阅读说明：本技术 工程化抗原递呈细胞及其在用于活化cd3+免疫细胞中的应用 (Engineered antigen presenting cells and their use for activating CD3+ immune cells ) 是由 蒋俊 文高柳 林鑫 于 2019-11-21 设计创作，主要内容包括：本发明公开工程化抗原递呈细胞及其在用于活化CD3+免疫细胞中的应用。本发明的工程化抗原递呈细胞通过工程化手段在野生型抗原递呈细胞内引入外源基因从而过表达重组白介素-12和可溶性CD80而得到。本发明通过重组白介素-12和可溶性CD80的组合协同作用促进CD3+免疫细胞的活化。(The invention discloses an engineered antigen presenting cell and application thereof in activating CD3+ immune cells. The engineered antigen presenting cell is obtained by introducing exogenous genes into a wild-type antigen presenting cell through an engineering means so as to over-express recombinant interleukin-12 and soluble CD 80. The invention promotes the activation of CD3+ immune cells through the combined synergistic effect of recombinant interleukin-12 and soluble CD 80.)

1. An engineered antigen presenting cell obtained by introducing a foreign gene into a wild-type antigen presenting cell by engineering means to overexpress recombinant interleukin-12 and soluble CD 80.

2. The engineered antigen presenting cell of claim 1, wherein the exogenous genes comprise a gene encoding recombinant interleukin-12 and a gene encoding soluble CD 80.

3. The engineered antigen presenting cell of claim 1, wherein the recombinant interleukin-12 is a fusion protein of two subunits, p40 and p35, and the gene sequence encoding the recombinant interleukin-12 is shown as SEQ ID No.1 or has more than 95% homology with the gene sequence and is derived from the same species.

4. The engineered antigen presenting cell of claim 1, wherein the soluble CD80 comprises an extracellular region of CD80, and the gene sequence encoding the soluble CD80 is as shown in SEQ ID No.2 or has more than 95% homology thereto and is derived from the same species.

5. The engineered antigen presenting cell of claim 1, further comprising an antigen or a nucleic acid encoding the antigen.

6. The engineered antigen presenting cell of claim 5, wherein the antigen is the GPC3 antigen.

7. The engineered antigen presenting cell of any one of claims 1 to 6, which is a dendritic cell.

8. An immunomodulatory composition comprising an engineered antigen presenting cell according to any one of claims 1-7 and/or a secretion thereof.

9. A method for activating CD3+ immune cells, comprising the step of contacting an engineered immune cell according to any one of claims 1-7 or an immunomodulatory composition according to claim 8 with an immune-tolerant CD3+ immune cell.

10. The method for activating CD3+ immune cells according to claim 9, wherein the CD3+ immune cells are in vitro cells.

Technical Field

The invention relates to the fields of immunology and medicine, in particular to an engineered antigen presenting cell and application thereof in activating CD3+ immune cells.

Background

Interleukin-12 (IL-12) is produced by antigen presenting cells and B cells, is a pro-inflammatory cytokine in the form of a heterodimer, and is secreted extracellularly in this form. IL-12 can induce IFN-gamma production, in vivo immune response (especially in bacterial or parasitic infection) it is also IFN-gamma production required.

Different cytokines such as IFN-gamma, TNF- β, GM-CSF enhance the ability of cells to produce IL-12, IFN-gamma enhances transcription of genes encoding p40 and p35, which have particularly pronounced effects on heterodimer production, and IFN-gamma forms a positive feedback mechanism with the ability of IL-12 production in inflammatory and Th1 responses.

Research shows that tumor patients have the characteristics of reduced DC number and function defect, and the number and the function of tumor tissues and DC infiltrated around the tumor tissues have close relation with the occurrence, development, metastasis and prognosis of tumors.

In recent years, it has become clear that the immune system does recognize tumor antigens, but despite the presence of tumor antigens, T cells are assured to remain quiescent. There is a hypothesis that: antigen presenting cells in the patient, which fail to correctly recognize the tumor antigen, present it to T lymphocytes, causing a tumor-specific immune response. Therefore, how to activate immune cells with immune tolerance and induce tumor-specific immune response becomes a key point in the current tumor immunization research.

Disclosure of Invention

The invention provides an engineered immune cell which is used for solving at least part of technical problems in the prior art. The invention discovers that the capacity of antigen presenting cells for activating CD3+ immune cells can be greatly improved by modifying the antigen presenting cells through an engineering means so that the antigen presenting cells secrete soluble CD80 fragments while excessively expressing recombinant interleukin-12. The present invention has been accomplished based at least in part on this finding. Specifically, the present invention includes the following.

In a first aspect of the invention, there is provided an engineered antigen presenting cell which is obtained by introducing a foreign gene into a wild-type antigen presenting cell by engineering means to overexpress recombinant interleukin-12 and soluble CD 80.

The engineered antigen presenting cell according to the present invention, wherein the exogenous gene comprises a gene encoding recombinant interleukin-12 and a gene encoding soluble CD 80.

The engineered antigen presenting cell of the invention, wherein the recombinant interleukin-12 is a fusion protein of two subunits of p40 and p35, and the gene sequence coding the recombinant interleukin-12 is shown as SEQ ID No.1 or has more than 95% homology with the gene sequence and is derived from the same species.

The engineered antigen presenting cell of the invention, wherein the soluble CD80 comprises an extracellular region of CD80, and the gene sequence encoding the soluble CD80 is shown in SEQ ID No.2 or has more than 95% homology with the soluble CD80 and is derived from the same species.

An engineered antigen presenting cell according to the invention, further comprising an antigen or a nucleic acid encoding said antigen.

An engineered antigen presenting cell according to the invention, wherein said antigen is the GPC3 antigen.

An engineered antigen presenting cell according to the invention, wherein it is a dendritic cell.

In a second aspect of the invention, there is provided an immunomodulatory composition comprising an engineered antigen presenting cell of the first aspect and/or a secretion thereof.

In a third aspect of the invention, there is provided a method for activating a CD3+ immune cell, comprising the step of contacting an engineered immune cell of the first aspect or an immunomodulatory composition of the second aspect with an immune-tolerant CD3+ immune cell.

The method for activating a CD3+ immune cell, wherein the CD3+ immune cell is an in vitro cell.

The invention finds that the soluble CD80 can greatly enhance the ability of IL-12 to activate CD3+ cells on the basis of a large number of screening experiments, and the two can play a synergistic effect, promote the continuous amplification of CD3+ immune cells such as T cells, generate more lymphocytes with anti-tumor activity and improve the treatment of tumors.

Drawings

FIG. 1 shows the results of CD 8T cell immune responses in experiments in which DC cells were transfected with mRNA encoding each component protein of the composition of the present invention and GPC3 antigen mRNA, and then sensitized in vitro with T cells, and the columns of FIG. 1 show, from left to right, the proportion of CD8 IFN-. gamma. +, CD8 IFN-. gamma. +, TNF- α +, CD8 TNF- α + cells to total CD 8T cells.

FIG. 2 shows the results of CD 4T cell immune responses in experiments in which DC cells were transfected with mRNA encoding each component protein of the composition of the present invention and GPC3 antigen mRNA, and then sensitized in vitro with T cells, and the columns of FIG. 2 show, from left to right, the proportion of CD4 IFN-. gamma. +, CD4 IFN-. gamma. +, TNF- α +, CD4 TNF- α + cells to total CD 4T cells.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

[ engineered immune cells ]

In a first aspect of the invention, an engineered antigen presenting cell is provided that can be used as an immunomodulatory composition to activate an immune cell in conjunction with a corresponding antigen. The engineered antigen presenting cell is obtained by introducing exogenous genes into a wild-type antigen presenting cell through an engineering means so as to over-express recombinant interleukin-12 and soluble CD 80.

In the present invention, wild-type antigen presenting cells generally refer to natural antigen presenting cells isolated from the body of a subject. The subject is generally a mammal, and examples thereof include rats, mice, pigs, rabbits, and primates. The subject is also preferably a human.

In the present invention, the foreign gene refers to a gene derived from the outside of the wild-type antigen presenting cell, and includes a gene derived from a different subject or the same source as the wild-type antigen presenting cell, and preferably, the wild-type antigen presenting cell and the foreign gene are derived from the same source, for example, both of human origin.

In the present invention, the foreign gene comprises a gene encoding recombinant interleukin-12 and a gene encoding soluble CD80 IL-12 is known to be a heterodimer p70 consisting of two chains p35 and p40, the two subunits being linked together by disulfide bonds, in nature, an excess of p40, which is far in excess of p70, forms a p40 homodimer, which is biologically inactive but binds to IL-12R β, competes with p70 for the IL-12 receptor (IL-12R) to antagonize its biological effects.

In the invention, the recombinant interleukin-12 has similar functions of natural IL-12, can proliferate activated T cells and increase the cytotoxic activity of the activated T cells, induce NK cells and T cells to generate gamma-interferon (IFN-gamma), regulate the development of Th1/Th2 cells and promote the differentiation of the cells to Th1 cells. In the present invention, recombinant IL-12 also induces cytokines in CTL immune responses, initiating cell-mediated immune responses. The recombinant IL-12 can not only activate innate immune cells, promote APC functions, particularly DC maturation, and improve antigen presentation efficiency. In addition, the recombinant IL-12 also cooperates with the IL-18 to promote the activation of memory T cells, so that the infected cells can be rapidly eliminated, and simultaneously, the secretion of other cytokines is promoted, the virus replication is inhibited, and the immune function of other cells is regulated.

In certain embodiments, the invention of the recombinant IL-12 by SEQ ID No.1 shows the sequence of the gene coding, the recombinant IL-12 produced by this lacks IL-12p35 signal peptide sequence. It is readily understood by those skilled in the art that the sequence shown in SEQ ID No.1 is only exemplary, and modifications can be made on the basis of this sequence without affecting the achievement of the objects of the present invention. Such modifications include the use of biased codons to optimize SEQ ID No. 1. Therefore, the present invention can also use a sequence having 95% or more, preferably 97% or more, more preferably 99% or more homology with SEQ ID No.1 and derived from the same species.

In the present invention, the recombinant IL-12 amino acid sequence is not particularly limited, its examples include SEQ ID No.3 shows the sequence, or with its homology of 95% or more, preferably 97% or more, more preferably 99% or more and derived from the same species sequence.

The CD80 of the present invention belongs to immunoglobulin superfamily (IgSF), and exists in the form of oligomer in most antigen presenting cells, such as dendritic cells, activated T cells, B cells, macrophages, etc. CD80 is known to be an important costimulatory molecule on APC expression, and is a positive factor that has important promoiety regulatory effects on antigen-induced initiation of T cell activation, proliferation and effector function generation after binding to T cell CD 28. Under wild conditions, CD80 bound to the surface of cells as a membrane protein. The present inventors have found that in contrast to this, soluble CD80 is secreted outside the cell and, as a soluble fragment, synergistically enhances the ability of IL-12 to activate immune cells. The reason for this may be that soluble CD80, on the one hand, due to its increased freedom of solubility, binds more readily to its receptor, e.g. CD28, thereby enhancing the transduction of downstream signals. On the other hand, CD80 is used as a downstream factor of IL-12, and when the downstream factor is overproduced, the feedback inhibition is generated so as to negatively regulate the production or activation of IL-12, while the invention is not influenced by the negative regulation by simultaneously overexpressing recombinant IL-12 and soluble CD80, so that the capability of activating immune cells can be greatly enhanced.

In the present invention, soluble CD80 is a fragment comprising the extracellular domain of CD 80. Preferably, further comprising other fragments. Examples of other fragments include, but are not limited to, Fc fragments, which are useful for extending the half-life and improving stability of the protein. It is preferred to use longer Fc fragments to increase molecular volume. However, if the molecular size is too large, the activity of soluble CD80 may be affected. Thus, the length or size of the Fc fragment influences the achievement of the object of the present invention. For the purposes of the present invention, the length of the Fc fragment is generally 105-315AA, preferably 120-315 AA. The Fc fragment of the present invention may comprise a fragment naturally occurring in an immunoglobulin, and may further comprise a mutant Fc fragment modified by known genetic engineering means to obtain more superior performance. For example, the Fc fragment contains 3 mutated fragments of "YTE", i.e. methionine (Met, M), serine (Ser, S) and threonine (Thr, T) of 252, 254 and 256 are replaced by tyrosine (Tyr, Y), T and glutamic acid (Glu, E), respectively, thereby obtaining soluble proteins with longer half-lives. For another example, by genetic engineering and modification of disulfide bonds of an Fc fragment, Fc proteins can be aggregated into multimeric complexes, thereby obtaining soluble proteins with superior stability.

In certain embodiments, the soluble CD80 of the invention is encoded by the sequence shown in SEQ ID No.2 or a sequence having more than 95%, preferably more than 96%, more preferably more than 98%, even more preferably more than 99% homology thereto. In the present invention, the amino acid sequence of soluble CD80 is not particularly limited, and examples thereof include the sequence shown in SEQ ID No.4, or a sequence having 95% or more, preferably 97% or more, more preferably 99% or more homology thereto and derived from the same species.

In the present invention, an engineered cell refers to a cell that has been artificially modified. Herein, the antigen presenting cell refers to a cell having the functions of taking up, processing and transmitting antigen information in the body, presenting the antigen to immune cells and assisting and regulating T cells, B cells recognizing the antigen and inducing immune response. Examples include, but are not limited to, macrophages, dendritic cells, and syndactylic cells, pancreatic cells, and B cells. Preferably, the immune cells of the invention are dendritic cells, more preferably human dendritic cells. The dendritic cells of the present invention may be mature dendritic cells or immature dendritic cells. It should be noted that the dendritic cells are obtained by in vitro induction culture, i.e., Peripheral Blood Mononuclear Cells (PBMCs), which are induced to become DC cells under stimulation of various types of culture media and various types of cytokines. In specific embodiments, the culture medium used for in vitro culture includes AIM-V medium, iDC medium and mDC medium, and examples of cytokines used for in vitro induction culture include, but are not limited to, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4.

Preferably, the antigen presenting cell of the invention further comprises an antigen or a precursor capable of producing an antigen, and the composition of the first aspect. "antigen" refers to a substance that is recognized by the immune system and is capable of eliciting an antigen-specific immune response through the formation of antibodies or/and antigen-specific T cells. In general, an antigen can be a protein or polypeptide that contains at least one antigenic epitope, is captured by an APC and can be presented on the surface of a T cell. In the present invention, the antigen may be a product of translation of mRNA or a product of transcription and translation of DNA. In certain embodiments, the antigen of the invention is a GPC3 antigen that is closely associated with the development and progression of primary liver cancer.

[ immune modulating composition ]

In a second aspect of the invention, there is provided an immunomodulatory composition comprising an engineered cell of the first aspect and/or a secretion thereof, capable of providing one or more antigens (preferably an oncogene) to a subject in need thereof, and modulating (in particular enhancing) the immunity of the antigen, thereby alleviating, delaying or curing the condition or disorder in the subject. The pharmaceutical compositions of the invention have enhanced, increased or boosted immunocompetence as compared to a drug that provides the antigen alone, or as compared to a composition that provides the antigen and IL-12 or CD 80. The immunomodulatory compositions of the invention may optionally further comprise other ingredients, such as pharmaceutically acceptable carriers. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: 1) dulbecco phosphate buffered saline, pH about 7.4, with or without about 1mg/ml to 25mg/ml human serum albumin; 2) 0.9% saline (0.9% w/v sodium chloride), and 3) 5% (w/v) glucose; antioxidants such as tryptamine and stabilizers such as Tween20 may also be included.

[ method for activating CD3+ immune cells ]

In a third aspect of the invention, there is provided a method for activating a CD3+ immune cell, comprising the step of contacting an engineered immune cell of the first aspect or an immunomodulatory composition of the second aspect with a CD3+ immune cell. Wherein the CD3+ immune cells are preferably immune tolerant CD3+ immune cells. Herein, CD3+ immune cells refer to cells comprising CD3 on the surface, preferably CD3+ T cells

In an exemplary method of activating CD3+ immune cells, it comprises the steps of:

(1) preparing a nucleic acid construct, wherein the nucleic acid construct can be one or more, comprising a nucleic acid that expresses recombinant interleukin-12, a nucleic acid that expresses soluble CD80, and optionally a nucleic acid that expresses an antigen;

(2) performing in vitro transcription to obtain a corresponding ribonucleic acid molecule;

(3) performing in vitro induction culture to obtain antigen presenting cells for expressing the ribonucleic acid molecules of step (2);

(4) introducing the ribonucleic acid molecule of the step (2) into the antigen presenting cell of the step (3) through transfection and expressing, thereby obtaining an engineered immune cell; and

(5) the engineered immune cells are contacted or co-cultured with CD3+ immune cells.

Preparation example 1

This preparation example was used to prepare DNA and mRNA encoding an antigen and serving as a precursor of an interleukin complex

1. Preparation of DNA and mRNA constructs

DNA sequences encoding IL12 p70 and soluble CD80 mRNA were constructed separately and used for subsequent in vitro transcription reactions. The coding sequence is followed by a segment of polyadenylation. The DNA sequence information is shown in Table 1 below.

In addition, a coding sequence of human tumor antigen GPC3 for in vitro sensitization is constructed, the coding sequence of GPC3 of the invention consists of a sequence shown in SEQ ID No.5, and the amino acid sequence consists of a sequence shown in SEQ ID No. 6. The sequence of GPC3 can be obtained from the Genebank database. The antigen disclosed in CN107583042A was used in this example.

TABLE-1 Gene sequence Listing

Name (R)	Serial number
		IL-12	SEQ ID No.1
CD80	SEQ ID No.2
		GPC3	SEQ ID No.5

2. In vitro transcription

First, the corresponding DNA plasmid obtained by the preparation was linearized with a restriction enzyme, and mRNA was prepared by in vitro transcription using T7 RNA polymerase using the linearized plasmid as a template. The prepared mRNA was then purified by lithium chloride precipitation.