CN113307872B

CN113307872B - Engineered nucleic acid, T cell, application and production method thereof

Info

Publication number: CN113307872B
Application number: CN202110544048.9A
Authority: CN
Inventors: 李思
Original assignee: Guangdong Tiankeya Biomedical Technology Co ltd
Current assignee: Guangdong Tiankeya Biomedical Technology Co ltd
Priority date: 2018-08-11
Filing date: 2019-06-28
Publication date: 2022-12-06
Anticipated expiration: 2039-06-28
Also published as: CN110423757A; CN113307872A; CN110423757B

Abstract

The invention discloses an engineered nucleic acid, comprising nucleic acid of a genetically engineered antigen receptor, capable of specifically binding to an antigen from HPV; nucleic acid blocking an immune checkpoint receptor expressed in a tumor, which nucleic acid may encode a blocking protein; an engineered T cell comprising the nucleic acid sequence described above; use of an engineered T cell in the preparation of a medicament for the treatment of cancer; a method of generating an engineered T cell: introducing the vector into T cells to obtain TCR-T cells, and then amplifying to obtain engineered T cells; the vector comprises the nucleic acid. The invention relates to an engineered nucleic acid, a T cell and an application and a production method thereof, wherein the obtained T cell can identify HPV E6 tumor antigen and can secrete single-chain antibody for blocking programmed cell death protein PD-1; these engineered T cells exhibit greater anti-tumor effects and can reduce T cell depletion; the invention may also be used in the immunotherapy of cancers expressing HPV E6.

Description

Engineered nucleic acid, T cell, application and production method thereof

The application is a divisional application of Chinese patent application 201910573639.1, the application date of the original application is 2019, 6 months and 28 days, and the name is 'an engineered nucleic acid, a T cell and an application and a generation method thereof'.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an engineered nucleic acid, a T cell, application thereof and a production method thereof.

Background

Some cancers (e.g., cervical cancer) are primarily caused by Human Papillomavirus (HPV) infection. Despite the efficacy of treatments such as chemotherapy, the prognosis of many cancers (including HPV-related cancers) may be poor. Thus, there is a medical need for cancer, particularly HPV-associated cancer.

HPV16 is a subtype of HPV, often causing malignancies. HPV16 may act through its E6 protein, which may affect the cell cycle and thus initiate cancer. The E6 protein of HPV16 may be expressed in a variety of cancers, including but not limited to cervical, oropharyngeal, anal canal, anorectal, vaginal, vulvar, and penile cancers.

T cells can be made to specifically recognize the HPV 16E 6 antigen by engineering the TCR. Similarly, chimeric Antigen Receptor (CAR) genetically modified T cells (CAR-T) are an effective therapy to modify T cells, and their core is to specifically target T cells to Tumor Associated Antigens (TAAs), such as CD19 and GD2, with good results in clinical trials for the treatment of, for example, B-cell malignancies and neuroblastoma.

Unlike naturally occurring T Cell Receptors (TCRs), CARs are artificial receptors fused to intracellular T cell signaling and costimulatory domains. CARs can directly recognize TAAs in a Major Histocompatibility Class (MHC) -independent manner. Although CAR-T cell therapy has been successful in hematological malignancies, the expected effect is not achieved in most solid tumors. This may be due to an immunosuppressive microenvironment in solid tumors. This environment involves expression by immune checkpoint receptors (IR) in T cells.

Various immune checkpoints have been discovered to date that inhibit T cell function, such as CTLA-4, TIM-3, LAG-3, and PD-1. These molecules can contribute to the failure of T cell function in chronic infections and cancer, leading to tumor escape from immune surveillance. Wherein PD-1 is up-regulated after T cell activation, and further can interact with two ligands PD-L1 or PD-L2 thereof so as to inhibit the release of T cell cytokines. PD-L1 is expressed on T cells, B cells, macrophages, and dendritic cells. It is also abundantly expressed in various tumor cells. While PD-L1 is expressed only in low levels in normal tissues. PD-1 has been the focus of recent research, and clinical studies show that PD-1 blockers can be used for treating cancer species such as colon cancer, renal cancer, lung cancer, melanoma, and the like.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide an engineered nucleic acid, a T cell and an application and production method thereof, wherein the obtained T cell can identify HPV E6 tumor antigen and can secrete a single-chain antibody for blocking programmed cell death protein PD-1; these engineered T cells exhibit greater anti-tumor effects and can reduce T cell depletion; the invention is also useful for immunotherapy of cancers expressing HPV E6.

The purpose of the invention is realized as follows:

1. an engineered nucleic acid, comprising:

(a) A nucleic acid of a genetically engineered antigen receptor capable of specifically binding to an antigen from HPV; <xnotran> SEQ ID NO.1: GGACAGCAGCTGAACCAGAGCCCCCAGAGCATGTTCATCCAGGAGGGCGAGGACGTGTCCATGAATTGCACCAGCAGCAGCATCTTCAACACTTGGCTGTGGTACAAGCAGGACCCAGGAGAGGGACCAGTGCTGCTGATTGCCCTGTACAAGGCCGGAGAGCTGACCTCTAACGGCAGACTGACCGCTCAGTTCGGCATCACCAGGAAGGACAGCTTCCTCAACATCAGCGCCAGCATCCCCAGCGACGTCGGAATCTACTTTTGCGCCGGCCACCCTAGCAGCAATAGCGGCTACGCCCTGAACTTCGGCAAGGGCACAAGCCTGCTGGTGACACCA; </xnotran> The nucleic acid may encode an engineered receptor TCR which specifically recognizes an HPV antigen;

(b) A nucleic acid that blocks an immune checkpoint receptor expressed in a tumor, the nucleic acid comprising the following sequence modules:

heavy chain CDR1 (SEQ ID NO. 2): ggatacctttaccaacacaactatac;

heavy chain CDR2 (SEQ ID NO. 3): ATTAACCCAAGTAATGGGTTACA;

heavy chain CDR3 (SEQ ID NO. 4): ACACGGCGGAGATTACGATGGAGGTTCGACTAC;

light chain CDR1 (SEQ ID NO. 5): AAATCAGTCTCATACTTCTGGTTTTAAT;

light chain CDR2 (SEQ ID NO. 6): CTGGCGTCA;

light chain CDR3 (SEQ ID NO. 7): CAACATGGACGGAGCTGCCCTTGACG;

the nucleic acid may encode a blocking protein.

Preferably, the antigen of HPV is an E6 or E7 antigen.

Preferably, the above immune checkpoint receptor is the PD-1 receptor.

2. An engineered T cell which is characterized by: comprising said nucleic acid capable of encoding a genetically engineered antigen receptor capable of binding specifically to an antigen of HPV and encoding a protein blocking an inhibitory receptor having the function or expression of blocking an immune checkpoint receptor in a tumour. The engineered T cells can specifically target the E6 antigen of HPV while secreting antibodies that block programmed cell death proteins, and thus these engineered T cells can exhibit a stronger anti-tumor response and reduced T cell failure.

Preferably, the above antigen receptor amino acid sequence is SEQ ID No.8: gly Gln Gln Leu Asn Gln Ser Pro Gln Ser Met Phe Ile Gln GluGly Glu Asp Val Ser Met Asn Cys Thr Tyr Lys Ala Gly Glu Leu Thr Ser Asn Gly Arg Leu Thr Ala Gln Phe Gly Ile Thr Arg Lys Ser Phe Leu Asn Ile Ser Ala Ser Pro Ser Asp Val Gly Ile Tyr Phe Cys Ala Gly His Pro Ser Ser Asn Gly Tyr Ala Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro.

Preferably, the protein blocking inhibitory receptor is a single chain antibody against PD-1 which is expressed persistently. The scFv retains the specificity of the complete antibody from which it was derived, and the single chain antibody against PD-1 comprises the following amino acid sequence blocks:

heavy chain CDR1 SEQ ID No.9: gly Tyr Thr Phe Thr Asn Tyr;

heavy chain CDR2 SEQ ID No.10: asn Pro Ser Asn Gly Thr;

heavy chain CDR3 SEQ ID NO.11: thr Arg Asp Tyr Asn Tyr Asp Gly Phe Asp Tyr;

light chain CDR1 SEQ ID No.12: lys Ser Val Ser Thr Ser Gly Phe Asn;

light chain CDR2 SEQ ID No.13: leu Ala Ser;

light chain CDR3 SEQ ID No.14: gln His Gly Arg Glu Lue Pro Leu Thr;

the light and heavy chains may be in any order, e.g., VH-linker-VL or VL-linker-VH, as long as the target antigen specificity of the scFv is retained.

3. An application of an engineered T cell in preparing a medicine for treating cancer.

Preferably, the cancer is cervical cancer or head and neck cancer.

4. A method for generating engineered T cells, which is characterized by comprising the following steps: introducing the vector into T cells to obtain TCR-T cells, and then amplifying to obtain engineered T cells; the vector comprises the nucleic acid.

Preferably, the vector is a retroviral vector.

Has the beneficial effects that:

the invention relates to an engineered nucleic acid, a T cell, application and a production method thereof, wherein the obtained T cell can identify HPV E6 tumor antigen and can secrete a single-chain antibody for blocking programmed cell death protein PD-1; these engineered T cells exhibit greater anti-tumor effects and can reduce T cell depletion; the invention is also useful for immunotherapy of cancers expressing HPV E6.

Drawings

FIG. 1 is the structure of the vector, which includes nucleic acid fragments of three genes linked by P2A and T2A linker sequences: (a) The variable as well as the constant region of the TCR α chain capable of recognizing HPV E6 antigen; (b) The variable and invariant regions of the TCR β chain capable of recognizing HPV E6 antigen; (c) A single chain antibody composed of heavy chain and light chain variable regions of anti-PD-1 connected by GS connecting peptide.

FIG. 2 is the CDR sequences in the anti-PD 1scFv sequence.

FIG. 3 is a graph of the quantitative values for secreted anti-PD-1 scFv in culture supernatants of engineered TCR-T cells.

FIG. 4 shows the expression of HPV E6-specific TCR in human peripheral blood T cells engineered by TCR-T.

FIG. 5 shows that anti-PD-1 scFv secreted by TCR-T cells can bind to PD-1 expressed by target cells.

FIG. 6 shows that anti-PD-1 scFv secreted by TCR-T cells can compete for and block the binding of PD1 protein to PD-L1.

FIG. 7 shows that anti-PD-1 scFv-loaded TCR-T cells could counteract the inhibitory effect of PD-L1 and produce more cytokine IFN γ.

FIG. 8 shows that TCR-T cells loaded with anti-PD-1 scFv can produce more cytokine IFN γ at different T cell stimulation ratios and at different time nodes.

FIG. 9 shows that TCR-T cells loaded with anti-PD-1 scFv have a greater ability to kill target cells.

FIG. 10 is a tumor treatment effect and safety assessment in mouse HPV E6 tumor model loaded with anti-PD-1 scFv engineered TCR-T.

Detailed Description

In the description of the present invention, it is to be understood that the term "single chain variable fragment", "single chain antibody variable region fragment" or "scFv" antibody as used refers to a product comprising only a heavy chain (VH) and a light chain (VL) linked by a linker peptide. The scFv can be expressed as a single chain polypeptide. The scFv retains the specificity of the intact antibody from which it is derived. The light and heavy chains may be in any order, e.g., VH-linker-VL or VL-linker-VH, as long as the target antigen specificity of the scFv is retained.

The term "antigen" as used herein refers to a molecule capable of being bound by an antibody or T Cell Receptor (TCR), or presented by an MHC.

The term "HPV antigen" as used herein refers to a polypeptide molecule consisting of Human Papilloma Virus (HPV), wherein the HPV may be HPV1, HPV2, HPV3, HPV4, HPV6, HPV10, HPV11, HPV16, HPV18, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV41, HPV42, HPV43, HPV45, HPV49, HPV51, HPV52, HPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV68, HPV69. More often the HPV is selected from the group consisting of high risk HPV, such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV69. The antigens of HPV are derived from the E6 and E7 antigens.

The term "T cell" as used herein includes CD4+ T cells and CD8+ T cells. The term T cell also includes T cells which are helper type 1 cells and T cells helper type 2 cells. T cells express wild-type or recombinant T Cell Receptors (TCRs), chimeric Antigen Receptors (CARs), or any other surface receptor capable of recognizing portions of an antigen associated with a target cell. In general, TCRs have two protein chains (α and β chains) that are capable of recognizing antigen polypeptides presented by MHC.

"linker" (L) or "linker domain" or as used herein refers to a "linker region" of an oligopeptide or polypeptide region from about 1 to 100 amino acids in length. The linker may be composed of flexible residues such as glycine and serine so that adjacent protein domains may move freely with respect to each other. Longer joints may be used when it is desired to ensure that two adjacent zones do not spatially interfere with each other. The linker may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers or functional equivalents thereof, and combinations thereof. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative applications of the present invention.

The invention is further illustrated by the following examples and figures.

Example 1: preparation method

1. Constructing and designing a vector: an MP71 retroviral vector construct comprising three coding regions, wherein the three coding regions are: (A) The variable region of the human anti-E6 TCR α chain (designated aE6_ Va-Ca)) fused to the TCR α chain constant region; (B) The beta chain variable region of the same human anti-E6 TCR fused to the TCR beta chain constant region (designated aE6_ Vb-Cb); (C) Variable regions of the heavy chain (designated PD1_ VH) and light chain (designated PD1_ VL) of the novel anti-PD-1 antibody linked to the TCR with a GS linker. The resulting retroviral vector is generally designated as E6.α PD1 — m11. A schematic representation of the retroviral vector used in this study is shown in FIG. 1.

2. Cell lines and culture media. HEK-293T and Ca Ski cells were purchased from ATCC. Peripheral Blood Mononuclear Cells (PBMC) from anonymous donors were purchased from Hemacare. 293T-PD-1 cells were constructed by transducing 293T cells with a lentiviral vector overexpressing human PD-1. Cells were cultured in DMEM +10% FBS, RPMI +10% FBS or X-Vivo +5% human serum A/B +1% HEPES +1% GlutaMAX.

3. Preparation of retrovirus. Retroviral and viral packaging vectors were co-transfected into 293T cells using standard calcium phosphate precipitation transfection procedures. Viral supernatants were harvested after 48 hours and used for T cell transduction.

4. T cell transduction and culture. Prior to retroviral transduction, PBMCs were activated for 2 days using T cell stimulated magnetic beads and upon transduction, freshly harvested retroviral supernatants were centrifuged at 32 ℃ for 2 hours into non-tissue culture treated 24-well plates coated with 15ug RetroNectin per well (Clontech). Activated PBMC were loaded onto plates and centrifuged at 600g for 30 min at 32 ℃. (ii) T cells at 37 ℃ and 5% CO ² And (5) incubating. Media was supplemented every 2 days.

5. TCR and PD-1 staining. All antibodies were purchased from Biolegend. Cells were stained with anti-TCR β chain antibody 72 hours after transfection and then flow assayed for expression of recombinant TCR. Expression of PD-1 was detected by the PD-1 antibody after 72 hours of co-culture with CaSki target cells. Staining with CD3, CD4 and CD8 antibodies was performed simultaneously.

6. TCR-T is propagated in vitro. Ca Ski tumor cells were pre-stained with CFSE and co-cultured with E6, E6.α PD1_ m11 or E6.α PD1_5C4TCR-T cells for 72 hours before measuring CFSE signal intensity by flow. Non-transduced T (NT) cells were used as negative control.

7. Expression of PD-1 following antigen-specific stimulation of T cells in vitro. All TCR-T cells were co-cultured with CaSki cells for 72 hours before T cells were harvested and assayed for PD-1 expression by flow assay in CD8T or CD4T cell subsets. NT was in non-transduced T cells, which served as a negative control.

Example 2: anti-PD-1 scFv expression in vitro.

293T cells were transfected with retroviral vectors encoding E6, E6.APD1_ m11 or E6.APD1_ 5C4TCR and cell culture supernatants were collected 48 hours after transfection. The expression amount of anti-PD-1 scFv was measured in 20. Mu.l of the supernatant by ELISA.

FIG. 3 shows the expression level of anti-PD-1 scFv in the culture supernatant of expressing cells. E6 represents E6 TCR without anti-PD-1 scFV; aPD1_ m11 represents E6 TCR of the novel anti-PD-1 single-chain antibody; E6.α PD1_5C4 represents E6 TCR containing single chain antibodies from published anti-PD-1.

Example 3: e6 TCR-T is expressed in vitro.

The vectors shown in FIG. 4 were first transduced into T cells. After 72 hours of culture, expression of recombinant TCR was detected by antibody staining against TCR β. TCR β flow assays are based on the CD3 positive lymphocyte gate.

As a result: figure 4 shows that E6 TCR is highly expressed in the E6.α PD1_ m11 transduced T cells.

Example 4: affinity against PD-1 scFv.

Retroviral vectors encoding E6, E6. Alpha PD1_ m11 or E6. Alpha PD1_5C4TCR were transfected into 293T cells, 300. Mu.l of each supernatant was incubated with 293T-PD-1 cells at room temperature for 30 minutes 48 hours after transfection, and then HA-labeled anti-PD-1 antibodies bound to the 293T-PD-1 cells were detected using HA-tagged antibodies.

As a result: as shown in FIG. 5, both secreted anti-PD 1 antibodies strongly bound 293T-PD-1 cells. The E6.α PD1_ m11 and E6.α PD1_5C4 antibodies have similar affinity for PD-1 expressed on cells.

Example 5: competitive binding of recombinant PD-L1.

293T cell supernatants after transfection with 1. Mu.l of 100. Mu.g/ml rhPD-L1/Fc, 300. Mu.l of E6, E6. Alpha PD1_ m11 or E6. Alpha PD1_5C4TCR were incubated with 293T-PD1 cells for 30 min. Cells were then stained with PE-conjugated anti-human Fc.

As a result: as shown in fig. 6, cell supernatants of e6.α PD1_ m11 and e6.α PD1_5C4TCR-T were able to compete for binding with recombinant PD-L1, demonstrating that single chain anti-PD 1 antibodies can block the interaction between PD-1 and its ligand PD-L1.

Example 6: TCR-T cells IFN gamma secretion in vitro.

The 96-well assay plates were coated with 3. Mu.g/ml anti-human CD3 antibody overnight at 4 ℃. The following day the supernatant in the wells was aspirated and washed once with 100. Mu.l PBS per well. Mu.l/ml rhPD-L1/Fc diluted with 100. Mu.l PBS was added. Then add 10 to each wellMu.l of goat anti-human IgG Fc antibody at 100. Mu.g/ml diluted in PBS. The assay plates were incubated at 37 ℃ for 4 hours. Human T cells were transfected for 2 days and placed in coated 96-well plates, and 100. Mu.l of the supernatant of E6, E6. Alpha. PD1_ m11 or E6. Alpha. PD1_5C4 TCR-transfected 293T cell cultures was added with Golgi secretion inhibitors. 37 ℃ and 5% of CO ₂ Incubate overnight. After incubation, T cells were harvested for intracellular IFN-. Gamma.staining.

As a result: as shown in FIG. 7, E6 TCR-T cells can be activated by CD3 antibody and secrete IFN γ, and recombinant PD-L1 (rhPD-L1) can inhibit IFN γ secretion induced by CD3 antibody. However, in experiments with the addition of the supernatants of e6.α PD1_ m11 and e6.α PD1_5C4, PD-L1 did not inhibit CD3 antibody-induced IFN γ secretion.

Example 7: TCR-T cells IFN gamma secretion in vitro. TCR-T cells were contacted with Ca Ski at a rate of 1:0,1:2,1:1 and 3:1 ratio of effector to target the supernatants were collected after 48 or 72 hours of mixed co-incubation and IFN- γ expression was measured using the human IFN- γ ELISA kit according to the manufacturer's instructions.

As a result: the effect of secreted anti-PD-1 scFv on antigen stimulation of TCR-T cell IFN γ secretion is shown in FIG. 8 (NT is a non-transduced control used as a control). E6. IFN γ secretion was detectable in supernatants of α PD1_ m11 and e6.α PD1_5C4TCR-T cells, however, IFN γ secretion was significantly higher in the e6.α PD1_ m11z group.

Example 8: specific target cell killing. Ca Ski tumor cells were pre-stained with CFSE, and E6, E6.α PD1 — m11 or E6.α PD1 — 5C4TCR-T cells were stained with 1:2,1:1 and 3: mix at ratio 1 and co-incubate overnight. Cytotoxicity of T cells on Ca Ski cells was then measured by Annexin V/7-AAD staining. Non-target 293T cells served as controls for this experiment.

As a result: as shown in fig. 9, E6 TCR-T cells can specifically kill E6+ target cells Ca Ski. E6.α PD1_ m11 and E6.α PD1_5C4 can kill E6+ target cells Ca Ski more efficiently.

Example 9: TCR-T in vivo activity and toxicity assays. 6-8 weeks female NSG mice were injected subcutaneously with 2e ⁶ The Ca ski tumor cell of (1). After 24 hours, the mice were injected with 10e separately via tail vein ⁶ E6-TCR-T, E6. Alpha PD1_ m11-TCR-T transitionLead or non-transduced T cells. Tumor size was measured twice weekly to assess TCR-T anti-tumor efficacy, and mouse physical condition and body weight were measured twice weekly to assess TCR-T related toxicity.

Results of TCR-T in vivo assay: as shown in fig. 10, TCR-T cells engineered with HPV E6-specific TCR had a certain tumor therapeutic effect, while engineered T cells loaded with anti-PD 1scFv had a stronger therapeutic effect, relative to non-engineered T cells. In terms of safety, neither of the two engineered TCR-T cell products caused significant toxicity to mice, indicating that the product was safe.

Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that:

engineered cells in various application protocols, engineered cells can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pigs, or any other species. Mainly, the cells may be from human, rat or mouse. More generally, the cells are obtained from human T cells, B cells or NK cells.

In a preferred application, the cell is a T cell. Examples of T cells for use in the present invention include, but are not limited to, T cells isolated from a patient by in vitro culture (e.g., tumor infiltrating lymphocytes).

Recombinant vectors are tools that can be used for the delivery of genetic material into cells for expression, and include, but are not limited to, plasmid vectors, viral vectors, BAC, YAC, HACS, and the like. Viral vectors that may be used include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy viral vectors, recombinant adeno-associated virus (AAV) vectors, hybrid vectors and/or plasmid transposons (for example, sleeping beauty seat system) and like systems.

In the main application scheme, the recombinant vector used is a retroviral vector. The vector may be produced by a special viral vector medium. In embodiments of the invention, such media include various media substrates suitable for production of the viral vectors and nutritional supplements.

Genetically engineered receptors include, but are not limited to, T Cell Receptors (TCRs), killer cell immunoglobulin-like receptor family (KIRs), C-type lectin receptor family, leukocyte immunoglobulin-like receptor family, type I cytokine receptors, type II cytokine receptor family, tumor necrosis factor family, TGF β receptor family, chemokine receptors, igSF.

In a primary application, the receptor is a genetically engineered T Cell Receptor (TCR), and the T cells expressing the receptor are α β -T cells, and in an alternative application, the T cells expressing the receptor can also be γ δ -T cells.

Target antigens

In some embodiments, the antigen associated with the disease or disorder can be from a protein expressed by HPV, HIV, HCV, HBV, EBV, HTLV-1, CMV, adenovirus, polyoma, HHV-8, or other pathogen, or from a cell-expressed protein, including ROR1, EGFR, her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, erbB2,3 or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha 2, kdr, kappa light chain, lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MA RT-1, gp100, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2 and MAGE A3 and/or biotinylated molecules.

In the subject, the target antigen is selected from Human Papillomavirus (HPV) antigens. Subtypes of HPV include, but are not limited to, HPV1, HPV2, HPV3, HPV4, HPV6, HPV10, HPV11, HPV16, HPV18, HPV26, HPV27, HPV28, HPV29, HPV30, HPV31, HPV33, HPV34, HPV35, HPV39, HPV40, HPV41, HPV42, HPV43, HPV45, HPV49, HPV51, HPV52, HPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV68, HPV69.

In some embodiments, the HPV antigen is selected from, but not limited to, E1, E2, E3, E4, E6, and E7, L1, and L2 proteins. In a major application, the antigen is the E6 antigen. In another principal application, the antigen is the E7 antigen.

In some embodiments, the disorder is a virus-induced malignancy, including but not limited to HPV, HCV, EBV, HIV, HHV-8, HTLV-1, MCV-induced malignancy. In the present invention, the main application is HPV-induced malignancies, including but not limited to cervical, oropharyngeal, anal canal, anorectal, vaginal, vulvar and penile cancer.

Checkpoint inhibitors in various application protocols, the engineered cells express at least one immune checkpoint inhibitor (CPI). Wherein the immune checkpoints include, but are not limited to, PD-1, PD-L2, 2B4 (CD 244), 4-IBB, A2AR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, butyrophilins, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD 47), TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA, and combinations thereof.

In the primary application scenario, the checkpoint is PD-1 or PD-L1. In various application regimens, the checkpoint inhibitor is anti-PD-1 scFv. The protein can cause the expression of PD-1 or PD-L1 to be reduced and/or the function of a check point to be blocked.

Nucleic acid composition of vector

See fig. 1. As shown in FIG. 1, the nucleic acid sequence of the vector includes three sequences. These three sequences include: (a) A combined sequence of variable and invariant regions that specifically recognizes the TCR α chain of HPV E6 TCR, defined as "aE6_ Va-Ca", wherein aE6_ Va corresponds to the variable region and Ca corresponds to the invariant region; (b) A combined sequence that specifically recognizes the variable and invariant regions of the TCR β chain of HPV E6 TCR, defined as "aE6_ Vb-Cb", wherein aE6_ Vb corresponds to the variable region and Cb corresponds to the invariant region; (c) The heavy chain variable region of the anti-PD-1 scFv was defined as "aPD1_ VH", and the light chain variable region of the anti-PD-1 scFv was defined as "aPD1_ VL". Wherein, the key regions of the anti-PD-1 scFv sequence comprise: the framework FR1 region of the heavy chain variable region; comprises a heavy chain CDR1 with a sequence shown in SEQ ID NO. 1; framework FR2 region of the heavy chain variable region; comprises a heavy chain CDR2 having the sequence shown in SEQ ID NO. 2; the framework FR3 region of the heavy chain variable region; comprises a heavy chain CDR3 having the sequence shown in SEQ ID NO. 3; the framework FR1 region of the light chain variable region; comprises a light chain CDR1 having the sequence shown in SEQ ID NO. 1; framework FR2 region of the light chain variable region; comprises a light chain CDR2 having a sequence shown in SEQ ID No. 2; the framework FR3 region of the light chain variable region; comprises a light chain CDR3 having the sequence shown in SEQ ID NO. 3; and the framework FR4 region of the light chain variable region.

The nucleic acid composition of the vector also includes P2A and T2A sequences that link the sequences (a), (b), and (c) above. In addition, the variable regions of the heavy and light chains of the anti-PD-1 scFv (identified as aPD1_ VH and aPD1_ VL, respectively) were linked to a GS linker.

Vectors and nucleic acid compositions also include additional helper sequences for assisting in transfection, transduction, integration, replication, transcription, translation, and stable expression of the vector.

Methods of making engineered cells

The invention provides methods or processes for making and using engineered cells for treating pathological diseases or disorders. The method comprises the following steps: (I) isolating T cells from the blood of the patient; (II) transducing T cells with a TCR-T vector to engineer the cells; (III) expanding the engineered T cells in vitro; (IV) returning the cells to the patient, wherein the engineered T cells will seek out and destroy antigen expression as positive tumor cells. At the same time, these engineered T cells will block the PD-1/PD-L1 immune checkpoint and enhance the anti-tumor immune response.

Transfection of cells can be accomplished using any standard method, such as calcium phosphate, electroporation, liposome-mediated methods, microinjection, bioparticle delivery systems, or any other known method.

In accordance with various applications described herein, the present invention provides immunotherapeutic treatment of HPV associated cancers, particularly HPV 16E 6+ or HPV 16E 7+ cancers. The engineered T-cell recognizes the tumor antigen HPV E6 and simultaneously secretes a single chain antibody (scFv) blocking programmed cell death protein 1 (PD-1). These engineered T cells exhibit a stronger anti-tumor response and reduced T cell depletion.

It has been found by the above experiments that the blockade of the PD-1 checkpoint is more effective because: (1) anti-PD-1 drug delivery is localized to the tumor site and (2) the anti-PD-1 scFv of the present invention binds more strongly than current anti-PD-1 antibodies. Since anti-PD-1 drug delivery is localized at the tumor site, toxicity due to non-specific inflammation is reduced.

Furthermore, the invention can be used for personalized anti-tumor immunotherapy. anti-HPV E6 antigen specificity can be readily produced from the blood of patients, with anti-PD-1 functionally engineered T cells. These engineered T cells are then re-injected into the patient as a cell therapy product. The product can be applied to any patient with HPV E6 positive tumor, including cervical cancer, head and neck cancer, etc.

<110> Guangdong Tiankoya biomedical science and technology Co., ltd

<120> an engineered nucleic acid, T cell, and methods of use and production thereof

<150> 62717787

<151> 2018-8-11

<160> 14

<170> PatentIn Version 2.1

<210> 1

<211> 339

<212> DNA

<213> Artificial sequence

<400> 1

ggacagcagc tgaaccagag cccccagagc atgttcatcc aggagggcga ggacgtgtcc 60

atgaattgca ccagcagcag catcttcaac acttggctgt ggtacaagca ggacccagga 120

gagggaccag tgctgctgat tgccctgtac aaggccggag agctgacctc taacggcaga 180

ctgaccgctc agttcggcat caccaggaag gacagcttcc tcaacatcag cgccagcatc 240

cccagcgacg tcggaatcta cttttgcgcc ggccacccta gcagcaatag cggctacgcc 300

ctgaacttcg gcaagggcac aagcctgctg gtg aca cca 339

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<400> 2

ggatacacct ttaccaacta ttac

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<400> 3

attaacccaa gtaatggtgg taca

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence

<400> 4

acacggcgag attacaatta cgatggaggg ttcgactac

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<400> 5

aaatcagtct caacttctgg ttttaat

<210> 6

<211> 9

<212> DNA

<213> Artificial sequence

<400> 6

ctggcgtca

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<400> 7

caacatggac gggagctgcc cttgacg

<210> 8

<211> 88

<212> PRT

<213> Artificial sequence

<400> 8

Gly Gln Gln Leu Asn Gln Ser Pro Gln Ser Met Phe Ile Gln Glu

1 5 10 15

Gly Glu Asp Val Ser Met Asn Cys Thr Tyr Lys Ala Gly Glu Leu

1 20 25 30

Thr Ser Asn Gly Arg Leu Thr Ala Gln Phe Gly Ile Thr Arg Lys

35 40 45

Asp Ser Phe Leu Asn Ile Ser Ala Ser Ile Pro Ser Asp Val Gly

50 55 60

Ile Tyr Phe Cys Ala Gly His Pro Ser Ser Asn Ser Gly Tyr Ala

65 70 75

Leu Asn Phe Gly Lys Gly Thr Ser Leu Leu Val Thr Pro

80 85

<210> 9

<211> 8

<212> PRT

<213> Artificial sequence

<400> 9

Gly Tyr Thr Phe Thr Asn Tyr Tyr

1 5

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence

<400> 10

Asn Pro Ser Asn Gly Gly Thr

1 5

<210> 11

<211> 13

<212> PRT

<213> Artificial sequence

<400> 11

Thr Arg Arg Asp Tyr Asn Tyr Asp Gly Gly Phe Asp Tyr

1 5 10

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence

<400> 12

Lys Ser Val Ser Thr Ser Gly Phe Asn

1 5

<210> 13

<211> 3

<212> PRT

<213> Artificial sequence

<400> 13

Leu Ala Ser

1

<210> 14

<211> 9

<212> PRT

<213> Artificial sequence

<400> 14

Gln His Gly Arg Glu Lue Pro Leu Thr

1 5

Claims

1. An anti-PD-1 antibody, said anti-PD-1 antibody comprising the amino acid sequence:

the heavy chain CDR1 is shown in SEQ ID NO. 9;

CDR2 of heavy chain is shown in SEQ ID NO. 10;

CDR3 of heavy chain is shown in SEQ ID NO. 11;

CDR1 of the light chain is shown in SEQ ID NO. 12;

CDR2 of the light chain is shown in SEQ ID NO. 13; and

CDR3 of the light chain is shown in SEQ ID NO. 14.

2. The anti-PD-1 antibody of claim 1, which is an scFv.

3. The anti-PD-1 antibody according to claim 2, wherein said scFv comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein VH and VL are linked by a linker.

4. The anti-PD-1 antibody according to claim 3, characterized in that the order of linkage of VH and VL in the scFv is: VH-linker-VL.

5. The anti-PD-1 antibody according to claim 3, characterized in that the order of the linkage of VH and VL in the scFv is: VL-linker-VH.

6. The anti-PD-1 antibody according to any one of claims 3 to 5, the linker being a GS linker peptide.

7. A nucleic acid that blocks immune checkpoint function in a tumor, the nucleic acid comprising the sequence: nucleic acid encoding heavy chain CDR 1: as shown in SEQ ID NO. 2; nucleic acid encoding heavy chain CDR 2: as shown in SEQ ID NO. 3; nucleic acid encoding heavy chain CDR 3: as shown in SEQ ID NO. 4; nucleic acid encoding light chain CDR 1: as shown in SEQ ID NO. 5; nucleic acid encoding light chain CDR 2: as shown in SEQ ID NO. 6; and a nucleic acid encoding a light chain CDR 3: as shown in SEQ ID NO. 7; wherein the immune checkpoint is PD-1.

8. An engineered nucleic acid, comprising: (a) A nucleic acid of a genetically engineered antigen receptor, the antigen receptor expressed by the nucleic acid being capable of specifically binding an antigen from HPV, the nucleic acid sequence being SEQ ID NO:1; (b) A nucleic acid that expresses an anti-PD-1 antibody, the nucleic acid expressing an anti-PD-1 antibody comprising the amino acid sequence: CDR1 of heavy chain is shown as SEQ ID NO. 9; the heavy chain CDR2 is shown in SEQ ID NO.10, and the heavy chain CDR3 is shown in SEQ ID NO. 11; CDR1 of the light chain is shown as SEQ ID NO. 12; CDR2 of the light chain is shown as SEQ ID NO. 13; and CDR3 of the light chain is shown in SEQ ID NO. 14.

9. An engineered T cell, characterized by: comprising the engineered nucleic acid of claim 8.

10. Use of an engineered T cell according to claim 9 in the manufacture of a medicament for the treatment of cancer, said cancer being cervical cancer, head and neck cancer, oropharyngeal cancer, anal canal cancer, anorectal cancer, vaginal cancer, vulvar cancer, or penile cancer.

11. A method of producing engineered T cells, comprising the steps of: introducing the vector into isolated T cells to obtain TCR-T cells, and then performing in vitro amplification to obtain the engineered T cells; the vector comprising the engineered nucleic acid of claim 8.

12. The method of claim 11, wherein: the vector is a retroviral vector or a lentiviral vector.