阅读说明：本技术 核酸构建体 (Nucleic acid constructs ) 是由 S.科多巴 J.西尔利伯恩 M.普勒 于 2019-07-31 设计创作，主要内容包括：本发明提供了核酸构建体,其包含：第一目的核苷酸序列(NOI1)；框滑动基序或翻译连读基序(FSM/TRM)；和第二目的核苷酸序列(NOI2)。本发明还提供了表达此类构建体的载体和细胞。本发明还提供了调节核酸构建体中两个转基因的相对表达的方法,该方法包括以下步骤：在两个转基因之间包括框滑动基序或翻译连读基序,以减少下游转基因的表达。(The present invention provides a nucleic acid construct comprising: a first nucleotide sequence of interest (NOI 1); a frame sliding motif or a translation read-through motif (FSM/TRM); and a second nucleotide sequence of interest (NOI 2). The invention also provides vectors and cells expressing such constructs. The invention also provides a method of modulating the relative expression of two transgenes in a nucleic acid construct, the method comprising the steps of: a frame sliding motif or a translation read-through motif is included between the two transgenes to reduce expression of the downstream transgene.)

1. A nucleic acid construct comprising: a first nucleotide sequence of interest (NOI 1); a Frame Sliding Motif (FSM) or a translation read-through motif (TRM); and a second nucleotide sequence of interest (NOI 2).

2. The nucleic acid construct of claim 1, further comprising a nucleotide sequence encoding a cleavage site (CL) such that NOI1 and NOI2 are expressed as separate proteins.

3. The nucleic acid construct of claim 2, having the structure:

NOI 1-CL-FSM-NOI 2; or

NOI1-TRM-CL-NOI2。

4. The nucleic acid construct according to any of the preceding claims, comprising a Frame Sliding Motif (FSM) comprising repeats of uracil, thymine or guanine bases.

5. The nucleic acid construct according to claim 4, wherein the FSM comprises the sequence UUUUUUUUUU (SEQ ID No. 1).

6. The nucleic acid construct according to claim 4 or 5, wherein the FSM further comprises a stop codon.

7. The nucleic acid construct according to claim 6, wherein the FSM comprises one of the following sequences:

UUUUUUUGA(SEQ ID NO.2)

UUUUUUUAG(SEQ ID NO.3)

UUUUUUUAA(SEQ ID NO.4)。

8. the nucleic acid construct according to any one of claims 1 to 3, comprising a translation read-through motif (TRM) comprising the sequence STOP-CUAG or STOP-CAAUUA, wherein "STOP" is a STOP codon.

9. The nucleic acid construct of claim 8, wherein the translational read-through motif comprises one of the following sequences:

UGA-CUAG(SEQ ID No.5)

UAG-CUAG(SEQ ID No.6)

UAA-CUAG(SEQ ID No.7)

UGA-CAAUUA(SEQ ID No.8)

UAG-CAAUUA(SEQ ID No.9)

UAA-CAAUUA(SEQ ID No.10)。

10. the nucleic acid construct according to claim 8 or 9, having the 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

wherein:

TRM1 and TRM2, which may be the same or different, are a first translational read-through motif and a second translational read-through motif;

CL1 and CL2, which may be the same or different, are a first nucleic acid sequence and a second nucleic acid sequence, each encoding a cleavage site;

SP is a reduced signal peptide; and

NOI3 is a third nucleotide sequence of interest.

11. The nucleic acid construct of claims 2-10, wherein the cleavage site comprises a self-cleaving peptide, a furin cleavage site, or a tobacco etch virus cleavage site.

12. The nucleic acid construct of claim 11, wherein the cleavage site comprises a 2A self-cleaving peptide, or a 2A-like peptide, from an orthohoof virus or a cardiovirus.

13. The nucleic acid construct of any preceding claim wherein the NOI2 encodes a cytokine, chemokine or toxin.

14. The nucleic acid construct of claim 1, which when expressed in a cell is capable of producing two products:

a) a first product encoded by no i1 only; and

b) a second product encoded by NOI1 and NOI2, which second product is produced when a frame slip or translational read-through occurs.

15. The nucleic acid construct of claim 14, wherein the second product is a Chimeric Antigen Receptor (CAR) and the first product is a truncated form of the CAR that is incapable of inducing CAR-mediated cell signaling.

16. The nucleic acid construct of claim 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 co-stimulatory domains.

17. A vector comprising the nucleic acid construct of any one of the preceding claims.

18. A retroviral vector or a lentiviral vector according to claim 17.

19. A cell comprising the nucleic acid construct of any one of claims 1 to 16 or the vector of claim 17 or 19.

20. A method for preparing a cell according to claim 19, comprising the step of introducing a nucleic acid construct according to any one of claims 1 to 16 or a vector according to claim 17 or 18 into a cell.

21. A method for modulating the relative expression of two transgenes in a nucleic acid construct comprising the step of including a frame slip motif or a translation read-through motif between the two transgenes to reduce expression of a downstream transgene.

Technical Field

The present invention relates to constructs and methods for modulating the relative expression of transgenes.

Background

Expression of multiple transgenes

Gene therapy involves the modification of cells to express biomolecules to treat or correct pathological conditions. Obtaining physiologically or therapeutically relevant levels of biomolecules is critical to successful gene therapy.

Current methods for regulating gene expression typically rely on modifying the promoter region to increase or decrease the rate of transcription or inserting regulatory elements therein to make their expression inducible by the action of engineered transcription factors and small molecules.

Gene therapy approaches typically involve the expression of more than one transgene. Transduction of cells with multiple vectors to produce multiple products is difficult, expensive and unpredictable. Thus, various methods have been developed to allow co-expression of two proteins from a single vector (see FIG. 1).

The initial attempt was to use two different promoters in the same expression cassette, which resulted in two independent transcripts, each encoding an independent protein. This is a difficult process for a number of reasons. One key issue is "promoter interference," i.e., one promoter dominates and leads to silencing of a second promoter. In addition, different promoters operate differently in different cellular environments, which makes it difficult to achieve consistent "regulation" of the relative expression of each transgene.

An alternative approach is to use an Internal Ribosome Entry Sequence (IRES). Here, a single transcript is generated. The IRES sequence in the transcript is located between the open reading frames of the two transgenes and mimics the mRNA cap structure. Thus, the ribosome begins translation at the 5' cap or IRES, resulting in the expression of two separate proteins. The key limitation of this approach is the inability to control relative expression. The expression of the 3 'transcript is generally less than that of the 5' transcript, but the expression ratio is difficult to predict and adjust.

Another approach has been developed based on the characterization of the role of foot-and-mouth disease virus (FMDV)2A peptides in allowing FMDV (and related viruses) to express multiple proteins from a single Open Reading Frame (ORF) (Donnelly et al; J.Gen.Virol.; 82, 1027-1041 (2001)). The 2A peptide (and homologues) are cleaved with extremely high efficiency immediately after ORF translation, thereby allowing expression of multiple peptides from a single ORF. The use of self-cleaving peptides such as the 2A peptide results in the expression of the transgene at a 1:1 ratio.

WO2016/174409 describes the use of altered signal peptides to alter the proportion of transgene expression in nucleic acid constructs. In this system, mutations are introduced, for example, into the signal peptide motif of a type I transmembrane protein to reduce the efficiency of transport to the cell surface. This approach can result in about a 10-fold reduction in cell surface expression levels compared to the wild-type signal peptide motif.

However, this strategy is only suitable for reducing the expression of cell surface proteins, as it works by reducing the transport of proteins to the cell surface. Furthermore, a 10-fold reduction in expression may not be sufficient for certain applications, such as the expression of toxic compounds, where overexpression may lead to host cell death.

Thus, there is a need for alternative methods of altering the relative expression of two or more transgenes in a cell that are not associated with these limitations.

Chimeric Antigen Receptor (CAR)

Chimeric antigen receptor T cell (CAR-T cell) therapy redirects cytolytic T cells to target tumor cells by expressing on the cell surface a Chimeric Antigen Receptor (CAR) that recognizes a tumor specific antigen. Chimeric antigen receptors consist of an antibody or ligand binding motif, a spacer domain and a transmembrane domain, which are fused to the intracellular signaling domain of the CD3 zeta chain of the T Cell Receptor (TCR). The signaling domain from a co-receptor, such as CD28, OX40, or 4-1BB, may also be included in CAR 1 along with the CD3 zeta signaling domain.

CAR-T cell therapy has proven to be an effective therapy for the treatment of hematological malignancies, such as B-cell leukemia, which achieves impressive response rates. However, CAR-T cell therapy has not been as successful in the treatment of solid tumors, where several factors combine to limit CAR-T cell activity. These factors include expression of immunosuppressive ligands such as PD-L1, secretion of cytokines that inhibit favorable inflammatory responses, expression of ligands that induce tumor cell apoptosis and nutrient depletion. To overcome the immunosuppressive tumor microenvironment, CAR-T cells were engineered to express molecules that either block immunosuppressive signaling or enable T cell survival in hostile tumor microenvironments.

Cytokines that enhance the inflammatory response may improve the efficacy of CAR-T cell therapy. These cytokines include IL-7, IL-12, IL15, IL-17A, IL-18, and IL-21, which are shown to enhance CAR-T cell responses when provided exogenously or when released during CAR T cell therapy.

IL-12 is a potent immunomodulatory cytokine that is normally secreted by phagocytes and dendritic cells in response to: a pathogen; t cell signaling and Natural Killer (NK) cell signaling; and extracellular matrix components. CAR-T cells have been engineered to express IL-12 constitutively or from inducible promoters, which have been shown to improve the efficacy of CAR-T cell therapy when targeting solid tumors. IL-12 has systemic toxicity, so CAR T cells or other immune cells have been engineered to release IL-12 into the tumor microenvironment 1. However, transgenic T-cell IL-12 secretion can lead to high toxic systemic levels.

Although expression of cytokines, chemokines or toxins may improve the efficacy of CAR-T cell therapy, secretion of these biomolecules must be tightly regulated to limit their toxicity to safe levels that provide therapeutic benefit and minimal side effects. This can be achieved by inserting or deleting cis-acting sequences to achieve the desired level of transcription or introducing inducible elements into the promoter region to regulate the rate of transcription of the gene from the promoter region. Notable inducible promoter systems include the tetracycline-off (tet-off) system and the tetracycline-off (tet-off) system, which utilize a Tetracycline Responsive Element (TRE) in combination with an engineered form of the tetracycline repressor (TetR), which binds to the 19bp nucleotide sequence of the TRE 2. In the tetracycline-off system, TetR is fused to the transactivation domain of the viron protein 16(VP16) of herpes simplex virus, thereby generating the 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. Mutation of the TetR domain in tTA renders it tetracycline dependent for binding to TRE, thereby producing reverse tTA (rtta) and tetracycline on the system. In the presence of tetracycline, rtTA is able to bind to TRE and stimulate transcription, but not in the absence. Although the tetracycline inducible system can control transgene expression, it relies on the use of small molecules, the insertion of a TRE into the promoter sequence, and the expression of tTA or rtTA, which places a heavy burden on the cell.

An alternative method of controlling gene expression is to use an Internal Ribosome Entry Site (IRES). As described above, the IRES is placed downstream of the primary transgene and promotes constitutive expression of other transgenes, usually at levels lower than the primary transgene. This approach has been used to constitutively express IL-12 in CAR-T cells in an attempt to enhance the immune response and avoid pre-treatment of the host prior to infusion of CAR-T cells. However, the expression level of IL-12 is unpredictable and may still be toxic in vivo.

Therefore, there is a need for alternative methods for co-expression of chimeric antigen receptors with molecules such as cytokines, chemokines or toxins that are not associated with the above disadvantages.

Summary of The Invention

The present inventors have developed systems that use frame sliding or translational read-through as a means to modulate transgene expression. In particular, the system can be used to control the relative expression of two or more transgenes expressed from a single mRNA transcript.

Accordingly, in a first aspect, the present invention provides a nucleic acid construct comprising: a first nucleotide sequence of interest (NOI 1); a frame sliding motif or a translation read-through motif (FSM/TRM); and a second nucleotide sequence of interest (NOI 2).

The nucleic acid construct may further comprise a nucleotide sequence encoding a cleavage site (CL) such that NOI1 and NOI2 are expressed as separate proteins.

The nucleic acid construct may comprise the structure:

NOI 1-FSM/TRM-CL-NOI 2 or NOI1-CL-FSM/TRM-NOI 2.

In particular, the nucleic acid construct comprises the following structure:

NOI 1-CL-FSM-NOI 2; or

NOI1-TRM-CL-NOI2。

The nucleic acid construct may comprise more than 2 nucleotide sequences of interest. For example, the nucleic acid construct may have the following structure:

NOI 1-FSM 1/TRM1-CL 1-NOI 2-FSM2/TRM2-CL2-NOI3, or

NOI1–CL1-FSM1/TRM1–NOI2-CL2-FSM2/TRM2-NOI3

Wherein:

NOI1, NOI2 and NOI3 are nucleotide sequences of interest;

FSM1/TRM1 and FSM2/TRM2, which may be the same or different, are box sliding motifs or translation readthrough motifs; and

CL1 and CL2, which may be the same or different, are the nucleic acid sequences that each encode a cleavage site.

The Frame Sliding Motif (FSM) may comprise a repeat of uracil, thymine or guanine bases, for example the sequence UUUUUUU (SEQ ID No. 1).

The frame sliding motif can further comprise a stop codon. For example, the FSM may comprise one of the following sequences:

UUUUUUUGA(SEQ ID NO.2)

UUUUUUUAG(SEQ ID NO.3)

UUUUUUUAA(SEQ ID NO.4)。

the translation read-through motif (TRM) may comprise the sequence STOP-CUAG or STOP-CAAUUA, where "STOP" is a STOP codon. For example, a translational read-through motif can comprise one of the following sequences:

UGA-CUAG(SEQ ID No.5)

UAG-CUAG(SEQ ID No.6)

UAA-CUAG(SEQ ID No.7)

UGA-CAAUUA(SEQ ID No.8)

UAG-CAAUUA(SEQ ID No.9)

UAA-CAAUUA(SEQ ID No.10)

a nucleic acid construct comprising a translation read-through motif can comprise two adjacent TRMs that can be the same or different.

A nucleic acid construct comprising a translation read-through motif can comprise two nucleic acid sequences that each encode a cleavage site. The TRM may be located between the two cleavage site coding sequences, or there may be two TRMs, each located upstream of the cleavage site.

Nucleic acid constructs comprising a translational read-through motif can comprise an attenuated or inefficient signal peptide located upstream of a second (or subsequent) nucleotide of interest.

A nucleic acid construct comprising a translation read-through motif can have one of the following sequences:

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

wherein:

TRM1 and TRM2, which may be the same or different, are a first translational read-through motif and a second translational read-through motif;

CL1 and CL2, which may be the same or different, are a first nucleic acid sequence and a second nucleic acid sequence, each encoding a cleavage site;

SP is a reduced signal peptide; and

NOI3 is a third nucleotide sequence of interest.

The cleavage site may, for example, comprise a self-cleaving peptide, a furin cleavage site, or a tobacco etch virus cleavage site.

The cleavage site may, for example, comprise a 2A self-cleaving peptide or a 2A-like peptide from an orthohoof virus or a cardiovirus.

The second (or subsequent) nucleotide of interest may encode a cytokine, chemokine or toxin.

The nucleic acid construct, when expressed in a cell, may be capable of producing two products:

a) a first product encoded by no i1 only; and

b) a second product encoded by NOI1 and NOI2, which second product is produced when a frame slip or translational read-through occurs.

In this embodiment, the second product may be a Chimeric Antigen Receptor (CAR), and the first product may be a truncated form of the CAR that is incapable of inducing CAR-mediated cell signaling.

Alternatively, the first product may be a chimeric antigen receptor (CAR, i.e., a first generation CAR) comprising an intracellular signaling domain, and the second product is a CAR comprising an intracellular signaling domain and one or more co-stimulatory domains (i.e., a second or third generation CAR).

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

The vector may be, for example, a retroviral vector or a lentiviral vector.

In a third aspect, the invention provides a cell comprising a nucleic acid construct according to the first aspect of the invention or a vector according to the second aspect of the invention.

In a fourth aspect, the present invention provides a method for preparing a cell according to the third aspect of the invention, comprising the step of introducing a nucleic acid construct according to the first aspect of the invention or a vector according to the second aspect of the invention into a cell.

In a fifth aspect, the present invention provides a method for modulating the relative expression of two transgenes in a nucleic acid construct, the method comprising the steps of: a frame sliding motif or a translation read-through motif is introduced between the two transgenes to reduce expression of the downstream transgene.

The obvious advantage of using frame sliding or translational read-through as a means to control transgene expression is that broad expression can be achieved, and in the case of two transgenes being expressed, the level of expression of the second transgene can be reduced to less than 1% of the first transgene. This is well below the level achievable with the altered signal peptide and makes this technique useful for expressing proteins such as toxic compounds where extremely low expression levels are necessary.

The use of frame sliding or translational read-through allows the transgene to be expressed at a predictable and determined rate. The expression level of the transgene can also be modulated by using different frame sliding motifs or read-through motifs.

The system can be used to control the expression of any transgene and is not limited to the expression of cell surface proteins.

Drawings

FIG. 1: method for expressing different proteins from the same vector

(a) Two different promoters within the same expression cassette produce two different transcripts, each of which produces an independent protein. (b) The use of an Internal Ribosome Entry Sequence (IRES) results in a single transcript that is translated into two separate proteins. (c) The use of FMDV 2A peptides results in a single transcript and a single polyprotein that rapidly cleaves into two separate proteins.

FIG. 2: frame sliding motif and construct design

(A) A diagram of a frame sliding motif is illustrated in which a series of seven uridines are inserted into the transgene sequence, thereby promoting frame sliding. Frame slipping results in the alternative reading frame, usually in the-1 direction, continuing transcription/translation and the production of functional proteins. (B) Construct structures showing the position of the transgene, the frame SLIP motif (SLIP), and the 2A self-cleaving peptide sequence. (C) Flow cytometry analysis SupT1 cells transduced with a construct containing RQR8 sorting marker followed by a control sequence (6xU) or a frame sliding motif (7xU) and a CD22-CD19 chimeric protein consisting of the extracellular domain of CD22 fused to the transmembrane and truncated intracellular domains of CD19 were analyzed. Introduction of the frame slip motif resulted in a significant reduction in expression of the CD22-CD19 chimera, while similar levels of RQR8 sorting marker were observed.

FIG. 3: translation read-through motifs and construct design.

(A) Examples of known translation read-through motifs. (B-G) Structure of translation readthrough motif construct. (B) A translation read-through construct consisting of two transgenes, wherein the translation read-through motif is located 3 'and 2A self-cleaving peptide sequence of the first transgene and 5' of transgene 2. The expression of transgene 1 will be significantly higher than that of transgene 2. (B') a dual stop codon translation read-through construct wherein two stop codons are incorporated into the translation read-through motif to reduce the expression level below that obtained using a single stop codon. (C) A universal translational read-through construct consisting of a translational read-through motif flanked by two 2A self-cleaving peptide sequences. This construct mitigates sequence-dependent effects that can lead to unpredictable levels of translational read-through. (D) A combination protocol wherein a translational read-through motif is combined with an attenuated signal peptide sequence to further reduce the expression level of transgene 2. (E and E') composite translation read-through motifs, in which multiple motifs are placed in tandem to produce a cascade of reduced transgene expression. (E) Multiple transgenes were expressed from a single expression cassette using a translational read-through motif and a 2A self-cleaving peptide sequence. (E ') isolating the translational read-through motif 5' of transgene 2 using a self-cleaving peptide sequence. (F and F') a functional translation read-through construct. (F) Secretion of the antigen binding domain (scFv/VHH) and functional CAR can be achieved by placing a translation read-through motif 3' to the CAR spacer domain. Functional read-through results in the expression of a functional CAR, while termination of translation at the stop codon produces a secreted antibody. (F ') A combination of a first generation CAR, a second generation CAR, and a third generation CAR can be generated by placing a translation read-through motif 3' to the first endodomain (either the CD3 ζ endodomain or the co-receptor endodomain). Functional translation read-through will switch expression from a first generation CAR to a second or third generation CAR.

FIG. 4: comparison of attenuated signal peptides and the translational read-through motif protocol for controlling expression of CD22-CD19 chimeras.

(A) Flow cytometric analysis of HEK293T cells transfected with constructs encoding RQQ8 sorting marker and CD22-CD19 chimera. A signal peptide mutant (line 1), a translational read-through motif construct (lines 2 and 3), and a double stop codon translational read-through motif (line 4). The expression of the CD22-CD19 chimera was reduced in the lysine 11 signal peptide mutant (L11K mutation) compared to the wild-type signal peptide. Similar results were produced when stop-CUAG and stop-CAAUUA translational read-through motifs were placed 5' to the CD22-CD19 transgene, both motifs reducing the expression level of the chimera to about 2% or less of the RQR8 sorting marker. Lower levels of CD22-CD19 chimera expression were obtained using a double stop codon translation read-through motif (row 4). (B) Quantification of mean fluorescence intensity of CD22-CD19 chimeras on the surface of transfected HEK293T cells, attenuated signal peptide mutants were compared to the translation read-through constructs. Substantially lower levels of CD22-CD19 chimera were obtained using the translational read-through construct compared to the attenuated signal peptide mutant.

FIG. 5: tandem translation read-through motifs

A) Map of the resulting construct placing the stop codon and the translation read-through motif in tandem. For simplicity, only four examples are shown, which show the structure of constructs with tandem translation read-through motifs. The cell surface marker, the HA epitope presented on the stem of CD8a (CD8a talk), is always located at the first position in the expression cassette, followed by a tandem Clover3 or EBFP with different coding sequences, these sequences being separated by the self-cleaving peptide sequence (2A). Two control constructs were cloned in which the stop codon was replaced by UGG (encoding tryptophan) and tandem mClover or EBFP was located at the second or third position. For constructs with tandem translational read-through motifs, all three stop codons (UGA, UAG and UAA) were tested, and the translational read-through motif used was CUAG. The same stop codon was used at both positions. B) Flow cytometry of PBMCs transduced with constructs showing tandem Clover3 fluorescence intensity from constructs with 0, 1 or 2 stop codons/translation read-through motif (control without stop codon and tandem Clover3 at position 2). The histograms show that tandem Clover3 expression from constructs with translation read-through motifs was reduced, with levels varying depending on the stop codon present. C) Normalization of tandem Clover3 fluorescence intensity levels to cell surface markers. Levels were reduced 6-fold, 24-fold, or 33-fold when placed directly downstream of the UGA, UAG, or UAA stop codon and CUAG translation read-through motif, respectively. When the tandem Clover3 sequence was placed downstream of the two stop codons and the translation read-through motif, a 100-fold reduction in tandem Clover3 levels was observed.

FIG. 6: controlled expression of human flexi-IL-12 downstream of the translational read-through motif

A) A diagram showing the structure of the construct, in which human flexi-IL-12, consisting of a fusion between IL-12 α (p35) and IL-12 β (p40) subunits, was placed downstream of the sorting marker RQR8, with self-cleaving peptides promoting the expression of both proteins. All three stop codons were tested with the translation read-through motif CAAUUA. B) Representative flow cytometry plots of PBMCs transduced with the constructs and stained with antibodies against CD3 epsilon and RQR 8. C) IL-12 secretion from transduced PBMCs was quantified at 24, 48 and 72 hours after determination of transduction efficiency. The results indicate that IL-12 secretion from the translation read-through construct is significantly reduced.

FIG. 7: controlled expression of murine IL-12 downstream of a translational read-through motif

A) Diagram of a tricistronic construct with suicide gene (Rapacap 9), cell surface marker Thy1.1 and murine flexi-IL-12. Murine flexi-IL-12 levels are regulated by an Internal Ribosome Entry Site (IRES), an attenuated signal peptide motif, or a translation read-through motif. B) Representative flow cytometry plots of BalbC splenocytes transduced with the construct and stained with antibodies to CD3 epsilon and thy 1.1. C) The level of IL-12 secreted from the transduced splenocytes was quantified by ELISA. D) Supernatants from transduced splenocytes were used to restimulate activated splenocytes. Supernatants from restimulation were analyzed for the presence of IFNg by ELISA. Importantly, these results indicate that supernatants of splenocytes transduced with translation read-through motifs can cause secretion of IFN γ by restimulated splenocytes.

FIG. 8: toxicity testing of translation read-through IL-12 constructs

A) Splenocytes were co-transduced with the previously described constructs encoding RapaCasp6, thy1.1, and IL-12 and a second construct comprising truncated murine CD34 (muccd 34), firefly luciferase (FLuc), and anti-GD 2 Chimeric Antigen Receptor (CAR). Transduced splenocytes were stained with antibodies to CD34 and thy1.1 and flow cytometry performed to determine transduction efficiency. B) Mice were injected with transduced splenocytes and sacrificed after 15 days and spleens removed. Splenomegaly was only observed in mice injected with splenocytes transduced with constitutively expressed IL-12 (labeled as group 2A).

FIG. 9: flow cytometry analysis of splenocytes

A) Splenocytes from dead 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) existing CD11B⁺(macrophage), CD3⁺(T cell), CD4⁺(helper T cell), CD8⁺(cytotoxic T cells) and C19⁺(B cells). The number of macrophages and T cells in the spleen of mice injected with splenocytes transduced with a construct constitutively expressing IL-12 or a construct derived from an IRES was increased. A reduction in the number of B cells was observed in mice with splenocytes constitutively expressing IL-12.

Detailed Description

The present invention relates to nucleic acid constructs comprising motifs that reduce transgene expression, such as frame sliding motifs or translation read-through motifs.

Frame sliding

During transcription, RNA polymerase catalyzes the incorporation of nucleotides into the growing RNA strand based on complementarity with the DNA template. However, when RNA polymerase encounters a repeated stretch of bases, slippage or "slippage" occurs. Transcriptional glide is used in nature to regulate, for example, the E.coli pyrBI and codBA operon, the expression of the P gene in paramyxoviruses, and the decoding of the cellular dnaX gene of Thermus thermophilus.

When the RNA polymerase slips, it can lead to the synthesis of mRNA encoding an alternative reading frame due to the lack of one (or possibly two) repeat bases.

The nucleic acid constructs of the invention may comprise a transcription frame slipping site such that transcription of a downstream transgene only occurs in the presence of RNA polymerase slippage, thereby producing mRNA encoding an alternative reading frame.

Translation of mRNA sequences into proteins is a complex process involving the orchestration of ribosomes, initiation and elongation factors, aminoacyl transfer RNAs (aa-trnas), aminoacyl tRNA synthetases, and release factors. Translation begins when multiple factors bind to the 5' end of the mRNA, which then leads to recruitment of 40S ribosomes and scanning of the mRNA. When the complex of the initiation factor and the 40S ribosome encounters a codon (triplet of nucleotides) encoding the amino acid methionine (AUG codon), 60S ribosome is recruited and polypeptide synthesis begins with pairing of homologous trnas loaded with the appropriate amino acid. Initiation of translation may occur at initiation codons other than AUG, but AUG is the most commonly used initiation codon. Extension of the polypeptide occurs in a cyclic manner, where the tRNA binds to its cognate codon, a peptide bond is formed between the newly added amino acid and the extended polypeptide, and the polypeptide translocates to expose the next codon.

During translation, ribosome pauses may occur, at which time the ribosome stalls at a particular codon. Ribosome pausing can promote mRNA degradation by the nuclear cell pathway, or can induce slippage in the-1 or-2 direction. It is known that repeated sequences such as UUUUUUA within mRNA cause translation frame slipping, and the sequences are present at the 3' end of group-specific antigen gene (gag) and polymerase (pol) genes of Human Immunodeficiency Virus (HIV). Translation frame slip-1 of the HIV Gag/Pol gene leads to expression of Gag-Pol polyprotein.

The nucleic acid construct of the invention may comprise a translation frame slip site such that translation of the downstream transcript occurs only when frame slip of the ribosome is present.

The frame sliding site may comprise a stretch of the same type of base.

The box sliding motif can be placed upstream and/or downstream of a cleavage site in a nucleic acid construct. The box sliding motif can be located between the first transgene and the second transgene in the nucleic acid construct.

The box sliding motif can be used alone. In this embodiment, the second transgene may be placed out of frame downstream of the frame slip site, such that frame slip is required for transcription or translation of the second transgene.

The motif may, for example, comprise an extension of 5, 7, 8, 10 or 11 bases. The site may, for example, comprise repeated uracil, adenine or guanine bases. The site may for example comprise the sequence shown as SEQ ID No. 1.

UUUUUUU(SEQ ID No.1)。

Alternatively, a frame slip site may be used in combination with a stop codon. In this embodiment, the stop codon is located in-frame downstream of the frame slip site such that frame slip is required to ignore the stop codon.

The stop codon can be UGA, UAG or UAA. A box sliding site may comprise a multiple of three repeats of uracil, adenine or guanine bases, for example 3, 6, or 9 repeats of uracil, adenine or guanine bases.

Examples of frame sliding site/stop codon combinations are shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

UUUUUUUGA(SEQ ID NO.2)

UUUUUUUAG(SEQ ID NO.3)

UUUUUUUAA(SEQ ID NO.4)。

Translation read-through

Translation is terminated when the ribosome encounters a UGA, UAG, or UAA stop codon. At this time, the release factor recognizes a stop codon and promotes dissociation and recycling of ribosomes. Translation termination usually occurs with high fidelity due to competition between the release factor and the close cognate tRNA, with recoding of the stop codon, while sustained extension of the polypeptide occurs only 0.1% of the time. However, increased levels of recoded stop codons leading to translational read-through have been reported in certain genes. In some cases, this results in the production of longer polypeptides with additional functional motifs, a process known as functional read-through.

Translational read-through occurs when release factor 1(RF1) fails to recognize the stop codon and the near homologous aa-tRNA inserts an amino acid into the extended polypeptide, thereby suppressing the stop codon. Local concentrations of release factor and aa-tRNA affect the level of stop codon suppression and translation read-through, with low concentrations of release factor promoting translation read-through. In mammals, suppression of the UAG stop codon results in the insertion of a tryptophan, arginine, or cysteine residue.

One of the earliest examples of stop codon suppression found was the rabbit beta globin gene, the translational read-through of which resulted in the addition of 22 amino acids to the C-terminal 3 of the protein.

The frequency of translation read-through depends on several factors, including: 1) the stop codon used (UGA, UAG or UAA); 2) the sequence immediately flanking the stop codon, six nucleotides upstream and downstream of the stop codon being of particular importance; and 3) the presence of a cis-acting sequence at the 3' end of the mRNA.

The termination efficiencies of the three termination codons differ from each other, wherein UAA is the strongest termination codon and UGA is the weakest termination codon, and the termination efficiencies are rated UAA > UAG > UGA. Thus, the UGA stop codon shows the highest level of translational read-through, while the UAA stop codon shows the lowest level of translational read-through.

Sequence analysis of genes exhibiting translational read-through has identified at least two different motifs that promote stop codon suppression and sustained translation: STOP-CUAG and STOP-CAAUUA (wherein STOP can be UGA, UAG or UAA). The level of translational read-through depends on the stop codon used: UGA supports the highest level of translational read-through, and the level of read-through obtained from UAG and UAA stop codons is progressively lower, so the overall ranking of read-through is UGA > UAG > UAA.

The nucleic acid construct of the invention may comprise one of the sequences shown as SEQ ID No.5 to SEQ ID No. 10.

UGACUAG(SEQ ID No.5)

UAGCUAG(SEQ ID No.6)

UAACUAG(SEQ ID No.7)

UGACAAUUA(SEQ ID No.8)

UAGCAAUUA(SEQ ID No.9)

UAACAAUUA(SEQ ID No.10)

The translational read-through site can be located between the first transgene and the second transgene in the nucleic acid construct. The translation read-through site may be placed upstream and/or downstream of the cleavage site in the nucleic acid construct. The translational read-through site may flank the cleavage site (FIG. 3C). Two or more translational read-through sites, e.g., placed adjacent to each other (FIG. 3B ') or flanking the cleavage site (FIG. E'), can be used.

To further reduce the expression level of the downstream transgene, multiple stop codons can be inserted 5' of the translation read-through motif (fig. 2B). An exemplary multiple stop codon translation read-through motif is shown as SEQ ID NO.11, with two UGA stop codons located 5' of the CUAG read-through motif.

UGAUGACUAG(SEQ ID No.11)。

When the nucleic acid construct comprises more than two transgenes, the composite translational read-through motif can be placed in tandem 5' to the sequence encoding the cleavage site. This allows multiple transgenes to be expressed at different ratios. With each additional read-through motif, the expression level should be reduced by 10 to 50 fold relative to the upstream transgene.

A nucleic acid construct having a composite translational read-through motif can have the following structure:

NOI1-TRM1-CL1-NOI2-TRM2-CL2-NOI3

read-through sites can be placed within the coding sequence to obtain two versions of the protein: short versions obtained by translating the transcript up to the read-through site (i.e. no read-through occurs) and long versions obtained by translating the transcript outside and downstream of the read-through site (read-through occurs). Examples of such arrangements are shown in fig. 3F and 3F'.

Altered signal peptides

Although the translation read-through motif significantly reduced transgene expression, in some cases it may be necessary to further reduce the expression level. For type I transmembrane and secreted proteins, further reduction of expression can be achieved by combining a translation read-through motif with an altered signal peptide sequence. This method is suitable for type I transmembrane proteins and secreted proteins with a signal peptide sequence. In this case, the altered signal peptide and the second transgene are located 3' of the translational read-through motif and self-cleaving peptide sequence (fig. 3D).

The signal peptide is a short peptide, usually 5-30 amino acids long, present at the N-terminus of most newly synthesized proteins, which are destined to be directed towards the secretory pathway. These proteins include those within certain organelles (e.g., endoplasmic reticulum, golgi apparatus, or endosomes), secreted from cells, and transmembrane proteins.

Signal peptides typically contain a core sequence, which is a long stretch of hydrophobic amino acids with a tendency to form a single alpha-helix. The signal peptide may start with a short positively charged amino acid stretch, which helps to enforce the correct topology of the polypeptide during translocation. At the end of the signal peptide, there is typically a stretch of amino acids recognized and cleaved by the signal peptidase. Signal peptidases can cleave during translocation or after completion to produce free signal peptide and mature protein. The free signal peptide is then digested by a specific protease.

Although certain carboxy-terminal signal peptides are known, the signal peptide is typically located at the amino terminus of the molecule.

Altered signal peptides are described in detail in WO2016/174409, which is incorporated herein by reference. The altered signal peptide may comprise one or more mutations, such as substitutions or deletions, such that it has fewer hydrophobic amino acids than the wild-type signal peptide from which it is derived. The term "wild-type" refers to the sequence of the signal peptide that is present in the native protein from which it is derived.

When the nucleic acid construct comprises two transgenes, both encoding transmembrane proteins, the protein encoded by the downstream transgene (which has lower relative expression) may comprise fewer hydrophobic amino acids than the protein encoded by the upstream transgene (which has higher relative expression).

The hydrophobic amino acids mutated to alter the efficiency of the signal peptide may be: alanine (a); valine (V); isoleucine (I); leucine (L); methionine (M); phenylalanine (P); tyrosine (Y); or tryptophan (W).

The altered signal peptide may comprise a deletion of 1, 2, 3, 4, or 5 amino acids or a substitution of a hydrophobic amino acid. Hydrophobic amino acids may be replaced by non-hydrophobic amino acids, such as hydrophilic or neutral amino acids.

Nucleic acid constructs

The present invention relates to a nucleic acid construct comprising: a first nucleotide sequence of interest (NOI 1); a frame sliding motif or a translation read-through motif (FSM/TRM); and a second nucleotide sequence of interest (NOI 2).

The "nucleotide of interest" may be RNA or DNA. The nucleotide of interest (NOI) encodes a polypeptide of interest (POI) which may be all or part of a protein.

When transcribed and translated together, NOI1 and NOI2 (optionally together with subsequent NOIs) may encode a protein. For example, when expressed in a cell, the nucleic acid construct may be capable of producing two products:

a) a first product encoded by no i1 only; and

b) a second product encoded by NOI1 and NOI2, which product is produced when a frame slip or translational read-through occurs.

The relative expression levels of the full-length product encoded by NOI1 and NOI2 may be less than the expression levels of the truncated product encoded by NOI1 alone.

Alternatively, the nucleic acid construct may comprise one or more cleavage sites such that the nucleotide sequence of interest is expressed as an independent protein.

The nucleic acid construct of the invention can encode a polyprotein comprising the first and second polypeptides. The polymeric protein can be cleaved at the cleavage site to produce two separate polypeptides.

The NOI may encode an intracellular protein, a transmembrane protein or a secreted protein.

The NOI may, for example, encode a Chimeric Antigen Receptor (CAR) or a portion thereof, or a factor, e.g., a cytokine, that affects the activity of the CAR or a cell expressing the CAR.

The transgene may encode a target antigen. In this regard, the technology can be used to generate target antigens with varying and extremely low levels. These may be used in functional assays against T cells expressing, for example, CARs or engineered T Cell Receptors (TCRs).

The NOI may encode a protein involved in the synthesis of another entity of the cell. For example, cells can be induced to express the cancer antigen bis-sialylganglioside (GD2) by transgenic expression of two enzymes, GM3 synthase and GD2 synthase (WO 2015/132604). By reducing the expression levels of these enzymes in cells, it is possible to produce GD2^{Is low in}A target cell.

The NOI may encode a cytokine, for example a cytokine that enhances an inflammatory response and/or increases the efficacy of CAR-T cell therapy. The cytokine may be selected from the following: IL-7, IL-12, IL15, IL-17A, IL-18 and IL-21. In particular, the cytokine may be IL-12.

Interleukin 12(IL-12) is an interleukin that is naturally 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 stimulating factor and can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-. gamma.) and tumor necrosis factor-alpha (TNF-. alpha.) by T cells and Natural Killer (NK) cells, and reduces IL-4 mediated inhibition of IFN-. gamma..

IL-12 in natural killer cells and T lymphocytes activity plays an important role in. IL-12 mediates NK cells and CD8+ cytotoxic T lymphocytes cytotoxic activity enhancement.

IL-12 is a potent immunomodulatory cytokine, particularly useful for modulating tumor microenvironments, thereby redirecting immune responses against cancer. IL-12 has systemic toxicity, so for local IL-12 production method is received attention.

IL-12 is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p 40). The active heterodimer (termed "p 70") is formed after protein synthesis.

The NOI may encode IL-12A and/or IL-12B. The sequence of human IL-12A is available from Uniprot accession number P29459. The portion of the sequence lacking the signal peptide is shown as SEQ ID No. 53. The sequence of human IL-12B is available under Uniprot accession number P29460. The portion of the sequence lacking the signal peptide is shown as SEQ ID No. 54.

SEQ ID No.53 (human IL-12A)

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

SEQ ID No.54 (human IL-12B)

WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPC

The NOI may encode "flexi-IL-12", which is a fusion between human IL-12 α (p35) and IL-12 β (p40) subunits, joined by a linker. A suitable flexi-IL-12 sequence is shown in SEQ ID No. 55.

SEQ ID No.55(Flexi-IL-12 sequence)

In SEQ ID No.55, the signal peptide, which is the signal peptide from MOPC 63 in the V-III region of the murine kappa chain (Uniprot P01661), is shown in bold; the serine-glycine linker is in bold and underlined.

The NOI may comprise one of the sequences shown as SEQ ID nos. 53, 54 or 55 or a variant thereof. The variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No.53, 54 or 55, provided that the variant sequence retains IL-12 function when expressed in vivo. For example, the variant sequence may retain the ability to enhance cytotoxic T cell activity in vivo and/or the variant sequence may stimulate production of interferon-gamma (IFN- γ) by T cells.

The sequence encoding IL-12 or fliixi-IL 12 may be placed downstream of a frame sliding motif or a translation read-through motif. This provides a means to control cytokine expression and reduce cytokine expression levels relative to CARs.

The NOI may encode a chemokine, for example a chemokine that improves the efficacy of CAR-T cell therapy. The chemokine may be CCL 19. In particular, the nucleic acid construct may co-express CCL19 and IL-7.

The NOI may encode an antibody or portion thereof. For example, the NOI may encode an immunomodulatory antibody or antibody fragment. The antibody may block inhibitory signals (e.g., PD1) or activate the immune system (e.g., OX40 agonist, 41BB agonist, or ICOS agonist).

The transgene may encode a toxic compound, such as a botulinum toxin, a diphtheria toxin, or a pseudomonas toxin.

Chimeric antigen receptors

Classical Chimeric Antigen Receptors (CARs) are chimeric type I transmembrane proteins that link an extracellular antigen recognition domain (binder) to an intracellular signaling domain (endodomain). The conjugate is typically a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it may be based on other forms that comprise an antibody-like antigen binding site. Spacer domains are often necessary to separate the conjugate from the membrane and allow it to assume the proper orientation. A common spacer domain used is the Fc of IgG 1. Depending on the antigen, more compact spacers may suffice, such as stems from CD8a and even just IgG1 hinges. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the intracellular domain.

Early CAR designs had intracellular domains derived from the intracellular part of the gamma chain of fcer 1 or CD3 ζ. As a result, these first generation receptors transmit an immune signal 1 that is sufficient to trigger killing of the associated target cells by T cells, but is unable to fully activate T cells for proliferation and survival. To overcome this limitation, a complex endodomain was constructed: the fusion of the intracellular portion of the T cell costimulatory molecule to the intracellular portion of CD3 ζ creates a second generation receptor capable of simultaneously transmitting activation and costimulatory signals upon antigen recognition. The most commonly used co-stimulatory domain is that of CD 28. This provides the most potent co-stimulatory signal, i.e., immune signal 2, which triggers T cell proliferation. Several receptors have also been described, including the intracellular domains of the TNF receptor family, such as the closely related OX40 and 41BB, which transmit survival signals. An even more potent third generation CAR has now been described, having an endodomain capable of transmitting activation, proliferation and survival signals.

When the CAR binds to the target antigen, this results in transmission of an activation signal to the T cell on which it is expressed. The CAR thus directs the specificity and cytotoxicity of T cells to tumor cells expressing the targeted antigen.

Thus, a CAR typically comprises: (i) an antigen binding domain; (ii) a spacer region; (iii) a transmembrane domain; and (iii) an intracellular domain comprising or associated with a signaling domain.

The CAR may have the following general structure:

antigen binding domain-spacer domain-transmembrane domain-intracellular signaling domain (endodomain).

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

Functional read-through can be utilized to increase the functionality of the CAR. This can be achieved by inserting a translation read-through motif downstream of the antigen binding domain or between the signalling domains of the CAR (fig. 3F and F').

In certain instances, secretion of an antigen binding domain (scFv/VHH) may be required to mitigate off-target effects in which CAR T cells target normal tissues expressing low levels of the target antigen. The secreted antigen binding domain will bind to an antigen expressed on the surface of a normal cell, thereby preventing the CAR T cell from recognizing the normal cell. By placing a translation read-through motif directly upstream of the CAR transmembrane domain, functional read-through can be used to engineer T cells to secrete the antigen binding domain.

The nucleic acid construct may have the general structure:

scFv/VHH-TRM-spacer-TM domain-endodomain, or

scFv/VHH-spacer-TRM-TM Domain-endodomain

Wherein:

scFv/VHH is a nucleotide sequence encoding an antigen binding domain

The spacer is a nucleotide sequence encoding the spacer

The TM domain is a nucleotide sequence encoding the TM domain, and

an intracellular domain is a nucleotide sequence that encodes an intracellular domain, which can be, for example, a first, second or third generation intracellular domain.

Functional readthrough can also be used to generate a combination of first, second, or third generation CARs. In this case, a translational read-through motif can be placed between CD3z and the co-receptor endodomain, resulting in high expression levels of the first generation CAR (CD3 zeta signaling domain alone) and significantly lower expression levels of the second or third generation CARs.

The nucleic acid construct may have the general structure:

scFv/VHH-spacer-TM domain-CD 3 ζ endodomain TRM-costimulatory domain, or

scFv/VHH-spacer-TM Domain-CD 3 zeta intracellular Domain TRM 1-Co-stimulatory Domain 1-TRM 2-Co-stimulatory Domain 2

Wherein:

scFv/VHH is a nucleotide sequence encoding an antigen binding domain

The spacer is a nucleotide sequence encoding the spacer

The TM domain is a nucleotide sequence encoding the TM domain, and

the CD3 zeta endodomain is a nucleotide sequence encoding a CD3 zeta endodomain and

a co-stimulatory domain is a nucleotide sequence encoding a co-stimulatory domain, for example, an intracellular domain from a co-receptor such as CD28 or from a member of the TNF receptor superfamily.

Different iterations can be generated by switching the positions of CD3 zeta and the co-receptor endodomain such that the involvement of the CAR will primarily provide a costimulatory signal (signal 2) to the cell and a reduced antigenic signal (signal 1) to the cell, as less CARs will have both the co-receptor and CD3 zeta signaling domains.

Chimeric cytokine receptors

WO2017/029512 describes chimeric cytokine receptors that graft the binding specificity of a non-cytokine binding molecule onto the intracellular domain of the cytokine receptor. It also describes chimeric transmembrane proteins comprising a dimerization domain and an intracellular domain of a cytokine receptor.

Dimerization can occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active. Alternatively, dimerization may occur only in the presence of a dimerization Chemical Inducer (CID), in which case the transmembrane protein causes cytokine-type signaling only in the presence of a CID.

Constitutively active chimeric cytokine receptors may comprise the Fab portion of an antibody as the extracellular domain. In this regard, the dimerization domain may comprise the dimerization portions of a heavy chain constant domain (CH) and a light chain constant domain (CL).

A chimeric transmembrane protein may comprise two polypeptides:

(i) a first polypeptide comprising:

(a) a first dimerization domain; and

(b) a first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide comprising:

(a) a second dimerization domain that dimerizes with the first dimerization domain; and

(b) a second chain of the intracellular domain of the cytokine receptor.

In particular, the chimeric transmembrane protein may comprise:

(i) a first polypeptide comprising:

(a) heavy chain constant Domain (CH)

(b) A first chain of the cytokine receptor endodomain; and

(ii) a second polypeptide comprising:

(a) light chain constant Domain (CL)

(b) A second chain of the intracellular domain of the cytokine receptor.

The first and second chains of the cytokine receptor endodomain may be selected from the group consisting of type I cytokine receptor endodomain alpha, beta, and gamma chains.

The cytokine receptor endodomain may comprise:

(i) IL-2 receptor beta chain endodomains

(ii) The intracellular domain of the alpha chain of the IL-7 receptor; or

(iii) The intracellular domain of the alpha chain of the IL-15 receptor; and/or

(iv) The gamma chain receptor endodomain is shared.

IL-2

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

These three receptor chains are expressed separately and differently on various cell types, and can be assembled in different combinations and sequences to produce low, medium and high affinity IL-2 receptors.

The alpha chain binds IL-2 with low affinity, and the beta and gamma combine to form a complex that binds IL-2 with medium affinity (mainly on memory T cells and NK cells); and all three receptor chains on activated T cells and regulatory T cells form a complex that binds IL-2 with high affinity (Kd 10-11M).

The three IL-2 receptor chains span the cell membrane and extend into the cell, delivering biochemical signals to the interior of the cell. The alpha chain is not involved in signaling, but the beta chain is complexed with tyrosine phosphatase JAK 1. Similarly, the gamma chain is complexed with another tyrosine kinase known as JAK 3. These enzymes are activated by binding of IL-2 to the outer domain of IL-2R.

When naive T cells are also stimulated by antigens, IL-2 signaling promotes T cell differentiation into effector and memory T cells. They also play a key role in long-term cell-mediated immunity through their role in T cell immune memory formation, which depends on the expansion of the number and function of antigen-selected T cell clones.

The chimeric cytokine receptors of the present invention may comprise an IL-2 receptor beta chain and/or an IL-2 receptor (i.e., common) gamma chain.

The amino acid sequences of the intracellular domains of the IL-2 beta chain and the consensus gamma chain are shown as SEQ ID Nos. 56 and 57.

SEQ ID No.56 intracellular domain derived from the human consensus gamma chain:

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

SEQ ID No.57 intracellular domain derived from human IL-2R β:

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

the term "derived from" means that the intracellular domain 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 and activate one of the above signaling pathways.

A "variant" sequence has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the wild-type sequence (e.g., SEQ ID No.56 or 57), provided that the variant sequence retains the function of the wild-type sequence, i.e., retains the ability to form a complex with JAK-1 or JAK-3 and activate, for example, the JAK-STAT signaling pathway.

The percent identity between two polypeptide sequences can be readily determined by programs such as BLAST, which is freely available from http:// BLAST.

Constant region domains

There are two types of light chains in humans: kappa chain and lambda chain. The λ class has 4 subclasses: λ 1, λ 2, λ 3 and λ 4. The light chain constant region of a Fab-type chimeric receptor can be derived from any of these light chain types.

The light chain constant domain of the chimeric cytokine receptor may have the sequence shown in SEQ ID No.58, which is a kappa chain constant domain.

SEQ ID No.58

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

There are five types of mammalian immunoglobulin heavy chains: γ, δ, α, μ and ε, which define the classes of immunoglobulins IgG, IgD, IgA, IgM and IgE, respectively. Heavy chains γ, δ and α have a constant domain consisting of three tandem Ig domains and have hinges for increased flexibility. Heavy chains μ and ε are composed of four domains.

The CH domain of the chimeric cytokine receptor of the present invention may comprise the sequence shown in SEQ ID No.59, which is derived from a gamma immunoglobulin heavy chain.

SEQ ID No.59

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

In a preferred embodiment, the present invention provides a nucleic acid construct comprising: a first nucleotide sequence encoding a Chimeric Antigen Receptor (CAR); a Frame Sliding Motif (FSM) or a translation read-through motif (TRM); and a second nucleotide sequence encoding a Chimeric Cytokine Receptor (CCR).

Cleavage site

The nucleic acid construct of the first aspect of the invention may comprise a sequence encoding a cleavage site located between the nucleic acid sequences encoding the first and second polypeptides, such that the first and second polypeptides may be expressed as separate entities.

The cleavage site may be any sequence that enables the separation of the polypeptides comprising the first and second polypeptides.

The term "cleavage" is used herein for convenience, but cleavage sites may separate the first and second polypeptides into separate entities by mechanisms other than classical cleavage. For example, for the Foot and Mouth Disease Virus (FMDV)2A self-cleaving peptide (see below), various models have been proposed to explain the "cleaving" activity: proteolytic, autoproteolytic or translational effects by host cell proteases (Donnelly et al (2001) J.Gen.Virol.82: 1027-1041). The exact mechanism of such "cleavage" is not important for the purposes of the present invention, as long as the cleavage site located between the nucleic acid sequences encoding the first and second polypeptides results in the expression of the first and second polypeptides as separate entities.

The cleavage site may be a furin cleavage site.

Furin is an enzyme belonging to the subtilisin-like proprotein convertase family. Members of this family are proprotein convertases which process potential precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include parathyroid prohormone, transforming growth factor β 1 precursor, proprotein, β -secretase, membrane type 1 matrix metalloproteinase, the β subunit of pro nerve growth factor, and Willebrand factor. Furin cleaves proteins just downstream of the basic amino acid target sequence, typically Arg-X- (Arg/Lys) -Arg' (SEQ ID No.50), and is enriched in the Golgi.

The cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.

TEV proteases are highly sequence-specific cysteine proteases, which are chymotrypsin-like proteases. It is very specific to its target cleavage site and is therefore often used for the controlled cleavage of fusion proteins in vitro and in vivo. The consensus TEV cleavage site was ENLYFQ \ S (SEQ ID No.51) (where "\" indicates a peptide bond that was cleaved). Mammalian cells such as human cells do not express TEV protease. Thus, in embodiments where the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell, it is necessary to also express the exogenous TEV protease in the mammalian cell.

The cleavage site may encode a self-cleaving peptide.

"self-cleaving peptide" refers to a peptide that functions such that when a polypeptide comprising first and second polypeptides and a self-cleaving peptide is produced, it is immediately "cleaved" or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

The self-cleaving peptide may be a 2A self-cleaving peptide from an orthodontics virus or a cardiovirus. The major 2A/2B cleavage of both orthohoof and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In foot and mouth viruses such as Foot and Mouth Disease Virus (FMDV) and equine rhinitis type a virus, the 2A region is a short segment of about 18 amino acids which, together with the N-terminal residue of protein 2B (the conserved proline residue), represents an autonomous element capable of mediating "cleavage" at its own C-terminus.

The C-terminal 19 amino acids of the longer cardiovirus proteins together with the N-terminal proline of 2B mediate "cleavage" with an efficiency approximately equivalent to the ungulate FMDV 2a sequence. Cardioviruses include encephalomyocarditis virus (EMCV) and Taylor's Murine Encephalopathy Virus (TMEV).

Mutational analysis of EMCV and FMDV 2A revealed that the motif DxExNPGP (SEQ ID No.52) is closely related to the "cleavage" activity (Donelly et al (2001), supra).

The cleavage site of the present invention may comprise the following amino acid sequence:

Dx₁Ex₂NPGP, wherein x₁And x₂Is any amino acid. X₁Can be selected from the following group: I. v, M and SX₂。X₂Can be selected from the following group: t, M, S, L, E, Q and F.

For example, the cleavage site may comprise one of the amino acid sequences shown in table 2.

TABLE 2

The cleavage site based on the 2A sequence may be, for example, 15 to 22 amino acids in length. The sequence may comprise the C-terminus of the 2A protein followed by a proline residue (which corresponds to the N-terminal proline of 2B).

Mutation studies have also shown that certain variants are active in addition to the naturally occurring 2A sequence. The cleavage site may correspond to a variant sequence of a naturally occurring 2A polypeptide, the mutant sequence having one, two, or three amino acid substitutions that retain the ability to induce the polyprotein sequence to "cleave" into two or more separate proteins.

The cleavage sequence may be selected from the following sequences, all of which have shown some degree of activity (Donnelly et al (2001), supra):

LLNFDLLKLAGDVESNPGP(SEQ ID No.18)

LLNFDLLKLAGDVQSNPGP(SEQ ID No.19)

LLNFDLLKLAGDVEINPGP(SEQ ID No.20)

LLNFDLLKLAGDVEFNPGP(SEQ ID No.21)

LLNFDLLKLAGDVESHPGP(SEQ ID No.22)

LLNFDLLKLAGDVESEPGP(SEQ ID No.23)

LLNFDLLKLAGDVESQPGP(SEQ ID No.24)

LLNFDLLKLAGDVESNPGG(SEQ ID No.25)

based on the sequence of the DxExNPGP motif, "2A-like" sequences have been found in picornaviruses, "picornavirus-like" insect viruses, C-type rotavirus, repeats in trypanosoma species and bacterial sequences in addition to the mouth hoof virus or cardiovirus (Donnelly et al (2001), supra). The cleavage site may comprise one of these 2A-like sequences, for example:

YHADYYKQRLIHDVEMNPGP(SEQ ID No.26)

HYAGYFADLLIHDIETNPGP(SEQ ID No.27)

QCTNYALLKLAGDVESNPGP(SEQ ID No.28)

ATNFSLLKQAGDVEENPGP(SEQ ID No.29)

AARQMLLLLSGDVETNPGP(SEQ ID No.30)

RAEGRGSLLTCGDVEENPGP(SEQ ID No.31)

TRAEIEDELIRAGIESNPGP(SEQ ID No.32)

TRAEIEDELIRADIESNPGP(SEQ ID No.33)

AKFQIDKILISGDVELNPGP(SEQ ID No.34)

SSIIRTKMLVSGDVEENPGP(SEQ ID No.35)

CDAQRQKLLLSGDIEQNPGP(SEQ ID No.36)

YPIDFGGFLVKADSEFNPGP(SEQ ID No.37)

the cleavage site may comprise a 2A-like sequence (RAEGRGSLLTCGDVEENPGP) as shown in SEQ ID No. 31.

It has been shown that an N-terminal "extension" comprising 5 to 39 amino acids increases activity (Donnelly et al (2001), supra). In particular, the cleavage sequence may comprise one of the following or a variant thereof, having, for example, up to 5 amino acid changes, which retains cleavage site activity:

VTELLYRMKRAETYCPRPLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID No.38)

LLAIHPTEARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID No.39)

EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP(SEQ ID No.40)

APVKQTLNFDLLKLAGDVESNPGP(SEQ ID No.41)

carrier

The invention also provides a vector comprising a nucleic acid construct according to the first aspect of the invention.

Such vectors can be used to introduce the nucleic acid construct into a host cell such that it expresses the first and second polypeptides.

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

The vector may be capable of transfecting or transducing a mammalian cell, such as a T cell or a target cell.

Cells

The invention further provides a cell comprising a nucleic acid construct or vector of the invention expressing a first and a second polypeptide encoded by the nucleic acid sequences.

The cell may be any eukaryotic cell, such as an immune cell.

The cell may be a cytolytic immune cell, such as a T cell or a natural killer cell.

When the transgene expresses the target antigen, the cell may be a T cell or a target cell of a CAR-T cell.

Method of producing a composite material

In another aspect, the invention provides a method of making a cell according to the invention, comprising the step of introducing into the cell a nucleic acid construct or vector of the invention.

The nucleic acid construct may be introduced by transduction or transfection.

The cell may be a cell isolated from a subject, e.g., a T cell or NK cell isolated from a subject.

The invention also provides a method of modulating the relative expression of two transgenes in a nucleic acid construct, the method comprising the steps of: a frame sliding motif or a translation read-through motif is introduced between the two transgenes to reduce expression of the downstream transgene.

The nucleic acid construct may comprise a sequence encoding a cleavage site located between the two transgenes. The relative expression of the two transgenes can be adjusted by selecting different frame sliding motifs or translation read-through motifs and/or by mutating the sequences immediately 5 'and/or 3' to the motif.

The invention also provides a method of modulating the relative expression of two transgenes in a nucleic acid construct, the method comprising the steps of: a frame sliding motif or a translation read-through motif is introduced between the two transgenes to reduce expression of the downstream transgene.

The box sliding motif or the translation read-through motif may be as described in the previous aspect of the invention.

The invention will now be further described by way of examples, which are intended to assist those of ordinary skill in the art in carrying out the invention, and are not intended to limit the scope of the invention in any way.

Examples

Example 1 preparation and testing of frame slide constructs

The following constructs were transduced into SupT1 cells.

·SFGmR.RQR8-2A-SKIP_7xU-CD22ecto-CD19tm-dCD19endo

SFGmR.RQR8-2A-SKIP-6 xU-CD22ecto-CD19tm-dCD19endo (control)

The construct encodes RQR8 and a chimeric protein having a CD22 ectodomain and CD19 transmembrane and endodomain, RQR8 is a sort-suicide transmembrane protein described in WO 2013/153391.

The first construct, which contains a transcription frame slip motif with seven thymine bases (uracil in mRNA) that promotes transcription slip and base loss of mRNA, puts the CD22 coding sequence in frame and results in expression of the CD22-CD19 chimera.

Flow cytometry analysis showed that the introduction of the box-sliding motif resulted in a significant reduction in expression of the CD22-CD19 chimera, while similar levels of RQR8 sorting marker were observed (fig. 2C).

Example 2 preparation and testing of translation read-through constructs

The 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 downstream translational read-through from cleaved 2A peptide sequences and transgene coding sequences (CD22 and BCMA).

To test the function of these readthrough motifs in combination with different stop codons, the following constructs were transduced into HEK293T cells.

Read-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

The read-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 mutants

SFGmR.RQR8-2A-Signal peptide K9-hCD22ecto-CD19TM-dCD19endo

SFGmR.RQR8-2A-Signal peptide K10-hCD22ecto-CD19TM-dCD19endo

SFGmR.RQR8-2A-Signal peptide K11-hCD22ecto-CD19TM-dCD19endo

The wild-type signal peptide sequence as well as the sequences of the altered signal peptides K19, K10 and K11 are shown below as SEQ ID nos. 42 to 45.

SEQ ID No. 42-murine Ig kappa chain V-III region Signal peptide (wild type)

METDTLILWVLLLLVPGSTG

SEQ ID No. 43-murine Ig kappa chain V-III region Signal peptide (K9 mutant)

METDTLILKVLLLLVPGSTG

SEQ ID No. 44-murine Ig kappa chain V-III region Signal peptide (K10 mutant)

METDTLILWKLLLLVPGSTG

SEQ ID No. 45-murine Ig kappa chain V-III region Signal peptide (K11 mutant)

METDTLILWVKLLLVPGSTG

Expression of the CD22-CD19 chimera was analyzed by flow cytometry and the results are shown in FIG. 4A. The expression of the CD22-CD19 chimera was reduced in the lysine 11 signal peptide mutant (L11K mutation) compared to the wild-type signal peptide. Similar results were produced when the stop-CUAG and stop-CAAUUA translational read-through motifs were placed 5' to the CD22-CD19 transgene, both of which reduced the expression level of the chimera to about 2% or less of the RQR8 sorting marker. Lower levels of CD22-CD19 chimera expression were obtained using a double stop codon translation read-through motif (row 4).

The mean fluorescence intensity of the CD22-CD19 chimeras was quantified on the surface of transfected HEK293T cells and the results are shown in fig. 4B. Substantially lower levels of CD22-CD19 chimera were obtained using the translational read-through construct compared to the attenuated signal peptide mutant.

Example 3-functional translation read-through: secretion of soluble anti-CD 22 Fab

A nucleic acid construct was designed that encodes the RQR8 sorting marker followed by a 2A self-cleaving peptide sequence and an anti-CD 22 CAR with CL and CH1 spacer to generate soluble Fab fragments as shown below:

·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

these constructs were transduced to PBMCs and cytotoxicity assays were performed using target cells expressing low or high levels of CD22 to determine the efficacy of the CARs. This mimics the situation where low levels of target antigen are expressed on normal tissues and a cytotoxic response needs to be fine-tuned to prevent CAR T cells from targeting these cells.

Example 4-functional translation read-through: switching from first generation to second or third generation CAR

To investigate whether functional translational read-through can be used to switch between first and second generation CARs, the following constructs were generated:

·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

this first set of constructs expressed the sorting marker RQR8 and a first generation anti-CD 19 CAR having an Fc spacer domain and a CD3 ζ intracellular domain followed by a translational read-through motif and a 4-1BB or CD28 costimulatory intracellular domain. A restimulation assay was established using transduced PBMCs cultured in plates coated with an anti-Fc antibody that binds to the spacer domain of the CAR and triggers proliferation. The control first generation CAR with only CD3 ζ showed limited proliferation, while the other CARs proliferated extensively due to the presence of costimulatory signals.

A second set of constructs was generated, similar to the first set, as follows:

·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

in these constructs, the costimulatory domain from 4-1BB or CD28 was placed before the translational read-through motif, while the CD3 ζ endodomain was placed after the translational read-through motif. Cytotoxicity assays and proliferation assays were established using PBMCs transduced with these constructs. Translational read-through of these constructs is necessary for the generation of functional CARs (i.e., CARs with intracellular signaling domains).

Example 5 preparation and testing of composite read-through motifs

To reduce the expression of the multiple transgene expression cassette to extremely low levels, constructs with two STOP codons (STOP) -translation read-through motifs were designed as follows:

marker (RQR8 or HA8) -STOP _ read-through motif-2A-GD 3 synthase-STOP _ read-through motif-2A-GD 2 synthase

Ganglioside GD2 was synthesized by two enzymes GD2 synthase and GD3 synthase. The above constructs produced GD3 synthase with reduced expression, and GD2 synthase with even lower expression, compared to the marker gene.

Example 6 preparation and testing of Universal read-through constructs

To overcome the problem of background-dependent expression from read-through motifs, a set of universal constructs was created into which a second transgene could be cloned and expressed at a defined level. The universal construct consists of a self-cleaving 2A peptide sequence followed by a stop codon and a read-through motif and a second self-cleaving peptide sequence. Flanking the stop codon read-through motif with a self-cleaving peptide sequence reduces the effect of background dependence on translation read-through and results in consistent and predictable transgene expression levels.

The constructs tested were as follows:

read-through motif STOP CUAG

marker-2A-transgene 1-2A-UGA-CUAG-2A-transgene 2

marker-2A-transgene 1-2A-UAG-CUAG-2A-transgene 2

marker-2A-transgene 1-2A-UAA-CUAG-2A-transgene 2

The read-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 1-2 Ae-UAA-CAAUUA-2A-transgene 2

Example 7 ultra-Low expression by tandem Placement of translational read-through motifs

To determine whether it was possible to obtain ultra-low levels of transgene expression, a construct was created in which two translational read-through motifs were placed in tandem in a tricistronic cassette (fig. 5A). The first transgene in the cassette, always a cell surface marker, consisting of an HA epitope presented on the CD8a stem (CD8a talk) for identification of transduced cells, was followed by a stop codon, a translational read-through motif and a self-cleaving peptide sequence. The second transgene in the expression cassette was one of two fluorescent proteins, Clover3 (green fluorescent protein) or Enhanced Blue Fluorescent Protein (EBFP) in tandem, followed by a stop codon, a translational read-through motif and a self-cleaving peptide sequence. The third transgene in the expression cassette is a replacement fluorescent protein. By placing the stop codon in tandem with the translation read-through motif, the level of expression of the fluorescent protein at the third position should be lower than the level of expression at the second position.

Peripheral Blood Mononuclear Cells (PBMCs) were transduced with this construct and flow cytometry was performed to quantify the expression levels of fluorescent proteins relative to cell surface markers. The results of these experiments showed that the expression level of the fluorescent protein at the third position downstream of the two stop codons and the translation read-through motif was significantly lower than at the second position (fig. 5B). Consistent with previous experiments, the level of translational read-through depends on the stop codon, which is rated in stringency as UAA > UAG > UGA. In the case of tandem Clover3, the expression level was reduced 33-fold in the presence of UAA, 24-fold in the presence of UAG, and 6-fold in the presence of UGA compared to the control without the stop codon. When placed in the third position, tandem Clover3 expressed a > 100-fold decrease in water average for all three stop codons (fig. 5C).

Example 8 ligation by translationRead control of IL-12 secretion

IL-12 is a potent proinflammatory cytokine secreted by phagocytes and dendritic cells in response to pathogens, signals from T cells and Natural Killer (NK) cells, and inflammatory extracellular matrix components. The primary target of IL-12 is cytotoxic T cells, T_H1 helper T cells and NK cells, which express the IL-12 receptor on their cell surface. In response to IL-12, T_H1 helper T cells release IFN γ and TNF α.

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

We designed constructs in which a fusion between human IL-12 α (p35) and IL-12 β (p40) subunit flexi-IL-12 was cloned downstream of the sorting marker RQR8 and the self-cleaving peptide sequence. The stop codon or control sequence (UGG encoding tryptophan) and the translational read-through sequence CAAUUA were placed immediately downstream of the RQR8 coding sequence (fig. 6A). PBMCs were transduced with this construct and stained with antibodies against CD3 epsilon and RQR8 to determine transduction efficiency and verify the presence of the transgene (fig. 6B). After determination of transduction efficiency, PBMCs were placed back into the culture and supernatants were collected at 24, 48 and 72 hours and analyzed for the presence of IL-12 by high sensitivity ELISA (fig. 6C). The data indicate that the inclusion of a stop codon and a translation read-through motif reduced the expression level by more than 40-fold (fig. 6C). There is a trend in the stringency rating of stop codons from the most stringent to the least stringent in the order UAA > UAG > UGA. These data indicate that it is possible to significantly reduce the level of IL-12 secretion by transduced cells.

To demonstrate that secreted levels of flexi-IL-12 are able to trigger an immune response, we generated another set of constructs. These constructs were tricistronic constructs containing the suicide gene (Rapacap 9), the murine cell surface marker Thy1.1 and murine flexi-IL-12 (FIG. 7A). The murine flexi-IL-12 sequence was placed at the end of the tricistronic cassette and a self-cleaving peptide sequence was used to facilitate expression of the polypeptide. The translation read-through construct (UAA-CAAUUA) was compared to a construct containing murine flexi-IL-12 with a reduced signal peptide sequence and another construct that uses an Internal Ribosome Entry Site (IRES) to control cytokine expression. Splenocytes obtained from BalbC mice were transduced with the constructs and flow cytometry was performed using antibodies against CD3 and the cell surface marker thy1.1 to determine transduction efficiency (fig. 7B). After determination of transduction efficiency, splenocytes were returned to culture and supernatants were collected at 24, 48 and 72 hours and analyzed for the presence of murine IL-12 by ELISA (fig. 7C). The results indicate that the expression of IL-12 from IRES was reduced by about 3-fold. In contrast, the expression of IL-12 from the translation read-through motif UAA-CAAUUA was reduced to such an extent that it was no longer detectable by ELISA (FIG. 7C). To determine whether IL-12 is present in the culture medium, the supernatant is recovered and used to restimulate activated splenocytes. Supernatants from these restimulated splenocytes were analyzed for the presence of IFN γ, as IFN γ is secreted by T cells and NK cells stimulated with IL-12 (fig. 7D). IFN γ was detected in all supernatants harvested from restimulated splenocytes. According to the IL-12ELISA results, from the control or IRES transduction of splenocytes in the supernatant re-stimulation of splenocytes observed the highest level of IFN gamma secretion. Importantly, secretion of IFN γ was observed from splenocytes restimulated with supernatant from splenocytes transduced with the translation read-through construct (fig. 7D). These results indicate that it is possible to obtain significantly reduced levels of IL-12 secretion from transduced cells, thereby potentially alleviating the toxic effects of this potent cytokine, while still maintaining therapeutic benefit.

To demonstrate the efficacy and safety of secretion of IL-12 from a translational read-through construct in a more therapeutic setting, splenocytes were co-transduced with constructs encoding truncated murine CD34, an anti-GD 2 chimeric antigen receptor, and firefly luciferase, and the cytokine expression constructs described previously. Transduced splenocytes were stained with antibodies to CD34 and thy1.1 and analyzed by flow cytometry to determine transduction efficiency (fig. 8A). Mice were injected with 5x10⁶Transduced splenocytes, and mice were sacrificed after 15 days and their spleens removed. Comparison of spleen sizes revealed that splenomegaly was observed only in the case of constitutive expression of IL-12 (2A), while spleen sizes of the other groups were comparable to those of the control group (FIG. 8B). To investigate in more detail the cell types recruited to the spleen of injected mice, splenocytes were stained with antibodies against CD11b, CD3, CD4, CD8, and CD19 and analyzed by flow cytometry (fig. 9A). The results showed that macrophages (CD11 b)⁺) Recruited to the spleen of a mouse injected with splenocytes transduced with constitutively expressed IL-12, of which about 20% are CD11b⁺. Consistent with previous ELISA data, it was shown that IRES constructs produced higher levels of IL-12 than the translation read-through constructs, and that more CD11b was present in the spleen of mice injected with these cells⁺Cells (fig. 9B). T cell CD4 was observed in mice injected with splenocytes⁺And CD8⁺Is transduced via constructs constitutively expressing IL-12 or constructs derived from IRES (FIGS. 9C, D and E). In contrast, CD11b in the spleen of mice injected with splenocytes transduced with the translation read-through construct⁺And CD3⁺The number of cells was comparable to the control group. A reduction in the number of B cells was observed only in the spleen of mice injected with spleen cells constitutively expressing IL-12. Together, these results indicate that expression of IL-12 from a translation readthrough construct does not cause any toxicity or recruitment of immune cells in the absence of antigen.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.