WO2017107353A1

WO2017107353A1 - Cancer treatment agent, preparation method and use thereof employing il-12 with stable membrane expression

Info

Publication number: WO2017107353A1
Application number: PCT/CN2016/080418
Authority: WO
Inventors: 杨世成
Original assignee: 杨世成
Priority date: 2015-12-23
Filing date: 2016-04-27
Publication date: 2017-06-29
Also published as: CN105585637A; CN105585637B

Abstract

Provided are an IL-12-mutant CD62L fusion protein, preparation method and use thereof, wherein the mutant CD62L lacks an ADAM17 cleavage site.

Description

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

Technical field

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

Background technique

IL-12 is a very important immune stimulating factor. IL-12 is a heterodimeric cytokine linked by a covalent bond, consisting of the p35 and p40 subunits, secreted by activated immune cells in vivo. IL-12 is an important cellular immune regulatory factor that acts through NK cells and CTLs in immunity against infection and malignancy.

Although IL-12 has a significant antitumor effect, it is extremely limited due to systemic systemic administration which causes systemic toxic side effects. Prior to the present invention, the clinical application of IL-12 was greatly limited in clinical application due to the systemic toxic side effects induced by it and the accidental death of two patients caused by systemic administration in early clinical trials.

In order to avoid the toxic side effects of systemic administration and achieve clinical application, clinical researchers have applied to clinical trials of tumors through local injection of tumors, transient expression and multiple injections, but the therapeutic effects of IL-12 are the same. The dose of IL-12 returned was directly related, and the above-mentioned local clinical protocol did not achieve significant tumor inhibition.

In order to solve this problem, the present inventors have attempted to maximize the antitumor effect by genetically modifying anti-tumor T cells to continuously secrete IL-12, but for unknown reasons (a possible cause is the side effects of IL-12) T cells that have been genetically modified by sustained secretion of the IL-12 gene are not efficiently amplified in vitro and cause a large number of T cell apoptosis.

In summary, there is a lack of IL-12-based antitumor drugs that are highly effective and have low toxic side effects. Therefore, there is an urgent need in the art to develop a tumor therapeutic drug that is highly effective and has low toxic side effects.

Summary of the invention

The object of the present invention is to provide a tumor therapeutic drug with high efficiency and low toxic 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) an optional signal peptide and/or leader peptide at the N-terminus;

(ii) a first protein component;

(iii) a second protein component;

(iv) an optional linker element located 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 a protein element of the mutant CD62L, and the protein element of the mutant CD62L lacks a cleavage site of ADAM17.

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

(1) Structure described by Formula Ia:

D-A-B (Ia), or

(2) Structure described in Formula IIa:

D-A-C-B (IIa),

among them,

A is an IL-12 protein element;

B is a mutant CD62L protein element;

C is an optional linker element;

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

Each "-" independently indicates a peptide bond or a peptide linker to which the above elements are attached.

In another preferred embodiment, the protein element of the mutant CD62L lacks a partial or total sequence in the KLDKSFS sequence, resulting in the inability to be cleaved by ADAM17.

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

In another preferred embodiment, the linker peptide element comprises a linker peptide having the sequence set forth in SEQ ID NO.: 7.

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

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

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

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

In another preferred embodiment, the subunit of the IL-12 protein is selected from the group consisting of the P40 and P35 subunits.

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

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

In another preferred embodiment, a linker is present or absent between the P40 and P35 subunits. Preferably, the linker is a flexible linker of 4-20 amino acids, more preferably, the linker is GGGGGGS (i.e., _{G 6 S) (SEQ ID NO.:8} ).

In another preferred embodiment, the sequence of the IL12 protein element is set forth in SEQ ID NO.: 4.

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

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 a full length, mature form of CD62L, or an active fragment thereof.

In another preferred embodiment, the sequence of the mutant CD62L protein element is set forth in SEQ ID NO.: 6.

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

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

In another preferred embodiment, the fusion protein is provided with a linker peptide, preferably a 218 peptide segment, between the single-stranded IL-12 (first protein element) and the mutant CD62L (second protein element) (SEQ ID NO.: 7).

In another preferred embodiment, in the fusion protein, the first protein element is a single-stranded IL-12, and in the single-stranded IL-12, a linker peptide G ₆ S is provided in the P40 subunit and the P35 subunit. (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 cleaved by the ADAM17 protein, thereby not releasing IL-12;

b) The fusion protein comprises two subunits of IL-12, namely the P40 and P35 subunits, and is joined 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 the fusion protein of the first aspect of the invention.

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

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

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

In another preferred embodiment, the viral vector comprises a lentiviral vector, an adenoviral vector, and a yellow fever virus vector.

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

In a fourth aspect of the invention, a host cell comprising the polynucleotide of the second aspect of the invention, or a polynucleotide of the second aspect of the invention, is provided.

In another preferred embodiment, the host cell comprises a prokaryotic cell and a eukaryotic cell.

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 the protein of the first aspect of the invention, Including steps:

(1) cultivating 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;

(2) Optionally isolating the fusion protein.

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

In another preferred embodiment, the immune cells of at least 10 ³ (preferably ¹⁰³ ^{to 109,} more preferably preferably ^{^104-108} th) the cell population of immune cells.

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

In another preferred embodiment, at least a portion or all of said fusion protein is located on the cell membrane of said immune cell, and said first protein element, i.e., 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 a MART-1 TCR.

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

The immune cell of 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 contains 1 x 10 ³ - 1 x 10 ⁷ of said immune cells/ml.

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

In another preferred embodiment, the tumor comprises: a brain tumor, a colorectal cancer tumor, a lung cancer tumor, a liver cancer tumor, a breast cancer tumor, a gastric cancer tumor, and a pancreatic cancer tumor.

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

In another preferred embodiment, the fusion protein is administered as a monomer and/or a dimer.

In another preferred embodiment, the object is a human.

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

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows the amino acid composition of the CD62L transmembrane region and the cleavage site of ADAM17. 1A and 1B show that the dKSCD62L mutant (with KLDKSFS deleted) cannot undergo cleavage and release based on a cleavage enzyme (such as Adam17) after T cell activation, resulting in dKSCD62L remaining on the surface of the T cell membrane. In Figure 1C, WT represents the wild type sequence and dKS represents the CD62L mutant knocked out of the ADAM17 sequence.

Figure 2 shows that dKSCD62L lentiviral gene-modified anti-tumor T cells result in stable expression of dKSCD62L.

Among them, as shown in the above figure, the anti-tumor T cell line JKF6 (an anti-tumor T cell line isolated and cultured from melanoma tissue can directly recognize the tumor) can be detected by lentiviral gene, and the expression of WTCD62L and dKSCD62L can be detected.

As shown in the figure below, CD62L can undergo tumor antigen-specific cleavage and release after T (wild-type) CD62L is activated by tumor antigen 526. However, after expression of dKSCD62L membrane, it loses the cleavage and release of tumor antigen activation. The phenotype of expression of the stable membrane surface dKSCD62L.

JKF6T cells were co-cultured with

tumor cells

938 and 526 expressing the MART-1 antigen. Among them, co-cultured T cells were detected by flow cytometry on the surface of the membrane CD45RO and CD62L, and analyzed by FlowJo software, each group of T cells were sorted into CD8 cell subsets. The antibodies used for flow cytometry were CD62L FITC, CD45RO APC, MART-1PE and CD8PerCP.

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

Among them, in Figure 3A, after Lentiviral gene modification of JKF6 cells, T cells were activated by PMA/Ionomycin (PMA/Ion, a T cell activator) for 4 h, and then the CD62L series and IFNγ on the cell membrane surface were fixed and stained by routine. And the expression level of CD62L and intracytoplasmic IFNγ on the membrane surface was detected (top panel). Figure 3B shows the statistical analysis treatment of IFNy and CD62L expression. The data are analytical results of 3 independent experiments. The expression level of dKSCD62L is consistently stable, and IFNγ is similar to other groups, showing similar high levels. Results are expressed as mean ± standard deviation, and T-test was used for statistical processing. * indicates that dKSCD62L is compared with the other two groups of CD62L expression levels, P < 0.01.

Figure 4 shows the construction of a lentiviral vector expressing dKSCD62L fusion protein and its expression in human T cells. The lentivirus is a third generation lentiviral vector and the promoter is MSCV. The 5' and 3' LTR (long terminal repeat) of this vector was engineered into SIN-LTR (self-inactivating-LTR) in order to reduce the probability of lentiviral recombination and enhance safety performance.

The above figure shows the structural diagram of the LVV plasmid and the gene expression constructs of human IL-12/WTCD62L (WT, wild type), IL-12/dKSCD62L and IL-12 (human single chain IL-12), wherein hscIL-12 The LE-12 seq is fused to a lentiviral expression vector by gene cloning, and CD62L or dKSCD62L and hscIL-12 are linked by a peptide G ₆ S.

As shown in Figure 5, T cells pass two transductions, namely MART-1 TCR (first day) and three in the illustration. The cells were sequentially transduced (Day 5). On the 14th day, they were co-cultured with 526 and 938 in a 1:1 ratio for 4 hours, and then the expression of hscIL-12 on the surface of T cell membrane was detected by flow cytometry.

Figure 5 shows that co-culture of lentiviral vector IL-12/mutant CD62L-transduced T cells with tumor cells enhances IFNy expression and tumor antigen-dependent release of IL-12. In the figure, the mock is a blank control group and the tumor is a tumor group.

T cells were sequentially transduced with anti-tumor TCR and IL-12/mutant CD62L series fusion proteins for 14 days, and co-cultured with

tumor cells

526 and 938. After 16 hours, the expression levels of IFNγ and IL-12 in the supernatant were passed through ELISA kit. Detection. The results showed that the sustained IL-12 secretion group, IL-12/WTCD62L group and IL-12/dKSCD62L group could significantly enhance the reactivity of anti-tumor T cells with tumors compared with the T cell group, which showed a significant enhancement of IFNγ secretion. Level, P < 0.001. The stable expression on the surface of dKSCD62L membrane was consistent. IL-12/dKSCD62L also lost the cleavage and release of tumor antigen reactivity. Compared with IL-12/WTCD62L group and persistent secretion IL-12 group, the secreted IL- could be detected. The level of 12 was significantly reduced, P < 0.001.

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

After T cells were transduced with anti-tumor TCR and IL-12/CD62L series fusion proteins for 14 days, the expansion ratios of the cells in each group were compared with the control transduction group (T-cell), and the transduction of IL-12 group was compared with other groups. The amplification factor of the group was significantly reduced, p < 0.001. Among them, the transduction IL-12/dKSCD62L fusion protein group was the same as the other groups, and no significant difference was observed. The data analysis was performed by setting the amplification factor of the T-cell group to 100%, and the values of the other groups were compared with the T-cell group.

Figure 7 shows that lentiviral vector IL-12/dLSCD62L series transduced murine T cell-mediated cell transfusion therapy significantly prolonged survival of tumor-bearing mice.

Female pmel mice (7 mice per group) were implanted intracranial (IC) for 5 days by B16F10 cells (5000 cells/only), and the mice received 5 Gy whole body radiotherapy 1 day before cell return. Mouse T cells were obtained from mouse spleen cells and activated by 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); on day 2, transduction of T with lentiviral vector The cells were then cultured for 6 days, cells were collected, and (X) ⁵ ×10 ⁶ T cells were injected through the tail vein of the mice. The DC cells of the DC group were obtained from bone marrow cells, and induced to differentiate and mature for 8 days in vitro, and 1×10 ⁶ cells were inoculated intraperitoneally. The group description of DC and T cells is shown on the right. An asterisk (*) indicates that the experimental group was compared with the other groups, p < 0.001.

detailed description

Through extensive and intensive research, the present inventors have for the first time developed a novel IL-12 fusion protein with high activity to kill tumor cells with little toxic side effects. The fusion protein of the present invention is a fusion protein of IL-12/mutant CD62L, wherein the mutant CD62L lacks the cleavage site of ADAM17. Experiments show that when the fusion protein of the present invention is expressed in T cells, it is effectively displayed in T The surface of the cell. Unexpectedly, the IL-12/mutant CD62L fusion protein has little effect on the viability of T cells, and the anti-tumor T cells carrying the fusion protein of the present invention are unable to pass specificity when approaching tumor cells. CD62L is cleaved to release IL-12, resulting in IL-12 on the cell membrane surface acting more efficiently and safely on tumor cells. In the present invention, the T cell-targeted tumor is localized and the immune micro-environment of the T cell-tumor tissue is changed by the IL-12 on the cell surface, thereby synergistically and effectively achieving the maximization of the anti-tumor immunity effect, and extremely significantly reducing The toxic side effects of IL-12. The present invention has been completed on this basis.

Specifically, the present inventors developed a novel fusion protein of IL-12/dKSCD62L using a mutant CD62L (dKSCD62L) lacking the cleavage site of ADAM17, and modified anti-tumor T cells by a lentiviral gene. The experimental data show that IL-12/dKSCD62L lentiviral gene-modified anti-tumor T cells, the expression of membrane surface is not regulated by T cell activation, can maintain high membrane surface expression, and enhance the response to tumor antigens. The effect of the immune response. The preclinical tumor-bearing mouse model showed that the modified T cells could significantly prolong the survival of tumor-bearing mice, and no obvious cytotoxic effects were found. In other words, the T cells expressing the fusion protein by the membrane are effective in killing tumor cells on the one hand, and have been confirmed 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 substantially unaffected by the expressed IL-12 (in vitro, the fusion protein of the invention is directed against T cells compared to T cells that continue to secrete IL-12) Amplification has no significant effect).

the term

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, "containing", "having" or "including" includes "including", "consisting essentially of", "consisting essentially of", and "consisting of"; The subordinate concepts of "consisting of", "consisting essentially of" and "consisting of" are "contained," "having," or "including."

CD62L

CD62L is widely expressed on the surface of T cells and is an important immunoregulatory factor. It can regulate the migration of T cells to the lymph nodes of the whole body and is an important T cell homing factor. When CD62L+T cell migrates to the lymph node and is exposed to the tumor antigen, CD62L can be tested for tumor antigen reactivity by ADAM17 on the peptide K283-S284 at the transmembrane region. (Yang, Liu et al. 2011) In the present invention, the inventors confirmed that CD62L cleavage has the specificity of a tumor antigen reaction accompanied by CD107a (the molecule is a molecular protein on the lysosome, which is normally present in the cell) In the pulp, in the T In the cell-activated state, the molecule migrates to the cell surface accompanied by degranulation of T cells; therefore, membrane surface detection of this molecule is an important indicator of membrane migration of T cell killing.

Mutant CD62L

As used herein, the terms "mutant CD62L", "CD62L mutant", "CD62L lacking the ADAM17 cleavage site", "CD62L mutant lacking the ADAM17 cleavage site" or "CD62L mutant lacking the cleavage site", etc., etc. "Interchangeable use" refers to a CD62L polypeptide or active fragment thereof or a polypeptide derived therefrom which lacks a cleavage site for the cleavage enzyme ADAM17, i.e., a partial or total sequence of the KLDKSFS sequence is deleted, such that the CD62L polypeptide or The active fragment or derivative thereof cannot be cleaved by the cleavage enzyme ADAM17.

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

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

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

IL-12

IL-12 is a very important immune stimulating factor. IL-12 is a heterodimeric cytokine linked by a covalent bond, consisting of the p35 and p40 subunits, secreted by activated immune cells in vivo.

It will be understood that in the present invention, the IL-12 may be derived from a human or non-human mammal, and may be a full-length, mature form of IL-12, or an active fragment thereof. Furthermore, 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 may comprise one or more (e.g., two) subunits of an IL-12 protein.

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

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

Peptide linker

The bifunctional fusion proteins of the invention may optionally contain a peptide linker. The size and complexity of the peptide linker may affect the activity of the protein. In general, peptide linkers should be of sufficient length and flexibility to ensure connectivity. The two proteins have enough freedom in space to perform their function. At the same time, the effect of the formation of an alpha helix or a beta sheet in the peptide linker on the stability of the fusion protein is avoided.

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

Examples of preferred linker peptides include, but are not limited to, the linker peptide set forth in SEQ ID NO.: 7 or 8.

Signal peptide and leader peptide

The fusion protein of the present invention may also contain other elements including, but not limited to, signal peptides, leader peptides and the like.

In one embodiment of the invention, the fusion protein contains a signal peptide. Representative examples include, but are not limited to, a signal peptide of the human IL-12P40 subunit.

Bifunctional fusion protein and preparation thereof

As used herein, unless otherwise stated, the fusion protein is an isolated protein that is not associated with other proteins, polypeptides or molecules and is a product expressed by recombinant host cells, or isolated or purified.

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" is used interchangeably and refers to a structure having the structure of Formula Ia, or the structure described in IIa, ie, a fusion protein comprising a protein element comprising a IL-12 protein element, a mutant CD62L, and a linker element. A representative example is IL12-dKSCD62L. The protein of the invention may be a monomer or a multimer (e.g., a dimer) formed from a monomer. Furthermore, it is to be understood that the term also encompasses active fragments and derivatives of fusion proteins.

A preferred sequence of the fusion protein as shown in SEQ ID NO.:2, wherein the first bit is 1-328 of IL-12 P40 subunit; 329-335 G ₆ S bit linker peptide; position 336-532 of It is the P35 subunit of IL-12; the 218-linker peptide (SEQ ID NO.: 7) at positions 533-547; and the dKSCD62L amino acid sequence at positions 548-913.

As used herein, "isolated" means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present 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. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or Non-coding chain.

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. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.

As used herein, the term "primer" refers to a generic term for a oligodeoxynucleotide that, in pairing with a template, can be used to synthesize a DNA strand complementary to a template under the action of a DNA polymerase. The primer may be native RNA, DNA, or any form of natural nucleotide. The primer may even be a non-natural nucleotide such as LNA or ZNA. The primer is "substantially" (or "substantially") complementary to a particular sequence on a strand on the template. The primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer need not be fully complementary to the sequence of the template. For example, a sequence that is not complementary to the template is added to the 5' end of a primer complementary to the template at the 3' end, such primers are still substantially complementary to the template. As long as there are sufficiently long primers to bind well to the template, the non-fully complementary primers can also form a primer-template complex with the template for amplification.

According to the amino acid sequence provided by the present invention, one skilled in the art can conveniently prepare the fusion protein of the present invention by various known methods. These methods are, for example but not limited to, recombinant DNA methods, artificial synthesis, and the like.

The full length nucleotide sequence of the element of the fusion protein of the present invention (e.g., IL12 or mutant CD62L) or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or a synthetic method. For PCR amplification, primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art. The template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.

Once the relevant sequences are obtained, the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short. Usually, a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragment 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 host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.

The polynucleotide sequences of the present invention can be utilized to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally there are the following steps:

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

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

(3). Separating 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 of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be operably linked to an appropriate 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 for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, or 293 cells, and the like.

A particularly preferred cell is a cell of a human and a non-human mammal, especially an immune cell, including T cells, NK cells.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl ₂ . Conversion can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.

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 cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.

The protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include but not Limited to: conventional renaturation treatment, treatment with protein precipitant (salting method), centrifugation, osmotic bacteria, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high efficiency Liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Modified immune cell

The present invention also provides an immune cell (abbreviated as "the immune cell of the present invention") expressing the fusion protein of the present invention, which carries the fusion protein on the cell surface.

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

A preferred class of immune cells includes T cells, particularly human T cells. Preferably, said T cell surface carries a MART-1 TCR.

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

Furthermore, the T cells may optionally also express wild type CD62L, hscIL-12 (human single chain IL-12) or a combination thereof.

Mechanism of membrane expression of dKSCD62L mutant

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

The mechanism of action of the membrane expressing dKSCD62L mutant is shown in Figure 1. The CD62L transmembrane region contains the cleavage site of ADAM17, and in the case of T cell activation, ADAM7 can cleave and release CD62L. In the present invention, the KLDKSFS sequence (i.e., the cleavage site of ADAM17) is knocked out by genetic engineering, thereby realizing the stable membrane expression of exogenous CD62L from a molecular mechanism.

A typical mutant CD62L is dKSCD62L. After T cells were activated (including tumor antigen-induced T cell activation), ADAM17 was unable to cleave dKSCD62L on the membrane surface. In other words, after activation of T cells, the dKSCD62L mutant is not cleaved by the ADAM17 enzyme and cannot be released, resulting in the retention of dK-SCD62L on the surface of the T cell membrane, which in turn causes the IL-12 element in the fusion protein to remain on the surface of the T cell membrane. Most importantly, the fusion protein retains both the biological activity of Il-12 and the function of T cells.

Pharmaceutical composition and method 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.

In general, the fusion proteins of the invention can be formulated in non-toxic, inert and pharmaceutically acceptable water In a carrier medium, wherein the pH is usually from about 5 to about 8, preferably, the pH is from about 6 to about 8.

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

When the pharmaceutical composition of the present invention contains immune cells, "effective amount" or "effective amount" means 1 x 10 ³ - 1 x 10 ⁷ of said immune cells/ml.

As used herein, a "pharmaceutically acceptable" ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a fusion protein of the invention and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, 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 present invention can also be formulated 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 and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc. In general, when the fusion protein of the present invention is administered at a dose of about 5 mg to 20 mg/kg of animal body weight per day (preferably 5 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.

The fusion proteins of the invention are particularly suitable for use in the treatment of diseases such as tumors. Representative tumors include, but are not limited to, brain tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, breast cancer tumors, gastric cancer tumors, and pancreatic cancer tumors.

The main advantages of the invention include:

(a) The fusion protein of the present invention has no significant toxicity to T cells. This may be due to the fact that IL-12 is prominent outside the T cell membrane and cannot be effectively contacted or acted on T cells, or it may be because the interstitial effect of CD62L reduces the toxicity of IL-12 to T cells.

(b) When the anti-tumor T cells carrying the fusion protein of the present invention are close to the tumor cells, since the CD62L cannot be specifically cleaved, free IL-12 is not released (i.e., IL-12 remains on the cell membrane surface). This makes IL-12 on the surface of the membrane more effective and can only act on close-range tumor cells, from And significantly improved security.

(c) Maximizing the anti-tumor immune effect synergistically and effectively by attacking the local part of the tumor by T cells and changing the immune microenvironment of the T cell-tumor tissue by IL-12 on the cell surface.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

Materials and general methods

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

The map of the plasmid LVV used in the examples of the present invention is shown in Fig. 4, which is an engineering vector pLenti-MSCV containing a gene of interest, which is a promoter which is optimized for transduction of T cells by using MSCV as a promoter. The envelope protein particle pMD2.G contains VSV-G; gag/pol helper plasmid; and 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 the examples, the mutant CD62L refers to dKSCD62L.

hscIL-12/CD62L or IL-12/WTCD62L refers to hscIL-12/wild type CD62L.

Wild type CD62L is expressed as WTCD62L.

Tumor cell and T cell culture

Tumor cells

938 and 526 are conventional melanoma cell lines (supplied by Dr. Rosenberg of the National Cancer Institute) and subcultured in vitro with 10% FCS RPMI medium and 0.25% pancreas every 2-3 days. Enzyme subculture. Both tumors express 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-1 T cells recognize only A2+ cell line, ie 526 cells.

PBMC were derived from healthy human peripheral blood, and T cells were stimulated for growth with CD3/CD28 magnetic beads or CD3 antibody for 1 day, and cultured in IL-2 (100 IU/ml) X-VIVO medium. After recombinant lentivirus transduction of T cells, the fluorescence intensity of MART-1 in T cells was detected by flow cytometry through the MART-1 tetramer peptide. At the same time, the expression of CD62L on the cell surface, hscIL-12/mutant CD62L and CD107a on the membrane surface were detected by flow cytometry.

Construction of lentiviral vector

Using molecular biology techniques to carry a specific recognition of human melanoma-associated antigen MART-1 The recombinant lentiviral vector of human TCRα and β chain genes is transfected into autologous lymphocytes of peripheral blood, so that recombinant TCR is expressed in T lymphocytes to achieve the purpose of effectively killing tumors. A TCR expression vector containing a self-cleaving 2A peptide, furin (Furin) and a spacer sequence was constructed using a lentiviral vector with an optimized promoter.

Construction of a lentiviral expression vector expressing wild-type CD62L (abbreviated as "WTCD62L") and mutant CD62L (abbreviated as "dKSCD62L") is based on published literature (Yang, Cohen et al. 2008, Yang, Liu et al. 2011). The construction of hscIL-12 is based on published literature (Zhang, Kerkar et al. 2011). The fusion proteins of hscIL-12/wild type CD62L and hscIL-12/mutant CD62L were ligated with peptide G ₆ S and 218 peptides, respectively.

Preparation of fusion protein expression in lentiviral expression system

293T cells were cultured, and the cell density was adjusted in DMEM medium containing 10% fetal bovine serum one day before transfection. Then, 25×10 ⁶ 293T cells were inoculated per 15 cm cell culture dish, and cultured at 37 ° C, 5% CO ₂ . The culture in the box can be used for transfection after the cell density is increased to 80% to 90% after 16h to 24h. On the day of transfection, the medium was changed to complete medium without antibiotics (P/S) (DMEM + 10% FBS). The lentiviral backbones of LVV-MSCV-MART-1TCR, CD62L, hscIL-12L, and hscIL-12/mutant CD62L fusion proteins were co-transfected into 293T cells together with three other packaging plasmids, using commercially available calcium phosphate. medium. After 6 hours of culture, the medium was discarded, washed 3 times with PBS and replaced with 20 ml of fresh complete medium (DMEM + 10% FBS + P / S). The culture supernatant of 30-72 h after transfection was collected, centrifuged at 6000 rpm for 10 min, and the cell debris was discarded. The supernatant was filtered through a 0.45 μm PVDF filter into a 50 ml round bottom centrifuge tube, centrifuged at 50,000 g for 2 h at 4 ° C, and carefully discarded. Clear, DMEM (free of serum, double antibody) resuspended virus precipitate, according to the amount of virus used each time into a clean 15ml centrifuge tube, stored in a -80 ° C refrigerator, used to infect T cells. The titer of the virus detected by the Lentivirus-Associated p24 ELISA Kit was 5×10 ⁷ -1.5×10 ⁸ IFU. For details, see the instructions of the Lentivirus-Associated p24 ELISA Kit. (Yang, Cohen et al. 2008).

Establishment of co-culture system of tumor cells/T cells

T cells modified by anti-MART-1 TCR gene were co-cultured with 526, 938 cell lines, placed in a 1:1 ratio, ie 1×10 ⁶ per cell, in a 14 ml round bottom polypropylene culture tube, total volume 1 ml, transfer The mixture was incubated in a CO ₂ incubator at 37 ° C for 4 h. After 4 h, the cells were centrifuged at 800 x g for 10 minutes, the supernatant was collected, and the cells were lysed by RIPA lysate. The content of IFNγ and IL-12 in the supernatant was detected by an ELISA reagent. IL-12/wild-type CD62L or IL-12/mutant CD62L on the membrane surface was manipulated by live cell staining of a conventional flow cytometer. 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 by flow cytometry

Cell surface CD3, CD8, CD62L, CD107a, IL-12 and CD45RO are detected by fluorescently labeled corresponding antibodies, including isothiocyanate (FITC), allophycocyanin (APC), phycoerythrin (PE), PE-Cy7. , and APC-Cy7 (BD Biosciences, San Jose, CA). The MART-1:27-35 tetramer was designed by the company (iTAg MHC Tetramer, Beckman Coulter, Fullerton, CA) to detect gene-modified TCR expression levels. The specific procedure is as follows. First, the cells were washed twice with FACS staining solution (PBS containing 2% FBS), then 0.2 ml (10 ⁶ /ml) was added to the flow tube, incubated at 4 ° C for 30 minutes, and then washed twice. The dead cells were separated by adding 20 μl PI (l5 μg/ml propidium iodide) (Sigma-Aldrich, Saint Louis, MO) and cell subpopulation before the sample was taken to achieve the purpose of separation. The streaming data is analyzed by FlowJo 8.1.1 for post-processing (FlowJo, Ashland, OR).

Establishment of tumor-bearing mouse model

The Pmel experimental mouse model uses conventional methods, for example, see the literature (Overwijk, Tsung et al. 1998). The method involved in this experiment was as follows: Female pmel mice (6-8 weeks, 7 mice per group) were selected for intracranial tumor inoculation (IC). B16F10-MART-1 tumor cells were stopped by 0.25% trypsinization with 0.02% EDTA and washed once with serum-containing medium to wash the trypsin reaction, followed by washing twice with PBS. Tumor cells were finally mixed with methylcellulose in zinc option medium in a volume of 1:1, and 5000 cells were diluted in a 5 μl liquid and loaded onto a 250-μl syringe (Hamilton, Reno, NV) using a 25-gauge needle. The Quinesential Stereotaxic Injector System (Stoelting Co. Wood Dale, IL) was injected into the right brain caudate nucleus of the mice. Five days after inoculation of the tumor cells, the mice received whole body 5 Gy radiation. On day 2, the mice received a 0.5-1×10 ⁶ DC vaccine subcutaneously, or a 1X 10 ⁷ retransmission of anti-MART-1 TCR, IL-12/wild-type CD62L or IL-12/mutation by tail vein IV. Type CD62L lentiviral gene-modified T cells. Mouse T cells were obtained from mouse spleen cells, activated with 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); day 2, transduced with Lentiviral vector T The cells were then cultured for 6 days, cells were collected, and T cells were injected through the tail vein of the mice. DC cells of DC group mice were obtained from mouse bone marrow cells, induced differentiation and maturation in vitro for 8 days, and inoculated intraperitoneally. The deaths of the mice were then recorded daily, growth curves were recorded and plotted by Prism mapping software. Asterisks indicate that the experimental group was compared with the other groups, p < 0.001.

Example 1

Fusion gene construction

The fusion gene was synthesized by Invitrogen, and the length and sequence of the fusion gene was passed through 1% agar. Glycoelectrophoresis and sequencing confirmed.

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

The obtained IL-12/wild type CD62L fusion gene was constructed at the same time, and the IL-12/wild type CD62L fusion gene was very similar to SEQ ID NO.: 1, except that the nucleotide encoding the cleavage site was retained. The sequence, so the encoded fusion protein also retains the ADAM17 cleavage site (ie KLDKSFS).

Example 2

Construction of lentiviral expression vector

The method described in "General Construction of Lentiviral Vector and Gene Modification of T Cells" in a general method: humanized 293T cells cultured in a low-generation DMEM (containing 10% FBS) medium were counted and passed to 15 CM. In the culture dish, the bottom of the culture dish was treated with poly-D-Lysine, and 20×10 ⁶ cells were plated in each dish. The next day, a mixture of DNA mixture and Lipofectamine was added to each transfection dish: mixing The composition of the solution was as follows. Take 2 ml of Optimum I and add pLenti-MSCV (22.5 ug), pMD2.G (7.5 ug), gag/pol (15 ug), pRev (10 ug), and mix; take 2 ml of Optimum I and add Lipofectamine. 160ul (Invitrogen), mix. The two suspensions were mixed, incubated at room temperature for 5 minutes, and then uniformly added dropwise to the Petri dish. After 48-72 hours, the supernatant containing the genetically engineered vector was harvested, centrifuged to remove cell debris at 2000 g, and the supernatant was collected and filtered through a 0.45 uM filter to remove possible contamination, dispensed and stored in a negative 80 refrigerator. According to different needs, the collected virus supernatant can be subjected to ultracentrifugation at 50,000 g to obtain a higher concentration of the viral vector.

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

Example 3

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

The method is as follows: CD3/CD28 magnetic beads or anti-CD3 antibody activates PBMC, and on day 2, the T cells are modified by lentiviral gene. The brief method is as follows: Wash T cells three times in PBS buffer, according to virus titer and T cell The appropriate amount of lentivirus was added in a ratio of 3:1, centrifuged at 2000×g for 2 h, and after 6 h, the culture was continued by adding 100 IU/ml IL-2; the second transduction was performed on the 5th day, or combined co-transduction. The flask was divided according to the growth of the cells, and two weeks later, the cells modified by the T cells were examined by flow cytometry.

Example 4

dKSCD62L lentiviral gene-modified anti-tumor T cells achieve stable expression of dKSCD62L

In order to better observe the expression of exogenous CD62L and dKSCD62L proteins mediated by lentiviral genes, To distinguish the expression of endogenous physiological CD62L, a tumor infiltrating T cell line JKF6 isolated and cultured in melanoma tissue was selected. The JKF6 cell line can be subcultured in vitro for a long time, and the membrane surface loses the CD62L membrane molecule, and JKF6 can effectively recognize melanoma cells.

JKF6 can detect high levels of exogenous WTCD62L and dKSCD62L expression by lentiviral gene modification. Wild-type CD62L and mutant dKSCD62L gene-modified JKF6 obtained stable membrane surface CD62L series expression.

After activation with tumor antigen 526, wild-type CD62L can undergo tumor antigen-specific cleavage and release; however, after expression of dKSCD62L membrane, it loses the cleavage and release of tumor antigen activation, which is expressed as stable membrane surface dKSCD62L expression. Phenotype, as shown in Fig. 2 and Fig. 3, the co-cultured T cells were detected by flow cytometry on the surface of the membrane CD45RO and CD62L, and the membrane stability of the CD62L mutant was fully verified by FlowJo software analysis.

Example 5

dKSCD62L lentiviral gene modification against tumor JFK6 cells does not affect the antitumor activity of T cells

In order to verify the reactivity of the dKSCD62L lentiviral gene-modified JKF6, it is also considered that there may be other pathways for cleavage in CD62L in vivo, and in this example, the anti-tumor activity of T cells is further verified. Methods as below:

A broad-spectrum T cell activator PMA/Ionomycin (abbreviated as "PMA/Ion", a T cell activator) was used, and after activation of T cells with the activator for 4 hours, we detected CD62L on the cell membrane ( Expression of wild type or mutant type). In addition, it was also found that for the mutant dKSCD62L, activation-induced membrane cleavage disappeared, that is, stable membrane surface expression of dKSCD62L was obtained; for wild-type CD62L, T cell activation cleavage remained.

To verify the reactivity of T cells, the reactivity after modification with the CD62L gene was determined. As a result, as shown in Fig. 3, JKF6 modified by the mutant CD62L gene (or fusion protein gene) can express high levels of IFNγ after activation. The results of this experiment suggest that after modification of the fusion protein gene, the anti-tumor JKF6 has normal reactivity and is not affected by genetic modification.

Example 6

Construction of lentiviral expression dKSCD62L fusion protein vector and its expression in human T cells

In this example, a lentiviral vector (structure shown in Figure 4) of IL-12/CD62L, IL-12/dKSCD62L was constructed, in which the IL-12 gene and the mutant or wild type CD62L gene pass the amino acid peptide G ₆ S link, the lentivirus is a third generation lentiviral vector, and the promoter is MSCV. The 5' and 3' LTR (long terminal repeat) of the vector was transformed into SIN-LTR (self-inactivating-LTR) to reduce the probability of lentiviral recombination and enhance safety performance.

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

tumor cells

526 and 938 after 14 days of in vitro culture.

As a result, the phenotype of stable membrane expression of IL-12/dKSCD62L was observed, and the tumor antigen-dependent specific cleavage and release of IL-12/CD62L was further confirmed.

Example 7

Lentiviral vector IL-12/CD62L transduced T cells co-culture with tumor can enhance the expression of IFNγ

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

As shown in Fig. 5, human T cells were sequentially transduced with anti-tumor TCR and IL-12/CD62L series fusion proteins for 14 days, and co-cultured with

tumor cells

526 and 938. After 24 hours, the supernatants were IFNγ and IL-12. Expression levels were detected by ELISA kit.

The results showed that the sustained IL-12 secretion group, IL-12/wild type CD62L group and IL-12/dKSCD62L group could significantly enhance the reactivity of anti-tumor T cells with tumors compared with the T cell group, which showed a significant enhancement of IFNγ. The level of secretion, P < 0.001.

Consistent with the stable expression of the surface of dKSCD62L membrane, IL-12/dKSCD62L also lost the cleavage and release of tumor antigen reactivity, which was detectable compared with IL-12/wild type CD62L group and persistent secretion IL-12 group. The level of secreted IL-12 was significantly reduced, P < 0.001.

Therefore, this confirmed that it is feasible to express IL-12/dKSCD62L in a stable membrane, and further confirmed that the tumor antigen reactivity of T cells did not occur after co-culture of anti-tumor T cells expressing IL-12/dKSCD62L with the tumor cells. Changes can help maximize the anti-tumor effect of IL-12.

Example 8

Lentiviral vector IL-12/dKSCD62L transduced T cells can effectively avoid the side effects of in vitro T cell expansion caused by persistent secretion of IL-12.

After solving the problem of stable membrane expression and enhancing anti-tumor reactivity, this example observes the effect of transduction of the IL-12/dKSCD62L fusion protein on T cell expansion in vitro.

As shown in Figure 6, after T cells were sequentially transduced with anti-tumor TCR and wild-type or mutant CD62L/hscIL-12 fusion protein for 14 days, the expansion ratios of the cells in each group were compared with the control transduction group (T-cell). The data analysis was performed by setting the amplification factor of the T-cell group to 100%, and the values of the other groups were compared with the T-cell group.

The results showed that the number of amplifications in the transduced IL-12 group was significantly lower than that in the other groups, p < 0.001. Among them, the transduced hscIL-12/dKSCD62L fusion protein group was the same as the other groups, and no significant difference was observed. In addition, the amplification fold of the hscIL-12/dKSCD62L fusion protein group was slightly higher than that of the hscIL-12/wild type CD62L fusion protein group. This suggests that the transduction is due to the release of free IL-12. This resulted in almost no significant toxicity of IL-12 to T cells.

Example 9

Lentiviral vector hscIL-12/dLSCD62L series transduced mouse T cell-mediated cell transfusion therapy significantly prolongs survival of tumor-bearing mice

Based on the clear expression of IL-12/dKSCD62L, membrane stability, synergistic effect of tumor antigen response and in vitro expansion, in this example, a pre-clinical tumor-bearing mouse model was further used to observe the inhibition in vivo. Tumor effect. Methods as below:

Female pmel mice (7 mice per group) were selected for intracranial (IC) implantation by B16F10 cells for 5 days, and mice received 5 Gy whole body radiotherapy 1 day before cell return. Mouse T cells were obtained from mouse spleen cells and activated by 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); on day 2, transduction of T with lentiviral vector The cells were then cultured for 6 days, cells were collected, and (X) ⁵ ×10 ⁶ T cells were injected through the tail vein of the mice. The DC cells of the DC group were obtained from bone marrow cells, and induced to differentiate and mature for 8 days in vitro, and 1×10 ⁶ cells were inoculated intraperitoneally. The group description of DC and T cells is shown on the right. An asterisk (*) indicates that the experimental group was compared with the other groups, p < 0.001.

As shown in Figure 7. The results showed that compared with the other control groups, the IL-12/dKSCD62L group significantly prolonged the survival of tumor-bearing mice; the sustained secretion of IL-12 group showed significant systemic cytotoxicity, after the mice returned to the cells. All died in 4-7 days.

discuss

CD62L is a homing factor for T cell surface expression, which can achieve adhesion and release of T cells along the vessel wall by activation-induced cleavage of T cells. Studies have found that T cells lacking the tumor necrosis factor-converting enzyme 17 cause CD62L to be cleavable and released.

In the present invention, the present inventors knocked out the cleavage point of ADAM17 of CD62L (designated as dKSCD62L in the present invention), and the experimental results indicate that the expression of a novel IL-12/mutant CD62L molecule by genetically modifying anti-tumor T cells is not only Efficient killing of tumor cells can significantly reduce the side effects of IL-12.

Compared with anti-tumor T cells that continuously secrete IL-12 gene, IL-12/mutant CD62L fusion protein gene-modified anti-tumor T cells can not only prolong the survival of tumor-bearing mice, but also avoid the sustained secretion of IL-12. Induced systemic cytotoxicity is a brand new immune cell therapy strategy that expresses IL-12 through cell membrane and cleaves and releases tumor antigen reactivity through CD62L. It will play a role in the treatment of tumor immune cells. Important role.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that after reading the above teachings of the present invention, A person skilled in the art can make various changes or modifications to the invention, and such equivalents are also within the scope defined by the appended claims.

references

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

2. Overwijk, WW et al. (1998). "gp100/pmel 17is a murine tumor rejection antigen: induction of "self"-reactive,tumoricidal T cells using high-affinity,altered peptide ligand."J Exp Med 188(2) :277-286.

3. Yang, S. et al. (2008). "Development of optimal bicistronic lentiviral vectors facilitats high-level TCR gene expression and robust tumor cell recognition." Gene Ther 15(21): 1411-1423.

4.Yang, S., et al. (2011). "The shedding of CD62L(L-selectin)regulates the acquisition of lytic activity in human tumor reactive T lymphocytes."PLoS One 6(7):e22560.

Claims

A fusion protein characterized in that the fusion protein comprises the following elements fused together:

(i) an optional signal peptide and/or leader peptide at the N-terminus;

(ii) a first protein component;

(iii) a second protein component;

(iv) an optional linker element located 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 a protein element of the mutant CD62L, and the protein element of the mutant CD62L lacks a cleavage site of ADAM17.
The fusion protein according to claim 1, wherein said fusion protein has a structure selected from the group consisting of:

(1) Structure described by Formula Ia:

D-A-B (Ia), or

(2) Structure described in Formula IIa:

D-A-C-B (IIa),

among them,

A is an IL-12 protein element;

B is a mutant CD62L protein element;

C is an optional linker element;

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

Each "-" independently indicates a peptide bond or a peptide linker to which the above elements are attached.
An isolated polynucleotide, characterized in that the polynucleotide encodes the fusion protein of claim 1.
A vector comprising the polynucleotide of claim 3.
A host cell comprising the vector of claim 4 or a polynucleotide in which the polynucleotide of claim 3 is integrated.
A method of producing the protein of claim 1 comprising the steps of:

(1) cultivating the host cell of claim 5 under conditions suitable for expression, thereby expressing the fusion protein of claim 1;

(2) Optionally isolating the fusion protein.
An immune cell characterized in that said immune cell carries the fusion protein of claim 1 on the surface of the membrane.
A pharmaceutical composition, characterized in that the composition comprises:

The immune cell of claim 7;

A pharmaceutically acceptable carrier.
Use of the fusion protein of claim 1 and/or the immune cell of claim 7 for the preparation of a medicament for treating a tumor.
A non-therapeutic method for killing tumor cells in vitro, comprising the steps of: contacting the immune cells of claim 7 with tumor cells to kill the tumor cells.