Adam 17) after T cell activation, resulting in the retention of dKSCD62L on the surface of T cell membrane. In FIG. 1C, WT represents the wild-type sequence, and dKS represents a CD62L mutant with a knockout of the ADAM17 sequence.

FIG. 2 shows that the dKSCD62L lentivirus gene modified anti-tumor T cells resulted in stable expression of dKSCD 62L.

As shown in the above figure, the expression of WTCD62L and dKSCD62L was detected after the anti-tumor T cell line JKF6 (tumor-recognizing T cell line isolated and cultured from melanoma tissue) was modified with lentivirus gene.

As shown in the following figure, after T (wild type) CD62L is activated by tumor antigen 526, CD62L can undergo tumor antigen-specific cleavage and release; however, membrane expression of dKSCD62L lost tumor antigen-activated cleavage and release, and was characterized by a stable membrane-surface phenotype of expression of dKSCD 62L.

JKF6T cells were co-cultured with MART-1 antigen expressing

tumor cells

938 and 526. Wherein the co-cultured T cells are detected by flow cytometry for molecules CD45RO and CD62L on the membrane surface, and the T cells of each group are arranged into CD8 cell subset by analysis processing of FlowJo software. The antibodies used for flow cytometry were CD62L FITC, CD45RO APC, MART-1 PE and CD8 PerCP.

FIG. 3 shows that the dKSCD62L lentiviral gene modified anti-tumor JFK6 cells did not affect the anti-tumor activity of T cells.

In FIG. 3A, after the JKF6 cell is modified by lentivirus gene, the T cell is activated by PMA/Ionomycin (PMA/Ion, a T cell activator) for 4h, then the CD62L series and IFN gamma on the surface of the cell membrane are fixed and stained by routine, and the expression level of CD62L on the surface of the membrane and the expression level of IFN gamma in cytoplasm are detected (upper graph). Figure 3B shows the IFN γ and CD62L expression statistical analysis treatment. The data are the analytical statistics of 3 independent experiments, the expression level of dKSCD62L is stable continuously, and IFN γ also shows similar high level as other groups. Results are expressed as mean ± sd and were statistically processed using T-test. Denotes that the expression level of dKSCD62L compared to the other two groups of CD62L, P < 0.01.

FIG. 4 shows the construction of lentiviral vectors expressing the dKSCD62L fusion protein and its expression in human T cells. The lentivirus is a third generation lentivirus vector, and the promoter is MSCV. The 5 'and 3' LTRs of the vector were engineered into SIN-LTR (self-inactivating-LTR) in order to reduce the probability of lentiviral recombination and enhance safety.

Wherein the figure shows the schematic structure diagram of LVV plasmid and gene expression components of human IL-12/WTCD62L (WT, wild type), IL-12/dKSCD62L and IL-12(human single chain IL-12), wherein hscIL-12 is fused with IL-12 secretion peptide (lead seq) and is connected to lentivirus expression vector by gene cloning method, CD62L or dKSCD62L and hscIL-12 are connected by peptide G₆And S linking.

As in the scheme of fig. 5, T cells were transduced by two transductions, i.e., MART-1TCR (day one) and three vectors in the figure sequentially (day 5), at day 14, with 526 and 938 expressed in 1: after co-culturing for 4h at a ratio of 1, expression of hscIL-12 on the surface of T cell membrane was detected by flow cytometry.

FIG. 5 shows that co-culture of T cells transduced with a series of lentiviral vector IL-12/mutant CD62L with tumor cells can enhance IFN γ expression and tumor antigen-dependent release of IL-12. In the figure, mock is a blank control group and tumor is a tumor group.

T cells were sequentially transfected with anti-tumor TCR and IL-12/mutant CD62L fusion protein 14 days later, co-cultured with

tumor cells

526 and 938, and after 16 hours, IFN γ and IL-12 expression levels in the supernatant were detected by ELISA kit. The results show that the persistent IL-12 secretion group, the IL-12/WTCD62L group and the IL-12/dKSCD62L group can obviously enhance the responsiveness of anti-tumor T cells and tumors compared with the T cell group, and the reactivity is expressed by obviously enhancing the secretion level of IFN gamma, wherein P is less than 0.001. The stable expression of dKSCD62L on the membrane surface is consistent, IL-12/dKSCD62L also loses the cutting and releasing of tumor antigen reactivity, and compared with an IL-12/WTCD62L group and a sustained secretion IL-12 group, the level of the secreted IL-12 can be detected to be remarkably reduced, and P is less than 0.001.

FIG. 6 shows that T cells transduced with the lentiviral vector IL-12/dKSCD62L series can effectively avoid the toxic side effects of in vitro T cell expansion caused by continuous IL-12 secretion.

After the T cells sequentially transduce the anti-tumor TCR and the IL-12/CD62L series of fusion proteins for 14 days, the amplification fold of each group of cells is compared with that of a control transduction group (T-cell), the amplification fold of the group which continuously secretes IL-12 by transduction is obviously reduced compared with that of other groups, and p is less than 0.001. Among them, the group of transduced IL-12/dKSCD62L fusion proteins had the same amplification fold compared with other groups, and no significant difference was observed. The data analysis was carried out by setting the amplification factor of the T-cell group to 100%, and the values of the other groups were the ratios to the T-cell group.

FIG. 7 shows that the lentiviral vector IL-12/dLSCD62L series of transduced murine T cell mediated cell reinfusion treatments significantly prolonged the survival of tumor bearing mice.

Female pmel mice (7 mice per group) were implanted Intracranially (IC) with B16F10 cells (5000 cells/mouse) for 5 days, and mice received 5Gy of systemic radiotherapy 1 day prior to cell reinfusion. Murine T cells were obtained from mouse spleen cells and activated by 10ug/ml concanavalin (Con A) in the presence of IL-2(5 IU/ml); on day 2, T cells were transduced with lentiviral vectors, followed by continued culture for 6 days, cells were harvested and injected via the tail vein of mice (IV)5X 10⁶And (4) T cells. DC cells of the mice in the DC group are taken from bone marrow cells, are induced, differentiated and matured in vitro for 8 days, and pass through the abdomenChamber inoculation 1X 10⁶A cell. See the right hand panel for DC and T cell profiles. Asterisks indicate comparison of this experimental group with other groups, p<0.001。

Detailed Description

The inventor of the invention has extensively and deeply studied, developed for the first time a novel structure, with high efficiency killing tumor cell activity and small toxic and side effects of IL-12 fusion protein. The fusion protein is a fusion protein of IL-12/mutant CD62L, wherein the mutant CD62L lacks a cleavage site of ADAM 17. Experiments show that the fusion protein of the invention can be effectively displayed on the surface of T cells after being expressed in the T cells. Unexpectedly, the IL-12/mutant CD62L fusion protein has no influence on the vitality (vitality) of T cells, and when anti-tumor T cells carrying the fusion protein of the invention approach tumor cells, the IL-12 can not be released by specifically cutting CD62L, so that the IL-12 positioned on the surface of a cell membrane acts on the tumor cells more effectively and safely. In the invention, the immune microenvironment of T cell-tumor tissues is changed by attacking the local part of the tumor by T cells and changing the IL-12 on the cell surface, so that the maximization of the anti-tumor immune effect is synergistically and effectively realized, and meanwhile, the toxic and side effects of the IL-12 are extremely and remarkably reduced. The present invention has been completed based on this finding.

Specifically, the inventors developed a novel fusion protein of IL-12/dKSCD62L using mutant CD62L (dKSCD62L) lacking the cleavage site of ADAM17, and modified anti-tumor T cells with lentiviral genes. Experimental data show that after the IL-12/dKSCD62L lentivirus gene modifies the anti-tumor T cells, the expression of the membrane surface is not regulated and controlled by the activation of the T cells, the higher expression of the membrane surface can be maintained, and the effect of enhancing the immune response is shown in the response to tumor antigens. A preclinical tumor-bearing mouse model shows that the T cells modified by the scheme can obviously prolong the life cycle of tumor-bearing mice, and no obvious cytotoxic effect is found. In other words, T cells expressing the fusion protein on the membrane can effectively kill tumor cells on the one hand, and have proved to have safety in vitro and in vivo on the other hand. Furthermore, the modified T-cells themselves appear to be only slightly adversely affected or not substantially affected by the expressed IL-12 (the fusion proteins of the invention have no significant effect on the in vitro expansion of T-cells compared to T-cells which continue to secrete IL-12).

Term(s) for

As used herein, the term "head" refers to the N-terminus of a polypeptide or fragment thereof, particularly the N-terminus of a wild-type polypeptide or fragment thereof.

As used herein, the term "tail" refers to the C-terminus of a polypeptide or fragment thereof, particularly the C-terminus of a wild-type polypeptide or fragment thereof.

As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

CD62L

CD62L is widely expressed on the surface of T cells, is an important immune regulator, can regulate the migration of T cells to lymph nodes of the whole body, and is an important T cell homing factor. In the case of CD62L + T cell migrating to lymph nodes and contacting with tumor antigen, CD62L can test the reactive cleavage of tumor antigen by ADAM17 at the peptide fragment K283-S284 of the transmembrane region. (Yang, Liu et al.2011) in the present invention, the present inventors have confirmed that CD62L cleavage has specificity for tumor antigen response, accompanied by membrane migration of CD107a (the molecule is a molecular protein on lysosomes, normally present in the cytoplasm, and in the T cell activated state, the molecule migrates to the cell surface accompanied by degranulation of T cells; therefore, membrane surface detection of the molecule is an important indicator of T cell killing).

Mutant CD62L

As used herein, the terms "mutant CD 62L", "CD 62L mutant", "ADAM 17 cleavage site-deleted CD 62L", "ADAM 17 cleavage site-deleted CD62L mutant" or "cleavage site-deleted CD62L mutant" and the like are used interchangeably to refer to a CD62L polypeptide or an active fragment or derivative thereof that lacks the cleavage site of the cleavage enzyme ADAM17, i.e., lacks some or all of the sequence of KLDKSFS, such that the CD62L polypeptide or active fragment or derivative thereof is not cleaved by the cleavage enzyme ADAM 17.

A preferred mutant CD62L is a CD62L polypeptide deleted of the entire KLDKSFS sequence.

It is understood that in the present invention, the CD62L may be derived from a mammal (human or non-human mammal) or from other eukaryotic species. Preferably, CD62L is wild-type CD62L from humans.

In the present invention, a particularly preferred mutant CD62L is dKSCD62L, a human CD62L polypeptide having a deletion of the entire KLDKSFS sequence.

IL-12

IL-12 is a very important immune stimulator. IL-12 is a covalently linked heterogeneous dimeric cytokine, composed of p35 and p40 subunits, secreted in vivo by activated immune cells.

It is understood that in the present invention, the IL-12 can be from a human or non-human mammal, can be the full-length, mature form of IL-12, or its active fragment. In addition, the IL-12 (or IL-12 protein element) can be a single subunit or multiple subunits. For example, in the present invention, the first protein element can include one or more (e.g., two) subunits of the IL-12 protein.

In another preferred embodiment, the linkage between the P40 and P35 subunits is not particularly limited, and includes "head-to-head", "head-to-tail", "tail-to-head" and "tail-to-tail" linkages, wherein "head" refers to the N-terminus and "tail" refers to the C-terminus of the polypeptide.

In addition, a linker (linker) may or may not be present between the P40 and P35 subunits. Preferably, the linker is a flexible 4-20 amino acid linker, more preferably the linker is GGGGGGS (G)₆S)(SEQ ID NO.:8)

Peptide linker

Generally, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient freedom in space to function, while avoiding the formation of α helices or β folds in the peptide linker, etc., which may affect the stability of the fusion protein.

The length of the linker peptide is generally 0 to 15 amino acids, preferably 1 to 15 amino acids.

Examples of preferred linker peptides include (but are not limited to): a linker peptide represented by SEQ ID No. 7 or 8.

Signal peptides and leader peptides

The fusion proteins of the invention may also contain other elements, representative elements including (but not limited to): signal peptides, leader peptides, and the like.

In one embodiment of the invention, the fusion protein comprises a signal peptide. Representative examples include (but are not limited to): a signal peptide of IL-12P 40 subunit of human origin.

Bifunctional fusion proteins and their preparation

As used herein, unless otherwise indicated, the fusion protein is an isolated protein, unrelated to other proteins, polypeptides or molecules, expressed by a recombinant host cell, or an isolated or purified product.

In the present invention, "recombinant bifunctional fusion protein", "protein of the present invention", "fusion protein of the present invention", "bifunctional fusion protein", "IL-12-mutant CD62L fusion protein", "IL-12/mutant CD62L fusion protein" are used interchangeably and refer to a fusion protein having a structure according to formula Ia or IIa, i.e., a structure comprising a protein element including IL-12 protein element, mutant CD62L and a linker peptide element. A representative example is IL12-dKSCD 62L. The proteins of the invention may be monomers or multimers (e.g., dimers) formed from monomers. Furthermore, it is to be understood that the term also includes active fragments and derivatives of the fusion protein.

A preferred fusion protein has the sequence shown in SEQ ID No. 2, wherein the 1-328 position is the P40 subunit of IL-12; 329-Position is G₆An S-linker peptide; position 336-532 is the P35 subunit of IL-12; the 218 th connecting peptide (SEQ ID NO: 7) in the position 533-547; 548-913 is the dKSCD62L amino acid sequence.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

As used herein, "isolated recombinant fusion protein" means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

As used herein, the term "primer" refers to a generic term for an oligonucleotide that, when paired with a template, is capable of synthesizing a DNA strand complementary to the template from its origin by the action of a DNA polymerase. The primer can be natural RNA, DNA, and any form of natural nucleotide. The primers may even be non-natural nucleotides such as LNA or ZNA etc. A primer is "substantially" (or "substantially") complementary to a particular sequence on one strand of the template. The primer must be sufficiently complementary to one strand of the template to begin extension, but the sequence of the primer need not be completely complementary to the sequence of the template. For example, a primer that is complementary to the template at its 3 'end and has a sequence that is not complementary to the template at its 5' end remains substantially complementary to the template. Primers that are not perfectly complementary can also form a primer-template complex with the template, so long as there is sufficient primer binding to the template, allowing amplification to occur.

Based on the amino acid sequences provided by the present invention, the fusion protein of the present invention can be conveniently prepared by various known methods by those skilled in the art. Such methods are for example but not limited to: recombinant DNA methods, artificial synthesis, etc.

The full-length nucleotide sequence of the element (e.g., IL12 or mutant CD62L) of the fusion protein of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells encoded with the vector or fusion protein coding sequences of the invention, and methods for producing the proteins of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.

One particularly preferred cell is a cell of human and non-human mammals, particularly immune cells, including T cells, NK cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Modified immune cells

The invention also provides an immune cell expressing the fusion protein (referred to as the immune cell of the invention) carrying the fusion protein on the cell surface.

In the present invention, at least a portion or all of the fusion protein is located on the cell membrane of the immune cell, and the first protein element, the IL-12 protein element, is located extracellularly.

One preferred class of immune cells includes T cells, particularly human T cells. Preferably, the T cell surface carries MART-1 TCR.

For example, in a preferred embodiment, a lentiviral expression system genetically modified T cell is provided that expresses an anti-tumor TCR (T-cell receptor) while expressing an hscIL-12/mutant CD62L fusion protein.

In addition, the T cells can optionally also express wild-type CD62L, hscIL-12 (human single chain IL-12) or their combination.

Mechanism for membrane expression of dKSCD62L mutant

For ease of understanding, the inventors provide reference to the following mechanisms. It is to be understood that the scope of the present invention is not limited by the mechanism.

The mechanism of action of the membrane-expressed dKSCD62L mutant is shown in FIG. 1. The CD62L transmembrane region contains an ADAM17 cleavage site, and ADAM7 can cleave and release CD62L under the condition of T cell activation. In the invention, KLDKSFS sequence (namely the cutting site of ADAM 17) is knocked out through genetic engineering modification, so that stable membrane expression of exogenous CD62L is realized from a molecular mechanism.

A typical mutant CD62L is dKSCD 62L. ADAM17 was unable to cleave dKSCD62L on the membrane surface when T cells were activated, including tumor antigen-induced T cell activation. In other words, the dKSCD62L mutant is not cleaved by ADAM17 enzyme and can not be released after T cell activation, so that dK-SCD62L is retained on the surface of T cell membrane, and IL-12 element in the fusion protein is also retained on the surface of T cell membrane, and most importantly, the fusion protein retains both the biological activity of Il-12 and the function of T cells.

Pharmaceutical compositions and methods of administration

The invention also provides a composition comprising (a) an effective amount of a fusion protein of the invention and/or an effective amount of an immune cell of the invention, and a pharmaceutically acceptable carrier.

Typically, the fusion proteins of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to about 8, preferably about 6 to about 8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to humans and/or animals, such as 0.001 to 99 wt%; preferably 0.01 to 95 wt%; more preferably, 0.1 to 90 wt%.

When the pharmaceutical composition of the present invention contains immune cells, "effective amount" or "effective dose" means 1X 10³-1×10⁷The immune cells are used per ml.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the fusion protein of the present invention is administered at a dose of about 5mg to 20mg per kg of animal body weight per day, preferably 5mg to 10mg per kg of animal body weight per day. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The fusion protein is particularly suitable for treating diseases such as tumors and the like. Representative tumors include (but are not limited to): brain tumor, carcinoma of large intestine tumor, lung cancer tumor, hepatocarcinoma tumor, breast cancer tumor, gastric cancer tumor, and pancreatic cancer tumor.

The main advantages of the invention include:

(a) the fusion protein of the invention has no obvious toxicity to T cells. This may be due to IL-12 protruding from the T cell membrane and not being able to effectively contact or act on T cells, and may also be due to the spacer effect of CD62L which reduces the toxicity of IL-12 on T cells.

(b) When the anti-tumor T cells carrying the fusion protein of the present invention are in proximity to tumor cells, free IL-12 is not released (i.e., IL-12 remains on the surface of the cell membrane) because CD62L cannot be specifically cleaved. This allows the membrane surface IL-12 to act more efficiently and only on tumor cells in close proximity, thereby significantly improving safety.

(c) The maximization of the antitumor immune effect is synergistically and effectively achieved by the T cell attacking the tumor locally and changing the immune microenvironment of T cell-tumor tissue by IL-12 on the cell surface.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Materials and general methods

The primers and DNA sequences used were synthesized by Invitrogen corporation.

The map of the plasmid LVV used in the embodiment of the invention is shown in FIG. 4, and is an engineering vector pLenti-MSCV containing a target gene, and the vector takes MSCV as a promoter, which is an optimized promoter capable of effectively transducing T cells; the envelope protein plasmid pMD2.G contains VSV-G; a gag/pol helper plasmid; and the pRev plasmid system. The plasmids pRRLSIN, cPPT, MSCV/GFP and 293FT cells used were commercially available, and the reagents used were also commercially available.

In an example, mutant CD62L refers to dKSCD 62L.

HscIL-12/CD62L or IL-12/WTCD62L refers to hscIL-12/wild type CD 62L.

Wild-type CD62L is designated WTCD 62L.

Culture of tumor cells and T cells

Tumor cells

938 and 526 were conventional melanoma cell lines (as donated by dr. rosenberg, national cancer institute) subcultured in vitro in 10% FCS RPMI medium by 0.25% trypsin subculture every 2-3 days. Both tumors expressed MART-1 antigen, of which 938 is MHC I A2- (negative) and 526 is MHC I A2+ (positive), and the genetically modified anti-tumor MART-1T cells recognized only the A2+ cell line, 526 cells.

PBMC are derived from peripheral blood of healthy human, and T cells are stimulated to grow for 1 day with CD3/CD28 magnetic beads or CD3 antibody and maintained in IL-2(100IU/ml) in X-VIVO medium. After the T cells are transduced by the recombinant lentivirus, the fluorescence intensity of MART-1 in the T cells is detected by a MART-1 tetrameric peptide fragment by using a flow cytometer. Simultaneously, the expression of cell surface CD62L, membrane surface hscIL-12/mutant CD62L and CD107a is detected by flow.

Construction of Lentiviral vectors

The technical method of molecular biology is adopted to transfect peripheral blood autologous lymphocytes with recombinant lentiviral vectors carrying human TCR α and β chain genes for specifically recognizing human melanoma associated antigen MART-1, so that the recombinant TCR is expressed in the T lymphocytes to achieve the purpose of efficiently killing tumors.

The construction of lentiviral expression vectors expressing wild-type CD62L (abbreviated as "WTCD 62L") and mutant CD62L (abbreviated as "dKSCD 62L") is referred to published literature (Yang, Cohen et al 2008, Yang, Liu et al 2011) and the construction of hssil-12 is referred to published literature (Zhang, Kerkar et al 2011). Wherein the fusion protein of the hscIL-12/wild type CD62L and the hscIL-12/mutant CD62L uses peptide fragment G₆S and 218 peptide fragments are connected.

Preparation of lentivirus expression system expression fusion protein

293T cells were cultured, the cell density was adjusted by DMEM medium containing 10% fetal bovine serum the day before transfection, and 25X 10 cells were plated per 15cm cell culture dish⁶293T cells, incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 16-24 h, and then using for transfection when the cell density grows to 80-90%. The medium was changed to complete medium without antibiotic (P/S) (DMEM + 10% FBS) on the day of transfection. Separately, the lentiviral backbone of LVV-MSCV-MART-1TCR, CD62L, hscIL-12L and hscIL-12/mutant CD62L fusion proteins were co-transfected into 293T cells with three additional packaging plasmids using commercially available calcium phosphate as a vehicle. After 6h incubation the medium was discarded and washed 3 times with PBS and replaced with 20ml fresh complete medium (DMEM + 10% FBS + P/S). Collecting the culture supernatant 30-72h after transfection, centrifuging at 6000rpm for 10min, discarding cell debris, filtering the supernatant with 0.45 μm PVDF filter into 50ml round-bottom centrifuge tube, centrifuging at 4 deg.C and 50000g for 2h at high speed, carefully discarding the supernatant, DMEM (without serum and double antibody) resuspending virus precipitate, subpackaging into clean 15ml centrifuge tube according to the amount of virus used each time, and preserving in refrigerator at-80 deg.C for infecting T cells. The titer of the Lentivirus-Associated p24 ELISA Kit virus detection is 5X 10⁷-1.5×10⁸IFU, see Lentivirus-Associated p24 ELISA Kit instructions for specific procedures.(Yang,Cohen et al.2008)。

Establishment of Co-culture System of tumor cells/T cells

T cells modified by the anti-MART-1 TCR gene were co-cultured with the 526, 938 cell line, at a rate of 1: 1 ratio, i.e. 1X 10 of each cell⁶Placed in a 14ml round-bottomed polypropylene culture tube in a total volume of 1ml, transferred to CO at 37 ℃₂The incubator is used for 4 hours. After 4h, the cells were centrifuged at 800 Xg for 10min, the supernatant was collected and the cells were lysed by RIPA lysate. The content of IFN gamma and IL-12 in the supernatant is detected by ELISA reagent. The membrane surface IL-12/wild type CD62L or IL-12/mutant CD62L by conventional flow cytometry live cell staining. Wild type or mutant CD62L in the supernatant and wild type or mutant CD62L in the cells were detected by ELISA kit (R)&D Systems,Minneapolis,MN)。

Analysis of flow cytometry assays

Cell surface CD3, CD8, CD62L, CD107a, IL-12 and CD45RO were detected by fluorescently labeled corresponding antibodies including isothiocyanate (FITC), Allophycocyanin (APC), Phytoerythrin (PE), PE-Cy7, and APC-Cy7(BDbiosciences, San Jose, Calif.). MART-1: 27-35 Tetramer was synthesized by the company (iTAg MHC Tetramer, Beckman Coulter, Fullerton, Calif.) by design to detect the level of expression of the genetically modified TCR. The specific procedure is as follows, first the cells are washed twice with FACS staining solution (PBS staining 2% FBS) and then 0.2ml (10 ml) is added⁶/ml) in flow cell tubes, incubated at 4 ℃ for 30 minutes and then washed twice. Dead cells were isolated by pre-machine addition of 20. mu.l PI (l 5. mu.g/ml propidium iodide) (Sigma-Aldrich, Saint Louis, Mo.) to the sample and partitioning of the cell subpopulations. The streaming data was analyzed by on-machine post-processing by FlowJo 8.1.1 (FlowJo, Ashland, OR).

Establishment of tumor-bearing mouse model

The Pmel experimental mouse model is routinely used, for example, as described in the literature (Overwijk, Tsung et al 1998). The method involved in this experiment was to select female pmel mice (6-8 weeks, 7 mice per group) for intracranial tumor Inoculation (IC). B16F10-MART-1 tumor cells by containingThe reaction of pancreatin was terminated by digesting 0.25% pancreatin with 0.02% EDTA and washing once with a serum-containing culture solution, followed by washing twice with PBS. Tumor cells are finally differentiated by 1: 1 volume was mixed with Methylellulose in zinc option medium and 5000 cells were diluted in 5. mu.l of liquid and loaded to 250- μ l syring (Hamilton, Reno, NV) using a 25-gauge needle. The mice were injected into the right brain caldate nuclei using the quantitative stereotaxic Injector System (Stoelting co. 5 days after tumor cell inoculation, mice received full body 5Gy radiation. On day 2, mice received 0.5-1X 10 subcutaneously⁶DC vaccine, or 1X 10 by IV infusion through tail vein⁷T cells modified by sequential transduction of anti-MART-1 TCR, IL-12/wild type CD62L or IL-12/mutant CD62L lentiviral genes. Murine T cells were obtained from mouse spleen cells and activated with 10ug/ml of concanavalin (Con A) in the presence of IL-2(5 IU/ml); on day 2, T cells were transduced with lentiviral vectors, followed by culture for another 6 days, cells were harvested and injected via tail vein of mice. DC cells of the mice in the DC group were obtained from bone marrow cells of the mice, matured by in vitro induced differentiation for 8 days, and inoculated via the abdominal cavity. Mice were then recorded daily for mortality, growth curves were recorded and graphed by Prism mapping software. Asterisks indicate the experimental group compared to other groups, p<0.001。

Example 1

Construction of fusion Gene

The fusion gene was synthesized by Invitrogen corporation, and the length and sequence of the fusion gene were confirmed by 1% agarose electrophoresis and sequencing.

The structure of the constructed IL-12/mutant CD62L fusion gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded fusion protein is shown in SEQ ID No. 2.

And simultaneously constructing an obtained IL-12/wild type CD62L fusion gene, wherein the IL-12/wild type CD62L fusion gene is very similar to the fusion gene in SEQ ID NO. 1, and the difference is only that: the nucleotide sequence encoding the cleavage site is retained, so the encoded fusion protein also retains the ADAM17 cleavage site (i.e., KLDKSFS).

Example 2

Construction of Lentiviral expression vectors

The method of "construction of lentiviral vector and genetic modification of T cell" in the general method was adopted: humanized 293T cells cultured in vitro in low DMEM (10% FBS) -containing medium were counted and transferred to 15CM dishes, the bottom of which was treated with poly-D-Lysine, and 20X 10 cells were plated on each dish⁶Cells, the next day, a mixture of DNA mixture and Lipofectamine was added to each transfection dish, the mixture consisted of 2ml of Optimum I and pLenti-MSCV (22.5ug), pMD2.G (7.5ug), gag/pol (15ug), pRev (10ug) were added and mixed well; simultaneously, 2ml of Optimum I is taken and added with Lipofectamine 160ul (Invitrogen) and mixed evenly. The two suspensions were mixed, incubated at room temperature for 5 minutes, and then added dropwise into a petri dish. After 48-72 hours, the supernatant containing the engineered vector was harvested, centrifuged at 2000g to remove cellular debris, collected, filtered through a 0.45uM filter to remove potential contamination, aliquoted and stored in a minus 80 freezer. According to different requirements, the collected virus supernatant can be subjected to 50000g ultracentrifugation to obtain virus vectors with higher concentration.

The obtained lentiviral expression vectors were designated LV-hscIL-12/wild type CD62L (expressing IL-12/CD62L), LV-hscIL-12/mutant CD62L (expressing IL-12/dKSCD62L), and LV-hscIL-12, respectively.

Example 3

Preparation of T cells expressing IL-12/mutant CD62L fusion protein

The method comprises the following steps: PBMC were activated with CD3/CD28 magnetic beads or anti-CD 3 antibody, and T cells were modified with lentiviral genes on day 2, in a brief manner as follows: the T cells were washed 3 times with PBS buffer, and the ratio of virus titer to T cells was 3: 1 adding a proper amount of lentivirus, centrifuging for 2h at 2000X g, and after 6h, adding 100IU/ml IL-2 to continue culturing; 2 nd transduction, or combined co-transduction, was performed on day 5. The flasks were split according to cell growth, and after two weeks, the genes modified by the T cells were examined by flow cytometry.

Example 4

DKSCD62L lentivirus gene modified anti-tumor T cells realize stable expression of dKSCD62L

In order to better observe the exogenous CD62L and dKSCD62L protein expression mediated by lentivirus genes and distinguish the endogenous physiological CD62L expression, a tumor infiltrating T cell line JKF6 separately cultured in melanoma tissues is selected. The JKF6 cell line can be subcultured in vitro for a long time, and the CD62L membrane molecule is lost on the membrane surface, and JKF6 can effectively and specifically recognize melanoma cells.

After the JKF6 was modified by lentivirus genes, high levels of exogenous WTCD62L and dKSCD62L expression could be detected. The wild type CD62L and the mutant dKSCD62L gene modified JKF6 obtain stable membrane surface CD62L series expression.

After activation with the tumor antigen 526, the wild-type CD62L can be specifically cut and released by the tumor antigen; however, the membrane expression of dKSCD62L lost the cleavage and release of tumor antigen activation, and showed a phenotype of stable expression of dKSCD62L on the membrane surface, as shown in fig. 2 and fig. 3, co-cultured T cells detected the molecules CD45RO and CD62L on the membrane surface by flow cytometry, and the membrane stability of the CD62L mutant was fully verified by the analysis and treatment of FlowJo software.

Example 5

The dKSCD62L lentivirus gene modified anti-tumor JFK6 cells do not influence the anti-tumor activity of T cells

To verify the reactivity of the modified JKF6 with the dKSCD62L lentivirus gene, it was also considered that there may be other pathways for the cleavage of CD62L in vivo, and in this example, the anti-tumor activity of T cells was further verified. The method comprises the following steps:

a broad spectrum of T cell activators PMA/Ionomycin (referred to as "PMA/Ion" for short) was selected, which is a T cell activator, and 4 hours after activation of T cells with the activator, we detected the expression of CD62L (wild type or mutant) on the cell membrane. Furthermore, it was found that for mutant dKSCD62L, activation-induced membrane cleavage disappeared, i.e. stable membrane surface expression of dKSCD62L was obtained; for wild-type CD62L, T cell activation cleavage still existed.

To verify the reactivity of T cells, the reactivity was determined after modification with the CD62L gene. As a result, as shown in FIG. 3, JKF6 modified with the mutant CD62L gene (or fusion protein gene) was activated to express high levels of IFN γ. The experimental result indicates that after the fusion protein is subjected to gene modification, the reactivity of the anti-tumor JKF6 is normal and is not influenced by the gene modification.

Example 6

Construction of lentivirus expression dKSCD62L fusion protein vector and expression in human T cell

In this example, lentiviral vectors for IL-12/CD62L and IL-12/dKSCD62L were constructed (structure shown in FIG. 4), in which the IL-12 gene and the mutant or wild-type CD62L gene were separated by the amino acid peptide fragment G₆S-linking, wherein the lentivirus is a third generation lentivirus vector, and the promoter is MSCV. The 5 'and 3' LTRs of the vector are modified to SIN-LTR (self-inactivating-LTR) to reduce the probability of lentivirus recombination and enhance safety.

In order to clarify the expression of IL-12/dKSCD62L fusion protein in anti-tumor T cells, anti-tumor TCR and IL-12/dKSCD62L fusion protein were sequentially transduced into T cells, and the anti-tumor T cells were co-cultured with

tumor cells

526 and 938 after being cultured in vitro for 14 days.

The results observed IL-12/dKSCD62L stable membrane expression phenotype, and further confirmed IL-12/CD62L tumor antigen dependent specific cutting and release.

Example 7

The expression of IFN gamma can be enhanced by culturing T cells transduced by a lentivirus vector IL-12/CD62L series with tumors

In the present invention, a fusion protein vector that stably membrane-expresses IL-12/dKSCD62L was constructed in order to reduce systemic cytotoxicity induced by IL-12 release.

As shown in FIG. 5, after the anti-tumor TCR and IL-12/CD62L fusion protein were sequentially transferred to human T cells for 14 days, they were co-cultured with

tumor cells

526 and 938, and after 24 hours, the IFN γ and IL-12 expression levels in the supernatant were detected by ELISA kit.

The results show that the persistent IL-12 secretion group, the IL-12/wild type CD62L group and the IL-12/dKSCD62L group can obviously enhance the reactivity of the anti-tumor T cells and the tumor compared with the T cell group, and the reaction is shown to obviously enhance the secretion level of IFN gamma, wherein P is less than 0.001.

Consistent with the stable expression of dKSCD62L on the membrane surface, IL-12/dKSCD62L also lost tumor antigen-reactive cleavage and release, and a significant decrease in the level of secreted IL-12 was detected with P <0.001, as compared to the IL-12/wild-type CD62L group and the sustained secretion IL-12 group.

Therefore, it was confirmed that stable membrane expression of IL-12/dKSCD62L is feasible, and further that after co-culturing the anti-tumor T cells expressing IL-12/dKSCD62L with tumor cells, the tumor antigen reactivity of the T cells is not changed, which contributes to maximizing the anti-tumor effect of IL-12.

Example 8

T cells transduced by the lentiviral vector IL-12/dKSCD62L can effectively avoid toxic and side effects of in vitro T cell expansion caused by continuous IL-12 secretion

After addressing the issues of stable membrane expression and enhanced anti-tumor reactivity, this example observed the effect of transduction of the IL-12/dKSCD62L fusion protein on T cell expansion in vitro.

As shown in FIG. 6, after the anti-tumor TCR and the wild-type or mutant CD62L/hscIL-12 fusion protein are sequentially transduced by the T cells for 14 days, the amplification factor of each group of cells is compared with that of a control transduction group (T-cell), and the data analysis adopts that the amplification factor of the T-cell group is set to be 100%, and the values of other groups are the ratio of the T-cell group to the T-cell group.

The results show that the amplification fold of the group which continuously secretes IL-12 by transduction is obviously reduced compared with other groups, and p is less than 0.001. Among them, the hscIL-12/dKSCD 62L-transduced fusion protein group has the same amplification fold compared with other groups, and no significant difference is found. In addition, the amplification factor was slightly higher in the case of the hscIL-12/dKSCD 62L-transduced fusion protein group than in the case of the hscIL-12/wild-type CD 62L-transduced fusion protein group. This suggests that transduction results in little or no significant toxicity of IL-12 to T cells, since free IL-12 is not released.

Example 9

Transduction of murine T cell mediated cell feedback therapy with the lentiviral vector hscIL-12/dLSCD62L series significantly prolonged survival of tumor-bearing mice

Based on the clear expression, membrane stability, tumor antigen response synergy and in vitro amplification of IL-12/dKSCD62L, in this example, a preclinical tumor-bearing mouse model was further used to observe in vivo tumor suppression effects. The method comprises the following steps:

female pmel mice (7 mice per group) were selected for Intracranial (IC) implantation by B16F10 cells for 5 days, and the mice received 5Gy of systemic radiotherapy 1 day prior to cell reinfusion. Murine T cells were obtained from mouse spleen cells and activated by 10ug/ml concanavalin (ConA) in the presence of IL-2(5 IU/ml); on day 2, T cells were transduced with lentiviral vectors, followed by continued culture for 6 days, cells were harvested and injected via the tail vein of mice (IV)5X 10⁶And (4) T cells. DC cells of DC group mice are taken from bone marrow cells, induced, differentiated and matured in vitro for 8 days, and inoculated with 1X 10 cells through abdominal cavity⁶A cell. See the right hand panel for DC and T cell profiles. Asterisks indicate comparison of this experimental group with other groups, p<0.001。

As shown in fig. 7. The results indicate that the IL-12/dKSCD62L group significantly prolonged the survival of tumor-bearing mice compared to the other control groups; the group that continuously secreted IL-12 showed significant systemic cytotoxicity, with mice all dying 4-7 days after cell return.

Discussion of the related Art

CD62L is a homing factor expressed on the surface of T cells, and adhesion and release of T cells along the vessel wall can be achieved by activation-induced cleavage of T cells. It was found that T cells lacking ADAM17 enzyme (tumor Necrosis factor-converting enzyme 17) resulted in the inability of CD62L to cleave and release.

In the invention, the inventor knocks out the cutting point of ADAM17 of CD62L (named as dKSCD62L in the invention), and experimental results show that the novel IL-12/mutant CD62L molecule expressed by genetically modified anti-tumor T cells can not only kill tumor cells efficiently, but also remarkably reduce the toxic and side effects of IL-12.

Compared with the anti-tumor T cell modified by the gene continuously secreting IL-12, the anti-tumor T cell modified by the IL-12/mutant CD62L fusion protein gene not only can obviously prolong the survival of a tumor-bearing mouse, but also can avoid systemic cytotoxicity induced by the continuous secretion of IL-12, is a brand-new immune cell treatment strategy for expressing IL-12 through a cell membrane and realizing the cutting and releasing of tumor antigen reactivity through CD62L, and plays an important role in the treatment of the immune cell of the tumor.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Reference to the literature

Zhang, L. et al (2011), "stimulating adaptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment," Mol Ther 19(4):751-759.

Overwijk, W.W. et al (1998) ' gp100/pmel 17 is a membrane tumor rejection inhibitor: indication of ' self ' -reactive, tumor T cells using high-affinity, absolute peptide ligand, J Exp Med 188(2):277-286.

Yang, S. et al (2008), "Development of optimal biological molecules defects high-level TCR Gene expression and robust molecular recognition," Gene Ther 15(21):1411- "1423.

Yang, S.et al (2011), "The formatting of CD62L (L-selectin) ligands The acquisition of a kinetic activity in human reactor reactivity T lymphocytes," PLoS One6(7): e22560.

Claims

1. A fusion protein comprising the following elements fused together:

(i) a signal peptide and/or leader peptide absent, or at the N-terminus;

(ii) a first protein element;

(iii) a second protein element; and

(iv) a linker peptide element absent from, or located between, the first protein element and the second protein element;

and the first protein element is an IL-12 protein element; the second protein element is a protein element of mutant CD62L, which lacks the ADAM17 cleavage site of mutant CD 62L;

and said fusion protein has a structure selected from the group consisting of:

(1) the structure of formula Ia:

D-A-B (Ia), or

(2) The structure of formula IIa:

D-A-C-B (Ⅱa)，

wherein,

a is an IL-12 protein element;

b is a mutant CD62L protein element;

c is nothing, or a linker peptide element;

d is nothing, a signal peptide and/or a leader peptide sequence;

each "-" independently represents a peptide bond or a peptide linker connecting the above elements; wherein the protein element of mutant CD62L lacks part or all of the sequence of KLDKSFS, thereby rendering it incapable of being cleaved by ADAM 17;

wherein the sequence of the IL12 protein element is shown as SEQ ID No. 4; and the sequence of the mutant CD62L protein element is shown in SEQ ID No. 6.

2. The fusion protein of claim 1, wherein the linker peptide element has the sequence shown in SEQ id No. 7 or 8.

3. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is as set forth in SEQ id No. 2.

4. An isolated polynucleotide encoding the fusion protein of claim 1.

5. A vector comprising the polynucleotide of claim 4.

6. A host cell comprising the vector or genome of claim 5 having integrated therein the polynucleotide of claim 4.

7. A method of producing the protein of claim 1, comprising the steps of:

(1) culturing the host cell of claim 6 under conditions suitable for expression, thereby expressing the fusion protein of claim 1; and

(2) the fusion protein is absent or isolated.

8. An immune cell carrying the fusion protein of claim 1 on a membrane surface.

9. A pharmaceutical composition, said composition comprising:

the immune cell of claim 8, and

a pharmaceutically acceptable carrier.

10. Use of the fusion protein according to claim 1 and/or the immune cell according to claim 8 for the preparation of a medicament for the treatment of tumors.

11. A non-therapeutic in vitro method for killing tumor cells, comprising the steps of: contacting the immune cell of claim 8 with a tumor cell, thereby killing the tumor cell.