CN116496417B - Fusion proteins and T cells containing membrane IL7 - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to fusion proteins containing membrane type IL7 and T cells. The fusion protein contains IL7 and a membrane protein transmembrane peptide segment. The fusion protein containing the membrane IL7 and the T cells provided by the invention can enable the IL7 generated by the T cells input into a patient to play roles in promoting cell proliferation and the like, reduce the safety risk of secreted IL7 on the patient, and have excellent application prospects in medicine.

Description

Fusion proteins and T cells containing membrane IL7

Technical Field

The invention relates to the technical field of biological medicines, in particular to fusion proteins containing membrane type IL7 and T cells.

Background

Treatment of malignant tumors has been one of the leading challenges in the medical field. With the development of biomedical technology, immunotherapy is one of the very effective treatment strategies for malignant tumors, and the biggest feature of immunotherapy is to use the immune system itself to combat cancers, including antibody therapy, cell therapy, and immunomodulator therapy. Among cellular immunotherapy, CAR-T therapy has proven to be one of the most effective therapeutic approaches. In CAR-T therapy, it is necessary to isolate T cells from a patient, edit and expand the T cells, and then re-import the enhanced T cells into the patient to complete cellular immunotherapy.

In the case of in vitro expansion of T cells, the addition of IL7 to the T cell culture medium can increase the in vitro expansion of T cells, and if IL7 is used in vivo for patients (direct intravenous injection or infusion of IL7 into T cells of patients) it can enhance the expansion of T cells in vivo, but at the same time it can present serious safety risks to patients.

How to make the IL7 generated by T cells input into a patient play the roles of promoting cell proliferation and the like, and simultaneously reduce the safety risk of the patient is a technical problem to be solved in the field.

Disclosure of Invention

In order to solve the technical problems, the invention provides the following scheme.

In a first aspect, the invention provides a fusion protein comprising IL7 and a transmembrane domain peptide segment of a membrane protein.

According to the invention, the secreted cell factor IL7 is connected with the membrane protein transmembrane region, and the membrane protein transmembrane region can fix the IL7 on the surface of a T cell, so that the IL7 is expressed on the T cell membrane, and the safety risk of the secreted IL7 on a patient can be reduced after the T cell is returned to the patient.

In a second aspect, the invention provides a fusion protein comprising IL7, a membrane protein hinge region and a transmembrane region peptide segment.

Further, the invention discovers that the hinge region can enable IL7 to have better activity freedom degree by directly connecting IL7 with the hinge region and the transmembrane region peptide segment of the membrane protein, and is beneficial to promoting the IL7 to stimulate T cells.

Preferably, the membrane protein is CD8 alpha or B7-1.

Preferably, the membrane protein hinge region and the transmembrane region peptide segments are independently selected from the same or different membrane proteins.

Furthermore, the hinge region and the transmembrane region of different membrane proteins are combined with IL7, which may result in different ability of IL7 to be immobilized on T cell membrane, so that IL7 can be immobilized on T cell membrane well after the two membrane proteins are combined with IL7 after screening the membrane proteins.

Since IL7 itself is a secreted protein, even if constructed with the transmembrane region of the membrane protein, a portion of IL7 is secreted outside the cell, and it has been found through experiments that the amount of IL-7 secreted outside the cell is low and the expression efficiency of IL7 is high after the CD 8. Alpha. Membrane protein or B7-1 membrane protein is combined with IL 7.

Preferably, the amino acid sequence of IL7 is shown in SEQ ID NO. 1.

Preferably, the amino acid sequence of the CD8 alpha transmembrane region peptide fragment is shown as SEQ ID NO. 2;

the amino acid sequence of the peptide segment of the B7-1 transmembrane region is shown as SEQ ID NO. 4;

the amino acid sequences of the CD8 alpha hinge region and the transmembrane region peptide fragment are shown in SEQ ID NO. 3.

Preferably, the fusion protein further comprises a signal peptide and/or a CAR peptide fragment.

Preferably, the CAR peptide fragment is a CD19 CAR peptide fragment.

Preferably, the fusion protein comprises an IL7, a membrane protein hinge region and a transmembrane region peptide segment, a 2A peptide and a CAR peptide segment, which are sequentially linked.

When the 2A peptide is selected for connection, the expression rates of the membrane type IL7 and the CAR peptide fragment in T cells are good.

Preferably, the 2A peptide is F2A shown in SEQ ID NO. 16.

Preferably, the signal peptide is a signal peptide of CD8 a.

Preferably, the fusion protein further comprises a tag peptide.

In a third aspect, the invention provides a nucleic acid encoding a fusion protein of any of the embodiments described above.

In a fourth aspect, the invention provides a biomaterial comprising a fusion protein or nucleic acid according to any one of the embodiments above.

Preferably, the biological material is an expression cassette, a vector or a recombinant microorganism.

In a fifth aspect, the invention provides a T cell comprising a fusion protein, nucleic acid or biological material according to any one of the embodiments described above.

Compared with the prior art, the invention has the beneficial effects that:

the fusion protein containing the membrane type IL7 and the T cells are provided, so that the IL7 generated by the T cells input into a patient plays roles in promoting cell proliferation and the like, simultaneously reduces the safety risk of secreted IL7 on the patient, and has excellent application prospect in medicine.

Drawings

FIG. 1 is a schematic representation of the membrane-type IL7 and CD19 CAR construction of the present invention.

Figure 2 is a statistical plot of expression efficiency for different constructs of CD19 CAR.

FIG. 3 is a graph showing statistics of expression efficiency of different constructed membrane-type IL 7.

FIG. 4 is a statistical plot of the concentration of IL7 secreted extracellularly from different constructs.

FIG. 5 is a line graph of proliferation potency in vitro following infection of T cells with different constructs.

FIG. 6 is a statistical plot of IFN- γ secretion following co-incubation with RAJI after infection of T cells with different constructs.

FIG. 7 is a flow assay result for different constructs of CAR-T cell CD45RA and CCR7 expression.

FIG. 8 is a flow assay result for different constructs of CAR-T cell CD45RA and CCR7 expression.

In all figures "ns" indicates no statistically significant difference, and asterisks indicate statistically significant difference (×p <0.05, ×p <0.01, ×p < 0.001).

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.

Lentiviral packaging plasmids (pLP 1, pLP2, VSVG) were purchased from Invitrogen, and the vector pCDH-EF1-Luc2-T2A-tdTomato plasmid was purchased from Wuhan vast, biotechnology Co. Xba I and Sal I endonucleases were purchased from New England Biolabs (Beijing) LTD. X-VIVO15 medium was purchased from Lonza company. PEI was purchased from Sigma. IL-2 and OKT3 were purchased from ACRO Biosystems, inc. CD28 antibodies were purchased from synechiae. 293ft cells and Raji cells were purchased from ATCC. CD3, CD45RA, CCR7, etc. streaming antibodies were purchased from BD, FLAG antibodies were purchased from Sigma, CD19 protein was purchased from ACRO Biosystems, and ELISA kits for IFN-. Gamma.and IL7 were purchased from R & D systems and Ekesai BioCo.

In the following examples, membrane-type IL7 and CD19 CAR were connected by F2A, so that T cells simultaneously expressed membrane-type IL7 and CD19 CAR, and the use of F2A ensured that the expression rates of membrane-type IL7 and CD19 CAR in T cells were relatively uniform, and thus the fixation of membrane-type IL7 to the cell membrane was indirectly evaluated. At the same time, the effect of IL7 on CD19 CAR-T cell function can be evaluated.

EXAMPLE 1 construction of lentiviral vectors simultaneously expressing CD19 CAR and Membrane IL 7-positive T cells

Two membrane proteins (CD 8 alpha and B7-1) were obtained by screening in a large number, the hinge region and the transmembrane region of CD8 alpha were shown as SEQ ID NO.3, the transmembrane region of B7-1 was shown as SEQ ID NO.4, and then the above sequences were added to the rear of IL7 (the amino acid sequence of IL7 was shown as SEQ ID NO. 1), the present example replaced the IL7 signal peptide with CD8 alpha signal peptide to obtain the sequence shown as SEQ ID NO. 5), IL7-CD8 (SEQ ID NO. 6), IL7-B7 (SEQ ID NO. 7) were formed, respectively, and in order to facilitate the expression of the above fusion proteins, a flag tag (SEQ ID NO. 12) was added to the rear of IL-7, respectively, IL7-flag-CD8 (SEQ ID NO. 8), IL7-flag-B7 (SEQ ID NO. 9) were formed, and the above sequences were linked to CD19 (SEQ ID NO. 17) by F2A (SEQ ID NO. 16), respectively, to form mI 7-C8-BBz (SEQ ID NO. 10-37) and m7-B35 (SEQ ID NO. 11). SEQ ID No.17, SEQ ID No.10, SEQ ID No.11, respectively, were subjected to total gene synthesis, the structure is schematically shown in FIG. 1, and the obtained product was cloned into lentiviral vectors pCDH-EF1-Luc2-T2A-tdTomato by double digestion with Xba I and Sal I, and named pCDH-EF1-19BBz, pCDH-EF1-mI7C8-19BBz, and pCDH-EF1-mI7B7-19BBz, respectively.

The construction method of the pCDH-EF1-19BBz recombinant lentiviral vector comprises the following steps:

(1) The nucleotide sequence of 19BBz (SEQ ID NO. 13) was synthesized, and Xba I and Sal I cleavage sites were added at both ends of the nucleotide sequence, respectively, and cloned into pUC57 vector;

(2) The pUC57 vector containing the target gene is digested with Xba I and Sal I, and the target gene fragment is recovered by gel cutting;

(3) The original vector pCDH-EF1-Luc2-T2A-tdTomato was digested with Xba I and Sal I, and the vector fragment of about 6.5kb was recovered by cutting;

(4) And connecting the recovered target gene fragment and the vector fragment by using DNA ligase to obtain the recombinant lentiviral vector carrying pCDH-EF1-19 BBz.

The construction method of the pCDH-EF1-mI7C8-19BBz recombinant lentiviral vector comprises the following steps:

(1) Synthesizing a nucleotide sequence (SEQ ID NO. 14) of mI7C8-19BBz, adding Xba I and Sal I restriction sites at two ends of the nucleotide sequence, and cloning the nucleotide sequence onto a pUC57 vector;

(2) The pUC57 vector containing the target gene is digested with Xba I and Sal I, and the target gene fragment is recovered by gel cutting;

(3) The original vector pCDH-EF1-Luc2-T2A-tdTomato was digested with Xba I and Sal I, and the vector fragment of about 6.5kb was recovered by cutting;

(4) And connecting the recovered target gene fragment and the vector fragment by using DNA ligase to obtain the recombinant lentiviral vector carrying pCDH-EF1-mI7C8-19 BBz.

The construction method of the pCDH-EF1-mI7B7-19BBz recombinant lentiviral vector comprises the following steps:

(1) Synthesizing a nucleotide sequence (SEQ ID NO. 15) of mI7B7-19BBz, adding Xba I and Sal I restriction sites at two ends of the nucleotide sequence, and cloning the nucleotide sequence onto a pUC57 vector;

(2) The pUC57 vector containing the target gene is digested with Xba I and Sal I, and the target gene fragment is recovered by gel cutting;

(3) The original vector pCDH-EF1-Luc2-T2A-tdTomato was digested with Xba I and Sal I, and the vector fragment of about 6.5kb was recovered by cutting;

(4) And connecting the recovered target gene fragment and the vector fragment by using DNA ligase to obtain the recombinant lentiviral vector carrying pCDH-EF1-mI7B7-19BBz.

EXAMPLE 2 preparation of lentiviruses

The recombinant lentiviral vector of example 1 was transfected into 293ft cells by transfection reagent (PEI) to generate lentiviruses, respectively. The specific method comprises the following steps:

the packaged plasmid mixture (pLP 1: pLP2: vsvg=2:2:1, mass ratio) and each lentiviral vector were added to 500 μl of serum-free medium Opti-MEM in a mass ratio of 2:1, vortexed until well mixed. 32g PEI was added to 500. Mu.L of serum free medium Opti-MEM and vortexed until well mixed. Then 500ul of plasmid mixture was mixed with 500ul PEI and added to 293ft cells with a confluency of about 90%, virus supernatant was collected after 48 hours, and after ultracentrifugation, the virus was 100-fold concentrated to obtain concentrated virus.

Example 3 preparation of CAR-T cells

Healthy volunteers were infected with the above lentiviruses and cultured in T cell medium (the formulation of the medium is X-VIVO15 medium+100U/mL IL-2+50 ng/. Mu.L OKT3+1. Mu.g/mL CD28 antibody), and the CD19 protein and FLAG antibody were assayed by flow-through for the infection efficiency of CD19 CAR and membranous IL7, respectively.

As a result, it was found that CD19 CAR without F2A linkage was expressed most efficiently, and mI7B7-19BBz was more efficient than mI7C8-19BBz (FIG. 2). mI7B7-19BBz was more efficient than mI7C8-19BBz in membrane-type IL7 expression (FIG. 3).

Further, since IL7 itself is a secreted protein, even the addition of a transmembrane region may still result in secretion of part of IL7 outside the cell, and thus the amount of IL7 secreted to the cell by different constructs was examined by ELISA, and as a result, it was found that the amount of IL7 secreted by mI7C8-19BBz was significantly lower than that of mI7B7-19BBz (fig. 4).

Taken together, the results show that the membrane IL7 expression efficiency of mI7B7-19BBz is slightly higher than that of mI7C8-19BBz, but that the IL-7 secreted outside the cell by mI7C8-19BBz is significantly lower than that of mI7B7-19BBz, so that the safety of clinical application is ensured in order to ensure that IL7 is secreted outside the cell as little as possible, and mI7C8-19BBz is the optimal construction.

Example 4 functional verification of cells

In vitro culture of mI7C8-19BBz CAR-T, mI7B7-19BBz CAR-T and 19BBz CAR-T in X-vivo15 medium for 5 days and 7 days, respectively, the number of mI7C8-19BBz CAR-T and mI7B7-19BBz CAR-T was significantly higher than 19BBz CAR-T (FIG. 5), demonstrating that membranous IL7 has the ability to promote CAR-T proliferation.

The mI7C8-19BBz CAR-T, mI7B7-19BBz CAR-T and 19BBz CAR-T were incubated in vitro with CD19 positive Raji cells for 12h, respectively, and the concentration of IFN-gamma was detected using ELISA kits, and the amounts of mI7C8-19BBz CAR-T and mI7B7-19BBz CAR-T secreted IFN-gamma were found to be significantly higher than 19BBz CAR-T (FIG. 6), demonstrating that membrane type IL7 can enhance the CAR-T secretion IFN-gamma capacity.

After 7 days of in vitro culture of mI7C8-19BBz CAR-T, mI B7-19BBz CAR-T and 19BBz CAR-T in T cell culture medium, respectively, by flow testing CD45RA and CCR7 expression, mI7C8-19BBz CAR-T and mI7B7-19BBz CAR-T were found to have a higher proportion of na ve cells (CD45RA+CCR7+) (FIG. 7) and a lower proportion of effect memory cells (CD 45RA-CCR 7-) (FIG. 8), demonstrating that mI7C8-19BBz CAR-T and mI7B7-19BBz CAR-T have a greater ability to promote na ve cell proliferation and to attenuate T cell terminal differentiation.

The experiment proves that in the T cells containing the membrane type IL7, the IL7 can be well fixed on the surface of a T cell membrane, the secreted IL7 is lower, the in vivo safety is good, and the expression of the membrane type IL7 can enhance multiple functions of the CD19 CAR-T and further enhance the anti-tumor capability of the T cells.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (2)

1. A T cell comprising a fusion protein; the fusion protein contains IL7, a membrane protein hinge region and a transmembrane region peptide segment, and the membrane protein is CD8 alpha; the IL7 is directly connected with a membrane protein hinge region and a transmembrane region peptide segment;

the amino acid sequence of the IL7 is shown as SEQ ID NO. 1;

the amino acid sequences of the CD8 alpha hinge region and the transmembrane region peptide fragment are shown in SEQ ID NO. 3.

2. The T cell of claim 1, wherein said fusion protein further comprises a signal peptide;

and/or wherein a CAR peptide fragment is also contained.