WO2017071173A1 - Tumor therapeutic agent modified by il-12/cd62l fusion protein and preparation method and use thereof - Google Patents

Abstract

Provided are an IL-12/CD62L fusion protein and preparation method and use thereof. In particular, the release of the IL-12 on the surface of cell membrane activated by tumor antigen is achieved by modifying antitumor T cells with lentivirus containing IL-12/CD62L fusion gene as well as a specific cleavage and release induced by a CD62L tumor antigen-dependent activation.

Description

Tumor therapeutic agent modified by IL-12/CD62L fusion protein, preparation method and use thereof Technical field

The invention relates to the field of medicine, in particular to a tumor therapeutic agent modified by an IL-12/CD62L fusion protein, a preparation method thereof and use thereof. The present invention provides a class of IL-12/CD62L gene-modified T cells with enhanced anti-tumor effects.

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.

Individuals who are congenitally deficient in IL-12 or do not reflect IL-12 have serious defects in their anti-infective immunity. A series of preclinical experimental models have demonstrated that IL-12 intervention can significantly prolong the survival of tumor-bearing animal models.

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 order to solve this problem, the present inventors have also succeeded in the effective amplification of T cells in vitro by TCR activation of a gene modification scheme that specifically regulates the expression of IL-12, and have also achieved certain antitumor effects, but at the same time, IL exists. The toxic side effects caused by -12 out of regulation cannot be effectively applied to clinical applications.

In summary, there is a lack of IL-12-based antitumor drugs that are highly effective and have no 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 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 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) 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 CD62L.

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 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 CD62L protein element;

C is an optional linker element;

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

"-" means a peptide bond or a peptide linker to which the above elements are attached.

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 (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 CD62L protein element has a cleavage site for K283-S284.

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

In another preferred embodiment, the CD62L protein comprises wild type and mutant form.

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

In another preferred embodiment, the sequence of the 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, 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.: 7).

In another preferred embodiment, the fusion protein is in single-stranded IL-12 (first protein element) and CD62L (second protein) A linker peptide is provided between the elements), preferably a peptide 218 (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 cleaved by the ADAM17 protein to release 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, an isolated polynucleotide is provided, the polynucleotide encoding the fusion protein of the first aspect.

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, there is provided a host cell comprising the vector of the third aspect of the invention or the polynucleotide of the second aspect integrated in the genome.

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, a method of producing the protein of the first aspect, comprising the steps of:

(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;

(2) Optionally isolating the fusion protein.

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

The fusion protein of the first aspect, and

A pharmaceutically acceptable carrier.

In a seventh aspect of the invention, there is provided an immune cell carrying (or present on the cell surface thereof) the fusion protein of the first aspect.

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, 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 an eighth aspect of the invention, a pharmaceutical composition is provided, the composition comprising:

The immune cell of the seventh 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 a ninth aspect of the invention, the use of the fusion protein of the first aspect and/or the immune cell of the seventh aspect, for the preparation of a medicament for treating a tumor, is provided.

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 tenth aspect of the invention, a method of treating a tumor comprising the step of administering the fusion protein of the first aspect and/or the immune cell of the seventh aspect to a subject in need thereof.

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.

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 membrane surface cleavage and release induced by antigen-specific activation by CD62L/L-selectin.

Among them, FIG. 1A and FIG. 1B show schematic diagrams of the molecular mechanism of membrane molecule CD62L cleavage and release. As shown in the figure, CD62L on the surface of T cells undergoes directed aggregation of microfilaments (actin, etc.) under the action of specific activators such as antigens, resulting in specific cleavage membrane molecules of the cleavage enzyme (Adam17, etc.) on the surface of the cell membrane. , ie CD62L;

Figure 1C shows tumor antigen-induced membrane molecule-specific cleavage and release. Tumor cell 526 is a related antigen MART-1 expressing MHC I A2 molecule, which is a positive target cell; 938 tumor cell does not express MHC A2 molecule, and is a negative control cell. After PBMC was activated by anti-CD3/CD28 magnetic beads for 24 hours, T cells were transduced with lentiviral engineering supernatant expressing anti-tumor MART-1 TCR; 14 days later, T cells were associated with MART-1 antigen-expressing tumor cells 938 and 526 Co-culture. As shown in the figure, co-cultured T cells were tested for molecular CD45RO and CD62L on the surface of the membrane by flow cytometry. The T cells of each group were sorted into CD8 cell subsets (column 1 and column 3) by FlowJo software analysis. And CD8/MART-1 cell subsets (column 2 and column 4). The time points noted on the right indicate the time of collection and analysis of cells after co-culture, for a total of 4 time points: 1 h, 2 h, 4 h and 6 h. The antibodies used in flow cytometry were CD62L FITC, CD45RO APC, MART-1PE and CD8 PercP.

Figure 2 shows that the cleavage and release of CD62L is dependent on tumor antigen stimulation.

Among them, PBMCs were transactivated by anti-CD3/CD28 magnetic beads for 24 hours, and T cells were transduced with lentiviral engineering supernatant expressing anti-tumor MART-1 TCR; 14 days later, T cells were associated with MART-1 antigen-expressing tumor cells 938 and 526 was co-cultured for 4 h. As shown in the figure, the 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, CD8/MART- 1 cell subpopulation. CD62L levels in free CD62L and co-cultured cells in the supernatant were detected by ELISA kit. As shown in the figure, the amount of CD62L in the supernatant of the T+526 group was set to 100%, and the other groups calculated the percentage of the CD62L content by comparison.

Figure 3 shows that the cleavage of CD62L is a prerequisite for CD107a expression.

Among them, T cells were transduced by anti-tumor MART-1 TCR, co-cultured and analyzed and organized by FACS (as described above). FACS The antibodies used for the assay were as follows, CD107a FITC, MART-1PE, CD8 PercP5.5, CD45RO PE Cy-7, CD62L APC, CD3APC Cy-7.

As shown in the image to the right, the expression of CD107a in each T cell subset is indicated by the arrow. The lower panel EM represents the effector memory cells of CD62L-CD45RO+; CM represents the central memory cells of CD62L+CD45RO+.

Figure 4 shows the specific cleavage and release of IL-12 in the fusion protein CD62L/IL-12 induced by tumor antigen. Among them, after CD62L is activated by tumor antigen, CD62L undergoes tumor antigen-induced cleavage and release. The mechanism diagram shows how IL-12 in CD62L/IL-12 fusion protein is cleaved and released, ie, fusion protein IL-12 occurs. Partial release. According to this effector mechanism, the release of biologically active IL-12 can increase the recognition of tumor cells by anti-tumor T cells, thereby maximizing the anti-tumor effect. TCR, CD107a, Perforin, Granzyme, CD62L and IL-12 are shown in the figure.

Figure 5 shows the construction of a lentiviral vector expressing a CD62L/IL-12 fusion protein and its expression in human T cells.

Figure 6 shows that co-culture of lentiviral vector hscIL-12/CD62L transduced T cells with tumors enhances IFNy expression and tumor antigen-dependent release of IL-12.

Figure 7 shows that lentiviral vector hscIL-12/CD62L-transduced murine T cell-mediated cell transfusion therapy significantly prolongs survival in tumor-bearing mice.

Figure 8 shows that the lentiviral vector hscIL-12/CD62L transduced T cells can effectively avoid the toxic side effects of in vitro T cell expansion caused by persistent secretion of IL-12.

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 invention is an IL-12/CD62L fusion protein. Experiments have shown that when the fusion protein of the present invention is expressed in T cells, it is effectively displayed on the surface of T cells. Unexpectedly, the IL-12/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 can be tumor antigen-dependent when approaching tumor cells. The induced specific cleavage of CD62L is activated to release IL-12 at a site near the tumor cells, ie, release of IL-12 on the surface of the cell membrane that activates the tumor antigen. In the present invention, by changing the immune microenvironment of T cell-tumor tissue by release of IL-12 from the localized T cell attack tumor, maximization of anti-tumor immunity effect can be achieved very effectively, and IL- is extremely significantly reduced. 12 toxic side effects. The present invention has been completed on this basis.

Specifically, in the present invention, the present inventors adopted a protocol for connecting IL-12 by expressing a cell membrane, and in view of the specific release of tumor antigen reactivity of IL-12, the present invention innovatively employs IL-12 and CD62L. (L-selectin) A strategy of fusion that regulates the specific release of tumor immunity by IL-12 by cleavage of CD62L tumor antigen reactivity.

In the present invention, IL-12/CD62L lentiviral gene-modified T cells are used to enhance the anti-tumor effect, and the tumor antigen-dependent cleavage and release of CD62L is demonstrated by in vitro experiments, further confirming lentiviral-mediated fusion. The protein IL-12/CD62L can not only effectively modify the anti-tumor T cell expression fusion protein, but also can release the IL-12 on the membrane surface by CD62L cleavage. T cell/tumor cell co-culture experiments demonstrated that IL-12/CD62L lentiviral gene-modified anti-tumor T cells can significantly increase the intensity of the immune response, which is shown to significantly enhance the secretion level of IFNγ. Preclinical experimental models show that the treatment regimen avoids the systemic cytotoxicity of mice produced by the release of secreted IL-12 by genetically modified T cells, which is safe, effective, and can significantly prolong the survival time of tumor-bearing mice. Is a new anti-genetic resistance Tumor T cells achieve an immune cell treatment regimen that maximizes anti-tumor effects.

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 plasma, in the activated state of T cells, 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.

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 first protein element comprises the IL-12 protein P40 and P35 subunits joined together.

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, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins attached have sufficient 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): human sources Signal peptide of the 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-CD62L fusion protein", "IL-12/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 an IL-12 protein element, CD62L, and a linker element. A representative example is IL12-CD62L. 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 CD62L amino acid sequence at positions 548-920.

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. The DNA can be a coding strand or a 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. 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 (such as IL12 or CD62L) or a fragment thereof can usually be amplified by PCR. Obtained by law, recombinant method or 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 such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance 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 human T cells. Preferably, said T cell surface carries a MART-1 TCR.

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

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 may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably 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, 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 is not significantly toxic to T cells;

(b) The fusion protein of the present invention effectively inhibits the immune microenvironment of T cell-tumor tissue by locally and fixedly and periodically releasing IL-12 when T cells attack tumors, thereby inhibiting tumors very effectively and significantly reducing toxic side effect.

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.

A map of the plasmid LVV used in the examples of the present invention is shown in Fig. 5, 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.

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 and hscIL-12/CD62L and CD107a on the membrane surface were detected by flow cytometry.

Construction of lentiviral vector

The recombinant lentiviral vector carrying the human TCRα and β chain genes which specifically recognize the human melanoma-associated antigen MART-1 was transfected into autologous peripheral blood lymphocytes by molecular biological techniques, and the recombinant TCR was expressed in T lymphocytes. To To 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 the CD62L lentiviral expression vector was based on published literature (Yang, Cohen et al. 2008, Yang, Liu et al. 2011), and the construction of hscIL-12 was referenced to published literature (Zhang, Kerkar et al. 2011). The fusion protein of hscIL-12/CD62L is linked by the 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 the LVV-MSCV-MART-1 TCR, CD62L, hscIL-12L, and hscIL-12/CD62L fusion proteins were co-transfected into 293T cells together with three other packaging plasmids, using commercially available calcium phosphate as a vehicle. 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/CD62L on the membrane surface was manipulated by live cell staining of a conventional flow cytometer. CD62L in the supernatant and 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, diluted in 5000 cells and loaded into 250-μl syringe (Hamilton, Reno, NV) in 5 μl of liquid, 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, mice received a 0.5-1× ^{10 6} DC vaccine subcutaneously, or a 1× ^{10 7} trans-transfusion of anti-MART-1 TCR and IL-12/CD62L lentiviral gene-modified T cells via tail vein IV. Mouse T cells were obtained from mouse spleen cells and activated with 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); on day 2, the lentiviral vector was shown T cells were transduced, and 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 groups of DCs and T cells are illustrated in the right side of Figure 7. 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 were confirmed by 1% agarose electrophoresis and sequencing.

The structure of the obtained 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.

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: mixture The composition is as follows, take 2ml Optimum I and add pLenti-MSCV (22.5ug), pMD2.G (7.5ug), gag/pol (15ug), pRev (10ug), mix; take 2ml 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 designated as LV-CD62L, LV-hscIL-12/CD62L, LV-hscIL-12, respectively.

Example 3

Preparation of T cells expressing IL-12-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 A suitable amount of lentivirus was added in a ratio of 3:1, centrifuged at 2000 xg for 2 h, and after 6 h, culture was continued by adding 100 IU/ml IL-2; the second transduction was performed on days 5 to 10, 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

Membrane surface cleavage and release of CD62L is dependent on specific activation of tumor antigens

Anti-tumor TCR can directly produce anti-tumor T cells in vitro by modifying the PBMC of tumor patients with lentiviral genes. This kind of T cells does not require DC induction and has the specific effect of killing tumors.

In vitro short-term culture of anti-tumor T cells (generally referred to as T cells cultured for 2-3 weeks in vitro) mimics the immune cells produced by the in vivo immune response, and can be divided into CD45RO+/CD62L2 (Tem, effector memory T cells) by flow cytometry. CD45RO+/CD62L+ (Tcm, central memory T cells) and CD45ROlow/CD62L+ (Tn, initial T cells).

As shown in Figures 1, 2 and 3, when MART-1TCR gene-modified T cells were co-cultured with tumor cells corresponding to MHC class I antigen molecules, the cleavage and release of CD62L molecular antigen reactivity was observed, and CD62L was also found. The expression of CD107a on the membrane surface can be detected in the subpopulation of exfoliated cells (the molecule is a molecular protein on the lysosome, which is normally present in the cytoplasm. In the activated state of T cells, the molecule is accompanied by T cell depletion. The particles migrate to the cell surface; therefore, membrane surface detection of this molecule is an important indicator of T cell killing).

In vitro experiments confirmed that the cleavage and release of CD62L can be detected in the supernatant of the culture, and the free CD62L is present only in the experimental group in which the tumor antigen is reacted. Since the main molecular phenotype of anti-tumor T cells cultured in vitro for short-term is Tcm, that is, a large amount of CD62L on the surface of T cells, the material basis becomes an important theoretical cornerstone of the present invention. Briefly summarized as: the local release of the antigen-reactive tumor immune response of IL-12 can be achieved by genetically modifying anti-tumor T cells to express new IL-12/CD62L molecules and utilizing the characteristics of CD62L tumor antigen reactive cleavage and release. Enhance the local anti-tumor immune response.

Example 5

Specific release mechanism of IL-12/CD62L and expression of fusion protein

In this embodiment, the lentivirus used is a third generation lentiviral vector, the promoter is MSCV, and the expression gene component can be replaced by a gene to be expressed. The 5' and 3' LTR (long terminal repeat) of this vector was transformed into SIN-LTR (self-inactivating-LTR), aiming at reducing the probability of lentiviral recombination, enhancing safety performance, and combining WPRE to terminate RNA transcription. It is currently widely used in clinical carrier structures. As shown in Figure 5, human CD62L, hscIL-12 (human single chain IL-12) and CD62L/hscIL-12 genes, wherein hscIL-12 fused IL-12 secretory peptide (lead seq), linked by gene cloning In the lentiviral expression vector, CD62L and hscIL-12 are linked by a linker peptide.

T cells were transduced by two transductions, namely MART-1 TCR (first day) and sequential transduction of the three vectors (Day 10). On day 14, the surface of CD cells was detected by flow cytometry. Expression and expression of hscIL-12 on the membrane surface.

The result is shown in Figure 5. The CD62L/IL-12 fusion protein was expressed by the third generation lentiviral vector, and the selected promoter was MSCV, which has been widely used in tumor immunocytotherapy of human body, and has the stability and safety of expression, and the expression of gene components can be The gene is replaced by a gene to be expressed, namely, CD62L, MART-1 TCR, and IL-12/CD62L fusion protein. The 5' and 3' LTR (long terminal repeat) of the vector were modified into SIN-LTR (self-inactivating-LTR), The aim is to reduce the probability of lentiviral recombination, enhance safety performance, and combine WPRE to terminate RNA transcription. It is the latest generation of genetic engineering vector structure widely used in clinical practice. In the recombinant genes of human CD62L, hscIL-12 (human single chain IL-12) and CD62L/hscIL-12, hscIL-12 fused IL-12 secreting peptide (lead seq) and linked to lentivirus by gene cloning In the expression vector, CD62L and hscIL-12 are linked by the peptide SGSG. T cells were transduced by two transductions, namely MART-1 TCR (first day) and sequential transduction of the three vectors (Day 10). On day 14, the surface of CD cells was detected by flow cytometry. Expression and expression of hscIL-12 on the membrane surface. The experimental data showed that CD62L can be expressed in a large amount on the surface of anti-tumor T cells. IL-12 on the surface of membrane cells can be detected only in the fusion protein group expressing IL-12/CD62L, and the surface of secreted IL-12 membrane is not expressed. The mechanism shown in Figure 4 is fully verified.

As shown in Figure 4, the mechanism of the invention is as follows: IL-12 cleaves and releases from the membrane surface by CD62L, and indicates that this release is dependent on the tumor immune response. According to this effect mechanism, the release of biologically active IL-12 can increase the anti-tumor response of anti-tumor T cells, improve the microenvironment of local tumor immunity, and achieve an optimized anti-tumor immune response.

Example 6

Enhancement of immune response by IL-12/CD62L fusion protein and tumor antigen-dependent IL-12 release

T cells were sequentially transduced with anti-tumor TCR and CD62L/hscIL-12 fusion protein for 20 days, and co-cultured with tumor cells 526 and 938. After 24 hours, the expression levels of IFNγ and IL-12 in the supernatant were detected by ELISA kit.

The result is shown in Figure 6. Co-culture of lentiviral vector hscIL-12/CD62L transduced T cells with tumors can enhance the expression level of IFNγ and tumor antigen-dependent release of IL-12. T cells were sequentially transduced with anti-tumor TCR and CD62L/hscIL-12 fusion protein for 20 days, and co-cultured with tumor cells 526 and 938. After 24 h, the expression levels of IFNγ and IL-12 in the supernatant were detected by ELISA kit. As predicted, anti-tumor T cells modified by IL-12 soluble gene, like the IL-12/CD62L gene modified on the membrane surface, can significantly increase the secretion level of IFNγ compared with the anti-tumor T cell group. And there are significant statistical differences. The present invention further confirmed that only the cleavage and release of IL-12 in the IL-12/CD62L group is associated with activation of tumor antigens, and the sustained secretion of soluble IL-12 is not regulated by tumor antigens and is maintained at a high level. At the same time, the present invention also observes that the level of IL-12 cleavage and release on the surface of the membrane is lower than that of the continuously secreted IL-12, and we predict that the invention has high safety and clinical operability.

Example 7

Significant in vivo anti-tumor effect of IL-12/CD62L gene-modified anti-tumor T cells

Experimental methods: Female pmel mice (7 mice per group) were implanted intracranial (IC) 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 with 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); on day 2, the lentiviral vector was shown T cells were transduced, and 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 groups of DC and T cells are illustrated in the right side of Figure 7. Asterisks indicate that the experimental group was compared with the other groups, p < 0.001.

The result is shown in Figure 7. Lentiviral vector hscIL-12/CD62L-transduced murine T cell-mediated cell transfusion therapy significantly prolonged survival in tumor-bearing mice.

Example 8

Safety of IL-12/CD62L gene modified anti-tumor T cells

Experimental method: After T cell sequential transduction of anti-tumor TCR and CD62L/hscIL-12 fusion protein for 14 days, the expansion ratio of each group of cells and control transduction group (T-cell) was compared, and the transduction of hscIL-12 group was continuously transduced. The amplification factor was significantly lower than the other groups, p < 0.001. Among them, the hscIL-12/CD62L 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.

The result is shown in Figure 8. The results showed that the lentiviral vector hscIL-12/CD62L transduced T cells can effectively avoid the side effects of in vitro T cell expansion caused by persistent secretion of IL-12.

discuss

The present inventors have shown that by genetically modifying anti-tumor T cells to express new IL-12/CD62L molecules, and utilizing the characteristics of CD62L tumor antigen reactive cleavage and release, it is possible to achieve a partial immunoreactive tumor immune response of IL-12. Release, enhances the local anti-tumor immune response, and significantly reduces the toxic side effects of IL-12.

Compared with anti-tumor T cells that continuously secrete IL-12 gene, IL-12/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. 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 an important role in the treatment of tumor immune cells. effect.

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 various modifications and changes may be made by those skilled in the art in the form of 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 (10)

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 CD62L. 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 CD62L protein element; C is an optional linker element; D is an optional signal peptide signal peptide and/or leader peptide sequence; "-" means a peptide bond or a peptide linker to which the above elements are attached. The fusion protein according to claim 1 wherein said first protein element comprises IL-40 protein P40 and P35 subunits linked together; More preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.: 2. An isolated polynucleotide, characterized in that the polynucleotide encodes the fusion protein of claim 1. A vector comprising the polynucleotide of claim 4; More preferably, the viral vector comprises a lentiviral vector, an adenoviral vector, and a yellow fever virus vector. A host cell comprising the vector of claim 5 or a polynucleotide in which the polynucleotide of claim 4 is integrated. A method of producing the protein of claim 1 comprising the steps of: (1) cultivating the host cell of claim 6 under conditions suitable for expression, thereby expressing the fusion protein of claim 1; (2) Optionally isolating the fusion protein. A pharmaceutical composition, characterized in that the composition comprises: The fusion protein of claim 1 and A pharmaceutically acceptable carrier. An immune cell characterized in that the immune cell carries the fusion protein of claim 1. A pharmaceutical composition characterized in that said composition comprises The immune cell of claim 9; A pharmaceutically acceptable carrier.

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