CN117660358A - Engineered immune cells expressing secreted fusion proteins and uses thereof - Google Patents

Engineered immune cells expressing secreted fusion proteins and uses thereof Download PDF

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Publication number
CN117660358A
CN117660358A CN202410130289.2A CN202410130289A CN117660358A CN 117660358 A CN117660358 A CN 117660358A CN 202410130289 A CN202410130289 A CN 202410130289A CN 117660358 A CN117660358 A CN 117660358A
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Prior art keywords
til
cells
immune cell
engineered immune
fusion protein
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Inventor
于忠杰
许傲天
蒋彬
朱月姝
邵琳
刘文娜
李斌
宋宇
吴昊
赵毅
高青
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Qingdao Huasaiberman Medical Cell Biology Co ltd
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Qingdao Huasaiberman Medical Cell Biology Co ltd
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Abstract

The invention provides an engineering immune cell for expressing a secretion type fusion protein and application thereof. The invention relates to the technical field of genetic engineering and cell therapy. Specifically, the invention provides a fusion protein of IL-7 fused PD-L1 nano antibody, and expresses and secretes in TIL, which can effectively maintain the stem property of TIL cells, prevent TIL from exhaustion, shorten the distance between TIL cells and tumor cells, and enhance the killing capacity of TIL to tumor cells. In addition, the invention also constructs a promoter responding in a tumor microenvironment to start expression of the fusion protein, and simultaneously introduces TIGIT shRNA to effectively reduce TIL exhaustion and introduce a molecular switch to ensure the safety of TIL treatment.

Description

Engineered immune cells expressing secreted fusion proteins and uses thereof
Technical Field
The invention relates to the technical field of genetic engineering and cell therapy, in particular to an engineering immune cell for expressing secretory fusion protein and application thereof.
Background
Adoptive Cell Therapy (ACT) is a cancer immunotherapy that uses patient's own immune cells to recognize and eliminate tumor cells. Tumor Infiltrating Lymphocytes (TILs) are enriched for tumor-specific T cells, which are derived from the patient's tumor tissue, and are expanded in vitro and then returned to the patient as therapeutic agents. Clinical studies have been conducted worldwide with autologous TIL to treat tumors, and clinical results have demonstrated that TIL can lead to objective remission of various types of tumors, including melanoma, cervical squamous cell carcinoma, lung cancer, cholangiocarcinoma, and the like. However, the TIL treatment effect varies greatly from patient to patient due to tumor cell heterogeneity, tumor microenvironment, and the like.
Studies have shown that TIL composition and phenotype for ACT play an important role in determining therapeutic outcome. The ratio of CD8 and CD 4T cells in TIL, how many memory progenitor T cells have a stem cell-like phenotype, and the amount of stem TIL are highly correlated with clinical treatment outcome and patient prognosis. Therefore, how to generate more therapeutically effective TILs is a key to improving the therapeutic effectiveness of TILs.
At present, TIL treatment schemes need to clear stranguria on patients before TIL reinfusion and inject high dose IL-2 into the patients after TIL reinfusion to ensure the activity and the amplification capability of the TIL in the patients, but the stranguria-clearing drugs and the high dose IL-2 bring great side effects to the patients. Therefore, how to avoid the use of stranguria-clearing medicines and reduce the dosage of IL-2 used becomes the focus of next-generation TIL research and development.
Interleukin 7 (IL-7) is a cytokine essential for the adaptive immune system and is critical for B cell development, memory and proliferation and survival of naive T cells, and T cell development in the thymus. However, currently, in clinic IL-7 treatment of tumors is mainly performed by intravenous infusion, which may lead to toxic side effects caused by systemic exposure of high doses of IL-7; in addition, high doses of IL-7 or constitutive expression of IL-7 may lead to excessive accumulation of T cells. Therefore, how to use IL-7 at the inner part of tumor and control the usage of IL-7 in a proper range will be the focus of research on new therapies for treating tumor by IL-7.
Thus, there is an urgent need in the art to develop safely regulatable secreted IL-7 and its use in TIL cell therapy.
Disclosure of Invention
The invention aims to provide a safe and controllable secreted IL-7 fusion protein and application thereof in TIL cell therapy.
In a first aspect of the invention, there is provided an engineered immune cell expressing a secreted fusion protein comprising the following elements fused together:
(1) An IL-7 element, said IL-7 comprising full length IL-7 or an active fragment thereof; and
(2) anti-PD-L1 antibodies, including whole antibodies, fab fragments, single chain antibodies, nanobodies.
In another preferred embodiment, the immune cells are selected from the group consisting of: tumor Infiltrating Lymphocytes (TIL), T cells, NK cells.
In another preferred embodiment, the cells are Tumor Infiltrating Lymphocytes (TILs).
In another preferred embodiment, the tumor-infiltrating lymphocytes (TILs) are autologous.
In another preferred embodiment, the anti-PD-L1 antibody is a blocking anti-PD-L1 antibody.
In another preferred embodiment, the blocking anti-PD-L1 antibody blocks the binding of PD1 to PD-L1.
In another preferred embodiment, the blocking anti-PD-L1 antibody blocks the binding of CD80 to PD-L1.
In another preferred embodiment, the blocking anti-PD-L1 antibody blocks the binding of PD1 to PD-L1 and blocks the binding of CD80 to PD-L1.
In another preferred embodiment, the fusion protein has a structure as shown in formula (I):
X-L-Y (I)
wherein, X is the IL-7 element;
the L is none or a linker;
the Y is the antibody element;
"-" are each independently absent or linked peptides.
In another preferred embodiment, the IL-7 comprises full-length, mature forms of IL-7, or an active fragment thereof.
In another preferred embodiment, the IL-7 includes wild-type and mutant forms.
In another preferred embodiment, the sequence of IL-7 is shown in SEQ ID NO. 1.
In another preferred embodiment, the antibody is an anti-PD-L1 nanobody.
In another preferred embodiment, the anti-PD-L1 nanobody has a sequence as shown in SEQ ID NO. 2.
In another preferred embodiment, the connectors include flexible connectors and rigid connectors.
In another preferred embodiment, the connector is a flexible connector; preferably a GS linker consisting of glycine and serine.
In another preferred embodiment, the amino acid sequence of the linker is shown in SEQ ID NO. 3.
In another preferred embodiment, the fusion protein has the sequence shown in SEQ ID NO. 4.
In another preferred embodiment, the engineered immune cell contains a polynucleotide encoding the secreted fusion protein.
In another preferred embodiment, the polynucleotide comprises DNA, RNA, cRNA, or a combination thereof.
In another preferred embodiment, the engineered immune cell contains a nucleic acid construct, the first expression cassette of which comprises the coding sequence of the fusion protein.
In another preferred embodiment, the first expression cassette comprises a first promoter, which is a constitutive promoter or an inducible promoter.
In another preferred embodiment, the constitutive promoter is an SFFV promoter.
In another preferred embodiment, the first promoter is an inducible promoter, which is a promoter comprising the hypoxia responsive element HRE and/or the tumor microenvironment responsive element NFAT.
In another preferred embodiment, the inducible promoter is a promoter comprising n hypoxia responsive elements HREs, i.e. a promoter comprising n×hre elements, n being an integer selected from 1 to 9; preferably, n is an integer selected from 2 to 4; more preferably, n is 3.
In another preferred embodiment, the inducible promoter is selected from the group consisting of: TK-mini promoter containing n X HRE element, CMV-mini promoter containing n X HRE element, IL2-mini promoter containing n X HRE element.
In another preferred embodiment, the inducible promoter is the 3 XHRE TK-mini promoter.
In another preferred embodiment, the inducible promoter is a promoter comprising m tumor microenvironment responsive elements NFAT, i.e. a promoter comprising an mxnfat element, m being an integer selected from 1 to 12; preferably, m is an integer selected from 2 to 9; more preferably, m is 6.
In another preferred embodiment, the inducible promoter is an antigen inducible promoter comprising m NFAT elements, i.e., a promoter comprising m×nfat elements, m being an integer selected from 2 to 12; preferably, m is an integer selected from 3 to 9; more preferably, m is 6.
In another preferred embodiment, the inducible promoter is selected from the group consisting of: IL2-mini promoter containing an mNFAT element, TK-mini promoter containing an mNFAT element, CMV-mini promoter containing an mNFAT element.
In another preferred embodiment, the inducible promoter is an IL2-mini promoter containing a 6 XNFAT element.
In another preferred embodiment, the nucleic acid construct further comprises a coding sequence for an immunosuppressive molecular element selected from the group consisting of: TIGIT shRNA, LAG3, TIM3.
In another preferred embodiment, the immunosuppressive molecule is TIGIT shRNA.
In another preferred embodiment, the coding sequence of the immunosuppressive molecule element is located in a second expression cassette of the nucleic acid construct.
In another preferred embodiment, the second expression cassette comprises a second promoter, which is a constitutive promoter.
In another preferred embodiment, the second promoter is a U6 promoter.
In another preferred embodiment, the nucleic acid construct further comprises a coding sequence for a molecular switching element selected from the group consisting of: hEGFRt, BCMA, CD20.
In another preferred embodiment, the molecular switching element is hEGFRt.
In another preferred embodiment, the hEGFRt is a human EGFR extracellular segment third fourth domain.
In another preferred embodiment, the coding sequence of the molecular switching element is located in a first expression cassette of the nucleic acid construct and is linked to the coding sequence of the fusion protein of the first expression cassette by a cleavable linker peptide sequence.
In another preferred embodiment, the cleavable linking peptide is a self-cleaving 2A peptide; preferably a T2A peptide.
In another preferred embodiment, the coding sequence of the molecular switching element is located in a third expression cassette of the nucleic acid construct, the third expression cassette comprising a third promoter.
In another preferred embodiment, the third promoter is a constitutive promoter.
In another preferred embodiment, the engineered immune cell comprises a vector comprising a sequence selected from the group consisting of seq id no:
(Y1) the coding sequence of the fusion protein; or (b)
(Y2) the sequence of the nucleic acid construct.
In another preferred embodiment, the vector comprises a plasmid or a viral vector.
In another preferred embodiment, the viral vector comprises: lentiviral vectors, adenoviral vectors, yellow fever viral vectors.
In another preferred embodiment, the vector is a plasmid.
In a second aspect of the invention there is provided a composition comprising an engineered immune cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier.
In a third aspect of the invention, there is provided a kit comprising an engineered immune cell according to the first aspect of the invention, or a reagent for preparing an engineered immune cell according to the first aspect of the invention, wherein the reagent is selected from the group consisting of:
(Z1) a polynucleotide encoding said fusion protein; or (b)
(Z2) a vector comprising a polynucleotide encoding said fusion protein.
In a fourth aspect of the invention there is provided the use of an engineered immune cell according to the first aspect of the invention, or a kit according to the third aspect of the invention, for the preparation of a medicament for the treatment of a tumour.
In another preferred embodiment, the tumor is selected from the group consisting of: lung cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, ovarian cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a schematic representation of the activation of DC cells by engineered TIL cells expressing the invention and their killer tumor cells.
FIG. 2 shows a schematic structure of nucleic acid constructs expressed in 1#TIL, 2#TIL, 3#TIL and 4#TIL, respectively.
FIG. 3 shows the proportion of EGFR+ cells in each group of TIL cells detected by flow cytometry, ctr TIL representing the control non-engineered TIL cells.
FIG. 4 shows the efficiency of flow cytometry to detect TIGIT knockdown in each group of TIL cells, ctr TIL representing control TIL cells that were not engineered.
FIG. 5 shows the results of ELISA detection of IL-7 secretion by each group of TIL cells under different culture conditions, with Ctr TIL representing the non-engineered control TIL cells.
FIG. 6 shows the results of ELISA detection of IFN-gamma secretion after mixing of each group of TIL cells with tumor cells, ctr TIL representing the non-engineered control TIL cells.
FIG. 7 shows the results of cell proliferation experiments for each group of TIL cells, with Ctr TIL representing the control TIL cells that were not engineered.
FIG. 8 shows the proportion of stem cells in each set of TIL cells examined by flow cytometry, CD62L+CD45RA+ cells representing stem cells and Ctr TIL representing non-engineered control TIL cells.
FIG. 9 shows the effect and cytotoxicity of each group of TIL cells on tumor cell activity, with Ctr TIL representing the control TIL cells that were not engineered.
FIG. 10 shows the flow cytometry to detect activation of each set of TIL cells, CD25+CD69+ cells representing activated cells and Ctr TIL representing non-engineered control TIL cells.
FIG. 11 shows that flow cytometry detects apoptosis of 1#TIL cells, with Annxin V+ cells representing apoptotic cells and Ctr TIL representing non-engineered control TIL cells.
FIG. 12 shows apoptosis of # 1 in the presence of NK cells and antibodies.
Detailed Description
Through extensive and intensive studies, the present inventors have provided TIL cells expressing a secreted fusion protein, which is a fusion protein of an IL-7 fusion PD-L1 nanobody of novel structure. Experiments show that the fusion protein of the invention expresses and secretes in TIL, on one hand, can effectively promote the proliferation, activation and maintenance of TIL cell stem property of TIL cells, and on the other hand, can block the combination of PD-L1 and PD so as to prevent the depletion of TIL; meanwhile, one end of the fusion protein is combined with the TIL cells, and the other end of the fusion protein is combined with the tumor cells, so that the direct distance between the TIL cells and the tumor cells can be shortened, and the killing effect of the TIL on the tumor cells can be enhanced. The fusion protein of the invention not only can play the respective functions of IL-7 and anti-PD-L1 antibodies, but also can show the effect of synergistically enhancing the functions of TIL cells.
In addition, the fusion protein of the invention can be expressed in a tumor microenvironment by adopting an inducible promoter, such as a promoter containing an HRE element or an NFAT element, so that the fusion protein of the invention maintains higher concentration in a tumor part, and simultaneously reduces toxic and side effects caused by high-dose IL-7. In order to further reduce the depletion of TIL cells, TIGIT shRNA is also introduced, so that the expression level of TIGIT in TIL can be effectively knocked down, and the TIL depletion mediated by CD155/TIGIT channels is reduced. In order to further improve the safety, a hEGFRt molecular switch is also introduced into the TIL, and the apoptosis of the TIL secreting the IL-7 fusion PDL1 nano antibody can be specifically caused by ADCC/CDC action, so that the safety of TIL treatment is ensured. The present invention has been completed on the basis of this finding.
IL-7
Interleukin 7 (IL-7) is a glycoprotein with a molecular weight of 25 kDa, a cytokine essential for the adaptive immune system, and is critical for B cell development, memory and proliferation and survival of naive T cells, and T cell development in the thymus. IL-7 has strong immunoregulatory effect and is closely related to the development of tumors. Currently, IL-7 is clinically treated for tumors mainly by intravenous infusion, which may lead to toxic and side effects caused by systemic exposure of high doses of IL-7; in addition, high doses of IL-7 or constitutive expression of IL-7 may lead to excessive accumulation of T cells.
In the invention, IL-7 is fused with nano antibody, and the expression of the fusion protein is induced and started under the specific condition of tumor microenvironment, so that the IL-7 is enriched in tumor, higher concentration is maintained in local tumor, the growth of TIL in tumor tissue is effectively promoted, and the side effect of high dose IL-7 on normal tissue is reduced.
IL-7 as used herein includes full-length, mature forms of IL-7, or active fragments thereof, including wild-type and mutant forms.
In a preferred embodiment, the amino acid sequence of IL-7 is as follows:
MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 1)。
PD-L1
PD-L1 is expressed on cancer cells, dendritic cells, macrophages and B cells, especially at high levels on cancer cell membranes. The combination of the cancer cells PD-L1 and PD-1 triggers negative regulation signals, induces T cell apoptosis and damages immune functions, so that the cancer cells evade immune monitoring and killing. Blocking the combination of PD-L1 and PD-1 is a theoretical mechanism of PD-L1 inhibitor, and achieves the effects of eliminating negative regulation signals, recovering the functions of T cells and promoting the killing of cancer cells.
In the invention, an anti-PD-L1 antibody is fused with IL-7, and the anti-PD-L1 antibody is a blocking antibody, and the PD-L1 on the tumor cells cannot be combined with the PD-1 on the TIL cells by combining with the PD-L1 on the tumor cells, so that the TIL is effectively prevented from being exhausted, and the TIL activation state is maintained.
In addition, the blocking anti-PD-L1 antibodies of the invention also block the binding of PD-L1 on tumor cells to CD80 on DC cells, thereby allowing DC cells to activate TIL cells via the CD80/CD28 pathway (as shown in FIG. 1).
In a preferred embodiment, the anti-PD-L1 antibody is an anti-PD-L1 nanobody.
A typical anti-PD-L1 nanobody sequence is shown below:
QVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS (SEQ ID NO: 2)。
IL-7 fusion PD-L1 nanobody
As used herein, the terms "fusion protein of the invention", "secreted fusion protein of the invention" and "IL-7 fusion PD-L1 nanobody of the invention" are used interchangeably.
In a preferred embodiment, the fusion protein of the invention comprises: (1) an IL-7 element or an active fragment thereof; and (2) anti-PD-L1 antibody elements, including whole antibodies, fab fragments, single chain antibodies (scFv), nanobodies (single domain antibodies).
In a preferred embodiment, the antibody is an anti-PD-L1 nanobody.
In a preferred embodiment, the antibody is a blocking anti-PD-L1 nanobody.
In a preferred embodiment, the blocking anti-PD-L1 antibody blocks the binding of PD1 to PD-L1 and blocks the binding of CD80 to PD-L1.
A typical anti-PD-L1 nanobody has the sequence shown in SEQ ID NO. 2.
In a preferred embodiment, the fusion protein of the invention has a structure as shown in formula (I):
X-L-Y (I)
wherein, X is the IL-7 element;
the L is none or a linker;
the Y is the antibody element;
"-" are each independently absent or linked peptides.
Wherein the preferred connector is a flexible connector; more preferably a GS-linked peptide consisting of glycine and serine. In a preferred embodiment, the amino acid sequence of the linker is as follows:
SSGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 3)。
in a preferred embodiment, the fusion protein comprises from 5 'to 3' the following moieties: IL-7 shown in SEQ ID NO. 1, a linker shown in SEQ ID NO. 3, and a PD-L1 nanobody shown in SEQ ID NO. 2.
The fusion protein of the invention can fully exert the respective functions of IL-7 and PD-L1 antibodies, and has unexpected synergistic effect, namely, one end of the fusion protein of the invention is combined with IL-7 receptor (IL-7R) on TIL cells, and the other end is combined with PD-L1 on tumor cells, thereby shortening the distance between the TIL cells and the tumor cells (shown in figure 1) and enhancing the killing effect of the TIL cells on the tumor cells.
An exemplary fusion protein of the invention has the sequence shown below:
MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSSGGGSGGGGSGGGGSGGGGSGGGSQVQLVESGGGLVQPGGSLRLSCAASGKMSSRRCMAWFRQAPGKERERVAKLLTTSGSTYLADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAADSFEDPTCTLVTSSGAFQYWGQGTLVTVSS (SEQ ID NO: 4)。
TIGIT
TIGIT belongs to the poliovirus type 1 receptor (PVR), and is widely expressed in cd4+ T cells, cd8+ T cells and Treg cells as a co-inhibitory receptor. Ligands for TIGIT include CD155, CD113, CD112 and PVRL4. Functionally TIGIT/CD155 binding initiates an inhibitory signal through ITT-like motifs, TIGIT has been shown to play a crucial role in inducing immunosuppression. The activated CD155/TIGIT signaling pathway is significantly associated with tumor immune escape. Furthermore, due to the immunosuppressive properties of TIGIT, high levels of TIGIT are often predictive of a poor prognosis in solid tumors.
According to the invention, by introducing TIGIT shRNA, the expression level of T cell TIGIT is greatly knocked down, and the immune suppression induced by TIGIT is relieved.
Molecular switch
In order to further ensure the safety of the TIL after gene modification, the invention also provides a hEGFRt molecular switch. When TIL cells present a safety risk, the molecular switch element may be used as a target, and the at-risk TIL cells are cleared with the corresponding drugs according to ADCC and Complement Dependent Cytotoxicity (CDC) effect principles.
Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that regulates cell proliferation, apoptosis, angiogenesis and metastasis. EGFR is activated by EGF binding, further activating EGFR downstream signaling pathways. EGFR is highly expressed in various tumors, including lung cancer, colorectal cancer, head and neck cancer and the like, and becomes an important therapeutic target, and antibody drugs aiming at the EGFR target are widely applied to clinical tumor treatment, such as: cetuximab and imab. These antibodies inhibit tumor growth by inducing antibody-dependent cellular cytotoxicity (ADCC) and EGFR signaling inhibition.
In the present invention, the third and fourth domains of the extracellular region and the transmembrane region (hEGFRt) of human EGFR are expressed in TIL, and the activity of TIL is modified by cetuximab regulatory genes. When the safety risk of the TIL cells occurs, hEGFRt therapeutic monoclonal antibody cetuximab can be injected, and target cells are cleared through ADCC and CDC actions, so that the safety is improved; meanwhile, the flow detection of hEGFRt molecules can be utilized to indicate the cell positive rate of target gene integration.
Promoters
In the present invention, constitutive promoters and inducible promoters may be flexibly employed to promote expression of the fusion protein of the present invention as needed.
Constitutive promoters (Constitutive promoter) are capable of regulating gene expression to a degree that is substantially constant, so that there is no significant difference in the level of expression of the gene in different locations or tissues. Inducible promoters (Inducible promoter), i.e., under certain specific conditions or circumstances, the level of gene transcription is greatly increased under the control of such promoters.
In a preferred embodiment of the invention, the expression of the fusion protein of the invention is regulated using a hypoxia inducible promoter. Hypoxia is one of the features of tumor microenvironment, and is 2 to 9% O in normal tissues due to insufficient blood supply 2 Compared with the tumor microenvironment, the oxygen content is as low as 0.02% -2%. Hypoxia responsive elements (Hypoxia responsive element, HRE) are present in the promoters of hypoxia regulated target genes, sense changes in oxygen concentration in the environment, and regulate the expression levels of the target genes.
In a preferred embodiment, a promoter comprising n hypoxia responsive HRE elements is constructed, i.e. a promoter comprising n HRE elements, n being an integer selected from 1 to 9; preferably, n is an integer selected from 2 to 4; more preferably, n is 3.
In a preferred embodiment, the hypoxia inducible promoter is selected from the group consisting of: TK-mini promoter containing n X HRE element, CMV-mini promoter containing n X HRE element, IL2-mini promoter containing n X HRE element. In a preferred embodiment, the hypoxia inducible promoter is the 3 XHRE TK-mini promoter.
In a preferred embodiment of the invention, the expression of the fusion protein of the invention is regulated using a tumour antigen inducible promoter. Activated T cell Nuclear Factor (NFAT) can be activated by binding of a tumor antigen to a TCR, specifically responding to the tumor environment.
In a preferred embodiment, a promoter comprising m tumor antigen responsive elements NFAT, i.e. a promoter comprising m×nfat elements, m being an integer selected from 1 to 12; preferably, m is an integer selected from 2 to 9; more preferably, m is 6.
In a preferred embodiment, the tumor antigen responsive promoter is selected from the group consisting of: IL2-mini promoter containing an mNFAT element, TK-mini promoter containing an mNFAT element, CMV-mini promoter containing an mNFAT element. In a preferred embodiment, the tumor antigen responsive promoter is an IL2-mini promoter containing a 6 XNFAT element.
The invention utilizes the promoter containing HRE element and/or NFAT element to regulate the expression of the fusion protein, so that the fusion protein can be specifically and intensively expressed only in the tumor microenvironment, the local concentration of the tumor is increased, and the potential safety hazard caused by systemic exposure is avoided.
The nucleic acid construct of the invention
The invention also provides nucleic acid constructs that can express the fusion proteins, TIGIT shRNA and/or molecular switch of the invention.
The nucleic acid construct of the invention comprises a first expression cassette comprising a first promoter and a sequence encoding the fusion protein of the invention. In a preferred embodiment, the first promoter is a constitutive promoter or an inducible promoter, wherein the inducible promoter comprises a promoter comprising an HRE element and/or an NFAT element.
In a preferred embodiment, the nucleic acid construct of the invention further comprises a second expression cassette comprising a second promoter and a sequence encoding an immunosuppressive molecular element of the invention. The second promoter is a constitutive promoter and is a U6 promoter. Preferably, the immunosuppressive molecule is TIGIT shRNA.
In a preferred embodiment, the nucleic acid construct of the present invention further comprises a molecular switching element hEGFRt, the molecular switching element coding sequence being linked to the first expression cassette by a cleavable linker peptide, or the nucleic acid construct of the present invention comprises a third expression cassette comprising a third promoter and a coding element of a molecular switch, the third promoter being a constitutive promoter, including but not limited to SFFV promoters.
The vector of the invention
In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which other DNA segments may be ligated. Another class of vectors are viral vectors, wherein other DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating with the host genome, such as naked RNA polynucleotides that are not autonomously replicable, naked DNA polynucleotides, polynucleotides that consist of DNA and RNA in the same strand, poly-lysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, and the like. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector.
As used herein, "vector of the invention" refers to a vector containing a nucleic acid construct of the invention. Preferably, the vector of the invention is a plasmid.
Engineered TIL cells of the invention
As used herein, the term "engineered TIL cells of the invention" or "TIL cells of the invention" are used interchangeably, and each refer to a TIL cell capable of expressing a fusion protein of an IL-7 fusion PD-L1 nanobody of the invention and/or a TIL cell containing a vector of the invention.
The engineering TIL cell of the invention can express and secrete the IL-7 fusion PD-L1 nano antibody of the invention, which can effectively promote the proliferation of the TIL cell and promote T SCM The cell proportion is favorable for maintaining the stem property of TIL cells, and can also block the combination of PD-L1, PD1 and CD80, prevent the depletion of TIL and maintain the activation state of TIL. In addition, the IL-7 fusion PD-L1 nanobody can be secreted to be close to the distance between the TIL cells and the tumor cells, so that the TIL cells have more remarkable killing effect on the tumor cells.
Pharmaceutical compositions of the invention
The pharmaceutical compositions of the invention may comprise the fusion proteins of the invention or the immune effector cells of the invention (e.g., the engineered TIL cells of the invention) together with one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants. These compositions may be suitable for use in the treatment of the therapeutic indications described herein.
The composition may be a liquid solution, suspension, emulsion, sustained release formulation or powder, and may be formulated with a pharmaceutically acceptable carrier. The composition may be formulated as a suppository using conventional binders and carriers such as triglycerides. By "pharmaceutically acceptable carrier" is meant a carrier matrix or vehicle (vehicle) that does not interfere with the effectiveness of the biological activity of the active ingredient and does not produce toxicity to the host or subject.
The fusion protein or immune effector cell may be delivered with a pharmaceutically acceptable vehicle. In one embodiment, the vehicle may enhance stability and/or delivery properties. Vehicles such as artificial membrane vesicles (including liposomes, nonionic surfactant vesicles (noisomes), nanolipid vesicles, etc.), microparticles or microcapsules, or colloidal formulations comprising pharmaceutically acceptable polymers.
Pharmaceutical compositions comprising one or more fusion proteins or immune effector cells may be formulated into sterile injectable aqueous or oleaginous suspensions according to methods known in the art and using suitable dispersing or wetting agents and/or suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parent acceptable diluent or solvent.
In the present invention, the term "adjuvant" generally refers to any substance that aids or modulates the action of a drug, including but not limited to immunological adjuvants, which enhance or diversify the immune response to an antigen.
In the present invention, the term "subject" may be a mammal, such as a human or veterinary patient (e.g., a rodent, such as a mouse or rat, cat, dog, cow, horse, sheep, goat, or other livestock) in need of treatment. In some embodiments, the "subject" may be a clinical patient, a clinical trial volunteer, a laboratory animal, or the like. The subject may be suspected of having a disease characterized by cellular proliferation or having a disease characterized by cellular proliferation, diagnosed as having a disease characterized by cellular proliferation, or a control subject that is confirmed to not have a disease characterized by cellular proliferation, as described herein, diagnostic methods for a disease characterized by cellular proliferation and clinical demarcations of such diagnosis are known to those of skill in the art.
The pharmaceutical composition of the invention can be used for treating tumors. In the present invention, the term "tumor" or "tumor cell" generally refers to or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. Examples of tumors include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumor (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, and melanoma. "tumor cells" may further include "solid tumors", which refer to tumors selected from the group consisting of: gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatic carcinoma), anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumor, and head and neck cancer.
The main advantages of the invention include:
(1) IL-7 in the fusion protein can effectively promote proliferation of TIL cells and promote T SCM The cell proportion is favorable for maintaining the stem property of TIL cells.
(2) The anti-PD-L1 antibody in the fusion protein can be combined with PD-L1 on tumor cells, so that the combination of PD-L1 on tumor cells with PD-1 on TIL cells and CD80 on DC cells is blocked, TIL exhaustion can be effectively prevented, and a TIL activation state is maintained.
(3) The secreted IL-7 fusion PD-L1 antibody provided by the invention has one end combined with IL-7R on TIL cells and the other end combined with PD-L1 on tumor cells, so that the distance between the TIL cells and the tumor cells can be shortened, and the killing effect of the TIL on the tumor cells is enhanced.
(4) The nucleic acid construct adopts hypoxia inducible and antigen inducible promoters, and utilizes the tumor microenvironment to induce and express the fusion protein, so that the fusion protein can maintain higher concentration in tumor part, on one hand, the growth of TIL in tumor tissues is effectively promoted, and meanwhile, the side effect of high-dose IL-7 on normal tissues is reduced, and the controllability and the safety are increased.
(5) The TIGIT shRNA is introduced into the nucleic acid construct, so that the expression level of the TIGIT in the TIL can be effectively knocked down, and the TIL exhaustion mediated by CD155/TIGIT channels can be reduced.
(6) The hEGFRt molecular switch is introduced into the nucleic acid construct, and the apoptosis of the TIL secreting the IL-7 fusion PD-L1 nano antibody can be specifically caused by ADCC/CDC action, so that the safety of TIL treatment is ensured.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and fractions are percentages by number unless otherwise indicated.
The sequence information involved in the examples is shown in table a.
Nucleotide sequences of elements of Table A
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Example 1: lentivirus infected TIL cell containing target gene
1. Plasmid construction containing target Gene
The CDS sequences for IL-7, the third and fourth domains of the EGFR extracellular domain were downloaded from NCBI and the EGFR sequence-related cleavage site was mutated.
The schematic structure of nucleic acid constructs expressed on each of # 1, # 2, # 3 and # 4 TIL cells is shown in FIG. 2. The sequence of each nucleic acid construct is synthesized by gold Style company, and the synthesized gene sequence is cloned into a basic vector pLV-EF1a-c-MYC-IRES-EGFP (Wuhan vast ling) to obtain plasmids 1# and 2# and 3# and 4 #.
2. Transformation and plasmid extraction
Trans109 competent cells were removed from-80℃and rapidly inserted into ice, after thawing, the desired plasmids (1#, 2#,3#, 4#) were added, mixed well and allowed to stand in ice for 25 minutes. The mixture was heat-shocked in a 42℃water bath for 45 seconds, quickly put back on ice and left to stand for 2 minutes. 700. Mu.L of sterile LB medium without antibiotics was added, and after mixing well, resuscitated at 37℃for 70 minutes at 200 rpm. 5000 The cells were collected by centrifugation at rpm for 1 min and plated onto LB medium containing the corresponding antibiotics. The plates were placed in an incubator overnight at 37 ℃. The monoclonal was picked up and added to 1ml of LB medium containing antibiotics at 37℃at 200 rpm for 2 hours. The culture medium was grown overnight. 150 The cultured bacterial liquid was centrifuged for 10 minutes and the bacterial cells were collected. Resuspension of the bacterial pellet with 14ml solution P1; adding 14 and ml of solution P2 to fully lyse the thalli, and standing at room temperature for 2-3 minutes; add 14ml precooled solution P3, mix well, centrifuge for 5 minutes, collect filtrate. To the filtrate, 14ml of plasmid DNA conjugate was added, mixed well, centrifuged for 2 minutes, and the mixture was removed. 10ml of plasmid washing was added, centrifuged for 2 minutes, and the waste solution was discarded. 10ml of plasmid DNA washing 2 (absolute ethanol added) was added, centrifuged for 2 minutes, and the waste solution was discarded. The above steps are repeated. The residual ethanol was removed and the plasmid DNA eluted. The endotoxin-free plasmid DNA was eluted by centrifugation for 1 min through an endotoxin removal column. The resulting plasmid was transferred to a 1.5ml centrifuge tube.
3. Lentivirus packaging and harvesting
293T cells (100 mm dish culture) were observed under a microscope and virus packaging was performed at a cell fusion of 70% -80%. The transfection system was configured as follows: (1) centrifuge tube A: opti-MEM 500. Mu.l+Master plasmid 10. Mu.g+PMD2G5g+PSPAX 2 5. Mu.g+p 3000 40. Mu.l; (2) centrifuge tube B: OPti-MEM 500. Mu.l+lipo 3000 40. Mu.l. Removal of cells and addition of transfection System along the wall(1 ml per dish), mixing, and adding CO 2 Culturing is continued in the incubator. After 4 hours of transfection, cells were removed, the supernatant was discarded, and 8 ml of high-sugar DMEM medium containing 5% FBS and 1% diabody was added to each dish to continue culturing. 24 After h, the transfected 293T cell culture supernatant was centrifuged for 5 min, filtered with a 0.45 μm filter membrane and the resulting lentivirus venom was added to a ultrafiltration tube for ultrafiltration. Collecting the concentrated lentivirus, split charging, and storing in a refrigerator at-80deg.C.
4. Preparation of TIL cells, infection and activation of TIL cells by lentiviruses
Placing the lung cancer tumor mass in a 100 mm culture dish, and washing with PBS; removing necrotic areas and connective tissue from tumor mass with sterile ophthalmic scissors or surgical knife, and cutting into 1-3 mm pieces 3 The small blocks are placed in a culture medium for culture. Observing cells under a microscope, and changing liquid every other day if no obvious adherent cells exist; if the lymphocytes are not gradually increased or the cancer cells are not reduced, continuing to change the liquid; if lymphocyte density is increased obviously, if cell mass appears, the cell mass is placed at 37 ℃ after reaming operation is completed, and 5% CO is added 2 The culture incubator continues to culture; cell collection was performed by continuing the culture for no more than 10 days. The collected cell suspension is filtered by a filter screen with the size of 40 mu m, and tumor blocks are removed; 1000 Centrifuging at rpm for 5min, and adding appropriate amount of culture medium for resuspension.
Virus infects TIL cells at a ratio of moi=10, cell number 9×10 5 And the volume of the Lenntiboost is 6 mu l, and the total infection system is 600 mu l. Taking a 1.5ml centrifuge tube for each group of samples, respectively adding the Lenntiboost, the lentivirus and the TIL cells into the centrifuge tube, uniformly mixing, and putting into CO 2 Culturing in an incubator. 24 After h, cells were collected, centrifuged at 1000 rpm for 5 minutes, resuspended in 1 ml REP complete medium and transferred to a new 24-well plate for further culture. 72 After h, cells were collected, centrifuged at 1000 rpm for 5 minutes, resuspended in 1 ml REP complete medium and counted. Each group takes 5X 10 5 Transfer to a new 24-well plate, and perform conditioned culture: culturing normally 1# and 4 #; 2# 95% N in use 2 、5%CO 2 Replacement O 2 Simulating the culture in the anoxic microenvironment of the tumor; 3# to which CD3 (Yiqiaoshenzhou 10977-M001) is addedAnd anti-CD 28 (Yinqiao Shenzhou 11524-MM 06) monoclonal antibodies. Cell count after 24. 24 h culture was taken at 5X 10 5 Individual cells were subjected to flow assays and supernatants were subsequently subjected to ELISA assays. The remaining cells were continued to be cultured to obtain four kinds of engineered TIL cells of 1#TIL, 2#TIL, 3#TIL and 4#TIL, respectively, and the normal cultured TIL without engineering was used as a control (Ctr TIL).
Example 2: flow cytometry detection of lentiviral infection TIL efficiency
The cells prepared in example 1 were taken 5X 10 per group 5 And centrifuging for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 ml of 2% FBS in PBS and centrifuged at 400g for 5 minutes. The supernatant was discarded, cells were resuspended with 50 μl of 2% FBS in PBS, followed by the addition of Fc block to block Fc receptors (every 1×10 6 Each cell was incubated with 5 μl Fc block) for 10 minutes at room temperature. Half of the cells transferred out of the control group served as blank control, and the antibody group cells were added with a primary antibody cocktail (cetuximab-specific recognition of hEGFRt, merk Healthcare KGaA, C10010069), incubated at room temperature for 15 min, followed by centrifugation at 400g for 5 min. The supernatant was discarded, 1 ml of 2% FBS in PBS was added, and the mixture was centrifuged at 400g for 5 minutes. 50 μl of a secondary antibody cocktail prepared with PBS containing 2% FBS (FITC F (ab') 2 Goat anti-human IgG fcγ, biolegend, 398006), for 10 minutes at room temperature. 1 ml of PBS with 2% FBS was added and centrifuged at 400g for 5 minutes. The supernatant was discarded and 50 μl of 7-AAD dye (per 1×10) formulated in PBS with 2% FBS was added to the antibody panel cells 6 5 μl of each cell was used), and incubated at room temperature for 7 minutes. 1 ml of PBS with 2% FBS was added and centrifuged at 400g for 5 minutes. The supernatant was discarded, cells were resuspended in 200 μl of 2% FBS in PBS and checked on-machine.
The results are shown in FIG. 3, showing, for the flow data, EGFR positive rates for 1#TIL, 2#TIL and 3#TIL, respectively, as follows: 59.1%, 55.7%, 58.6%, all with high level of positive rate.
Example 3: flow cytometry to detect TIGIT expression levels in each group of TIL cells
The cells prepared in example 1 were taken 5X 10 per group 5 400 and g are centrifuged for 5 minutes; the supernatant was discarded, and the cells were resuspended in 1 ml of 2% FBS in PBS and centrifuged at 400g for 5 minutes. Discard supernatant, use 50 μl of 2% FBSPBS resuspended cells, followed by Fc block blocking Fc receptors (every 1×10 6 Each cell was incubated with 5 μl Fc block) for 10 minutes at room temperature. Half of the cells transferred from the control group served as blank, 50. Mu.l of diluted antibody (anti-human CD45RA, biolegend,304120; anti-human CD62L, biolegend,304810; anti-human TIGIT, biolegend, 372704, 1X 10) was added to the remaining cells 6 The individual cells used 5 μl antibody; wherein anti-human CD45RA and anti-human CD62L are indicators of cell stem property detection), for 8 minutes at room temperature. 50 μl of 7-AAD dye in PBS with 2% FBS (7-AAD cell viability staining solution, biolegend,420404. Per 1×10 6 5 μl of each cell was used), and incubated at room temperature for 7 minutes. 1 ml of PBS with 2% FBS was added and centrifuged at 400g for 5 minutes. The supernatant was discarded, cells were resuspended in 200 μl of 2% FBS in PBS and checked on-machine.
The results are shown in FIG. 4, which shows that 1#TIL, 2#TIL, 3#TIL and 4#TIL all significantly reduced the expression level of TIGIT in the cells compared to control TIL cells (Ctr TIL).
Example 4: cytokine secretion assay in TIL cells of each group
The secretion of IL-7 and IFN-gamma in each group of TIL cells was detected by ELISA using the following kits: human IL-7 pre-ELISA kit (Dake, 2307-1), human IFN-gamma pre-ELISA kit (Dake, 2307-3). All reagent consumables of the experiment are self-contained in the kit, and the operation is carried out according to the instruction of the kit
Sample adding: adding diluted cytokine standard to a standard hole, diluting a sample with a dilution buffer R (1×), and adding the sample to the sample hole, wherein the sample is 100 μl/well. Cover the sealing plate membrane and incubate for 2 hours at room temperature. Washing the plate: buckling and removing liquid in the holes, and adding 1X washing buffer working solution, wherein the volume is 300 [ mu ] l/well; after 1 minute of residence, the liquid in the wells was discarded. The procedure was repeated 3 times, each time with a filter paper for drying. Adding a detection antibody: biotinylated antibody working fluid, 100 μl/well, was added. Cover the sealing plate membrane and incubate for 1 hour at room temperature. The plate was repeatedly washed. Adding enzyme: and adding streptavidin-HRP working solution, wherein the ratio is 100 mu l/well. The plate was covered and incubated at room temperature (18-25 ℃) for 30 minutes. The plate was repeatedly washed. Color development: TMB,100 μl/well, was added, incubated at room temperature for 5-30 min in the dark, and termination was determined by the depth of color (dark blue) in the wells. Usually, the color development can be carried out for 10-20 minutes to achieve good effect. Terminating the reaction: and rapidly adding a stop solution, namely 100 mu l/well, and stopping the reaction. The OD value is read by a microplate reader at 450 nm. The data were analyzed by four-parameter method.
The results of the IL-7 secretion amount detection are shown in FIG. 5, and the 1#TIL adopts a constitutive promoter, so that the secretion amount is the highest; for 2#TIL and 3#TIL using inducible promoters, the amount of IL-7 secreted under normal conditions was comparable to the control TIL (Ctr TIL), whereas the amount of IL-7 secreted under conditioned culture was significantly increased.
The results of IFN-. Gamma.secretion assays are shown in FIG. 6, where the secretion amounts of 1#,2# and 3# TILs IFN-. Gamma.are significantly higher than the control TILs (Ctr TILs).
Example 5: expansion ability and Stem Properties of TIL cells of each group
Culturing TIL cells from each group obtained in example 1 to adjust the cell density to 2X 10 6 /ml. 200 μl of cell fluid was inoculated per well. Normal REP medium was supplemented every three days and the conditions were treated every 7 days. Cell counts were performed after 14 days and the proportion of stem cells in 1#til cells was examined using flow cytometry.
As shown in FIG. 7, the results of the cell expansion experiments show that the proliferation multiples of 1#TIL, 2#TIL and 3#TIL are significantly improved compared with the control TIL (Ctr TIL), and the 4#TIL has no significant difference, which indicates that the IL7 fusion PDL1 nanobody of the invention has unexpected effect of improving the proliferation capacity of cells.
The results of the cell stem property detection are shown in fig. 8, and the low differentiation and stem (cd45ra+cd62l+) phenotype in 1#til, 2#til and 3#til is unexpectedly and significantly increased, while 4#til has no significant difference, which indicates that the IL7 fusion PDL1 nanobody of the present invention has a significant effect of maintaining cell stem property.
Example 6: tumor killing ability of TIL of each group
Taking 1×10 5 Each tumor cell Calu-6 (ATCC, HTB-56 ™) was inoculated in a 24-well plate. After 24 hours, TIL cells were diluted to a density of 1.5X10 6 /ml. The culture medium in the original wells of the 24-well plate was pipetted off. The examples were added as TIL: tumor cells=5:1Each set of TILs prepared in 1. After 24 hours, the culture medium in the well was aspirated, and the cell supernatant was centrifuged for ELISA detection. The cells in the wells were washed with PBS and the tumor cell activity was measured by CCK8 (Meinauguration, MA 0218-5).
The results are shown in FIG. 9, table 1 and Table 2. The killing capacity of 1#, 2#, and 3# -TIL on tumor cells was unexpectedly and significantly higher than that of control TIL (Ctr TIL), while the killing capacity of 4# -TIL was not significantly different from that of control TIL, thus it can be demonstrated that IL7 fusion PDL1 nanobody expressed on 1#, 2#, and 3# -TIL can effectively enhance the killing capacity of TIL cells.
TABLE 1 percent survival of tumor cells (%)
TABLE 2 cytotoxicity (%)
Example 7: activation of TIL cells by DC cells
Control TIL (Ctr TIL) and 1#TIL cells of the invention were co-cultured with tumor cells and DC cells in a ratio of 1:2:2, respectively, for 24 hours; each group takes 1X 10 6 Centrifuging the cells for 5 minutes; discarding the supernatant, resuspending the cells, and centrifuging for 5 minutes; the addition of Fc block blocked Fc receptors (every 1X 10 6 Cells were incubated with 5 μl Fc block) for 10 min at room temperature; half of the cells transferred from the control group served as blank, 50. Mu.l of diluted antibody (anti-human CD69 antibody, biolegend,310910; anti-human CD25 antibody, biolegend,302622, every 1X 10) was added to the remaining cells 6 Cells using 5 μl antibody), 8 minutes incubation at room temperature; 7-AAD dye (7-AAD active staining solution, biolegend,420404. Per 1X 10) formulated with PBS with 2% FBS was added 50 μl 6 5 μl of cells were used), incubated at room temperature for 7 min; 1 ml of PBS with 2% FBS was added, and 400g was centrifuged for 5 minutes; the supernatant was discarded, cells were resuspended in 200 μl of 2% FBS in PBS, and flow cytometry was performed.
The results are shown in FIG. 10. Unexpectedly, 1#til was significantly increased in proportion to activated cells (cd25+cd69+) after co-culturing with DC cells and tumor cells compared to control TIL, which resulted in a 55.6% activated cells in 1#til and 16.5% control TIL cells, indicating that IL-7 fusion PD-L1 nanobody secreted by 1#til can block tumor cells PD-L1, blocking its binding to CD80 on DC cells, thereby allowing DC cells to activate TIL cells via CD80/CD28 pathway.
Example 8: hEGFRt specific molecular switch action validation
1. Complement dependent cytotoxicity assay (CDC)
Complement dependent cytotoxicity assays (CDCs) are processes in which the Fab fragment of an antibody binds to a target cell, the Fc fragment of an antibody binds to a complement molecule and activates the complement molecule to form an tapping complex, resulting in lysis of the target cell. In the invention, hEGFRt expressed by TIL cells is combined with cetuximab and combined with an added complement molecule, so that the TIL cells can be cracked.
Regulating TIL cell density to 2×10 6 Per ml, 1X 10 per group 6 TIL cells were then supplemented with 25% complement (young rabbit complement, tissue culture grade, frozen, RUO Cedarlane CL 3441-S-R), 100. Mu.g/mL cetuximab. And (5) after fully and uniformly mixing, placing the mixture in an incubator and standing the mixture. Apoptosis (PE-coupled Annexin-V apoptosis assay kit, bi di biopharmaceutical, 559763) levels were measured after 24 hours.
As shown in fig. 11, the TIL cell background apoptosis rate was 5.03%, the 1#til apoptosis rate was 43.4% in the presence of complement and cetuximab, and the apoptosis rate was increased about 8-fold, indicating that the hEGFRt expressed on the surface of the 1#til cells was able to act as a molecular switch, CDC effect occurred in the presence of antibody and complement, and no significant change in apoptosis rate was observed in the control TIL cells. Because 2#til and 3#til express substantially the same amount of hEGFRt on the cell surface, hEGFRt also has a high level of apoptosis rate in the presence of complement and cetuximab, and hEGFRt can also act as a molecular switch on 2#til and 3#til.
2. Antibody dependent cellular cytotoxicity Assay (ADCC)
Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to the binding of the Fab fragment of an antibody to an epitope of a virus-infected or tumor cell, and its Fc fragment binds to the Fc receptor on the surface of a killer cell (NK cell, macrophage, neutrophil, etc.), mediating the direct killing of the target cell by the killer cell, and is an important mechanism for the action of therapeutic antibody drugs against tumors. Binding of EGFR to cetuximab results in target cell death, whereas rituximab does not bind to EGFR and does not cause cell death, thus demonstrating the specificity of the design as a control.
NK cells were isolated from peripheral blood of healthy volunteers according to the protocol of the specification using NK cell culture kit (purchased from Fujian Sanyi blood manufacturing technologies Co., ltd., CT-001), amplified to obtain NK cells, and labeled with blue viable cell fluorescent probes. Target cell 1#TIL was resuspended with medium and the cell density was adjusted to 5X 10 5 Cells/ml, 1ml was added to a 6-well plate. Cetuximab and rituximab (roche diagnostics, gmbH, H0334) were added to a final concentration of 200 μg/ml, respectively, and after mixing, incubated at room temperature for 40 minutes. Resuspension of NK cells with medium, adjustment of cell density to 5X 10 6 1mL of cells/mL is added into a 6-hole plate, so that the effective target ratio E:T reaches 10:1, the final concentration of cetuximab in the system is 100 mug/mL, and the total volume of the system is 2mL. After the system is evenly mixed, the mixture is placed in an incubator for normal culture for 24 hours. Apoptosis (PE-coupled Annexin-V apoptosis assay kit, bi di biopharmaceutical, 559763) levels were measured after 24 hours.
As shown in fig. 12, the background apoptosis rate after adding 1#til to NK cells was about 5%, the apoptosis rate of 1#til in the presence of NK cells and cetuximab was about 38%, and the apoptosis rate of 1#til in the presence of NK cells and rituximab was only about 6%. Experimental results prove that the hEGFRt expressed on the surface of the 1#TIL cells has a specific molecular switching effect.
Discussion of the invention
The nucleic acid construct of the invention adopts various constitutive or inducible promoters to induce the expression of the fusion protein of the invention, and can be applied to different scenes.
The constitutive promoter can be used for killing tumor cells in blood circulation, inhibiting tumor diffusion, and also can be used for cancer patients with advanced systemic diffusion, such as constitutive promoter SFFV used in 1#TIL, and can stably express the IL-7 fusion PD-L1 nanobody for a long time;
The hypoxia inducible and antigen inducible promoters utilize tumor microenvironment to induce and express IL-7 fusion PD-L1 nano antibodies, so that the IL-7 fusion PD-L1 nano antibodies maintain higher concentration in tumor parts, reverse immunosuppression signals, and enrich IL-7 in tumor tissues locally, on one hand, effectively promote the growth of TIL in the tumor tissues, and simultaneously reduce possible side effects caused by IL-7. For example, the 3 XHRE promoter adopted in the 2#TIL and the 6 XNFAT promoter adopted in the 3#TIL can be used for expressing the IL-7 fusion PD-L1 nanobody of the invention locally in tumors, thereby increasing the controllability and reducing the potential side effects.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (11)

1. An engineered immune cell expressing a secreted fusion protein, wherein the fusion protein comprises the following elements fused together:
(1) An IL-7 element; and
(2) anti-PD-L1 antibody elements, including nanobodies, whole antibodies, fab fragments, single chain antibodies;
wherein the immune cells are selected from the group consisting of: tumor Infiltrating Lymphocytes (TIL), T cells, NK cells.
2. The engineered immune cell of claim 1, wherein the fusion protein has a structure according to formula (I):
X-L-Y (I)
in the method, in the process of the invention,
x is the IL-7 element;
l is none or a linker;
y is the antibody element, and the antibody is an anti-PD-L1 nano antibody;
"-" are each independently absent or linked peptides.
3. The engineered immune cell of claim 2, wherein the sequence of the IL-7 element is shown in SEQ ID No. 1 and the sequence of the anti-PD-L1 nanobody is shown in SEQ ID No. 2.
4. The engineered immune cell of claim 1, wherein the engineered immune cell comprises a nucleic acid construct, a first expression cassette of which comprises a coding sequence for the fusion protein.
5. The engineered immune cell of claim 4, wherein the first expression cassette comprises a first promoter that is an inducible promoter that is a promoter comprising the hypoxia-responsive element HRE and/or the tumor microenvironment-responsive element NFAT.
6. The engineered immune cell of claim 4, wherein the nucleic acid construct further comprises a coding sequence for an immunosuppressive molecular element comprising TIGIT shRNA.
7. The engineered immune cell of claim 4, wherein the nucleic acid construct further comprises a coding sequence for a molecular switching element selected from the group consisting of: hEGFRt, BCMA, CD20.
8. The engineered immune cell of claim 4, wherein the engineered immune cell comprises a vector comprising a sequence selected from the group consisting of seq id nos:
(Y1) the coding sequence of the fusion protein; or (b)
(Y2) the sequence of the nucleic acid construct.
9. A composition comprising the engineered immune cell of claim 1, and a pharmaceutically acceptable carrier.
10. A kit comprising the engineered immune cell of claim 1, or a reagent for preparing the engineered immune cell of claim 1, wherein the reagent is selected from the group consisting of:
(Z1) a polynucleotide encoding a secreted fusion protein; or (b)
(Z2) a vector comprising a polynucleotide encoding a secreted fusion protein.
11. Use of an engineered immune cell of claim 1, or a kit of claim 10, for the preparation of a medicament for the treatment of a tumor.
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姜茹斌等: ""肿瘤细胞中PD-L1表达调控机制的研究进展"", 《中国细胞生物学学报》, vol. 43, no. 12, 31 December 2021 (2021-12-31), pages 2421 - 2432 *

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