Nucleic acid structures

Abstract

This invention provides constructs and methods for modulating the relative expression of transgenes. [Solution] In a first aspect, the present invention provides a nucleic acid construct comprising a first-objective nucleotide sequence (NOI1); a flame slip motif or translational read-through motif (FSM / TRM); and a second-objective nucleotide sequence (NOI2). The nucleic acid construct may also include nucleotide sequences encoding cleavage sites (CLs) such that NOI1 and NOI2 are expressed as distinct proteins.

Description

[Technical Field]

[0001] The present invention relates to constructs and methods for modulating the relative expression of transgenes. [Background technology]

[0002] Expression of multiple transgenes Gene therapy involves modifying cells to express biomolecules that treat or correct pathological symptoms. Obtaining either physiologically or therapeutically relevant levels of biomolecules is crucial for the success of gene therapy.

[0003] Current methods for modulating gene expression generally rely on modifying promoter regions to make their expression inducible through the action of engineered transcription factors and small molecules, by increasing or decreasing the transcription rate or by inserting regulatory elements into them.

[0004] Gene therapy approaches often involve the expression of more than one transgene. Transduction of multiple vectors into cells to produce multiple products is difficult, costly, and unpredictable. For this reason, various methods have been developed to enable the co-expression of two proteins from a single vector (see Figure 1).

[0005] The initial attempt used two different promoters within the same cassette, which resulted in two distinct transcripts, each encoding a distinct protein. For several reasons, this is a challenging approach. A key issue is "promoter interference," where the first promoter is dominant, causing silencing of the second promoter. In addition, different promoters function differently in different cellular conditions, which makes it difficult to achieve consistent "tuning" of the relative expression of each transgene.

[0006] An alternative approach involves using an internal ribosome entry sequence (IRES), which generates a single transcript. The IRES sequence in the transcript is positioned between the open reading frames of two transgenes, mimicking an mRNA cap structure. Thus, the ribosome initiates translation at either the 5' cap or the IRES, resulting in the expression of two distinct proteins. A significant limitation of this method is the inability to control relative expression. While the 3' transcript is typically expressed less than the 5' one, the expression ratio is difficult to predict and adjust.

[0007] Following the elucidation of the role of the FMDV 2A peptide in enabling foot-and-mouth disease virus (FMDV) (and related viruses) to express multiple proteins from a single open reading frame (ORF), further approaches were developed (Donnelly et al., J. Gen. Virol.; 82, 1027-1041 (2001)). The 2A peptide (and its homolog) is cleaved with very high efficiency immediately after translation of the ORF, enabling the expression of multiple peptides from a single ORF. The use of self-cleaving peptides such as the 2A peptide results in the expression of transgenes in a 1:1 ratio.

[0008] International Publication No. 2016 / 174409 describes the use of modified signal peptides to alter the ratio of transgene expression within nucleic acid constructs. In this system, mutations are introduced, for example, into the signal peptide motif of a type I transmembrane protein, to reduce its effectiveness in transporting to the cell surface. This approach allows for an approximately 10-fold reduction in cell surface expression levels compared to the wild-type signal peptide motif.

[0009] However, this strategy works by reducing protein transport to the cell surface, and is therefore only suitable for reducing the expression of cell surface proteins. Furthermore, a tenfold reduction in expression may not be sufficient for some applications, such as the expression of toxic compounds whose overexpression can lead to host cell death.

[0010] Therefore, an alternative approach is needed for modifying the relative expression of two or more transgenes in cells, an alternative approach that does not have these limitations.

[0011] Chimeric antigen receptor (CAR) Chimeric antigen receptor T cell (CAR-T cell) therapy redirects cytolytic T cells to target tumor cells through the expression of chimeric antigen receptors (CARs) that recognize tumor-specific antigens on the cell surface. Chimeric antigen receptors consist of an antibody or ligand-binding motif, a spacer domain, and a transmembrane domain fused to an intracellular signaling domain derived from the CD3ζ chain of the T cell receptor (TCR). Co-receptor-derived signaling domains such as CD28, OX40, or 4-1BB may also be included in CAR1 along with the CD3ζ signaling domain.

[0012] CAR-T cell therapy has demonstrated itself to be an effective treatment for hematological malignancies such as B-cell leukemia, achieving impressive response rates. However, in the treatment of solid tumors, where multiple factors combine to limit CAR-T cell activity, CAR-T cell therapy has not achieved the same success. These include the expression of immunosuppressive ligands such as PD-L1, the secretion of cytokines that mitigate favorable inflammatory responses, the expression of ligands that induce apoptosis by tumor cells, and nutrient depletion. To overcome the immunosuppressive tumor microenvironment, CAR-T cells have been engineered to express molecules that either block immunosuppressive signaling or allow T cells to survive in the unfavorable tumor microenvironment.

[0013] Cytokines that enhance the inflammatory response may increase the efficacy of CAR-T cell therapy. These include IL-7, IL-12, IL-15, IL-17A, IL-18, and IL-21, which have been shown to enhance the CAR-T cell response when provided externally or released during CAR-T cell therapy.

[0014] IL-12 is a potent immunomodulatory cytokine, normally secreted by phagocytes and dendritic cells in response to T and natural killer (NK) cell signaling, as well as extracellular matrix components. CAR-T cells have been engineered to express IL-12 from constitutive or inducible promoters, which has been shown to improve the efficacy of CAR-T cell therapy when targeting solid tumors. Because IL-12 is systemically toxic, CAR-T cells or other immune cells have been engineered to release IL-12 into the tumor microenvironment. However, IL-12 secretion by transgenic T cells can result in highly toxic systemic levels.

[0015] The expression of cytokines, chemokines, or toxins can improve the efficacy of CAR-T cell therapy, but the secretion of these biomolecules must be strictly regulated to limit toxicity within safe levels that provide therapeutic benefits and minimal side effects. This can be achieved by inserting or deleting cis-acting sequences to obtain desired levels of transcription, or by regulating the rate of gene transcription from the promoter region by introducing inducible elements into the promoter region. Notable inducible promoter systems include the tetracycline-off and tetracycline-off systems, which utilize TRE in conjunction with an engineered form of tetracycline repressor protein (TetR) that binds to the 19 bp nucleotide sequence of tetracycline response element (TRE) 2. In the tetracycline-off system, TetR is fused to the transactivation domain of herpes simplex virus-derived viron protein 16 (VP16) to generate a tetracycline transactivator (tTA). In the absence of tetracycline, tTA binds to TRE in the promoter region and enhances gene transcription; conversely, in the presence of tetracycline, tTA cannot bind to TRE, and transcription does not occur. By mutating the TetR domain in tTA to make its binding to TRE tetracycline-dependent, inverse tTA (rtTA) and tetracycline were created on the system. In the presence of tetracycline, rtTA can bind to TRE and stimulate transcription, but not in its absence. While the tetracycline-inducible system allows for the regulation of transgene expression, it relies on the use of small molecules, insertion of TRE into the promoter sequence, and the expression of either tTA or rtTA to do so, which places a significant burden on the cell.

[0016] An alternative approach to controlling gene expression is to use internal ribosome entry sites (IRESs). As described above, IRESs are located downstream of the primary transgene and promote the constitutive expression of further transgenes, usually at lower levels than the primary transgene. To enhance the immune response and avoid the need for host preconditioning before CAR-T cell injection, such an approach has been used to constitutively express IL-12 in CAR-T cells. However, IL-12 expression levels are unpredictable and can still be toxic in vivo.

[0017] Therefore, alternative methods are needed to co-express chimeric antigen receptors with molecules unrelated to the aforementioned disadvantages, such as cytokines, chemokines, or toxins. [Overview of the project] [Means for solving the problem]

[0018] The inventors have developed a system that uses flame slip or translational read-through as a means of modulating the expression of a transgene. In particular, the system can be used to control the relative expression of two or more transgenes expressed from a single mRNA transcript.

[0019] Accordingly, in a first aspect, the present invention provides a nucleic acid construct comprising a first-objective nucleotide sequence (NOI1); a flame slip motif or translational read-through motif (FSM / TRM); and a second-objective nucleotide sequence (NOI2).

[0020] The nucleic acid construct may also contain nucleotide sequences encoding cleavage sites (CLs) so that NOI1 and NOI2 are expressed as distinct proteins.

[0021] Nucleic acid constructs have the following structure: NOI1-FSM / TRM-CL-NOI2 or NOI1-CL-FSM / TRM-NOI2 may include.

[0022] In particular, the nucleic acid construct has the structure: NOI1-CL-FSM-NOI2; or NOI1-TRM-CL-NOI2 includes.

[0023] The nucleic acid construct may contain nucleotide sequences for more than two purposes. For example, the nucleic acid construct has the structure: NOI1-FSM1 / TRM1-CL1-NOI2-FSM2 / TRM2-CL2-NOI3, or NOI1-CL1-FSM1 / TRM1-NOI2-CL2-FSM2 / TRM2-NOI3 (where NOI1, NOI2 and NOI3 are nucleotide sequences of interest; FSM1 / TRM1 and FSM2 / TRM2 may be the same or different and are frameshift motifs or translation readthrough motifs; and CL1 and CL2 may be the same or different and are nucleic acid sequences encoding cleavage sites respectively) may have.

[0024] The frameshift motif (FSM) may include a repeat of uracil, thymine or guanine bases, such as the sequence UUUUUUU (SEQ ID NO: 1).

[0025] The frameshift motif may also include a stop codon. For example, the FSM is the following sequence: UUUUUUUGA (SEQ ID NO: 2) UUUUUUUAG (SEQ ID NO: 3) UUUUUUUAA (SEQ ID NO: 4) may include one of.

[0026] The translation readthrough motif (TRM) may include the sequence STOP-CUAG or STOP-CAAUUA (where "STOP" is a stop codon). For example, the translation readthrough motif is the following sequence: UGA-CUAG (Sequence ID 5) UAG-CUAG (Sequence ID 6) UAA-CUAG (Sequence ID 7) UGA-CAAUUA (Sequence ID 8) UAG-CAAUUA (Sequence ID 9) UAA-CAAUUA (Sequence ID 10) It may include one of the following.

[0027] A nucleic acid construct containing a translation read-through motif may contain two adjacent TRMs, which may be the same or different.

[0028] A nucleic acid construct containing a translational read-through motif may contain two nucleic acid sequences, each encoding a cleavage site. The TRM may be located between the two cleavage site encoding sequences, or there may be two TRMs, each positioned upstream of the cleavage site.

[0029] Nucleic acid constructs containing translational read-through motifs may include attenuated or inefficient signal peptides located upstream of a second (or subsequent) target nucleotide.

[0030] Nucleic acid constructs containing translational read-through motifs have the following structure: NOI1-TRM-CL-NOI2 NOI1-TRM1-TRM2-CL-NOI2 NOI1-CL1-TRM-CL2-NOI2 NOI1-TRM-CL-SP-NOI2 NOI1-TRM1-CL1-NOI2-TRM2-CL2-NOI3 NOI1-TRM1-CL1-TRM2-CL2-NOI2 (Here, TRM1 and TRM2 may be the same or different, and are the first and second translated lead-through motifs; CL1 and CL2 may be the same or different, and are the first and second nucleic acid sequences encoding the cleavage site. SP is a decaying signal peptide; and NOI3 is the third target nucleotide sequence. It may have one of the following.

[0031] The cleavage sites may include, for example, self-cleaving peptides, furin cleavage sites, or tobacco etch virus cleavage sites.

[0032] The cleavage sites may include, for example, 2A self-cleaving peptides or 2A-like peptides derived from aft virus or cardiovirus.

[0033] The second (or subsequent) target nucleotide may encode a cytokine, chemokine, or toxin.

[0034] When nucleic acid constructs are expressed in cells, they produce two products: a) The first product encoded by NOI1 alone; and b) A second product encoded by NOI1 and NOI2, which is produced when a flame slip or translational read-through occurs. It may be capable of producing [something].

[0035] In this embodiment, the second product may be a chimeric antigen receptor (CAR), and the first product may be a shortened version of the CAR that cannot induce CAR-mediated cell signaling.

[0036] Alternatively, the first product may be a chimeric antigen receptor (CAR) containing an intracellular signaling domain (i.e., a first-generation CAR), and the second product may be a CAR containing an intracellular signaling domain and one or more costimulatory domains (i.e., a second-generation or third-generation CAR).

[0037] In a second aspect, the present invention provides a vector comprising a nucleic acid construct according to the first aspect of the present invention.

[0038] The vector could be, for example, a retroviral vector or a lentiviral vector.

[0039] In a third aspect, the present invention provides cells comprising a nucleic acid construct according to a first aspect of the present invention or a vector according to a second aspect of the present invention.

[0040] In a fourth aspect, the present invention provides a method for producing cells according to a third aspect of the present invention, comprising the step of introducing a nucleic acid construct according to a first aspect of the present invention or a vector according to a second aspect of the present invention into cells.

[0041] A fifth aspect provides a method for modulating the relative expression of two transgenes in a nucleic acid construct, the method comprising the step of including a flame slip motif or a translational read-through motif between the two transgenes in order to reduce the expression of a downstream transgene.

[0042] A key advantage of using flame slips or translational readthroughs as a means of controlling transgene expression is that a wide range of expression is achievable, and when two transgenes are expressed, the expression level of the second transgene can be reduced to less than 1% of that of the first transgene. This is considerably lower than the levels that can be achieved using modified signal peptides, making it possible to use the technique for expressing proteins such as toxic compounds where very low levels of expression are essential.

[0043] The use of flame slips or translational read-throughs allows for the expression of transgenes at predictable, predetermined ratios. It is also possible to adjust the level of transgene expression by using different flame slipping or read-through motifs.

[0044] This system can be used to control the expression of any transgene, and is not limited to the expression of cell surface proteins. In certain embodiments, for example, the following items are provided: (Item 1) A nucleic acid construct comprising a first target nucleotide sequence (NOI1); a flame slip motif (FSM) or translational read-through motif (TRM); and a second target nucleotide sequence (NOI2). (Item 2) A nucleic acid construct as described in item 1, including nucleotide sequences encoding cleavage sites (CLs) so that NOI1 and NOI2 are expressed as separate proteins. (Item 3) structure: NOI1-CL-FSM-NOI2; or NOI1-TRM-CL-NOI2 A nucleic acid construct as described in item 2, having the properties of item 2. (Item 4) A nucleic acid construct according to any of items 1 to 3, comprising a flame slip motif (FSM) containing a repeat of uracil, thymine, or guanine bases. (Item 5) The nucleic acid construct described in item 4, wherein the FSM contains the sequence UUUUUUU (sequence number 1). (Item 6) A nucleic acid construct according to item 4 or 5, wherein the FSM also includes a stop codon. (Item 7) The aforementioned FSM has the following sequence: UUUUUUUGA (Sequence ID 2) UUUUUUUAG (Sequence ID 3) UUUUUUUAA (Sequence ID 4) A nucleic acid construct as described in item 6, including one of the following. (Item 8) A nucleic acid construct as described in any of items 1-3, comprising a translation read-through motif (TRM) containing the sequence STOP-CUAG or STOP-CAAUUA (where "STOP" is a stop codon). (Item 9) The aforementioned translation read-through motif is arranged as follows: UGA-CUAG (Sequence ID 5) UAG-CUAG (Sequence ID 6) UAA-CUAG (Sequence ID 7) UGA-CAAUUA (Sequence ID 8) UAG-CAAUUA (Sequence ID 9) UAA-CAAUUA (Sequence ID 10) A nucleic acid construct as described in item 8, including one of the following. (Item 10) structure: NOI1-TRM-CL-NOI2 NOI1-TRM1-TRM2-CL-NOI2 NOI1-CL1-TRM-CL2-NOI2 NOI1-TRM-CL-SP-NOI2 NOI1-TRM1-CL1-NOI2-TRM2-CL2-NOI3 NOI1-TRM1-CL1-TRM2-CL2-NOI2 (Here, TRM1 and TRM2 may be the same or different, and are the first and second translated lead-through motifs; CL1 and CL2 may be the same or different, and are the first and second nucleic acid sequences that encode the cleavage site, respectively. SP is a decaying signal peptide; and NOI3 is the third target nucleotide sequence. A nucleic acid construct as described in item 8 or 9, having the following characteristics. (Item 11) A nucleic acid construct according to any one of items 2 to 10, wherein the cleavage site includes a self-cleaving peptide, a furin cleavage site, or a tobacco etch virus cleavage site. (Item 12) The nucleic acid construct according to item 11, wherein the cleavage site contains a 2A self-cleaving peptide or a 2A-like peptide derived from an aft virus or cardiovirus. (Item 13) NOI2 is a nucleic acid construct described in any of items 1-12, which encodes a cytokine, chemokine, or toxin. (Item 14) When expressed in cells, it produces two products: a) The first product encoded by NOI1 alone; and b) A second product encoded by NOI1 and NOI2, which is produced when a flame slip or translational read-through occurs. A nucleic acid construct as described in item 1, which can produce the following. (Item 15) The nucleic acid construct described in item 14, wherein the second product is a chimeric antigen receptor (CAR), and the first product is a shortened version of the CAR that is unable to induce CAR-mediated cell signaling. (Item 16) The nucleic acid construct according to item 14, wherein the first product is a chimeric antigen receptor (CAR) comprising an intracellular signaling domain, and the second product is a CAR comprising an intracellular signaling domain and one or more costimulatory domains. (Item 17) A vector containing a nucleic acid construct as described in any of items 1-16. (Item 18) A retroviral vector or lentiviral vector as described in item 17. (Item 19) Cells containing a nucleic acid construct as described in any one of items 1 through 16 or a vector as described in item 17 or 19. (Item 20) A method for producing the cells described in item 19, comprising the step of introducing a nucleic acid construct described in any of items 1 to 16 or a vector described in item 17 or 18 into the cells. (Item 21) A method for modulating the relative expression of two transgenes in a nucleic acid construct, comprising the step of including a flame slip motif or a translational read-through motif between the two transgenes in order to reduce the expression of a downstream transgene. [Brief explanation of the drawing]

[0045] [Figure 1]Methods used to express different proteins from the same vector: (a) Two different promoters within the same cassette result in two different transcripts, each producing a distinct protein. (b) The use of an internal ribosome entry sequence (IRES) results in a single transcript that translates into two distinct proteins. (c) The use of the FMDV2A peptide results in a single transcript and a single polyprotein that are rapidly cleaved into two distinct proteins. [Figure 2] Flame slip motif and construct design. (A) Figure showing a flame slip motif in which a series of seven uridines are inserted into the transgene sequence, thereby promoting flame slippage. Flame slippage typically results in the continuous transcription / translation of an alternative reading frame in the -1 direction, and the production of a functional protein. (B) Structure of a construct showing the locations of the transgene, flame slip motif (SLIP), and 2A self-cleaving peptide sequence. (C) Flow cytometry analysis of SupT1 cells transduced with a construct containing an RQR8 sorting selection marker followed by a CD22-CD19 chimeric protein consisting of either a regulatory sequence (6×U) or a flame slip motif (7×U) and the external domain of CD22 fused to the transmembrane and truncated end domains of CD19. Introduction of the flame slip motif resulted in a dramatic decrease in CD22-CD19 chimeric expression, and similar levels of the RQR8 sorting selection marker were observed. [Figure 3]Design of translational read-through motifs and constructs. (A) Examples of known translational read-through motifs. (B-G) Structures of translational read-through motif constructs. (B) A translational read-through construct consisting of two transgenes, with the translational read-through motif positioned at the 3' end of the first transgene and at the 5' end of the 2A self-cleaving peptide sequence and transgene 2. Expression of transgene 1 is significantly higher than that of transgene 2. (B') A double-stop translational read-through construct incorporating two stop codons into the translational read-through motif to further reduce the expression level beyond that achieved with a single stop codon. (C) A universal translational read-through construct consisting of a translational read-through motif sandwiched between two 2A self-cleaving peptide sequences. This construct mitigates sequence-dependent effects that can result in unpredictable levels of translational read-through. (D) A combinatorial approach combining the translational read-through motif with an attenuated signal peptide sequence to further reduce the expression level of transgene 2. (E and E') Complex translational read-through motifs with multiple motifs arranged in series to produce a cascade of reduced transgene expression. (E) Multiple transgenes are expressed from a single cassette using the translational read-through motif and a 2A self-cleaving peptide sequence. (E') The translational read-through motif on the 5' side of transgene 2 is isolated using the self-cleaving peptide sequence. (F and F') Functional translational read-through constructs. (F) By positioning the translational read-through motif on the 3' side of the spacer domain of the CAR, secretion of the antigen-binding domain (scFv / VHH) and a functional CAR can be achieved. The functional read-through results in the expression of a functional CAR, and termination of translation at the stop codon produces a secreted antibody. (F') By ​​positioning the translational read-through motif on the 3' side of the first end domain (either the CD3ζ end domain or the co-receptor end domain), combinations of first-generation, second-generation, and third-generation CARs can be produced. The functional translational read-through switches the expression from the first-generation CAR to the second or third-generation CAR. [Figure 4A]Comparison of attenuation signal peptide and translational read-through motif approaches to control CD22-CD19 chimera expression. (A) Flow cytometry analysis of HEK293T cells transfected with the RQR8 sorting selection marker and constructs encoding the CD22-CD19 chimera. Signal peptide variant (row 1), translational read-through motif constructs (rows 2 and 3), and double-stop translational read-through motif (row 4). Compared to the wild-type signal peptide, the lysine 11 signal peptide variant (L11K mutation) reduces CD22-CD19 chimera expression. The stop-CUAG and stop-CAAUUA translational read-through motifs yielded similar results when placed on the 5' side of the CD22-CD19 transgene, and both motifs reduced the chimera expression level to approximately 2% or less of the RQR8 sorting selection marker. A double-stop translational read-through motif (row 4) was used to obtain CD22-CD19 chimeras with lower expression levels. (B) Quantification of mean fluorescence intensity of CD22-CD19 chimeras on the surface of transfected HEK293T cells, comparing decaying signal peptide variants with translational read-through constructs. Using translational read-through constructs, substantially lower levels of CD22-CD19 chimeras were obtained compared to decaying signal peptide variants. [Figure 4B]Comparison of attenuation signal peptide and translational read-through motif approaches to control CD22-CD19 chimera expression. (A) Flow cytometry analysis of HEK293T cells transfected with the RQR8 sorting selection marker and constructs encoding the CD22-CD19 chimera. Signal peptide variant (row 1), translational read-through motif constructs (rows 2 and 3), and double-stop translational read-through motif (row 4). Compared to the wild-type signal peptide, the lysine 11 signal peptide variant (L11K mutation) reduces CD22-CD19 chimera expression. The stop-CUAG and stop-CAAUUA translational read-through motifs yielded similar results when placed on the 5' side of the CD22-CD19 transgene, and both motifs reduced the chimera expression level to approximately 2% or less of the RQR8 sorting selection marker. A double-stop translational read-through motif (row 4) was used to obtain CD22-CD19 chimeras with lower expression levels. (B) Quantification of mean fluorescence intensity of CD22-CD19 chimeras on the surface of transfected HEK293T cells, comparing decaying signal peptide variants with translational read-through constructs. Using translational read-through constructs, substantially lower levels of CD22-CD19 chimeras were obtained compared to decaying signal peptide variants. [Figure 5-1]Serialized translational read-through motifs. A) Diagram of a construct generated using a series-arranged stop codon and translational read-through motif. For simplicity, only four examples showing the structure of constructs with serialized translational read-through motifs are shown. The cell surface marker, which is an HA epitope presented on the CD8a stalk, is always in the first position in the cassette, followed by either a tandem Clover3 or EBFP with a different coding sequence, separated by a self-cleaving peptide sequence (2A). Two control constructs were cloned in which the stop codon was replaced with UGG (encoding tryptophan) and the tandem mClover or EBFP was in the second or third position. For constructs with serialized translational read-through motifs, all three stop codons (UGA, UAG, and UAA) were tested, and the translational read-through motif utilized was CUAG. The same stop codon was used in both positions. B) Flow cytometry of transduced PBMCs shows tandem Clover3 fluorescence intensity from constructs with 0, 1, or 2 stop codons / translation read-through motifs (control is without stop codons, with tandem Clover3 at position 2). Histograms show decreased tandem Clover3 expression from constructs with serial translation read-through motifs, with levels varying depending on the present stop codon. C) Normalization of tandem Clover3 fluorescence intensity levels compared to cell surface markers. When placed immediately downstream of UGA, UAG, or UAA stop codons and the CUAG ​​translation read-through motif, levels decreased 6-fold, 24-fold, or 33-fold, respectively. When the tandem Clover3 sequence was placed downstream of two stop codons and translation read-through motifs, a 100-fold decrease in tandem Clover3 levels was observed. [Figure 5-2]Serialized translational read-through motifs. A) Diagram of a construct generated using a series-arranged stop codon and translational read-through motif. For simplicity, only four examples showing the structure of constructs with serialized translational read-through motifs are shown. The cell surface marker, which is an HA epitope presented on the CD8a stalk, is always in the first position in the cassette, followed by either a tandem Clover3 or EBFP with a different coding sequence, separated by a self-cleaving peptide sequence (2A). Two control constructs were cloned in which the stop codon was replaced with UGG (encoding tryptophan) and the tandem mClover or EBFP was in the second or third position. For constructs with serialized translational read-through motifs, all three stop codons (UGA, UAG, and UAA) were tested, and the translational read-through motif utilized was CUAG. The same stop codon was used in both positions. B) Flow cytometry of transduced PBMCs shows tandem Clover3 fluorescence intensity from constructs with 0, 1, or 2 stop codons / translation read-through motifs (control is without stop codons, with tandem Clover3 at position 2). Histograms show decreased tandem Clover3 expression from constructs with serial translation read-through motifs, with levels varying depending on the present stop codon. C) Normalization of tandem Clover3 fluorescence intensity levels compared to cell surface markers. When placed immediately downstream of UGA, UAG, or UAA stop codons and the CUAG ​​translation read-through motif, levels decreased 6-fold, 24-fold, or 33-fold, respectively. When the tandem Clover3 sequence was placed downstream of two stop codons and translation read-through motifs, a 100-fold decrease in tandem Clover3 levels was observed. [Figure 6-1]Regulatory expression of human flexi-IL-12 downstream of a translational read-through motif. A) Diagram showing the structure of a construct in which human flexi-IL-12, consisting of a fusion between IL-12α(p35) and IL-12β(p40) subunits, is placed downstream of the selection marker RQR8 along with an autocleavage peptide that facilitates the expression of both proteins. All three stop codons were tested using the translational read-through motif CAAUUA. B) Representative flow cytometry plots of PBMCs transduced with the construct and stained with antibodies against CD3ε and RQR8. C) Quantification of secreted IL-12 from transduced PBMCs at 24, 48, and 72 hours after determining transduction efficiency. The results demonstrate that IL-12 secretion is significantly reduced by the translational read-through construct. [Figure 6-2] Regulatory expression of human flexi-IL-12 downstream of a translational read-through motif. A) Diagram showing the structure of a construct in which human flexi-IL-12, consisting of a fusion between IL-12α(p35) and IL-12β(p40) subunits, is placed downstream of the selection marker RQR8 along with an autocleavage peptide that facilitates the expression of both proteins. All three stop codons were tested using the translational read-through motif CAAUUA. B) Representative flow cytometry plots of PBMCs transduced with the construct and stained with antibodies against CD3ε and RQR8. C) Quantification of secreted IL-12 from transduced PBMCs at 24, 48, and 72 hours after determining transduction efficiency. The results demonstrate that IL-12 secretion is significantly reduced by the translational read-through construct. [Figure 7-1]Regulatory expression of mouse IL-12 downstream of translational read-through motifs. A) Diagram of a tricistronic construct containing the suicide gene (RapaCasp9), cell surface marker Thy1.1, and mouse flexi-IL-12. Mouse flexi-IL-12 levels were modulated by internal ribosome entry sites (IRES), decay signal peptide motifs, or translational read-through motifs. B) Representative flow cytometry plots of BalbC splenocytes transduced from the constructs and stained with antibodies against CD3 and Thy1.1. C) Quantification of secreted IL-12 levels from transduced splenocytes by ELISA. D) Activated splenocytes were restimulated using supernatant from transduced splenocytes. The supernatant from the restimulated cells was analyzed for the presence of IFNg by ELISA. Importantly, these results demonstrate that supernatant from transduced splenocytes with translational read-through motifs was able to induce IFNγ secretion from restimulated splenocytes. [Figure 7-2] Regulatory expression of mouse IL-12 downstream of translational read-through motifs. A) Diagram of a tricistronic construct containing the suicide gene (RapaCasp9), cell surface marker Thy1.1, and mouse flexi-IL-12. Mouse flexi-IL-12 levels were modulated by internal ribosome entry sites (IRES), decay signal peptide motifs, or translational read-through motifs. B) Representative flow cytometry plots of BalbC splenocytes transduced from the constructs and stained with antibodies against CD3 and Thy1.1. C) Quantification of secreted IL-12 levels from transduced splenocytes by ELISA. D) Activated splenocytes were restimulated using supernatant from transduced splenocytes. The supernatant from the restimulated cells was analyzed for the presence of IFNg by ELISA. Importantly, these results demonstrate that supernatant from transduced splenocytes with translational read-through motifs was able to induce IFNγ secretion from restimulated splenocytes. [Figure 8A]Toxicity testing of translational read-through IL-12 constructs. A) The RapaCasp6, Thy1.1, and IL-12 coding constructs were co-transduced into splenocytes with a second construct containing shortened mouse CD34 (mudCD34), firefly luciferase (FLuc), and anti-GD2 chimeric antigen receptor (CAR). Transduced splenocytes were stained with antibodies against CD34 and Thy1.1, and the transduction efficiency was determined by flow cytometry. B) Transduced splenocytes were injected into mice, and after 15 days, they were sacrificed and their spleens were removed. Splenomegaly was observed only in mice injected with splenocytes transduced to constitutively expressed IL-12 (group labeled 2A). [Figure 8B] Toxicity testing of translational read-through IL-12 constructs. A) The RapaCasp6, Thy1.1, and IL-12 coding constructs were co-transduced into splenocytes with a second construct containing shortened mouse CD34 (mudCD34), firefly luciferase (FLuc), and anti-GD2 chimeric antigen receptor (CAR). Transduced splenocytes were stained with antibodies against CD34 and Thy1.1, and the transduction efficiency was determined by flow cytometry. B) Transduced splenocytes were injected into mice, and after 15 days, they were sacrificed and their spleens were removed. Splenomegaly was observed only in mice injected with splenocytes transduced to constitutively expressed IL-12 (group labeled 2A). [Figure 9-1] Flow cytometry analysis of splenocytes. A) Splenocytes from sacrificed mice were stained with antibodies against CD11b, CD3, CD4, CD8, and CD19 and analyzed by flow cytometry. The gating strategy used is shown. B-F) Percentages of present CD11b+ (macrophages), CD3+ (T cells), CD4+ (helper T cells), CD8+ (cytotoxic T cells), and C19+ (B cells). Splenocytes of mice injected with transduced IL-12 or IRES constructs showed an increased number of macrophages and T cells. Mice with IL-12 constitutively expressing spleen cells showed a decrease in B cell count. [Figure 9-2]Flow cytometry analysis of splenocytes. A) Splenocytes from sacrificed mice were stained with antibodies against CD11b, CD3, CD4, CD8, and CD19 and analyzed by flow cytometry. The gating strategy used is shown. B-F) Percentages of present CD11b+ (macrophages), CD3+ (T cells), CD4+ (helper T cells), CD8+ (cytotoxic T cells), and C19+ (B cells). Splenocytes of mice injected with transduced IL-12 or IRES constructs showed an increased number of macrophages and T cells. Mice with IL-12 constitutively expressing spleen cells showed a decrease in B cell count. [Modes for carrying out the invention]

[0046] The present invention relates to nucleic acid constructs comprising motifs that reduce the expression of transgenes, such as flame slip motifs or translational read-through motifs.

[0047] Frame slip During transcription, RNA polymerase catalyzes the incorporation of nucleotides into the growing RNA chain based on complementarity with the DNA template. However, when RNA polymerase encounters a sequence of repeating bases, slippage or "stuttering" can occur. Transcriptional slippage is utilized in nature, for example, in the regulation of the E. coli pyrBI and codBA operons, the expression of the P gene in paramyxoviruses, and the sequencing of the cellular dnaX gene in Thermus thermophiles.

[0048] When RNA polymerase slips, one (or possibly two) repeat bases are lost, which can lead to the synthesis of mRNA encoding an alternative reading frame.

[0049] The nucleic acid constructs of the present invention may include a transcription frame slip site such that transcription of the downstream transgene occurs only if there is slippage of RNA polymerase resulting in mRNA encoding an alternative reading frame.

[0050] The translation of mRNA sequences into proteins is a complex process involving the orchestration of ribosomes, initiation and elongation factors, aminoacyltransfer RNA (aa-tRNA), aminoacyl-tRNA synthetase, and termination factors. Translation initiation begins when a complex of factors binds to the 5' end of the mRNA, which then leads to the recruitment of a 40S ribosome and scanning of the mRNA. When the complex of the initiation factor and the 40S ribosome encounters a codon (a triplet of nucleotides) encoding the amino acid methionine (AUG codon), a 60S ribosome is recruited, and polypeptide synthesis begins from the pairing of a congeneral tRNA loaded with the appropriate amino acid. Translation initiation can occur with alternative start codons other than AUG, but AUG is the most frequently used start codon. Polypeptide elongation occurs in a periodic manner, as the tRNA binds to its congeneral codon, a peptide bond is formed between the newly added amino acid and the elongating polypeptide, and the polypeptide moves to expose the next codon.

[0051] During translation, ribosome interruption can occur at specific codons where ribosomes stop. Ribosome interruption can accelerate mRNA degradation via the nucleocytic pathway, or it can induce slip in the -1 or -2 direction. Repeat sequences within mRNA, such as UUUUUUA, are known to induce translational frame slipping, and this sequence is present at the 3' end of the group-specific antigen (gag) and polymerase (pol) genes of human immunodeficiency virus (HIV). Translational frame slipping-1 of the HIV gag / pol genes results in the expression of the Gag-Pol polyprotein.

[0052] The nucleic acid construct of the present invention may include a translational flame-slipping site such that translation of the downstream transcript occurs only when ribosome-mediated flame-slip is present.

[0053] Flame slip sites may contain a sequence of bases of the same type.

[0054] The flame slip motif can be located upstream and / or downstream of the cleavage site in the nucleic acid construct. The flame slip motif is located between the first and second transgenes in the nucleic acid construct.

[0055] The flame slip motif can be used alone. In this embodiment, the second transgene may be positioned outside the flame downstream of the flame slip site, such that the flame slip is required for the transcription or translation of the second transgene.

[0056] A motif may, for example, contain a sequence of 5, 7, 8, 10, or 11 bases. A site may, for example, contain a repeat of uracil, adenosine, or guanine bases. A site may, for example, contain the sequence shown as Sequence ID No. 1. UUUUUUU (Sequence ID 1).

[0057] Alternatively, the frame slip region can be used in combination with a stop codon. In this embodiment, the stop codon is located in the frame downstream of the frame slip region such that frame slipping is required for the stop codon to be ignored.

[0058] The stop codon may be UGA, UAG, or UAA. The flame slip site may contain a repeat of uracil, adenosine, or guanine bases in multiples of 3, such as 3, 6, or 9 repeats of uracil, adenosine, or guanine bases.

[0059] Examples of frame slip site / stop codon combinations are shown as sequence numbers 2, 3, and 4. UUUUUUUGA (Sequence ID 2) UUUUUUUAG (Sequence ID 3) UUUUUUUAA (Sequence ID 4).

[0060] Translation Readthrough Translation terminates when a ribosome encounters a UGA, UAG, or UAA stop codon. At this point, the termination factor recognizes the stop codon, facilitating ribosome dissociation and reuse. Since competition between the termination factor and closely related tRNAs and continued polypeptide elongation occur at a rate of only 0.1%, translation termination usually occurs with high fidelity through stop codon recoding. However, in certain genes, an increased level of stop codon recoding resulting in translational read-through has been reported. In some cases, this has led to the generation of longer polypeptides with additional functional motifs in a process called functional read-through.

[0061] When termination factor 1 (RF1) fails to recognize a stop codon, a closely related aa-tRNA inserts an amino acid into the elongating polypeptide, thereby repressing the stop codon and resulting in translational read-through. Local concentrations of termination factor and aa-tRNA affect the levels of stop codon repression and translational read-through, with low concentrations of termination factor promoting translational read-through. In mammals, repression of the UAG stop codon results in the insertion of tryptophan, arginine, or cysteine ​​residues.

[0062] One of the earliest examples of stop codon repression discovered is the rabbit betaglobin gene, where translational read-through results in the addition of 22 amino acids to the C-terminus of protein 3.

[0063] The frequency of translational read-through depends on several factors, including 1) the stop codon used (UGA, UAG, or UAA); 2) the sequence directly adjacent to the stop codon (the six nucleotides upstream and downstream of the stop codon are particularly important); and 3) the presence of a cis-acting sequence at the 3' end of the mRNA.

[0064] The termination efficiencies of the three stop codons vary, with UAA being the strongest and UGA the weakest, and the termination efficiency hierarchy is defined as UAA > UAG > UGA. As a result, the highest level of translation readthrough is shown with the UGA stop codon, and the lowest with the UAA stop codon.

[0065] Sequence analysis of genes exhibiting translational readthrough identified at least two distinct motifs that promote stop codon repression and sustained translation: STOP-CUAG and STOP-CAAUUA (where stop can be UGA, UAG, or UAA). The level of translational readthrough depends on the stop codon used: UGA exhibits the highest level of translational readthrough, while decreasing levels of readthrough are obtained from UAG and UAA stop codons, so the overall hierarchy of readthrough is UGA > UAG > UAA.

[0066] The nucleic acid construct of the present invention may include one of the sequences shown as SEQ ID NOs. 5-10. UGACUAG(SEQ ID NO. 5) UAGCUAG (Sequence ID 6) UAACUAG (Sequence ID 7) UGACAAUUA (Sequence ID 8) UAGCAAUUA (Sequence ID 9) UAACAAUUA (Sequence ID 10)

[0067] Translational read-through sites may be located between the first and second transgenes in the nucleic acid construct. Translational read-through sites may be located upstream and / or downstream of cleavage sites in the nucleic acid construct. Translational read-through sites may be adjacent to cleavage sites (Figure 3C). Two or more translational read-through sites may be used, for example, adjacent to each other (Figure 3B') or adjacent to cleavage sites (Figure E').

[0068] To further reduce the expression level of downstream transgenes, multiple stop codons can be inserted into the 5' end of the translational read-through motif (Figure 2B). An example of a multiple-stop translational read-through motif is shown as SEQ ID NO: 11, in which two UGA stop codons are positioned on the 5' end of the CUAG ​​read-through motif. UGAUGACUAG (Sequence ID 11)

[0069] When a nucleic acid construct contains more than two transgenes, compound translation read-through motifs can be arranged in series with the 5' end of the sequence encoding the cleavage site. This allows multiple transgenes to be expressed in different ratios. With each additional read-through motif, the expression level should be reduced 10 to 50 times compared to the upstream transgene.

[0070] Nucleic acid constructs having a complex translation read-through motif have the following structure: NOI1-TRM1-CL1-NOI2-TRM2-CL2-NOI3 It may have.

[0071] Readthrough sites can be positioned within the coding sequence to produce two versions of the protein: a shorter version produced by translation of the transcript up to the readthrough site (i.e., where readthrough does not occur), and a longer version produced by translation of the transcript outside the readthrough site and downstream (i.e., where readthrough occurs). Examples of such arrangements are shown in Figure 3F and F'.

[0072] Modified lig peptide While translational read-through motifs significantly reduce transgene expression, in certain situations, further reductions in expression levels may be necessary. For type I transmembrane proteins and secreted proteins, further reductions in expression can be achieved by combining a modified signal peptide sequence with a translational read-through motif. Such an approach is suitable for type I transmembrane proteins and secreted proteins that have a signal peptide sequence. In this case, the modified signal peptide and the second transgene are positioned 3' to the translational read-through motif and the self-cleaving peptide sequence (Figure 3D).

[0073] Signal peptides are short peptides, typically 5–30 amino acids long, that reside at the N-terminus of most newly synthesized proteins destined for secretory pathways. These proteins include proteins and transmembrane proteins that reside within specific organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes) and are secreted from the cell.

[0074] Signal peptides generally contain a core sequence that is a long, sequential sequence of hydrophobic amino acids that tends to form a single alpha-helix. Signal peptides may begin with a short, positively charged sequence of amino acids, which helps to enhance the proper topology of the polypeptide during translocation. The ends of the signal peptide typically contain a sequence of amino acids that are recognized and cleaved by a signal peptidase. The signal peptidase may cleave the signal peptide either during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0075] Generally, signal peptides are located at the amino terminus of a molecule, but several carboxy-terminated signal peptides are known.

[0076] Modified signal peptides are described in detail in International Publication No. 2016 / 174409 (which is incorporated herein by reference). Modified signal peptides may contain one or more mutations, such as substitutions or deletions, to have fewer hydrophobic amino acids than the wild-type signal peptide from which they are derived. The term "wild-type" means the sequence of the signal peptide present in the native protein from which it is derived.

[0077] If a nucleic acid construct contains two transgenes, both encoding transmembrane proteins, the protein encoded by the downstream transgene (which has lower relative expression) may contain fewer hydrophobic amino acids than the protein encoded by the upstream transgene (which has higher relative expression).

[0078] The hydrophobic amino acids that are mutated to modify signal peptide efficiency may be alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (P), tyrosine (Y), or tryptophan (W).

[0079] The modified signal peptide may contain one, two, three, four, or five amino acid deletions or substitutions of hydrophobic amino acids. The hydrophobic amino acids may be replaced with non-hydrophobic amino acids, such as hydrophilic or neutral amino acids.

[0080] nucleic acid construct The present invention relates to a nucleic acid construct comprising a first-objective nucleotide sequence (NOI1); a flame slip motif or translational read-through motif (FSM / TRM); and a second-objective nucleotide sequence (NOI2).

[0081] The "target nucleotide" can be RNA or DNA. The target nucleotide (NOI) encodes the target polypeptide (POI), which can be all or part of a protein.

[0082] NOI1 and NOI2 (along with subsequent NOI(or more) as needed) can encode a protein when transcribed and translated together. For example, when a nucleic acid construct is expressed in a cell, it produces two products: a) The first product encoded by NOI1 alone; and b) A second product encoded by NOI1 and NOI2, which is produced when a flame slip or translational read-through occurs. It may be capable of producing [something].

[0083] The relative expression levels of the full-length products encoded by NOI1 and NOI2 may be lower than the expression level of the shortened product encoded by NOI1 alone.

[0084] Alternatively, a nucleic acid construct may contain one or more cleavage sites so that the desired nucleotide sequence is expressed as a separate protein.

[0085] The nucleic acid construct of the present invention may encode a polyprotein comprising a first and a second polypeptide. The polyprotein may be cleaved at a cleavage site to produce two distinct polypeptides.

[0086] NOIs can encode intracellular proteins, transmembrane proteins, or secretory proteins.

[0087] NOIs are, for example, chimeric antigen receptors (CARs) or parts thereof, or agents that affect the activity of CARs or CAR-expressing cells, such as cytokines. It is possible.

[0088] The transgene may encode a target antigen. In this respect, the technique can be used to produce target antigens using a variety of very low levels of target antigens. These can be used, for example, in functional assays of T cells expressing CARs or engineered T cell receptors (TCRs).

[0089] NOIs can encode proteins involved in the synthesis of other entities by cells. For example, cells can be induced to express the cancer antigen disialoganglioside (GD2) by transgenic expression of two enzymes: GM3 synthase and GD2 synthase (International Publication No. 2015 / 132604). By reducing the expression levels of these enzymes in cells, GD2 low It is possible to create target cells.

[0090] NOIs may encode cytokines, such as cytokines that enhance the inflammatory response and / or increase the effectiveness of CAR-T cell therapy. Cytokines may be selected from the following: IL-7, IL-12, IL-15, IL-17A, IL-18, and IL-21. In particular, the cytokine may be IL-12.

[0091] Interleukin-12 (IL-12) is an interleukin spontaneously produced by dendritic cells, macrophages, neutrophils, and human B lymphoblasts in response to antigen stimulation. IL-12 is involved in the differentiation of naive T cells into Th1 cells. It is known as a T cell stimulator that can stimulate T cell growth and function. It stimulates the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T cells and natural killer (NK) cells, and reduces the IL-4-mediated suppression of IFN-γ.

[0092] IL-12 plays a crucial role in the activity of natural killer cells and T lymphocytes. IL-12 mediates the enhancement of cytotoxic activity in NK cells and CD8+ cytotoxic T lymphocytes.

[0093] IL-12 is a particularly important and potent immunomodulatory cytokine for modulating the tumor microenvironment and redirecting the immune response against cancer. Since IL-12 is systemically toxic, methods for locally producing IL-12 are of interest.

[0094] IL-12 is a heterodimeric cytokine encoded by two distinct genes, IL-12A(p35) and IL-12B(p40). The active heterodimer (referred to as "p70") is formed after protein synthesis.

[0095] NOI can encode IL-12A and / or IL-12B. The sequence for human IL-12A is available from Uniprot accession number P29459. A portion of this sequence lacking the signal peptide is shown below as SEQ ID NO 53. The sequence for human IL-12B is available from Uniprot accession number P29460. A portion of this sequence lacking the signal peptide is shown below as SEQ ID NO 54. [ka]

[0096] The NOI may encode "flexi-IL-12," a fusion between human IL-12α(p35) and IL-12β(p40) subunits linked by a linker. The appropriate flexi-IL-12 sequence is shown below as sequence number 55. [ka] [ka]

[0097] In Sequence ID No. 55, the signal peptide derived from the mouse kappa chain V-III region MOPC63 (Uniprot P01661) is shown in bold; the serine-glycine linker is underlined and in bold.

[0098] NOI may contain one of the sequences shown as SEQ ID NOs. 53, 54, or 55, or a variant thereof. Provided that the variant sequence retains IL-12 function when expressed in vivo, the variant sequence may have at least 80, 85, 90, 95, 98, or 99% sequence identity with SEQ ID NOs. 53, 54, or 55. For example, the variant sequence may retain the ability to enhance the activity of cytotoxic T cells in vivo, and / or the variant sequence may stimulate interferon-gamma (IFN-γ) production by T cells.

[0099] Sequences encoding IL-12 or fliexi-IL12 may be located downstream of a flame slip motif or translational read-through motif. This provides a means to control cytokine expression and reduce cytokine expression levels compared to CARs.

[0100] NOIs may encode chemokines, such as those that improve the efficacy of CAR-T cell therapy. The chemokine could be CCL19. In particular, nucleic acid constructs may co-express CCL19 and IL-7.

[0101] NOIs can encode antibodies or parts thereof. For example, an NOI can encode an immunomodulatory antibody or an antibody fragment. Antibodies can block inhibitory signals (such as PD1) or activate the immune system (e.g., OX40 agonists, 41BB agonists, or ICOS agonists).

[0102] The introduced gene may encode toxic compounds, such as Clostridium botulinum, diphtheria, or Pseudomonas toxins.

[0103] Chimeric antigen receptor Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins that connect an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). The binder is typically a single-stranded variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats that include an antibody-like antigen-binding site. A spacer domain is usually required to detach the binder from the membrane and allow it to be properly oriented. A common spacer domain used is the Fc of IgG1. Depending on the antigen, a more compact spacer, such as a stalk derived from CD8α, or even just the IgG1 hinge, may be sufficient. The transmembrane domain anchors the protein to the cell membrane and connects the spacer to the endodomain.

[0104] Early CAR designs had endodomains derived from the intracellular portion of either the FcεR1 or CD3ζ γ-chain. As a result, these first-generation receptors transmitted immunological signal 1, which was sufficient to trigger T cell killing on congenital target cells, but not to fully activate T cells for proliferation and survival. To overcome this limitation, composite endodomains were constructed: fusion of the intracellular portion of a T cell costimulatory molecule to that of CD3ζ results in second-generation receptors capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28, which delivers the most potent costimulatory signal (i.e., immunological signal 2) that triggers T cell proliferation. Several receptors containing TNF receptor family endodomains, such as the closely related OX40 and 41BB, that transmit survival signals have also been described. Even more potent third-generation CARs with endodomains capable of transmitting activation, proliferation, and survival signals have now been described.

[0105] When a CAR binds to a target antigen, this results in the transmission of an activation signal to the T cells that express it. Thus, CARs induce T cell specificity and cytotoxicity toward tumor cells expressing the target antigen.

[0106] Therefore, CARs typically include (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) a signaling domain, or an intracellular domain associated therewith.

[0107] CAR is a general structure: Antigen-binding domain - Spacer domain - Transmembrane domain - Intracellular signal transduction domain (endodomain) It may have.

[0108] The NOI or combination of NOIs of the nucleic acid constructs of the present invention may encode all or part of a CAR.

[0109] Functional readthroughs can be used to enhance the functionality of CARs. This can be achieved by inserting a translational readthrough motif downstream of the antigen-binding domain or between the signaling domains of the CAR (Figure 3F and F').

[0110] In some situations, it may be desirable to induce the secretion of an antigen-binding domain (scFv / VHH) to mitigate the on-target off-tumor effect, where CART cells target normal tissue expressing low levels of the target antigen. The secreted antigen-binding domain should bind to the antigen expressed on the surface of normal cells, thereby preventing CART cells from recognizing the normal cells. Functional readthroughs can be used to manipulate T cells to secrete the antigen-binding domain by positioning a translational readthrough motif just upstream of the transmembrane domain of the CAR.

[0111] Nucleic acid constructs have a general structure: scFv / VHH-TRM-spacer-TM domain-end domain, or scFv / VHH-spacer-TRM-TM domain-end domain (Here, scFv / VHH is a nucleotide sequence that encodes an antigen-binding domain. A spacer is a nucleotide sequence that codes for a spacer. The TM domain is a nucleotide sequence that codes for the TM domain, and An endodomain is a nucleotide sequence that codes for an endodomain, and can be, for example, a first, second, or third-generation endodomain. It may have.

[0112] Functional readthrough can also be used to generate combinations of CARs that are first, second, or third generation. In this scenario, the translational readthrough motif may be located between the CD3z and coreceptor endodomains, which results in high levels of expression of first-generation CARs (CD3ζ signaling domain only) and substantially low levels of second or third-generation CARs. Nucleic acid constructs have a general structure: scFv / VHH-spacer-TM domain-CD3ζ end domain TRM-costimulatory domain, or scFv / VHH-Spacer-TM Domain-CD3ζ End Domain TRM1-Co-Stimulus Domain 1-TRM2-Co-Stimulus Domain 2 (Here, scFv / VHH is a nucleotide sequence that encodes an antigen-binding domain. A spacer is a nucleotide sequence that codes for a spacer. The TM domain is a nucleotide sequence that codes for the TM domain, and The CD3ζ end domain is the nucleotide sequence that codes for the CD3ζ end domain. The co-stimulatory domain is a nucleotide sequence that encodes a co-stimulatory domain, such as the endodomain derived from a co-receptor, for example, CD28 or a member of the TNF receptor superfamily. It may have.

[0113] Since few CARs possess both a co-receptor and a CD3ζ signaling domain, different repeats can be generated by swapping the positions of the CD3ζ and co-receptor endodomains so that CAR association primarily provides the cell with a co-stimulatory signal (signal 2) and a reduced antigen signal (signal 1).

[0114] Chimeric cytokine receptor International Publication No. 2017 / 029512 describes chimeric cytokine receptors in which the binding specificity of non-cytokine-binding molecules is transplanted into the endodomain of cytokine receptors. It also describes chimeric transmembrane proteins containing a dimerization domain and a cytokine receptor endodomain.

[0115] Dimerization can occur spontaneously, in which case the chimeric transmembrane protein is constitutively active. Alternatively, dimerization can occur only in the presence of a chemical dimerization inducer (CID), in which case the transmembrane protein triggers cytokine-type signaling only in the presence of the CID.

[0116] Constitutively active chimeric cytokine receptors may include the Fab portion of an antibody as an external domain. In this respect, the dimerization domain may include the dimerization portions of the heavy chain constant domain (CH) and the light chain constant domain (CL).

[0117] Chimeric transmembrane proteins consist of two polypeptides: (i) (a) the first dimerization domain; and (b) The first chain of the cytokine receptor endodomain A first polypeptide including; and (ii) (a) a first dimerization domain and a second dimerization domain that dimerizes; and (b) Second chain of the cytokine receptor endodomain The second polypeptide, which includes It may include.

[0118] In particular, chimeric transmembrane proteins, (i) (a) Heavy chain constant domain (CH) (b) The first chain of the cytokine receptor endodomain A first polypeptide containing; and (ii) (a) Light chain constant domain (CL) (b) Second chain of the cytokine receptor endodomain The second polypeptide, which includes It may include.

[0119] The first and second chains of the cytokine receptor endodomain can be selected from the α, β, and γ chains of the type I cytokine receptor endodomain.

[0120] The cytokine receptor endodomain is (i) IL-2 receptor β-chain endodomain (ii) IL-7 receptor α-chain endodomain; or (iii) IL-15 receptor α-chain endodomain; and / or (iv) Common gamma chain receptor endodomain It may include.

[0121] IL-2 IL-2 binds to the IL-2 receptor, which has three forms produced by different combinations of three different proteins often referred to as α, β, and γ "chains"; these subunits are also part of the receptors for other cytokines. The β and γ chains of IL-2R are members of the type I cytokine receptor family.

[0122] The three receptor chains can be expressed independently and differently on various cell types, and can assemble in different combinations and orders to generate low-affinity, medium-affinity, and high-affinity IL-2 receptors.

[0123] The α chain binds to IL-2 with low affinity, while the β and γ combination forms a complex that binds to IL-2 with moderate affinity, primarily on memory T cells and NK cells; all three receptor chains form a complex that binds to IL-2 with high affinity (Kd approximately 10-11M) on activated T cells and regulatory T cells.

[0124] These three IL-2 receptor chains spread across the cell membrane and extend into the cell, delivering biochemical signals to the intracellular interior. The alpha chain is not involved in signal transduction, while the beta chain forms a complex with the tyrosine phosphatase JAK1. Similarly, the gamma chain forms a complex with another tyrosine kinase called JAK3. These enzymes are activated by IL-2 binding to the external domain of IL-2R.

[0125] When early T cells are also stimulated by an antigen, IL-2 signaling promotes the differentiation of T cells into effector T cells and memory T cells. Through their role in the development of T cell immunological memory, which depends on the expansion of the number and function of antigen-selective T cell clones, they also play a crucial role in long-term cellular immunity.

[0126] The chimeric cytokine receptor of the present invention may include an IL-2 receptor β chain and / or an IL-2 receptor (i.e., common) γ chain.

[0127] The amino acid sequences of the endodomains of the IL-2β chain and common γ chain are shown as SEQ ID NOs. 56 and 57. [ka]

[0128] The term "derived from" means that the endodomain of the chimeric cytokine receptor of the present invention has the same sequence as the wild-type sequence of the endogenous molecule, or a variant thereof that retains the ability to form a complex with JAK-1 or JAK-3 to activate one of the above signaling pathways.

[0129] The "variant" sequence has at least 80, 85, 90, 95, 98, or 99% sequence identity with the wild-type sequence (e.g., sequence number 56 or 57), provided that the variant sequence retains the function of the wild-type sequence, i.e., the ability to complex with JAK-1 or JAK-3 to activate, for example, the JAK-STAT signaling pathway.

[0130] The percentage of identity between two polypeptide sequences can be easily determined by programs such as BLAST, which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0131] Steady-state domain In humans, there are two types of light chains: kappa (κ) chains and lambda (λ) chains. The lambda class has four subtypes: λ1, λ2, λ3, and λ4. The constant region of the light chain of Fab-type chimeric receptors can originate from any of these light chain types.

[0132] The light chain constant domain of the chimeric cytokine receptor may have the sequence shown as sequence number 58, which is the kappa chain constant domain. [ka]

[0133] There are five types of mammalian immunoglobulin heavy chains: γ, δ, α, μ, and ε, each defining the classes of immunoglobulins IgG, IgD, IgA, IgM, and IgE, respectively. Heavy chains γ, δ, and α have a constant domain composed of three tandem Ig domains and have a hinge to add flexibility. Heavy chains μ and ε consist of four domains.

[0134] The CH domain of the chimeric cytokine receptor of the present invention may include the sequence shown as SEQ ID NO: 59, which is derived from a γ-immunoglobulin heavy chain. [ka]

[0135] In preferred embodiments, the present invention provides a nucleic acid construct comprising a first nucleotide sequence encoding a chimeric antigen receptor (CAR); a flame slip motif (FSM) or translational read-through motif (TRM); and a second nucleotide sequence encoding a chimeric cytokine receptor (CCR).

[0136] Cutting site A nucleic acid construct according to a first aspect of the present invention may include sequences encoding cleavage sites located between nucleic acid sequences encoding the first and second polypeptides, such that the first and second polypeptides can be expressed as separate entities.

[0137] The cleavage site can be any sequence that allows the polypeptide, including the first and second polypeptides, to be separated.

[0138] The term “cleavage” is used herein for convenience, but cleavage sites can separate the first and second polypeptides into individual entities by mechanisms other than classical cleavage. For example, for the foot-and-mouth disease virus (FMDV) 2A autocleavage peptide (see below), various models have been proposed to explain “cleavage” activity: proteolysis by host cell proteinases, autoprotein degradation, or translational effect (Donnelly et al., (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such “cleavage” is not important for the purposes of this invention, as long as the cleavage site is located between the nucleic acid sequences encoding the first and second polypeptides and expresses the first and second polypeptides as separate entities.

[0139] The cleavage site may be a furin cleavage site.

[0140] Furin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta-1 precursor, proalbumin, probeta-secretase, membrane type 1 matrix metalloproteinase, pronervation growth factor beta subunit, and von Willebrand factor. Furin cleaves proteins immediately downstream of the basic amino acid target sequence (typically Arg-X-(Arg / Lys)-Arg' (SEQ ID NO: 50)) and is abundant in the Golgi apparatus.

[0141] The cleavage site may be a site of tobacco etch virus (TEV) cleavage.

[0142] TEV proteases are highly sequence-specific cysteine ​​proteases that are chymotrypsin-like proteases. Because they are highly specific to their target cleavage site, they are frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (SEQ ID NO: 51) (where "\" indicates the peptide bond to be cleaved). Mammalian cells, such as human cells, do not express TEV proteases. Therefore, in embodiments in which this nucleic acid construct contains a TEV cleavage site and is expressed in mammalian cells, the exogenous TEV protease must also be expressed in mammalian cells.

[0143] The cleavage site may encode a self-cleaving peptide.

[0144] A "self-cleaving peptide" refers to a polypeptide containing a first and second polypeptide, and a peptide that, once produced, functions to be immediately "cleaved" or separated into distinct first and second polypeptides without requiring any external cleavage activity.

[0145] Self-cleaving peptides can be 2A self-cleaving peptides derived from aftviruses or cardioviruses. The main 2A / 2B cleavage in aftviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In aftviruses such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids that, together with the N-terminal residue (conserved proline residue) of protein 2B, corresponds to an autonomous element that can mediate its own C-terminus "cleavage".

[0146] The C-terminal 19 amino acids of longer cardiovirus proteins, along with the N-terminal proline of 2B, mediate "cleavage" with nearly the same efficiency as the aftvirus FMDV2a sequence. Examples of cardioviruses include encephalomyocarditis virus (EMCV) and Tyler's mouse encephalitis virus (TMEV).

[0147] Mutagenicity analysis of EMCV and FMDV2A revealed that the motif DxExNPGP (SEQ ID NO: 52) is essentially involved in "cleavage" activity (Donelly et al. (2001), see above).

[0148] The cleavage site of the present invention is the amino acid sequence: Dx1Ex2NPGP (where x1 and x2 are any amino acids. X1 may be selected from the following groups: I, V, M, and S. X2 may be selected from the following groups: T, M, S, L, E, Q, and F).

[0149] For example, the cleavage site may include one of the amino acid sequences shown in Table 2. [Table 2-1] [Table 2-2]

[0150] The cleavage site based on the 2A sequence may be, for example, 15-22 amino acids long. The sequence may include the C-terminus of the 2A protein followed by a proline residue (corresponding to the N-terminal proline of 2B).

[0151] Mutation studies have also shown that, in addition to the naturally occurring 2A sequence, several variants are also active. The cleavage sites are variant sequences derived from naturally occurring 2A polypeptides having one, two, or three amino acid substitutions, and may correspond to variant sequences that retain the ability to induce the “cleavage” of a polyprotein sequence into two or more distinct proteins.

[0152] The cleavage sequence can be selected from the following, all of which have been shown to be active to some extent (Donnelly et al. (2001) above): [ka]

[0153] Based on the DxExNPGP sequence, the "motif," "2A-like" sequence was found in picornaviruses other than aftvirus or cardiovirus, "picornavirus-like" insect viruses, rotavirus type C, and repeat sequences within Trypanosoma species and bacterial sequences (Donnelly et al. (2001) above). The cleavage sites are these 2A-like sequences, e.g.: [ka] [ka] It may include one of the following.

[0154] The cleavage site may contain a 2A-like sequence, indicated as sequence number 31 (RAEGRGSLLTCGDVEENPGP).

[0155] It has been shown that activity can be increased by including an N-terminal "extension" of 5 to 39 amino acids (Donnelly et al. (2001), see above). In particular, the cleavage sequence may include one of the following sequences, or a variant thereof having, for example, up to 5 amino acid changes, and which retains cleavage site activity: [ka]

[0156] vector The present invention also provides a vector comprising a nucleic acid construct according to one aspect of the present invention.

[0157] Such vectors can be used to introduce nucleic acid constructs into host cells to express the first and second polypeptides.

[0158] The vector may be, for example, a plasmid or viral vector, such as a retroviral vector or lentiviral vector, or a transposon-based vector, or synthetic mRNA.

[0159] A vector may be capable of transfecting or transducing mammalian cells, such as T cells or target cells.

[0160] cell The present invention further provides cells comprising the nucleic acid construct or vector of the present invention, which express first and second polypeptides encoded by the nucleic acid sequence.

[0161] The cells could be any eukaryotic cells, such as immunological cells.

[0162] The cells may be cytotoxic immune cells, such as T cells or natural killer cells.

[0163] If the transgene expresses a target antigen, the cell may be a target cell for T cells or CAR-T cells.

[0164] method In a further embodiment, the present invention provides a method for producing cells according to the present invention, comprising the step of introducing a nucleic acid construct or vector of the present invention into cells.

[0165] Nucleic acid constructs can be introduced by transduction or transfection.

[0166] The cells may be cells isolated from the subject, such as T cells or NK cells isolated from the subject.

[0167] The present invention also provides a method for modulating the relative expression of two transgenes in a nucleic acid construct, comprising the step of including a flame slip motif or a translational read-through motif between the two transgenes in order to reduce the expression of a downstream transgene.

[0168] A nucleic acid construct may contain sequences encoding cleavage sites located between two transgenes. The relative expression of the two transgenes can be regulated by selecting different flame-slip motifs or translational read-through motifs, and / or by mutating sequences immediately 5' and / or 3' of the motifs.

[0169] The present invention also provides a method for modulating the relative expression of two transgenes in a nucleic acid construct, comprising the step of including a flame slip motif or a translational read-through motif between the two transgenes in order to reduce the expression of a downstream transgene.

[0170] The frame slip motif or translation lead-through motif may be as described in a prior aspect of the present invention.

[0171] Next, the present invention will be further illustrated by examples, which are intended to assist those skilled in the art in carrying out the present invention and are not intended to limit the scope of the invention. [Examples]

[0172] Example 1 - Fabrication and testing of a frame slip structure The following constructs were transduced into SupT1 cells. ·SFGmR.RQR8-2A-SKIP_7xU-CD22ecto-CD19tm-dCD19endo ·SFGmR.RQR8-2A-SKIP_6xU-CD22ecto-CD19tm-dCD19endo(control)

[0173] The construct encodes RQR8, a selective suicide transmembrane protein described in International Publication No. 2013 / 153391, and a chimeric protein having a CD22 external domain and CD19 transmembrane and endodomains.

[0174] The first construct includes a transcriptional frame slip containing seven thymine bases (uracil in the mRNA) that promote transcriptional slippage and loss of bases from mRNA, which positions the CD22 coding sequence within the frame, resulting in the expression of a CD22-CD19 chimera.

[0175] Flow cytometry analysis showed that the introduction of the flame slip motif resulted in a dramatic decrease in CD22-CD19 chimera expression, while similar levels of the RQR8 selection marker were observed (Figure 2C).

[0176] Example 2 - Fabrication and testing of translational lead-through structures Two translational read-through motifs, STOP-CUAG and STOP-CAAUUA, were shown to be functional in human T cells (SupT1) and human B cells (Raji), and to support the translational read-through of downstream autocleaved 2A peptide sequences and transgene coding sequences (CD22 and BCMA).

[0177] To test the functionality of these readthrough motifs in combination with different stop codons, the following constructs were transduced into HEK293T cells. Lead-through motif STOP-CUAG ·SFGmR.RQR8-STOP-TGACTAG-2A-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-STOP-TAGCTAG-2A-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-STOP-TAACTAG-2A-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-NO_STOP_TGGCTAG-T2A-hCD22ecto-CD19TM-dCD19endo Lead-through motif STOP-CAAUUA ·SFGmR.RQR8-STOP-TGACAATTA-2A-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-STOP-TAGCAATTA-2A-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-STOP-TAACAATTA-2A-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8_NO_STOP_TGGCAATTA-T2A-hCD22-CD19TM-dCD19endo Signal peptide variants ·SFGmR.RQR8-2A-Signal K9-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-2A-Signal K10-hCD22ecto-CD19TM-dCD19endo ·SFGmR.RQR8-2A-SignalK11-hCD22ecto-CD19TM-dCD19endo

[0178] The wild-type signal peptide sequences are shown below as SEQ ID NOs. 42-45, along with the sequences of modified signal peptides K19, K10, and K11. Sequence ID No. 42 - Mouse Ig Kappa Chain V-III Region Signal Peptide (Wild Type) METDTLILWVLLLLVPGSTG Sequence ID No. 43 - Mouse Ig kappa chain V-III region signal peptide (K9 mutant) METDTLILKVLLLLVPGSTG SEQ ID NO: 44 - Mouse Ig Kappa Chain V-III Region Signal Peptide (K10 Mutant) METDTLILWKLLLLVPGSTG Sequence ID No. 45 - Mouse Ig kappa chain V-III region signal peptide (K11 mutant) METDTLILWVKLLLVPGSTG

[0179] The expression of the CD22-CD19 chimera was analyzed by flow cytometry, and the results are shown in Figure 4A. Compared to the wild-type signal peptide, the lysine 11 signal peptide mutant (L11K mutant) showed reduced expression of the CD22-CD19 chimera. The stop-CUAG and stop-CAAUUA translational read-through motifs yielded similar results when placed on the 5' side of the CD22-CD19 transgene, and both motifs reduced the chimera expression level to approximately 2% or less of the RQR8 selection marker. A double-stop translational read-through motif (row 4) was used to obtain even lower expression levels of the CD22-CD19 chimera.

[0180] The mean fluorescence intensity of CD22-CD19 chimeras was quantified on the surface of transfected HEK293T cells, and the results are shown in Figure 4B. Using translational read-through constructs, substantially lower levels of CD22-CD19 chimeras were obtained compared to attenuated signal peptide mutants.

[0181] Example 3 - Functional translational readthrough: Secretion of soluble anti-CD22Fab As shown below, nucleic acid constructs encoding an RQR8 selection marker, followed by a 2A self-cleaving peptide sequence, and an anti-CD22 CAR having CL and CH1 spacer regions were designed to produce soluble Fab fragments. ·SFGmR.RQR8-2A-aCD22_FabCAR_9A8-1-STOP_SKIP_TAG-41BBz ·SFGmR.RQR8-2A-aCD22_FabCAR_9A8-1-STOP_SKIP_TAA-41BBz ·SFGmR.RQR8-2A-aCD22_FabCAR_9A8-1-STOP_SKIP_TGA-41BBz

[0182] These constructs were transduced into PBMCs, and cytotoxic assays were performed using target cells expressing low or high levels of CD22 to determine the efficacy of CAR. This mimics a situation where low levels of the target antigen are expressed on normal tissue, and fine-tuning of the cytotoxic response is necessary to prevent targeting of these cells by CAR cells.

[0183] Example 4 - Functional Translation Readthrough: Transition from 1st Generation to 2nd or 3rd Generation CAR To investigate whether it's possible to switch between first-generation and second-generation CARs using functional translation readthroughs, generate the following construct: ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-STOP_SKIP_TAA-41BB ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-STOP_SKIP_TGA-41BB ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-NO_STOP_SKIP_TCA-41BB ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-STOP_SKIP_TAG-41BB ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-STOP_SKIP_TAA-CD28 ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-STOP_SKIP_TAG-CD28 ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-NO_STOP_SKIP_TCA-CD28 ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-Zeta-STOP_SKIP_TGA-CD28

[0184] This first set of constructs expresses the selection marker RQR8, a first-generation anti-CD19 CAR having an Fc spacer domain and a CD3ζ endodomain, followed by a translational read-through motif and a 4-1BB or CD28 co-stimulatory endodomain. A restimulation assay is set up using transduced PBMCs cultured in plates coated with an anti-Fc antibody that binds to the CAR's spacer domain and triggers growth. A control first-generation CAR having only CD3ζ shows limited growth, while the other CARs grow more significantly in the presence of a co-stimulatory signal.

[0185] As shown below, the second set of constructs is generated in the same way as the first set of constructs: ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-41BB-STOP_SKIP_TAA-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-41BB-STOP_SKIP_TGA-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-41BB-NO_STOP_SKIP_TCA-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-41BB-STOP_SKIP_TAG-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-CD28-STOP_SKIP_TAA-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-CD28-STOP_SKIP_TGA-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-CD28-NO_STOP_SKIP_TCA-Zeta ·SFGmR.RQR8-2A-aCD19fmc63-HCH2CH3pvaa-CD28-STOP_SKIP_TAG-Zeta

[0186] In these constructs, the co-stimulatory domain derived from 4-1BB or CD28 is in front of the translation read-through motif, and the CD3ζ end domain is placed after it. Using PBMCs transfected with these constructs, cytotoxicity assays and proliferation assays are set up. The translation read-through from these constructs is necessary to produce a functional CAR (i.e., a CAR having an intracellular signaling domain).

[0187] Example 5 - Preparation and testing of composite read-through motifs To reduce the expression of the multi-transgene cassette to a very low level, constructs having two STOP - translation read-through motifs were designed as follows: Marker (RQR8 or HA8) - STOP_read-through - 2A - GD3 synthase - STOP_read-through - 2A - GD2 synthase

[0188] Ganglioside GD2 is synthesized by two enzymes, GD2 synthase and GD3 synthase. The above constructs result in reduced GD3 synthase expression compared to the marker gene and even lower GD2 synthase expression.

[0189] Example 6 - Fabrication and testing of a universal lead-through structure To overcome the problem of context-dependent expression from read-through motifs, a group of universal constructs can be generated, into which a second transgene expressed at a predetermined level can be cloned. The universal construct consists of a self-cleaved 2A peptide sequence, followed by a stop codon and a read-through motif, and a second self-cleaved peptide sequence. By aligning the stop read-through motif with the self-cleaved peptide sequence, the context-dependent effect on translational read-through is reduced, resulting in consistent and predictable levels of transgene expression.

[0190] The structures tested were as follows: Lead-through motif STOP CUAG • Marker-2A-transgene1-2A-UGA-CUAG-2A-transgene2 • Marker-2A-transgene1-2A-UAG-CUAG-2A-transgene2 • Marker-2A-transgene1-2A-UAA-CUAG-2A-transgene2 Lead-through motif STOP CAAUUA • Marker-2A-Transgene 1-2A-UGA-CAAUUA-2A-Transgene 2 • Marker-2A-Transgene 1-2A-UAG-CAAUUA-2A-Transgene 2 • Marker-2A-Transgene 1-2A-UAA-CAAUUA-2A-Transgene 2

[0191] Example 7 - Ultra-low expression by arranging translational lead-through motifs in series To determine if it was possible to obtain very low levels of transgene expression, constructs were generated in which two translational read-through motifs were arranged in series in a tricistron cassette (Figure 5A). The first transgene in the cassette was always a cell surface marker consisting of an HA epitope presented on a CD8a stalk for transduction, followed by a stop codon, a translational read-through motif, and a self-cleaving peptide sequence. The second transgene in the cassette was one of two fluorescent proteins, either tandem Clover3 (green fluorescent protein) or enhanced blue fluorescent protein (EBFP), followed by a stop codon, a translational read-through motif, and a self-cleaving peptide sequence. The third transgene in the cassette was alternating fluorescent proteins. By arranging the stop codon and translational read-through motif in series, the expression level of the fluorescent protein at the third position should be lower than that at the second position.

[0192] The constructs were transduced into peripheral blood mononuclear cells (PBMCs), and flow cytometry was performed to quantify the expression levels of the fluorescent protein compared to cell surface markers. These experiments demonstrated that the expression levels of the fluorescent protein were significantly lower when it was placed in a third position downstream of the two stop codons and translational read-through motifs compared to when it was placed in the second position (Figure 5B). Consistent with previous experiments, the level of translational read-through was stop codon-dependent, with a hierarchical stop codon stringency of UAA > UAG > UGA. For tandem Clover3, the reduction in expression levels compared to the control without stop codons was 33-fold for UAA, 24-fold for UAG, and 6-fold for UGA. When placed in the third position, the level of tandem Clover3 expression decreased more than 100-fold for all three stop codons (Figure 5C).

[0193] Example 8 - Control of IL-12 secretion by translational read-through IL-12 is a potent pro-inflammatory cytokine secreted by phagocytes and dendritic cells in response to pathogens, signals from T cells and natural killer (NK) cells, and components of the inflammatory extracellular matrix. The primary targets of IL-12 are cytotoxic T cells and T cells that express IL-12 receptors on their cell surface. H These are helper T cells and NK cells. In response to IL-12, T H Helper T cells release IFNγ and TNFα.

[0194] CAR-T cells have been engineered to express IL-12 constitutively or from inducible promoters, which has been shown to improve the efficacy of CAR-T cell therapy when targeting solid tumors. Systemic administration of IL-12 is toxic, and to circumvent this problem, CAR-T cells or other immune cells have been engineered to release IL-12 into the tumor microenvironment. However, transgenic T cell IL-12 secretion can result in highly toxic systemic levels of cytokines.

[0195] The inventors designed a construct by cloning flexi-IL-12, a fusion between human IL-12α(p35) and IL-12β(p40) subunits, downstream of the selection marker RQR8 and a self-cleaving peptide sequence. A stop codon or regulatory sequence (UGG encoding tryptophan) and a translational read-through sequence CAAUUA were placed immediately downstream of the RQR8 coding sequence (Figure 6A). The construct was transduced into PBMCs, and the transduction efficiency was determined by staining with antibodies against CD3ε and RQR8 to verify the presence of the transgene (Figure 6B). After determining the transduction efficiency, the PBMCs were returned to culture, and the supernatant was collected at 24, 48, and 72 hours and analyzed for the presence of IL-12 by high-sensitivity ELISA (Figure 6C). The data demonstrated that the level decreased by more than 40-fold by including the stop codon and translational read-through motif (Figure 6C). The hierarchy of stop codon stringency showed a trend from highest stringency to lowest: UAA > UAG > UGA. These data demonstrated that it was possible to significantly reduce the level of IL-12 secretion from transduced cells.

[0196] To demonstrate that secreted levels of flexi-IL-12 could trigger an immune response, the inventors generated another group of constructs. These were tricistronic constructs containing a suicide gene (RapaCasp9), the mouse cell surface marker Thy1.1, and mouse flexi-IL-12 (Figure 7A). The mouse flexi-IL-12 sequence was located at the terminal end of the tricistron cassette and facilitated polypeptide expression using an autocleavage peptide sequence. The translational read-through construct (UAA-CAAUUA) was compared to a construct containing mouse flexi-IL-12 with a decay signal peptide sequence, and another construct that controlled cytokine expression using an internal ribosome entry site (IRES). The constructs were transduced into splenocytes obtained from BalbC mice, and the transduction efficiency was determined by flow cytometry using antibodies against CD3 and the cell surface marker Thy1.1 (Figure 7B). After determining the transduction efficiency, splenocytes were returned to culture, and the supernatant was collected at 24, 48, and 72 hours and analyzed for the presence of mouse IL-12 by ELISA (Figure 7C). The results demonstrated an approximately 3-fold decrease in IL-12 expression from IRES. In contrast, IL-12 expression from the translational read-through motif UAA-CAAUUA decreased to the point where it was no longer detectable by ELISA (Figure 7C). To confirm the presence of IL-12 in the culture medium, the supernatant was collected and used to restimulate activated splenocytes. Since IFNγ is secreted by IL-12-stimulated T cells and NK cells, the supernatant from these restimulated splenocytes was analyzed for the presence of IFNγ (Figure 7D). IFNγ was detectable in all supernatants collected from restimulated splenocytes. Consistent with the IL-12 ELISA results, the highest levels of IFNγ secretion were observed from splenocytes restimulated with supernatant from control or IRES-transduced splenocytes. Importantly, IFNγ secretion was observed from splenocytes restimulated with supernatant derived from transduced splenocytes containing the translational read-through construct (Figure 7D). These results demonstrate that it is possible to obtain a significant reduction in IL-12 secretion levels from transduced cells, potentially mitigating the toxic effects of this potent cytokine while maintaining therapeutic benefits.

[0197] To demonstrate the efficacy and safety profile of IL-12 secretion from the translation read-through construct in a more therapeutic setting, constructs encoding truncated murine CD34, anti-GD2 chimeric antigen receptor and firefly luciferase, as well as the previously described cytokine expression construct, were co-transduced into splenocytes. The transduced splenocytes were stained with antibodies against CD34 and Thy1.1 and analyzed by flow cytometry to determine the transduction efficiency (Figure 8A). 5×10 6 transduced splenocytes were injected into mice, and after 15 days, the mice were sacrificed and their spleens were removed. Comparison of spleen sizes showed that splenomegaly was observed only when IL-12 was constitutively expressed (2A), and that the spleen sizes in the other groups were comparable to the control (Figure 8B).

[0198] To investigate in more detail the cell types recruited to the spleens of the injected mice, splenocytes were stained with antibodies against CD11b, CD3, CD4, CD8 and CD19 and analyzed by flow cytometry (Figure 9A). The results showed that macrophages (CD11b + ) were recruited to the spleens of mice injected with splenocytes transduced with constitutively expressed IL-12, and that approximately 20% of the cells were CD11b + . Consistent with the previous ELISA data showing that the IRES construct resulted in higher levels of IL-12 than the translation read-through construct, more CD11b + cells were present in the spleens of mice injected with these cells (Figure 9B). In mice injected with splenocytes transduced with constructs constitutively expressing IL-12 or IRES, an increase in the recruitment of both CD4 + and CD8 + T cells was observed (Figure 9C, D and E). In contrast, CD11b + and CD3 +The number of cells was similar to that of the control group. A decrease in B cell count was observed only in the spleens of mice injected with IL-12 constitutively expressing splenocytes. Taken together, these results suggest that, in the absence of the antigen, expression of IL-12 from the translational readthrough construct did not cause any toxicity or recruitment of immune cells.

[0199] All publications referenced herein are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in relation to certain preferred embodiments, it should be understood that the invention as described in the claims should not be unduly limited to such specific embodiments. In fact, various modifications of the embodiments of the invention described herein will be apparent to those skilled in the art in molecular biology or related fields and are intended to be within the scope of the following claims.

Claims

[Claim 1] The invention as shown in the drawings.

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