CN115807020A

CN115807020A - Use of interleukin 15 receptor alpha armored CAR-T cells to reduce interleukin 15-induced cytotoxicity

Info

Publication number: CN115807020A
Application number: CN202211166507.5A
Authority: CN
Inventors: 钟晓松; 白玥; 张莹
Original assignee: Carrizi Beijing Life Technology Co ltd
Current assignee: Carrizi Beijing Life Technology Co ltd
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2023-03-17
Also published as: WO2024060577A1

Abstract

The invention relates to an armored CAR-T cell expressing interleukin 15 receptor alpha and interleukin 15 and an immunotherapy application thereof.

Description

Use of interleukin 15 receptor alpha armored CAR-T cells to reduce interleukin 15-induced cytotoxicity

Technical Field

The invention relates to the field of medicine, in particular to an armored CAR-T cell expressing interleukin 15 receptor alpha and interleukin 15 and an immunotherapy application thereof.

Background

Chimeric Antigen Receptor (CAR) -T cell therapy is a powerful adoptive immunotherapy against hematological cancers, whereas Interleukin (IL) -15 is an important immunostimulant with the ability to induce long-lasting CAR-T cells (Yang X, et al, closely related T-memory cells with in-vivo expansion of CAR. CD19-T cells in tissues and are preserved by IL-7 and IL-15.Blood. (2014), https:// doi. Org/10.1182/blood-2014-01-552174). However, it was reported that higher baseline or peak serum IL-15 levels during CAR-T treatment, although associated with better antitumor response, were also associated with higher toxicity rates, including, for example, cytokine Release Syndrome (CRS), neurotoxicity, and Graft Versus Host Disease (GVHD). Thus, there is a need in the art for methods that can reduce the toxicity of such CAR-T cells, but maintain their anti-tumor effect and long-term persistence in vivo.

Summary of The Invention

Through intensive research, the inventors constructed armored CAR-T cells (armored CAR-T cells) loaded with both optimized IL-15 receptor alpha (IL-15 Ra) and IL-I5 with specific amino acid modifications using CD 19-specific CAR-T cells. As shown in the examples herein, CAR-IL-15T cells loaded with optimized IL-15Ra showed enhanced proliferative capacity and cell viability and maintained the Tscm phenotype under cell culture conditions similar to CAR-IL-15T cells; also, CAR-IL-15T cells loaded with the IL-15Ra reduced the release of IL-15 into the extracellular environment compared to CAR-IL-15T cells. Further, the present inventors demonstrated in a mouse model that CAR-IL-15T cells loaded with the IL-15Ra extended survival time of mice, have excellent antitumor activity, compared to conventional CAR-T cells and CAR-IL-15T cells; and at the same time results in reduced serum IL-15 levels and correspondingly lower toxicity after administration compared to CAR-IL-15T cells. These results indicate that armored CAR-T cells of the invention comprising optimized IL-15Ra play an important role in reducing adverse events during CAR-T therapy and enhancing anti-tumor capacity. Based on these findings, the present inventors have thus established the present invention.

Thus, in a first aspect, the invention provides a nucleic acid combination comprising a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule, wherein the first nucleic acid molecule comprises a polynucleotide encoding a chimeric antigen polypeptide (CAR), the second nucleic acid molecule comprises a polynucleotide encoding IL-15 and the third nucleic acid molecule comprises a polynucleotide encoding an optimized IL15Ra, wherein the optimized IL15Ra comprises double mutations S202R and D203E at amino acid positions 202 and 203, wherein the amino acid positions are numbered according to SEQ ID No. 6. Preferably, the optimized IL-15Ra comprises the amino acid sequence of SEQ ID NO 6; or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or 99.5% identity thereto. Still more preferably, said polynucleotide encoding optimized IL15Ra comprises the sequence depicted in SEQ ID No. 3 or a polynucleotide having at least 90%, 92%, 95%,96%,97%,98%, 99% or 99.5% identity thereto.

In some embodiments, two or all three of the first, second and third nucleic acid molecules are present in functional linkage on a single nucleic acid construct, e.g., a viral vector, such as a lentiviral vector. In other embodiments, the first, second and third nucleic acid molecules are each present on a different nucleic acid construct, e.g., a viral vector, such as a lentiviral vector.

In one embodiment, the nucleic acid combination according to the invention is a single nucleic acid construct comprising a first, a second and a third nucleic acid molecule, wherein said nucleic acid construct encodes a fusion protein having from N-terminus to C-terminus the structure of formula (I):

CAR-(L1)-E1-(L2)-E2 (I)

wherein, the first and the second end of the pipe are connected with each other,

CAR denotes a chimeric antigen receptor polypeptide which is,

l1 and L2 each independently represent a linker peptide (particularly a linker peptide comprising a self-splicing site),

e1 and E2 are different from each other and are independently selected from IL-15 and optimized IL-15Ra, and

wherein "-" represents a functional linkage between said components of formula (I).

In one embodiment, E1 represents IL-15 encoded by the second nucleic acid molecule, and E2 represents optimized IL-15Ra encoded by the third nucleic acid molecule. In another embodiment, E2 represents the second nucleic acid molecules encoding IL-15, and E1 represents the third nucleic acid molecules encoding optimized IL-15Ra.

In some embodiments, the linker peptides L1 and L2 are the same; in other embodiments, the linker peptides L1 and L2 are different. In some embodiments, L1 and L2 comprise self-splice sites, e.g., selected from: self splice sites for P2A, T2A, E2A or F2A. In one embodiment, L1 comprises a P2A site (preferably, comprises the amino acid sequence of SEQ ID NO: 17); l2 comprises a T2A site (preferably, comprising the amino acid sequence of SEQ ID NO: 18).

In one embodiment, the first nucleic acid molecule comprises a polynucleotide encoding a CAR polypeptide, wherein the CAR polypeptide comprises from N-terminus to C-terminus: optionally a signal peptide (e.g., a GM-CSFR α signal peptide), an extracellular antigen-binding domain that specifically binds a tumor antigen, optionally a hinge region or spacer, a transmembrane domain, and a cytoplasmic signaling domain, wherein the cytoplasmic signaling domain comprises a costimulatory domain and a primary signaling domain. In one embodiment, the antigen binding domain that specifically binds to a tumor antigen is an antibody or antibody fragment, particularly an scFv. In one embodiment, the antigen binding domain targets CD19, more preferably comprises LCDR1-3 in the VL amino acid sequence of SEQ ID No. 8 and HCDR1-3 in the VH amino acid sequence of SEQ ID No. 9 (especially the CDR sequences as defined by Kabat), even more preferably VL comprising SEQ ID No. 8 or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto and VH comprising SEQ ID No. 9 or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto, even more preferably scFv comprising SEQ ID No. 11 or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto. In one embodiment, the CAR polypeptide comprises a hinge/spacer region, preferably selected from the group consisting of: a hinge region from IgG or a spacer from a CD8 α or CD28 extracellular region, and preferably a human CD8 α hinge region or CD28 hinge region, e.g., a CD28 hinge region comprising the amino acid sequence shown in SEQ ID NO. 12 or an amino acid sequence at least 90%, 92%, 95%,96%,97%,98%, 99% or more identical thereto. In one embodiment, the CAR polypeptide comprises a transmembrane domain selected from: the transmembrane domains of CD4, CD8 α, CD28 and CD3 ζ, and preferably human CD8 transmembrane domain or CD28 transmembrane domain, for example, a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO. 13 or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto. In a further embodiment, the CAR polypeptide comprises one or more (especially two) co-stimulatory domains selected from the group consisting of: the costimulatory domains of CD28, CD27,4-1BB, ICOS and OX 40; and preferably comprises a combination of a human CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, e.g., comprising the amino acid sequences shown in SEQ ID NO. 14 and SEQ ID NO. 15 or amino acid sequences having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto. In one embodiment, the CAR polypeptide comprises a primary signaling domain that is a CD3 ζ primary signaling domain, e.g., an amino acid sequence comprising the amino acid sequence set forth in SEQ ID No. 16 or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto. In a preferred embodiment, the CAR polypeptide comprises a cytoplasmic signaling domain consisting of the costimulatory domain of CD28 and the costimulatory domain of 4-1BB and the CD3 ζ primary signaling domain, e.g., the cytoplasmic signaling domain comprises the amino acid sequence set forth in SEQ ID No. 21 or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto. In yet another embodiment, the CAR polypeptide comprises: from N-terminus to C-terminus, an antibody or antigen-binding fragment of an anti-tumor antigen (e.g., CD 19), such as a scFv, a CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3 ζ primary signaling domain. In a preferred embodiment, the CAR polypeptide comprises the amino acid sequence of SEQ ID No. 4, or an amino acid sequence that is at least 90%, 92%, 95%,96%,97%,98%, 99% or more identical thereto.

In one embodiment, the second nucleic acid molecule comprises a polynucleotide encoding IL-15. In one embodiment, the second nucleic acid molecule comprises a polynucleotide encoding SEQ ID NO. 5 or an amino acid sequence having at least 85%,90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto. In one embodiment, the second nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO. 2, or a nucleotide sequence having at least 85%,90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto.

In one embodiment, the third nucleic acid molecule comprises a polynucleotide encoding optimized IL-15Ra. In one embodiment, the optimized IL-15Ra comprises the amino acid sequence of SEQ ID NO 6.

As demonstrated in the examples herein, the nucleic acid combination of the invention, when introduced into an immune effector cell, such as a T cell, results in a reduced amount of release of IL-15 in the extracellular environment, and thus a reduced toxicity induced by IL-15, compared to a control immune effector cell into which only the first nucleic acid molecule encoding the CAR and/or the second nucleic acid molecule encoding the IL-15 is introduced; and preferably, the function of IL-15 to increase the persistence of CAR-T cells is maintained.

In a second aspect, the invention provides a polypeptide encoded by a nucleic acid combination of the invention comprising: (i) a Chimeric Antigen Receptor (CAR) polypeptide; (ii) an IL-15 polypeptide; and (iii) optimizing the IL-15Ra polypeptide. In one embodiment, two or all three of (i) - (iii) are functionally linked to each other, in particular by a connecting peptide as a single polypeptide chain. In another embodiment, the polypeptides of (i), (ii) and (iii) are encoded by a combination of nucleic acids of the invention, which are separate from each other. When two or more polypeptides are referred to as being separate from each other, it is meant that the referred polypeptides are not covalently linked to each other (either directly or through a linking peptide), but that there may or may not be non-covalent binding between the referred isolated polypeptides, e.g., there may be non-covalent binding between the isolated IL-15 and the optimized IL-15Ra as a complex, but not any non-covalent binding to the CAR polypeptide. In one embodiment, the polypeptide encoded by the nucleic acid combination of the invention is a single fusion polypeptide comprising functionally linked: (i) a Chimeric Antigen Receptor (CAR) polypeptide; (ii) an IL-15 polypeptide; and (iii) an optimized IL-15Ra polypeptide, more preferably the fusion polypeptide has a structure according to formula (I) of the present invention, even more preferably the fusion polypeptide can be cleaved after expression in a cell by self-splicing sites located in L1 and L2 to yield three isolated polypeptides, i.e., a CAR polypeptide, an IL-15 polypeptide and an optimized IL-15Ra polypeptide.

In a third aspect, the present invention provides nucleic acid constructs, particularly vectors, e.g., viral vectors, such as lentiviral vectors, comprising the nucleic acid combinations of the invention. In one embodiment, the first, second and third nucleic acid molecules comprised in the nucleic acid combination of the invention are present on the vector in the form of polycistrons.

In a fourth aspect, the invention provides a host cell comprising a nucleic acid combination or nucleic acid construct or vector of the invention. The host cell may be an immune effector cell, such as a T cell or NK cell. Thus, in one embodiment, the invention also provides an armored CAR-T cell and a method of making the same, wherein the armored CAR-T cell comprises a nucleic acid combination according to the invention, or incorporates a vector of the invention. In one embodiment, expression of the nucleic acid combination or vector of the invention comprised in said armored CAR-T cell produces a fusion polypeptide of formula (I) according to the invention, optionally said fusion polypeptide is cleaved in the cell into three separate polypeptides, i.e. a CAR polypeptide, an IL-15 polypeptide and an optimized IL-15Ra polypeptide, by the self-splice sites comprised in the linker peptide of formula (I). In another embodiment, expression of the nucleic acid combination or vector of the invention comprised in said armored CAR-T cell results in three polypeptides of the invention, i.e., a CAR polypeptide, an IL-15 polypeptide and an optimized IL-15Ra polypeptide, being isolated from each other.

In some embodiments, the armored CAR-T cells of the invention expressing IL-15 and optimized IL-15Ra exhibit enhanced proliferative capacity and cell viability, as well as increased proportion of T cell subpopulations of the Tscm phenotype, compared to CAR-T cells expressing only the CAR molecule, as determined as described in the examples. In some embodiments, the armored CAR-T cells of the invention that express IL-15 and optimize IL-15Ra reduce the release of IL-15 in the extracellular environment, and thus have reduced toxicity induced by IL-15, compared to CAR-T cells that express IL-15 alone. In still other embodiments, the armored CAR-T cells of the invention expressing IL-15 and optimized IL-15Ra, as determined as described in the examples, exhibit in vivo anti-tumor efficacy and reduced adverse events associated with IL-15 in CAR-T therapy.

In a fifth aspect, the invention provides a pharmaceutical composition comprising an armored CAR-T cell of the invention.

In a sixth aspect, the invention provides the use of an armored CAR-T cell of the invention in the preparation of a medicament for the prevention or treatment of cancer or for providing anti-tumor immunity, and a method of preventing or treating cancer or for providing anti-tumor immunity in a subject using an armored CAR-T cell of the invention. In some embodiments, the CAR-T cell is administered systemically (e.g., intravenously) or locally (e.g., intratumorally). In some embodiments, the tumor is a hematological tumor or a solid tumor.

In a seventh aspect, the invention provides an optimized IL15Ra polypeptide and nucleic acid molecules encoding the same, and their use in reducing toxicity of a CAR-T cell recombinantly expressing IL15, preferably said use comprises introducing and expressing in said CAR-T cell a nucleic acid molecule encoding said optimized IL-15Ra polypeptide, more preferably said CAR-T cell comprises a nucleic acid molecule encoding a fusion protein according to the structure of formula (I) of the invention.

In an eighth aspect, the invention provides a method for increasing the persistence and reducing the toxicity of a CAR-T cell comprising introducing and expressing in said CAR-T cell a nucleic acid combination or vector according to the invention.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

Figure 1 schematically shows the structure of an armored CAR-T cell according to the invention. Wherein: CD19-CAR represents a CD 19-targeted chimeric antigen receptor polypeptide comprising, from N-terminus to C-terminus, an anti-CD 19 scFv, a CD28 spacer/transmembrane region, a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain; SD represents a splice donor site; SA represents a splice acceptor site; LTR represents a long terminal repeat; P2A and T2A represent self-splicing peptides P2A and T2A, respectively; IL-15 represents IL-15 protein; IL-15Ra represents a modified IL-15Ra protein.

Figure 2 shows the characterization of CAR, CAR-IL15 and CAR-IL15 Ra T cells produced after transduction of T cells with a retroviral vector encoding a CAR. (A and B) transduction efficiency was determined by flow cytometry, quantifying the number of CD4+ and CD8+ T cells and the number of CD19+ CD8+ CAR T cells. (C) IL-15 and IL-15Ra expression levels in three CAR-T cells were confirmed by PCR. As a control, the housekeeping gene GAPDH was simultaneously amplified in the cells.

Figure 3 shows the detection of in vitro proliferation of armored CAR-T cells overexpressing IL-15 and/or IL-15Ra under stimulation by cytokines or target tumor cells (a) and cytokine IL-2 production (B).

FIG. 4 shows detection of differentiation phenotype of IL-15 and/or IL-15Ra overexpressing armored CAR-T cells 7 days after NAML-6-eGFP stimulation of target tumor cells, where flow cytometry detects Tsccm (CD 8) in the cell population ⁺ CD45RO ^- CCR7 ⁺ CD27 ⁺ CD95 ⁺ ) The ratio of (a) to (b). (A) The highest measured cell level of Tscm was 1.67%,9.23% and 4.84% for the three groups, respectively. (B) Histogram of measurements

Figure 5 shows cytokine IFN γ secretion (a) and percentage of apoptosis (B) and cell survival (C) of CAR, CAR-IL15 and CAR-IL15 Ra T cells after incubation with target tumor cells NAML-6-eGFP for a period of time.

Figure 6 shows IL-15 secretion (a) and CD132 cell surface expression (B) of CAR, CAR-IL15 and CAR-IL15 Ra T cells under in vitro culture conditions.

Figure 7 shows that after 24 hours of coculture of CAR-T cells with target cells NAML-6-eGFP cells (2); and (B) corresponding statistical result histogram.

FIG. 8 shows a xenograft mouse tumor model experiment. (A) animal experiment flow chart; (B) fluorescence plot of tumor burden in mice.

FIG. 9 shows the tumor burden over time for each group of mouse individuals in a xenograft mouse tumor model experiment. Quantitative bioluminescence imaging data (i.e., the absolute number of photons/sec/cm emitted from the surface of an animal per unit time, unit area, unit arc) for all mice was obtained using the IVIS imaging system ² /sr)). Higher values indicate greater tumor burden.

FIG. 10 shows the overall survival rate and serum IL-15 concentration of xenograft-bearing mice. (A) The overall survival of xenograft tumor-bearing mice was measured using the Kaplan-Meier method. (B) On day 50, blood was collected from each group of mice, and the concentration of human IL-15 was measured with serum.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

I. Definition of

For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to. When the term "comprising" or "includes" is used herein, unless otherwise specified, it also encompasses the presence of stated elements, integers or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, it is also intended to encompass antibody variable regions consisting of that particular sequence.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

As used herein, the term "and/or" means any one of the options or two or more of the options.

The term "IL-15" refers to interleukin 15 cytokine. An example of IL-15 is interleukin IL-15 of human origin, such as the protein under UniProtKB-accession number P40933, or a homologue thereof, such as interleukin IL-15 of non-human mammalian origin, such as non-human primates, rodents, livestock, sports animals and the like.

The term "IL-15Ra" or "IL-15Ra" refers to the interleukin 15 receptor alpha protein. An example of IL-15Ra is human-derived interleukin IL-15 receptor alpha, such as the protein under UniProtKB-accession number Q13261, or a homologue, or variant thereof. In one embodiment, the IL-15Ra of the present invention is an interleukin 15 receptor alpha protein that introduces modifications (e.g., double mutations S202R and D203E) in the parent IL-15Ra receptor protein, also referred to as "optimized IL-15Ra". In some embodiments, the parent IL-15Ra receptor protein may be of mammalian origin, e.g., human origin, or a native or wild-type IL-15Ra of a non-human mammal. In some embodiments, the parental IL-15Ra receptor protein comprises the motif YPQGHRET at positions 197-204.

The terms "chimeric receptor," "chimeric antigen receptor," or "CAR" are used interchangeably herein and refer to a recombinant polypeptide comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain from a stimulatory molecule as described below, e.g., a primary signaling domain of CD 3-zeta. In another aspect, the intracellular signaling domain further comprises one or more functional signaling domains from at least one, preferably two, costimulatory molecules, e.g., CD28 and 4-1 BB. The CAR polypeptide can be expressed on any cell, e.g., an immune effector cell such as a T cell or NK cell.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. In one aspect, a primary signal can be initiated (e.g., by binding of the TCR/CD3 complex to a peptide-loaded MHC molecule) and subsequently mediate T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence that functions in a stimulatory manner may comprise an Immunoreceptor Tyrosine Activation Motif (ITAM). Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, intracellular signaling domains from TCR zeta and CD3 zeta. In the present invention, the cytoplasmic domain of the CAR polypeptide of the invention comprises at least one functional cytoplasmic signaling sequence from a stimulatory molecule, e.g., the cytoplasmic signaling sequence of CD3 ζ.

The term "CD3 ζ" is defined as the protein provided under the UniProtKB-P20963 accession number, or an equivalent thereof. Herein, a "CD3 zeta signaling domain" is defined as a segment of amino acid residues from the cytoplasmic domain of the CD3 zeta chain sufficient to functionally transmit the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of CD3 ζ comprises residues 52 through 164 of the amino acid sequence under the UniProtKB-P20963 accession number or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) as a functional ortholog thereof. In one embodiment, the "CD3 zeta signaling domain" is the sequence provided in SEQ ID NO 16 or a variant thereof.

The term "costimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response (e.g., without limitation, proliferation) of the cell. Costimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to the antigen receptor or its ligand. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In some embodiments, a "co-stimulatory molecule" is CD28, 4-1BB (i.e., CD 137). As used herein, "costimulatory domain" refers to the intracellular portion of the costimulatory molecule.

The term "4-1BB" refers to a TNFR superfamily member, also known as CD137, that has the amino acid sequence provided under the UniProtKB-Q07011 accession number or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "4-1BB co-stimulatory domain" is defined herein as a cytoplasmic region from 4-1BB, e.g., amino acid residues 214-255 of UniProtKB-Q07011 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The term "CD28" refers to the amino acid sequence provided under the UniProtKB-P10747 accession number or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "CD28 co-stimulatory domain" is defined herein as amino acid residues 180-220 from the cytoplasmic region of CD28, e.g., uniProtKB-P10747, or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "CD28 transmembrane domain" is defined herein as a transmembrane region from CD28, e.g., amino acid residues 153-179 of UniProtKB-P10747 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.). The term "CD28 hinge domain" is used interchangeably herein with "CD28 spacer" and is defined as a hinge domain from the extracellular region of CD28, e.g., amino acid residues 114-152 of UniProtKB-P10747 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

The term "recombinant" when used herein in reference to, for example, a virus or cell or a nucleic acid or protein or vector, means that the virus, cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by altering an existing native nucleic acid or protein itself, or that material from the virus or cell so modified.

The terms "exogenous" or "heterologous" when used in reference to a nucleic acid or protein are used interchangeably to mean that the nucleic acid or protein is foreign to the host cell in which it is contained or is to be contained, i.e., its location in the host cell is not the natural location in which it naturally occurs. Heterologous nucleic acid sequences also refer to sequences that are derived from and introduced (e.g., introduced by infection with a viral vector) into the same host cell or subject and thus exist in a non-native state, e.g., the sequences are at different positions, are present in different copy numbers, or are under the control of different regulatory elements.

The term "expression cassette" refers to a DNA sequence that encodes and is capable of expressing one or more genes of interest (e.g., a CAR polypeptide of the invention, or an IL-15 protein, or an optimized IL-15Ra, or two or three thereof). In an expression cassette, typically, a heterologous polynucleotide sequence encoding a gene of interest is functionally linked to an expression control sequence. For example, an expression cassette can comprise two or more genes of interest in polycistronic form under the control of the same promoter, and thereby encode and express a single polypeptide chain in which the two or more proteins of interest encoded by the two or more genes of interest are functionally linked to each other.

The term "functionally linked," also known as "operatively linked," means that the specified components are in a relationship that allows them to function in the intended manner.

The terms "linker" or "linker" are used interchangeably herein and refer to a short amino acid sequence consisting of amino acids, such as alanine (a), glycine (G) and/or serine (S) and/or threonine residues (T), or self-splicing peptides comprising self-splicing sites, used alone or in combination. In one embodiment, the linker peptide has a length of 1-50 amino acids, e.g., 1,2,3,4,5 amino acids, or 10,15,20,25,30 amino acids. The linking peptide that may be used between the components of the CAR fusion polypeptide of the invention is not particularly limited. Computer programs can be used to mimic the three-dimensional structure of proteins and peptides to rationally design suitable linker peptides. For example, short oligopeptide linkers or polypeptide linkers may be used to form linkages between the component sequences as desired, e.g., glycine-serine doublets, or single amino acids, e.g., alanine, glycine, may be used as linkers.

The terms "amino acid change" and "amino acid modification" are used interchangeably to refer to addition, deletion, substitution, and other modification of an amino acid. Any combination of amino acid additions, deletions, substitutions, and other modifications can be made, provided the final polypeptide sequence has the desired properties. In some embodiments, the substitution of an amino acid is a non-conservative amino acid substitution, i.e., one amino acid is substituted with another amino acid having a different structural and/or chemical property. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).

The terms "conservative sequence modification" and "conservative sequence change" refer to an amino acid modification or change that does not significantly affect or alter the characteristics of the parent polypeptide or its constituent elements comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative modifications, especially conservative substitutions, can be introduced into the CAR fusion polypeptides of the invention or its constituent elements (e.g., CAR or IL-15 Ra) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

"percent (%) identity" of an amino acid sequence/nucleotide sequence refers to the percentage of amino acid/nucleotide residues in a candidate sequence that are identical to the amino acid residues/nucleotide residues in the specific amino acid/nucleotide sequence shown in the specification, after aligning the candidate sequence with the specific amino acid/nucleotide sequence shown in the specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and in the case of amino acid sequences, without considering any conservative substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the fusion polypeptides or nucleic acid molecules of the invention or the constituent elements thereof, relative to those described hereinThe fusion polypeptides or nucleic acid molecules disclosed in the body or constituent elements thereof (e.g., CAR polypeptide/encoding nucleic acid, or IL-15 protein/encoding nucleic acid, or optimized IL-15Ra protein/nucleic acid) have a substantial degree of identity with respect to their sequence, e.g., at least 80%,85%,90%,95%,97%,98%, or 99% or more identity. The variant may comprise a conservative modification. For the purposes of the present invention, percent identity appliesPublic available BLAST in https:// BLASTAnd the tool adopts default parameters for determination.

As used herein, the expression "variant" or "functional variant" polypeptide or protein refers to a polypeptide or protein that has substantially the same sequence or significant sequence identity as a reference polypeptide or protein and retains the desired biological activity of the reference polypeptide or protein.

The term "vector" as used herein when referring to a nucleic acid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

The term "lentivirus" refers to a genus of the family Retroviridae (Retroviridae). Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, and thus they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, and specifically includes self-inactivating lentiviral vectors as provided in Milone et al, mol. Ther.17 (8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but are not limited to, those from Oxford BioMedica

Gene delivery technology, LENTIMAXT from LentigenM vector systems, and the like. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

The term "immune effector cell" refers to a cell involved in an immune response, e.g., involved in promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α/β T cells and γ/δ T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, mast cells, and myeloid cell-derived phagocytes.

The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The terms "tumor" and "cancer" are used interchangeably herein to encompass solid tumors and liquid tumors.

The term "anti-tumor immunity" means that an immunological effect can be exhibited by a variety of means, including, but not limited to, causing, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

The nucleic acid combinations, polypeptides and armed CAR-T cells of the invention

The present invention has found in an intensive study that in CAR-based immune cells (e.g., CAR-T cells and CAR-NK cells), the persistence and/or anti-tumor immunity of said immune cells can be promoted by increasing IL-15 and optimizing the expression of the IL-15Ra gene, while limiting the toxicity induced by IL-15 released from the environment (e.g., serum).

Thus, in one aspect, the invention provides a CAR-based immune cell, wherein the immune cell comprises not only a heterologous polynucleotide encoding a CAR polypeptide, but also a heterologous polynucleotide encoding IL-15 and optimized IL-15Ra. Where the immune cell is a T cell, such CAR-based immune cells with recombinantly expressed IL15 and optimized IL-15Ra are also referred to herein as "Armored CAR-T cells" or "Armored CAR-T cells". In the immune cell, the heterologous polynucleotide encoding the CAR polypeptide and the heterologous polynucleotides encoding IL-15 and optimized IL-15Ra can be on a single nucleic acid molecule or on separate different nucleic acid molecules.

In a further aspect, the invention provides a combination of nucleic acids useful in forming a CAR immune cell according to the invention. In one embodiment, the nucleic acid combination of the invention comprises a first nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) molecule, a second nucleic acid molecule encoding an IL-15 protein, and a third nucleic acid molecule encoding an optimized IL-15Ra. While not being bound by any theory, it is believed that: the optimized IL-15Ra of the invention, due to the specifically designed double mutation, can combine with IL-15 to form a stable heterodimer structure and be displayed on the cell membrane when expressed in the same cell as IL-15, thereby limiting the amount of IL-15 released into the environment (e.g., animal serum) while ensuring that IL-15 performs its function in a stable functional conformation, thereby reducing toxicity induced by serum high IL-15 levels. As demonstrated in the examples herein, the armored CAR-T cells of the invention comprising optimized IL-15Ra demonstrated superior performance in reducing adverse events during CAR-T therapy and enhancing anti-tumor.

In the nucleic acid combination of the present invention, the CAR polypeptide encodes a polynucleotide, the IL-15 protein-encoding polynucleotide, and the optimized IL-15 Ra-encoding nucleic acid may be in the same expression cassette or in different expression cassettes for all three or any two of them, and may be expressed as separate polypeptides, or as a fusion polypeptide for any two or all three of them. In one embodiment, the nucleic acid combination is in the form of a single nucleic acid molecule encoding and expressing a single fusion polypeptide comprising the CAR polypeptide, IL-15 and optimized IL15Ra, preferably wherein IL-15 and IL15Ra proteins are functionally linked together and the CAR polypeptide is functionally linked to one of IL-15 and IL15Ra proteins by a linker peptide comprising a cleavable site. In some preferred embodiments, the fusion polypeptide has the structure of formula (I) from N-terminus to C-terminus:

CAR-(L1)-E1-(L2)-E2 (I)

wherein the content of the first and second substances,

CAR represents a chimeric antigen receptor polypeptide encoded by a first nucleic acid molecule,

l1 and L2 each independently represent a linker peptide (particularly a self-splicing peptide),

e1 and E2 are different from each other and independently of each other denote IL-15 or optimized IL-15Ra, respectively, encoded by the second or third nucleic acid molecule, and

wherein the components of formula (I) are functionally linked.

In yet another aspect, the present invention also provides a fusion polypeptide having the structure of formula (I) above.

The CAR-based immune cells, nucleic acid combinations, polypeptides, and components thereof of the invention are each described in detail below. It will be appreciated by those skilled in the art that any technical features and any combination thereof which are mentioned in the description of the components are within the contemplation of the present invention, unless the context clearly dictates otherwise; furthermore, it will be understood by those skilled in the art that the CAR-based immune cells of the invention may comprise any such combination of features, as may the nucleic acid constructs and CAR fusion polypeptides of the invention, unless the context clearly indicates to the contrary.

Optimized IL-15Ra polypeptides and nucleic acids encoding same

The IL-15Ra of the invention is a membrane-bound protein with characteristic double mutations S202R and D203E. In one embodiment, the IL-15Ra of the invention may be derived from any functional single transmembrane native full length IL-15Ra protein or variant thereof (including native allelic variants or species homologues) wherein double mutations S202R and D203E have been introduced at amino acid positions 202 and 203, wherein said amino acid positions are numbered according to SEQ ID NO: 6. In this context, when referring to the amino acid position of the IL-15Ra protein, "numbering according to SEQ ID NO: 6" means that this is determined by reference to the amino acid sequence of SEQ ID NO: 6. For any given IL-15Ra polypeptide, the corresponding amino acid position on that given IL-15Ra polypeptide can be identified by amino acid sequence Alignment with SEQ ID NO:6 (e.g., using BLAST; basic Local Alignment Search Tool available from http:// BLAST. Ncbi. Nlm. Nih. Gov, aligned using default parameters). Thus, for example, reference to amino acid positions 202 and 203 of a given IL-15Ra polypeptide refers to positions 202 and 203 of SEQ ID NO:6 (when the IL-15Ra comprises SEQ ID NO: 6), or the corresponding amino acid positions aligned on the given polypeptide sequence (when the IL-15Ra comprises an amino acid sequence having a percentage, e.g., 90-99.5%, identity to SEQ ID NO: 6). Herein, mutation S202R refers to a mutation of amino acid position 202 with serine (S) to arginine (R); mutation D203E refers to amino acid position 203 having a mutation of aspartic acid (D) to glutamic acid (E). In any embodiment according to the present invention, preferably, the IL-15Ra polypeptide comprises, in addition to said double mutation, i) the amino acid sequence of SEQ ID NO 6; ii) an amino acid sequence having at least one, two or three modifications but not more than 30,20 or 10 modifications to the amino acid sequence of SEQ ID NO 6; or iii) an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO 6. Preferably, the IL-15Ra of the invention comprises the motif YPQGHSDT at positions 197-204.

In a preferred embodiment, the IL-15Ra polypeptides of the invention retain the signal peptide from which they are derived from the native IL-15 parent polypeptide. In other embodiments, the IL-15Ra polypeptides of the invention have a heterologous signal peptide from another transmembrane eukaryotic protein, such as a mammalian protein, to direct their integration into the cell membrane after expression and processing in the cell. In one embodiment, the IL-15Ra polypeptide of the invention forms a non-covalent complex with an IL-15 polypeptide expressed in a cell following expression from the same cell and is transported to the surface of the cell membrane.

An optimized IL-15Ra polypeptide-encoding polynucleotide useful in the present invention may be any polynucleotide comprising a nucleotide sequence encoding an optimized IL-15Ra protein according to any of the above embodiments of the present invention. In one embodiment, the optimized IL-15 Ra-encoding polynucleotide comprises an amino acid sequence encoding SEQ ID NO 6 or a variant thereof, e.g., having at least 95%,96%,97%,98%, or 99% identity thereto. In one embodiment, the IL-15 Ra-encoding polynucleotide comprises the nucleotide sequence of SEQ ID NO. 3 or a variant thereof, e.g., an amino acid sequence at least 95%,96%,97%,98%, or 99% identical thereto; or a nucleotide sequence that hybridizes thereto under stringent hybridization conditions.

IL-15 polypeptides and nucleic acids encoding same

IL-15 polypeptides useful in the present invention include, but are not limited to, full-length native IL-15 protein or functional fragments thereof, or variants thereof (including natural allelic variants or species homologs). The amino acid sequence of IL-15 from human is given under the UniProtKB-P40933 accession number.

In any embodiment according to the present invention, preferably the IL-15 polypeptide comprises i) the amino acid sequence of SEQ ID NO. 5; ii) an amino acid sequence having at least one, two or three modifications but NO more than 30,20 or 10 modifications to the amino acid sequence of SEQ ID NO. 5; or iii) an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO. 5.

The IL-15 encoding polynucleotide useful in the present invention may be any polynucleotide comprising a nucleotide sequence encoding an IL-15 polypeptide according to any of the above embodiments of the present invention. In one embodiment, the IL-15 encoding polynucleotide comprises a nucleotide sequence encoding SEQ ID NO. 5 or an amino acid sequence that is a variant thereof, e.g., at least 95%,96%,97%,98%, or 99% identical thereto. In one embodiment, the IL-15 encoding polynucleotide comprises the nucleotide sequence of SEQ ID NO. 2 or a variant thereof, e.g., an amino acid sequence at least 95%,96%,97%,98%, or 99% identical thereto; or a nucleotide sequence that hybridizes thereto under stringent hybridization conditions.

In some embodiments, the amount of IL-15 released from a cell is reduced when the optimized IL15Ra polypeptide of the invention is expressed in the same cell as the IL-15 polypeptide. The released IL-15 may comprise the IL-15 polypeptide itself, or a heterodimer thereof with soluble IL-15Ra (e.g., soluble IL-15Ra shed from a cell membrane). In one embodiment, the reduction is relative to a control cell that expresses only IL-15 and not the optimized IL-15Ra polypeptides of the invention. In yet another embodiment, the control cell expresses a wild-type full length IL-15Ra polypeptide without mutations S202R and D203E.

Chimeric Antigen Receptor (CAR) polypeptides and polynucleotides encoding same

CAR polypeptides useful in the invention are not particularly limited. In one aspect, the CAR polypeptide of the invention comprises an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. In one embodiment, the cytoplasmic signaling domain of the CAR polypeptide of the invention comprises a primary signaling domain. In one embodiment, the cytoplasmic signaling domain of the CAR polypeptide of the invention comprises a costimulatory domain and a primary signaling domain. In one embodiment, the Chimeric Antigen Receptor (CAR) molecule according to the invention comprises, from N-terminus to C-terminus: consisting of (a) an antigen-binding domain that specifically binds to a tumor antigen and (b) a hinge or spacer region; (c) a transmembrane domain; and (d) a cytoplasmic signaling domain. In one embodiment, the CAR molecule according to the invention comprises, from N-terminus to C-terminus: (a) An antigen binding domain that specifically binds to a tumor antigen, (b) a hinge region or spacer region; (c) a transmembrane domain; (d) two co-stimulatory domains from CD28 and 4-1 BB; and (e) a primary signaling domain from CD 3.

In some embodiments, the target antigen for a CAR polypeptide of the invention is a membrane antigen, e.g., a tumor-specific antigen or a tumor-associated antigen, that is surface-expressed on a target cell, particularly a tumor cell. Tumors that may be mentioned include haematological tumors and solid tumors, including primary and metastatic tumors. In some embodiments, the target antigen is a tumor cell surface antigen comprising an antigenic cancer epitope that can be immunologically recognized by Tumor Infiltrating Lymphocytes (TILs) derived from a mammal. In other embodiments, the target antigen is a tumor cell surface antigen comprising one or more antigenic cancer epitopes associated with a malignant tumor. In a preferred embodiment, the extracellular antigen-binding domain of the CAR molecule of the invention targets a tumor antigen, preferably selected from the group consisting of: CD19, the EphA2 receptor (EphA 2), the folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof. More preferably, the tumor antigen is the membrane antigen CD19.

Depending on the antigen to be targeted, the CAR of the invention can be constructed to include an appropriate antigen binding domain specific for the desired antigen target to confer the CAR molecule, and the CAR-T cells comprising the CAR molecule, the ability to specifically recognize and bind the target antigen. In one embodiment, the extracellular antigen-binding domain of the CAR molecule according to the invention is a polypeptide molecule having binding affinity for a target antigen. In one embodiment, a CAR according to the invention comprises an antigen binding domain derived from an antibody or antibody fragment. In yet another embodiment, the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In a preferred embodiment, the antigen binding domain comprises a scFv joined by a VL and a VH via a linker.

The scFv can be produced by linking the VH and VL regions together using a flexible polypeptide linker according to methods known in the art. In some embodiments, the scFv molecule comprises a flexible polypeptide linker having an optimized length and/or amino acid composition. In some embodiments, the scFv comprises a linker between its VL and VH regions, wherein the linker comprises at least 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50 or more amino acid residues. The linker sequence may comprise any naturally occurring amino acid. In one embodiment, the peptide linker of the scFv consists of amino acids such as glycine and/or serine residues, used alone or in combination, to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and, for example, comprises the amino acid sequence (Gly-Gly-Gly-Ser) n, wherein n is a positive integer equal to or greater than 1 (SEQ ID NO: 40). For example, n =1,n =2,n =3.n =4, n =5 and n =6,n =7, n =8, n =9 and n =10. In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly 4 Ser) 4 (SEQ ID NO: 27) or (Gly 4 Ser) 3 (SEQ ID NO: 28). In another embodiment, the linker comprises multiple repeats of (Gly 2 Ser), (GlySer), or (Gly 3 Ser) (SEQ ID NO: 29). In yet another embodiment, the linker comprises the gstsggkpgsgagtkg amino acid sequence. In one embodiment, the scFv for use in the present invention comprises, from N-terminus to C-terminus: VL-linker-VH; or VH-linker-VL.

The CAR polypeptides of the invention comprise at least one transmembrane domain, which may be derived from natural or synthetic sources. For example, the transmembrane domain may be derived from a membrane-bound protein or transmembrane protein, such as a transmembrane domain from CD3 ζ, CD4, CD28, CD8 (e.g., CD8 α, CD8 β). In the Chimeric Antigen Receptor (CAR) polypeptides of the invention, the transmembrane domain confers membrane attachment to the CAR polypeptides of the invention. In some embodiments, the transmembrane domain in a CAR of the invention may be linked to the extracellular region of the CAR by a hinge/spacer region. For transmembrane and hinge/spacer regions useful in CAR polypeptides, see, e.g., kento Fujiwara et al, cells 2020,9,1182; doi:10.3390/cells9051182.

The cytoplasmic signaling domain comprised in the CAR polypeptide of the invention comprises at least a primary signaling domain. The primary signaling domain is capable of activating at least one immune effector function of an immune cell into which the CAR of the invention is introduced. Such immune effector functions include, but are not limited to, for example, enhancing or promoting the function or response of an immune attack on a target cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines.

Examples of cytoplasmic signaling domains for use in the CAR polypeptides of the invention include cytoplasmic sequences of T Cell Receptors (TCRs) and/or co-receptors that can function to initiate signal transduction upon binding of the extracellular domain to a target antigen, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capability. T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation by the TCR (i.e., the primary signaling domain) and those that function in an antigen-independent manner to provide a costimulatory signal (i.e., the secondary cytoplasmic domain, e.g., the costimulatory domain). In one embodiment, the CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary signal domain, e.g., an intracellular primary signal domain of CD3 ζ. In another embodiment, the cytoplasmic domain of a CAR polypeptide of the invention further comprises a secondary signal domain, e.g., a costimulatory domain from a costimulatory molecule. In one embodiment, the cytoplasmic region of a CAR polypeptide of the invention comprises one or more (particularly two) costimulatory domains, such as a combination of the costimulatory domains of 4-1BB (also known as CD 137) and CD28, in tandem with a CD3 ζ intracellular signaling domain.

In some embodiments, the CAR polypeptides of the invention may comprise a signal peptide or leader sequence N-terminal to the extracellular antigen-binding domain. Through the signal peptide/leader sequence, the nascent CAR polypeptide can be directed to the endoplasmic reticulum of the cell and then anchored to the cell membrane. Any signal peptide/leader sequence of eukaryotic origin may be used, for example a signal peptide/leader sequence of mammalian or human secretory protein origin.

In some embodiments, a Chimeric Antigen Receptor (CAR) polypeptide according to the invention comprises an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain.

In one embodiment, the antigen binding domain is an antigen binding domain that targets a tumor antigen. In one embodiment, the tumor antigen is a membrane antigen, such as CD19 or EphA2, and preferably CD19. In one embodiment, the extracellular antigen-binding domain is an antigen-binding domain that binds CD19. In one embodiment, the extracellular antigen-binding domain comprises a murine, human, or humanized antigen-binding domain that binds CD19. In one embodiment, the antigen binding domain that binds CD19 comprises heavy chain complementarity determining region 1 (HC CDR 1), heavy chain complementarity determining region 2 (HC CDR 2), and heavy chain complementarity determining region 3 (HC CDR 3) of the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 9; and/or light chain complementarity determining region 1 (LC CDR 1), light chain complementarity determining region 2 (LC CDR 2), and light chain complementarity determining region 3 (LC CDR 3) of the light chain variable region (VL) amino acid sequence of SEQ ID NO: 8. In one embodiment, the antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein,

the heavy chain variable region comprises: i) The amino acid sequence of SEQ ID NO 9; ii) an amino acid sequence having at least one, two or three modifications but not more than 30,20 or 10 modifications to the amino acid sequence of SEQ ID NO 9; or iii) an amino acid sequence having 95-99% identity to the heavy chain variable region amino acid sequence of SEQ ID NO 9; and/or

The light chain variable region comprises i) the amino acid sequence of SEQ ID NO 8; ii) an amino acid sequence having at least one, two or three modifications but NO more than 30,20 or 10 modifications to the amino acid sequence of SEQ ID NO 8; or iii) an amino acid sequence 95-99% identical to the heavy chain variable region amino acid sequence of SEQ ID NO 8.

In one embodiment, the antigen binding domain comprises i) the amino acid sequence of SEQ ID NO. 11; ii) an amino acid sequence having at least one, two or three modifications but NO more than 30,20 or 10 modifications to SEQ ID NO 11; or iii) a sequence substantially identical to SEQ ID NO:11 with 95-99% identity.

In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8 α, CD28, CD3 ζ, TCR ζ, fcRy, fcRβ, CD3 γ, CD3 δ, CD3 ε, CD5, CD9, CD16, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), fcε RI, CD66d, the α, β, or ζ chain of a T cell receptor, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, and an activating NK cell receptor. In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8 α, CD28, and CD3 ζ. In one embodiment, the transmembrane domain comprises i) the amino acid sequence of SEQ ID NO 13; ii) an amino acid sequence comprising at least one, two or three modifications but NO more than 5 modifications of the amino acid sequence of SEQ ID NO 13; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO 13.

In one embodiment, the cytoplasmic signaling domain comprises a functional signaling domain of a protein selected from the group consisting of TCR ζ, fcR γ, fcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the cytoplasmic signaling domain comprises a functional signaling domain of a CD3 ζ protein (also referred to herein as a CD3 ζ primary signaling domain). In one embodiment, the cytoplasmic signaling domain comprises i) the amino acid sequence of SEQ ID NO 16; ii) an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO 16; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO. 15.

In one embodiment, the cytoplasmic signaling domain further comprises a costimulatory domain of one or more proteins selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, CD8, ICOS, DAP10, DAP12, OX40, CD40, GITR, 4-1BB (i.e., CD 137), CD27, and CD28. In one embodiment, the cytoplasmic signaling domain comprises a co-stimulatory domain of one or both proteins selected from the group consisting of: the costimulatory domains of CD28, CD27,4-1BB, ICOS and OX 40. In one embodiment, the cytoplasmic signaling domain comprises a costimulatory domain of a protein selected from the group consisting of: CD28 and 4-1BB (i.e., CD 137), or a combination thereof. In one embodiment, the cytoplasmic signaling domain comprises a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, wherein preferably the CD28 co-stimulatory domain comprises i) the amino acid sequence of SEQ ID NO 14; ii) an amino acid sequence comprising at least one, two or three modifications but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO 14; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 14; and preferably, the 4-1BB co-stimulatory domain comprises i) the amino acid sequence of SEQ ID NO 15; ii) an amino acid sequence comprising at least one, two or three modifications but NO more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO 15; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 15.

In a preferred embodiment, the cytoplasmic signaling domain of the CAR polypeptide comprises: comprising a costimulatory signal from CD28 and 4-1BB and a primary signaling domain from CD3 ζ. In one embodiment, the cytoplasmic signaling domain comprises: i) 1, SEQ ID NO; ii) an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO. 1; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO. 1.

In one embodiment, the CAR polypeptide comprises a transmembrane domain and an extracellular antigen-binding domain, and further comprises a hinge region or spacer disposed between the transmembrane domain and the extracellular antigen-binding domain. In one embodiment, the hinge/spacer region is selected from the group consisting of a GS hinge, a CD8 hinge, an IgG4 hinge, an IgD hinge, a CD16 hinge, and a CD64 hinge. In one embodiment, the CAR polypeptide comprises a hinge region from the CD28 extracellular region. In one embodiment, the hinge/spacer region comprises i) the amino acid sequence of SEQ ID NO 12; ii) an amino acid sequence comprising at least one, two or three modifications but NO more than 5 modifications of the amino acid sequence of SEQ ID NO 12; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO 12. Herein, the expressions "hinge", "hinge region" and "hinge domain" are used interchangeably.

In a preferred embodiment, the CAR polypeptide of the invention comprises: (a) an antigen binding domain; (b) a hinge region/spacer region; (c) a transmembrane domain; (d) a co-stimulatory domain from CD28 and 4-1 BB; and (e) a primary signaling domain from CD3 ζ.

In one embodiment, the CAR polypeptide further comprises a leader peptide or signal peptide, such as a signal peptide from the human granulocyte-macrophage colony stimulating factor receptor alpha chain (GM-CSFR alpha). In one embodiment, the CAR polypeptide comprises a signal peptide having the amino acid sequence of SEQ ID NO. 7.

In one embodiment, a CAR polypeptide according to the invention comprises i) the amino acid sequence of SEQ ID NO 4; ii) an amino acid sequence having at least one, two or three modifications but not more than 30,20 or 10 modifications to the amino acid sequence of SEQ ID NO. 4; or iii) an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO. 4.

A CAR-encoding nucleic acid useful in the present invention may be any polynucleotide comprising a nucleotide sequence encoding a CAR polypeptide according to any of the above embodiments of the invention. In one embodiment, the CAR-encoding nucleic acid comprises a nucleotide sequence encoding SEQ ID No. 4 or a variant thereof, e.g., an amino acid sequence at least 95%,96%,97%,98%, or 99% identical thereto.

Nucleic acid constructs of the invention

In one aspect, the invention provides one or more nucleic acid constructs comprising one, two or all three of a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an IL-15Ra polypeptide according to the invention. In one embodiment, the polynucleotide encoding a CAR polypeptide according to the invention, the polynucleotide encoding an IL-15 polypeptide according to the invention, and the polynucleotide encoding an optimized IL-15Ra polypeptide according to the invention are located on three different nucleic acid constructs, respectively. In one embodiment, the polynucleotide encoding a CAR polypeptide according to the invention, the polynucleotide encoding an IL-15 polypeptide according to the invention, and the polynucleotide encoding an optimized IL-15Ra polypeptide according to the invention are provided in a single nucleic acid construct.

In one embodiment, the nucleic acid construct according to the invention is a plasmid or a viral vector comprising an expression cassette. In one embodiment, the polynucleotide encoding a CAR polypeptide according to the invention, the polynucleotide encoding an IL-15 polypeptide according to the invention, and the polynucleotide encoding an IL-15Ra polypeptide according to the invention are present on a single nucleic acid construct and are each located in different expression cassettes under the control of the same or different promoters. In one embodiment, the polynucleotide encoding a CAR polypeptide according to the invention, the polynucleotide encoding an IL-15 polypeptide according to the invention, and the polynucleotide encoding an IL-15Ra polypeptide according to the invention are present in a single expression cassette in polycistronic form.

In one embodiment, the polynucleotide encoding a CAR polypeptide according to the invention comprises a polynucleotide encoding a CAR polypeptide according to any of the preceding embodiments of the invention, in particular a polynucleotide encoding a CD 19-targeted CAR polypeptide.

In one embodiment, the polynucleotide encoding an IL-15 polypeptide according to the invention comprises a polynucleotide encoding an IL-15 according to any of the preceding embodiments of the invention. In one embodiment, the IL-15 encoding polynucleotide encodes the amino acid sequence of SEQ ID NO. 5. In one embodiment, the IL-15 encoding polynucleotide comprises: i) 2, the nucleotide sequence of SEQ ID NO; ii) a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleotide sequence of SEQ ID NO. 2; or iii) a nucleotide sequence having at least 90-99% identity to the nucleotide sequence of SEQ ID NO. 2.

In one embodiment, the polynucleotide encoding an IL-15Ra polypeptide according to the invention comprises a polynucleotide encoding an IL-15Ra according to any of the preceding embodiments of the invention. In one embodiment, the IL-15 Ra-encoding polynucleotide encodes the amino acid sequence of SEQ ID NO 6. In one embodiment, the IL-15Ra encoding polynucleotide comprises: i) 3, the nucleotide sequence of SEQ ID NO; ii) a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleotide sequence of SEQ ID NO. 3; or iii) a nucleotide sequence having at least 90-99% identity to the nucleotide sequence of SEQ ID NO. 2.

In one embodiment, the CAR polypeptide, IL-15 and IL-15Ra polypeptide are each expressed separately from a nucleic acid construct according to the invention. In another embodiment, a fusion polypeptide comprising the CAR polypeptide, IL-15, and IL-15Ra polypeptide is produced from expression of a nucleic acid construct according to the invention, wherein the fusion polypeptide comprises a cleavable linker peptide interposed between the CAR polypeptide, IL-15, and IL-15Ra polypeptide expressed, whereby upon expression in a cell the fusion polypeptide can be cleaved to produce the CAR polypeptide, IL-15, and IL-15Ra polypeptide alone. In some embodiments, a polynucleotide encoding an IL-15 or IL-15Ra polypeptide is genetically fused at one end thereof, in frame, to a polynucleotide encoding a CAR polypeptide using a self-cleaving peptide; and at the other end, is genetically fused in frame to a polynucleotide encoding IL-15Ra or IL-15 polypeptide using a self-cleaving peptide.

In one embodiment, a nucleic acid construct according to the invention comprises a polynucleotide encoding a fusion polypeptide having the structure of formula (I):

CAR-(L1)-E1-(L2)-E2 (I)

wherein CAR, L1, L2, E1 and E2 are as defined above.

Preferably, L1 and L2 comprise a self-cleavage site. Self-splicing sites that may be used in the present invention include, but are not limited to, those comprising P2A, T2A, E2A or F2A self-splicing sites. For the sequence and application of the 2A self-Cleavage site, see Jin Hee Kim et al, high Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell lines, zebraphish and Mice, PLoS ONE April 2011, DOI 10.1371/journal. Point. 0018556.

In some embodiments according to the invention, preferably, said L1 comprises a P2A site and said L2 comprises a T2A site. In one embodiment, the P2A site comprises i) the amino acid sequence of SEQ ID NO 17; ii) an amino acid sequence having at least one, two or three modifications but NO more than 5 modifications to the amino acid sequence of SEQ ID NO 17; or iii) an amino acid sequence having at least 95%,96%,97%,98%, or 99% identity to the amino acid sequence of SEQ ID NO 17. In one embodiment, the T2A site comprises i) the amino acid sequence of SEQ ID NO 18; ii) an amino acid sequence having at least one, two or three modifications but NO more than 5 modifications to the amino acid sequence of SEQ ID NO 18; or iii) an amino acid sequence having at least 95%,96%,97%,98%, or 99% identity to the amino acid sequence of SEQ ID NO. 18. In one embodiment, a GSG linker may be inserted at the N-terminus of the 2A peptide to further improve its cleavage efficiency.

Thus, in a further aspect, the invention also provides a fusion polypeptide according to formula (I), preferably said L1 and L2 may comprise any self-splicing 2A site according to the foregoing. The various components of the structure of formula (I) may be functionally linked, either directly or indirectly, by a linker (e.g., a single amino acid residue or a short peptide).

In some embodiments, to achieve expression of the nucleic acid combination of the invention upon transfer into a cell, the nucleic acid construct of the invention comprises a promoter operably linked to a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an IL-15Ra polypeptide according to the invention. In some embodiments, the nucleic acid construct is a vector. Vectors that may be suitable for use in the present invention include any vector suitable for replication and integration in eukaryotes; and contains transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequences.

Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviral vectors provide a convenient platform for gene delivery systems. The nucleic acid combinations of the invention can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, a lentiviral vector is used. For example, the nucleic acid sequences of the nucleic acid combination of the invention are cloned into a lentiviral vector in order to

(i) Producing a full-length CAR polypeptide in a single coding frame, an IL-15 polypeptide in a single coding frame, and an optimized IL-15Ra polypeptide in a single coding frame, or

(ii) Generating a full-length CAR polypeptide in a single coding frame and an IL-15 polypeptide and an IL-15Ra polypeptide linked by a self-splicing peptide in a single coding frame; or

(ii) A fusion polypeptide comprising a full-length CAR polypeptide, an IL-15 polypeptide, and an IL-15Ra polypeptide linked by a self-splicing peptide is produced in a single coding frame.

Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in progeny cells. Lentiviral vectors have the additional advantage over vectors derived from cancer-retroviruses (e.g.murine leukemia virus) in that they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. Retroviral vectors may also be, for example, gamma retroviral vectors. The gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a Primer Binding Site (PBS), one or more (e.g., two) Long Terminal Repeats (LTRs), and a transgene of interest, e.g., a gene encoding a CAR. The gamma retroviral vector may lack viral structural genes such as gag, pol and env.

An example of a promoter capable of expressing a transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to efficiently drive transgene expression from cloning into lentiviral vectors. See, e.g., milone et al, mol. Ther.17 (8): 1453-1464 (2009).

Another example of a promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutively strong promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. However, other constitutive promoter sequences may also be used, including, but not limited to, monkey virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, elongation factor-1 α promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.

Cells comprising the nucleic acid combinations of the invention

The invention also provides a cell into which a nucleic acid combination of the invention or a nucleic acid construct of the invention has been introduced. The nucleic acid combination of the invention or the nucleic acid construct of the invention can be introduced into a cell by any nucleic acid transfer method known in the art. In one embodiment, the cell is a mammalian cell, such as an immune effector cell. In one embodiment, the cell is an armored CAR-T cell comprising an IL-15 and optimized IL-15Ra polypeptide according to the invention and a CAR polypeptide.

In some embodiments, therefore, the invention also provides methods of introducing and expressing the nucleic acid combinations of the invention in mammalian immune effector cells (e.g., mammalian T cells or mammalian NK cells) and thereby generating immune effector cells, particularly armored CAR-T cells according to the invention. In some embodiments, the invention also provides immune effector cells obtainable by said method, in particular armored CAR-T cells according to the invention.

In some embodiments, to produce an immune effector cell according to the invention, a cell source, e.g., an immune effector cell, e.g., a T cell or NK cell, is obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) that can elicit an immune response. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

Any technique known to those skilled in the art (e.g., ficoll) can be used ^TM Isolation) of T cells from a blood component collected from a subject. In a preferred aspect, the cells from the circulating blood of the individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma fractions and to place the cells in an appropriate buffer or culture medium for subsequent processing steps. In one aspect of the invention, cells are washed with Phosphate Buffered Saline (PBS).

Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA +, and CD45RO + T cells, can be further isolated by positive or negative selection techniquesAnd (4) cells. For example, in one embodiment, by beads conjugated with anti-CD 3/anti-CD 28 (e.g., as described above)

M-450 CD3/CD 28T) for a time sufficient to positively select for the desired T cells, and isolating the T cells. In some embodiments, the period of time is between about 30 minutes and 36 hours or more. Longer incubation times can be used to isolate T cells in any situation where small numbers of T cells are present, such as for isolating Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. In addition, the efficiency of capturing CD8+ T cells can be increased using longer incubation times. Thus, by simply shortening or extending this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the bead to T cell ratio, T cell subsets can be preferentially selected at the beginning of the culture or at other time points during the culture process.

Enrichment of a population of T cells can be accomplished by a negative selection process with a combination of antibodies directed against surface markers unique to negatively selected cells. One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells.

In some embodiments, the immune effector cell may be an allogeneic immune effector cell, e.g., a T cell or an NK cell. For example, the cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking functional T Cell Receptor (TCR) and/or Human Leukocyte Antigen (HLA) (e.g., HLA class I and/or HLA class II) expression.

In some embodiments, the immune effector cell is an armored CAR-T cell according to the invention. In one embodiment, the armored CAR-T cell expresses a fusion polypeptide according to formula (I), and optionally the fusion polypeptide, upon expression, results from splicing in the CAR polypeptide of the invention, IL-15 polypeptide and optimized IL-15Ra polypeptide, which are isolated from each other. In one embodiment, the armored CAR-T cells according to the invention have at least one of the following properties:

-having enhanced proliferative capacity and cell viability, and an increased proportion of T cell subpopulations of a Tscm phenotype, compared to a control CAR-T cell expressing only said CAR polypeptide;

-reducing the amount of IL-15 released in the extracellular environment, and thereby having reduced IL-15-induced toxicity, compared to a control CAR-T cell expressing only said CAR polypeptide and said IL-15; and

in vivo anti-tumor efficacy and reduced adverse events associated with IL-15 in CAR-T therapy.

Use of the immune effector cells of the invention and methods of treatment using the immune effector cells of the invention

T cell therapy was first applied to the treatment of hematologic B cell malignancies and showed effective and encouraging results. However, the anti-tumor activity of CAR-T cell therapy is limited by the limited persistence of CAR-T cells. To address the use of CAR T cells in a variety of cancers, there is an urgent need for technical means to effectively modulate the persistence of CAR-T cells in vitro and in vivo. For this purpose, means are proposed for the recombinant expression of IL-15 in CAR-T cells. However, active IL-15 in the released serum has the potential to induce toxicity. In order to ensure the persistence of CAR-T cells and minimize IL-15 related toxicity or side effects, through intensive studies, the inventors propose that by simultaneously expressing IL-15 in CAR-T cells and the optimized IL-15Ra of the invention, we obtained armored CAR-T cells of the invention comprising more subpopulations of TCSM cells with reduced toxicity, facilitating the anti-tumor therapeutic application of said CAR-T cells in a subject.

In some embodiments, immune effector cells, e.g., T cells (e.g., patient-specific autologous T cells), are engineered to incorporate the nucleic acid combinations or vectors of the invention, thereby heterologously co-expressing the CAR polypeptide of the invention, IL-15, and optimized IL-15Ra polypeptides in the cells. Following expansion of the engineered immune effector cells (e.g., T cells or NK cells), they can be used for Adoptive Cell Therapy (ACT).

In some embodiments, the immune effector cells of the invention may be autologous or allogeneic T cells or NK cells when the patient is treated with the immune effector cells. In some embodiments, the immune effector cells of the invention can improve long-term survival of cells and/or the proportion of TSCM subpopulations after adoptive transfer compared to the use of control CAR-T or CAR-NK cells without heterologous IL-15 and IL-15Ra expression.

In one embodiment, the immune effector cells of the invention are used to treat cancer in a subject and are capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth.

The present invention provides a method of treating cancer in a subject comprising administering to an individual in need thereof a therapeutically effective amount of an immune effector cell expressing a nucleic acid combination of the invention. The invention also provides the use of an immune effector cell of the invention as hereinbefore described in the manufacture of a medicament for the treatment of cancer. The cancer includes hematologic cancers (e.g., leukemia) or solid tumors (e.g., glioma), including primary and metastatic cancers.

The various embodiments/aspects of the invention and the features of the various embodiments/aspects thereof described should be understood as being arbitrarily combinable with each other, each of which is included within the scope of the invention as if it were specifically and individually set forth herein, unless the context clearly indicates otherwise.

The following examples are described to aid in the understanding of the present invention. The examples are not intended to, and should not be construed as, limiting the scope of the invention in any way.

Examples

Materials and methods

Cell lines

The human NALM-6 cell line and the retroviral packaging cell line PG13 were purchased from the American Type Culture Collection (ATCC). NAML-6-eGFP cells expressing enhanced GFP were generated by retroviral infection. NAML-6-eGFP cells were maintained in RPMI-1640 (Lonza) containing 10% fetal bovine serum (Biosera) and 10,000IU/mL penicillin/10,000. Mu.g/mL streptomycin (EallBio Life Sciences). All cells were incubated at 37 ℃ and 5 deg.C％CO ₂ And cultured in a humidified incubator in 95% air.

Generation of retroviral vectors encoding CD 19-specific CAR

The third generation CD19-CAR gene is synthesized by biological companies, wherein the nucleotide sequence of the IL-15 gene is shown as SEQ ID NO:2 and the nucleotide sequence of the optimized IL-15Ra gene is shown as SEQ ID NO:3, respectively.

The synthesized nucleotide sequences were subcloned into the vector SFG vector (addge) by homologous recombination (Clonepress II One Step Cloning kit) as shown in FIG. 1 to construct the constructs CD19-CAR, CD19-CAR-IL15, and CD19-CAR-IL15-IL15Ra.

The third generation of proteins expressed from the CD19-CAR gene has the amino acid sequence (SEQ ID NO: 4) shown below, comprising, from N-terminus to C-terminus, a signal peptide (bold underline), CD19 scFv, a short linker peptide (bold underline), a CD28 spacer/transmembrane region (italics), a CD28 costimulatory domain (underlined), a 4-1BB costimulatory domain (bold italics with boxes), and a CD3 zeta signaling domain:

the nucleotide sequence of the IL-15 gene (SEQ ID NO: 2) is as follows:

ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGAGGAGCCAGAGCGAACTGGGTGAACGTGATCTCGGACCTGAAGAAGATCGAGGACCTCATCCAGTCGATGCACATCGACGCGACGCTGTACACGGAGTCGGACGTCCACCCGTCGTGCAAGGTCACGGCGATGAAGTGCTTCCTCCTGGAGCTCCAAGTCATCTCGCTCGAGTCGGGGGACGCGTCGATCCACGACACGGTGGAGAACCTGATCATCCTGGCGAACAACTCGCTGTCGTCGAACGGGAACGTCACGGAGTCGGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCGTTCGTGCACATCGTCCAGATGTTCATGAACACGTCG

the amino acid sequence (SEQ ID NO: 5) of the protein expressed by the IL-15 gene is shown as follows:

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFMNTS

the nucleotide sequence (SEQ ID NO: 3) of the optimized IL-15Ra gene is as follows:

ATGGCCCCGAGGCGGGCGCGAGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTCCTGCTGCTCCGGCCGCCGGCGACGCGGGGCATCACGTGCCCGCCCCCCATGTCCGTGGAGCACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGCGGTACATCTGCAACTCGGGTTTCAAGCGGAAGGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACGACCCCCTCGCTCAAGTGCATCCGCGACCCGGCCCTGGTTCACCAGCGGCCCGCGCCACCCTCCACCGTAACGACGGCGGGGGTGACCCCGCAGCCGGAGAGCCTCTCCCCGTCGGGAAAGGAGCCCGCCGCGTCGTCGCCCAGCTCGAACAACACGGCGGCCACAACTGCAGCGATCGTCCCGGGCTCCCAGCTGATGCCGTCGAAGTCGCCGTCCACGGGAACCACGGAGATCAGCAGTCATGAGTCCTCCCACGGCACCCCCTCGCAAACGACGGCCAAGAACTGGGAACTCACGGCGTCCGCCTCCCACCAGCCGCCGGGGGTGTACCCACAGGGGCATAGGGAGACAACCGTGGCGATCTCCACGTCCACGGTCCTGCTGTGTGGGCTGAGCGCGGTGTCGCTCCTGGCGTGCTACCTCAAGTCGAGGCAGACTCCCCCGCTGGCCAGCGTTGAGATGGAGGCCATGGAGGCTCTGCCGGTGACGTGGGGGACCAGCAGCAGGGATGAGGACTTGGAGAACTGCTCGCACCACCTATAA

the protein (SEQ ID NO: 6) for optimizing the expression of the IL-15Ra gene is as follows:

MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHRETTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

all CAR constructs constructed were verified by sequencing. After transient transfection, PG13 cells were used to produce retroviral particles.

Generating CAR-T cells

Primary T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors by centrifugation on a Lymphoprep (MP Biomedicals) gradient. To generate antigen-specific transgenic T cells, T cells in PBMCs were stimulated with anti-CD 3 and anti-CD 28 beads and then infected with retroviruses. 7 days after infection, on CAR-T cells were tested for CAR expression and then GemCell at 5% ^TM X-VIVO of human serum AB and IL-2 (138U/ml) ^TM 15 in serum-free medium. CD8 isolation Using CD8 Positive isolation kit (Thermo Fisher Scientific) ⁺ T cells. The study was approved by the review committee of the institution of the Beijing century Tan Hospital and informed consent was obtained from all participants.

Flow cytometry

Flow cytometry was performed on a facscan Plus instrument (BD Biosciences). FlowJo v.10 (FlowJo, LLC) was used for data analysis. The cells were stained with APC-cy 7-labeled mouse anti-human CD3 antibody (BD Biosciences), FITC-labeled mouse anti-human CD8 antibody (BD Biosciences), alexa Fluor 700-labeled mouse anti-human CD8 antibody (BD Biosciences), BV 421-labeled mouse anti-human CD4 antibody (BD Biosciences), V450-labeled mouse anti-human CD107a (BD Biosciences), BV 605-labeled mouse anti-human CD45RO (BD Biosciences), PE-cy 7-labeled mouse anti-human CCR7 (BD Biosciences), alexa Fluor 700-labeled mouse anti-human CD27 (BD Biosciences), PE-cy 5-labeled mouse anti-human CD95 (BD Biosciences), and Alexa Fluor 647-labeled goat anti-mouse IgG (Fab-specific) F (Jackson') 2 antibody (Jacson research) to detect CAR-T cells.

Cytotoxicity assays

CAR-T cells were co-cultured in 24-well plates at different effective target ratios (E: T) with target tumor cells NAML-6-EGFP expressing fluorescent protein GFP. After 24 hours, cells were harvested and tumor cells were detected by surface markers using a flow cytometer (BD facsconcto II Plus).

Proliferation assay

CD19-CAR-T cells, CD19-CAR-IL-15T cells, and CD19-CAR-IL15 Ra T cells were cultured with IL-2 and stimulated using NALM-6 (E: T =10. On days 0, 7, 14 and 21, the number of viable CELLs was counted by trypan blue exclusion using a Vi-CELL viability analyzer.

Cytokine production assay

CAR-T cells were co-cultured with target cells (NAML-6-eGFP) at an E: T ratio of 2. Supernatants were collected and tested for IFN-. Gamma., IL-15 and IL-2 by ELISA kits (DY 285B, D1500, DY202, R & D systems) according to the manufacturer's instructions.

PCR

IL-15 and IL-15Ra expression in antigen-stimulated CAR-T cells was confirmed by PCR. Total RNA was extracted from CAR-T cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The amount and purity of RNA was measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Only samples with appropriate absorbance measurements (A260/A280 of 2.0 and A260/A230 of 1.9-2.2) were considered for inclusion in the study. cDNA was synthesized using the High Capacity cDNA reverse transcription kit (Thermo Fisher Scientific).

IL-15 was amplified using primers 5 'ATGGATGCAATGAAGAGAGGG-3' (sense) and 5 'CGACGTGTTCATGAACATCTGGA-3' (antisense); IL-15Ra was amplified using primers 5 'ATGGCCGAGGCGGGCGCGAGG-3' (sense) and 5 'TAGGTGGTGCGAGCAGTT-3' (antisense); GAPDH was amplified using primers 5 'TGACCACAGTCCAATGCCATC-3' (sense) and 5 'GTGAGCTTCCCGTTCAGCTC-3' (antisense) as controls.

Xenograft mouse tumor model

6 to 8 week old NOD-SCID mice were purchased from Charles River Laboratories. Will be 1 × 10 ⁶ NAML-6-eGFP cells were injected intravenously into NOD-SCID mice to construct xenograft mouse models. Injection of tumor cells 1 day later, tail vein injection of 1X 10 ⁷ CAR-T cells, once daily for 3 days. IVIS (IVIS, xenogen, alameda, CA, USA) was used to monitor tumor development. All experiments, including mice, were approved by the institutional review board of the Beijing century Tan Hospital.

Statistical analysis

Charts and statistical analyses were performed using Graphpad Prism 8.0.2. Data were analyzed using student t-test, p-value <0.05 was considered significant; * p <0.05; * P <0.01; * P <0.001; * P <0.0001; ns, not significant. The overall survival of mice bearing NAML-6-eGFP xenografts was measured using the Kaplan-Meier method and comparisons between groups were made using Cox proportional hazards regression analysis. All experiments were repeated at least 3 times.

Example 1 construction and characterization of armored CD 19-specific CAR-T cells

As described in materials and methods, IL-15 gene and IL-15Ra gene linked to CD19-CAR gene were constructed (fig. 1), and retroviral vectors co-expressing these genes were transduced into T cells and the transduction efficiency was examined by flow cytometry. FIG. 2A shows that similar CAR transduction efficiencies were obtained for the three CAR constructs, with 35.4%, 26.1% and 17.4% CD8, respectively ⁺ T cells express CD19 specific CARs. FIG. 2B shows that there is a similar ratio of CD4+/CD8+ T cells between the three groups.

Next, successful expression of IL-15 and IL-15Ra in armored CAR-T cells was confirmed using PCR. Total RNA of CAR-T cells was extracted and PCR amplified. The results (FIG. 2C) show that CD19-CAR-IL-15T cells overexpress IL-15, CD19-CAR-IL-15-IL-15Ra T cells overexpress IL-15 as well as IL-15Ra.

Example 2 detection of in vitro proliferation and differentiation phenotype of IL-15 armored CAR-T cells

The proliferation of IL-15 and/or IL-15 Ra-overexpressing CAR-T cells in vitro was detected by direct cell counting. Briefly, CD19-CAR-T cells, CD19-CAR-IL-15T cells, and CD19-CAR-IL15 Ra T cells were cultured with IL-2. On days 0,3, 7 and 14, the number of viable CELLs was counted by trypan blue exclusion using a Vi-CELL viability analyzer. The results show that both armored CAR-T cells overexpressing IL-15 and/or IL-15Ra showed higher proliferative capacity than CAR-T cells (fig. 3A).

In addition, since IL-2 is a growth factor for T cells, the concentration of IL-2 in the supernatant was measured. Briefly, CAR-T cells were co-cultured with target cells (NAML-6-eGFP) at an E: T ratio of 2. Thereafter, the supernatant was collected and subjected to IL-2 detection by ELISA kit. The results indicate that CD19-CAR-IL-15 and CD19-CAR-IL-15-IL15Ra T cells released more IL-2 than CD19-CAR T cells (fig. 3B).

7 days after stimulation of NAML-6-eGFP cells, the differentiation phenotype of CAR-T cells was examined by flow cytometry. On Tsccm cells (CD 8) representing the Long-term persistence of CAR-T cells ⁺ CD45RO ^- CCR7 ⁺ CD27 ⁺ CD95 ⁺ ) A study was conducted. The results showed significantly more Tscm cells for CD19-CAR-IL-15 and CD19-CAR-IL-15 Ra T cells relative to CD8+ CD19-CAR T cells (fig. 4A highest measured Tscm cell level, 1.67%,9.23% and 4.84% for the three groups, respectively; fig. 4B shows a histogram of the measurements).

In addition, it has been reported that T cells with a lower degree of differentiation produce less IFN γ upon antigen stimulation. Thus, CD19-CAR-IL-15 and CD19-CAR-IL15 Ra T cells were stimulated with NAML-6-eGFP cells at an E: T ratio of 2 for 24 hours and the concentration of IFN γ was measured by ELISA. As shown in figure 5A, T cells expressing IL-15 and IL-15Ra produced less IFN γ, which means that CD19-CAR-IL-15 and CD19-CAR-IL-15 Ra T cells differentiated to a lesser degree.

Subsequently, the percentage of apoptotic cells and the cell survival of armored CAR-T cells after incubation with target cells for a certain time were analyzed, taking into account the effect of IL-15 on T cell proliferation. Briefly, two armored CAR-T and control CAR-T cells, co-cultured (10) with target cells NAML-6-eGFP, respectively, for 7 days, after which the cells were harvested, stained with the dyes Annexin V and 7ADD for 15 minutes, and CAR-T cell survival and apoptosis rate were measured by flow cytometry. The results show that IL-15 and IL-15Ra inhibited CAR-T cell apoptosis and enhanced cell viability (FIGS. 5B and 5C).

Example 3 IL-15Ra in vitro reduces IL-15-induced toxicity

To investigate the effect of IL-15Ra on IL-15 under cell culture conditions, CAR-T cells were co-cultured with NALM-6-eGFP for 24 hours (E: T = 2. Afterwards, armored CAR-T cell supernatants were collected and IL-15 concentrations were measured by ELISA. The results indicate (fig. 6A) that CD19-CAR-IL-15T cells have the highest IL-15 release, whereas CD19-CAR-IL-15-IL15Ra T cells are comparable to both CD19-CAR-T cells, with significantly lower levels of IL-15 release. Figure 2B has demonstrated that CAR-IL-15 Ra T cells can successfully express both IL-15 and IL-15Ra. Thus, while not being bound by any theory, it is believed that CAR-IL-15 Ra T cells can reduce the amount of IL-15 released into the culture medium and thereby reduce the cytotoxicity induced by the released IL-15 by binding the expressed IL-15 to the cell membrane surface expressed modified IL-15Ra.

Furthermore, given that CD132 is a co-receptor subunit chain for IL-2 and IL-15 and its high expression is associated with GVHD (graft versus host disease), cell surface CD132 expression of two armored CAR-T cells was detected and compared. Briefly, CAR-T cells were harvested after 7 days of coculture with NALM-6-eGFP (E: T =10. Cell surface CD132 expression was detected by flow cytometry. The results (fig. 6B) showed that CAR-IL-15 Ra T cells had the lowest CD132 expression (CAR-IL-15 Ra T cells 60.8% vs. CAR-IL-15T cells 65.5% vs. CAR-T cells 93.2%).

In addition, CAR-T cells were co-cultured with target cells NALM-6-eGFP cells (E: T = 2. The cells were harvested and the GFP signal was detected by flow to determine the number of viable NALM-6 cells. The results are shown in fig. 7A and 7B.

Example 4 IL-15 armored CAR-T cells expressing IL-15Ra show enhanced antitumor activity and reduced toxicity in vivo

To examine the in vivo anti-tumor activity of armored T cells, NAML-6-eGFP cells were injected intravenously into NOD-SCID mice to generate xenograft mouse models. One day later, CAR-T cells were injected intravenously and untransduced T cells (NTs) were used as negative controls. Mice were monitored for more than three months (fig. 8A).

Fig. 8B and fig. 9 show that mice treated with CD19-CAR-IL-15T cells and CD19-CAR-IL-15 Ra T cells had no tumor recurrence compared to the control group, indicating that IL-15 induced enhanced anti-tumor activity. However, despite no tumor recurrence, all mice in the CD19-CAR-IL-15T cell treated group died within 70 days with the lowest survival rate compared to the CD19-CAR and CD19-CAR-IL-15 Ra T cell treated group (fig. 8B). This indicates that IL-15 is toxic to the animals.

The overall survival of mice bearing NAML-6-eGFP xenografts was measured using the Kaplan-Meier method and comparisons between groups were performed using Cox proportional hazards regression analysis. As shown in figure 10A, approximately 40% of the mice of CD19-CAR-IL-15 Ra survived more than 90 days compared to the CD19-CAR group with only 20% of the mice surviving more than 90 days.

On day 50, blood was collected from each group of mice, and the concentration of human IL-15 was measured with serum. Figure 10B shows that CD19-CAR-IL-15T treated mice had the most human IL-15 in the blood, while CD19-CAR-IL-15 Ra T cell treated mice had significantly reduced blood IL-15 levels compared to control CAR-T cell treated mice. This indicates that co-expression of IL-15Ra in CD19-CAR-IL-15 Ra T cells blocks IL-15 blood release and thus IL-15 toxicity in serum, prolonging survival of the treated tumor-bearing mice.

Sequence listing

Claims

1. A nucleic acid combination comprising a first nucleic acid molecule, a second nucleic acid molecule, and a third nucleic acid molecule, wherein:

-the first nucleic acid molecule comprises a polynucleotide encoding a chimeric antigen polypeptide (CAR),

-the second nucleic acid molecule comprises a polynucleotide encoding IL-15,

-the third nucleic acid molecule comprises a polynucleotide encoding an optimized IL15Ra,

wherein the optimized IL15Ra comprises the double mutations S202R and D203E at amino acid positions 202 and 203, wherein the amino acid positions are numbered according to SEQ ID NO 6.

2. The nucleic acid combination according to claim 1, wherein the optimized IL-15Ra comprises the double mutation S202R and D203E, preferably comprises the motif YPQGHSDT at positions 197-204 and comprises the amino acid sequence of SEQ ID No. 6; or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or 99.5% identity thereto; preferably, the polynucleotide encoding optimized IL15Ra comprises the sequence set forth in SEQ ID No. 3, or a polynucleotide having at least 90%, 92%, 95%,96%,97%,98%, 99% or 99.5% identity thereto.

3. The nucleic acid combination of claims 1-2, wherein the first nucleic acid molecule comprises a polynucleotide encoding a CAR polypeptide, wherein the CAR polypeptide comprises from N-terminus to C-terminus: optionally a signal peptide, an extracellular antigen-binding domain that specifically binds a tumor antigen, optionally a hinge region or spacer, a transmembrane domain, and a cytoplasmic signaling domain, and wherein the cytoplasmic signaling domain comprises a combination of a costimulatory domain of CD28 and a costimulatory domain of 4-1BB and a CD3 ζ primary signaling domain.

4. The nucleic acid combination of claims 1-3, wherein the CAR polypeptide comprises: from the end of N to the end of C,

an antibody or antigen-binding fragment against a tumor antigen (e.g., CD 19), such as an scFv,

a CD28 hinge region, which is a CD-side hinge region,

a CD28 transmembrane domain of a polypeptide having a high affinity for,

(ii) a CD28 co-stimulatory domain,

a 4-1BB co-stimulatory domain, and

a CD3 zeta primary signaling domain,

preferably, the CAR polypeptide comprises the amino acid sequence of SEQ ID NO. 4, or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto.

5. The nucleic acid combination according to claims 1-4, wherein the second nucleic acid molecule comprises a polynucleotide encoding IL-15, and preferably the second nucleic acid molecule comprises a polynucleotide encoding SEQ ID No. 5 or an amino acid sequence having at least 85%,90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto, and more preferably the second nucleic acid molecule comprises the nucleotide sequence of SEQ ID No. 2, or a nucleotide sequence having at least 85%,90%, 92%, 95%,96%,97%,98%, 99% or more identity thereto.

6. The nucleic acid combination according to claims 1-5, wherein two or all three of the first, second and third nucleic acid molecules are present in a functional linkage on a single nucleic acid construct, e.g. a viral vector, such as a lentiviral vector.

7. The nucleic acid combination according to claims 1-6, wherein the nucleic acid combination is a single nucleic acid construct comprising a first, a second and a third nucleic acid molecule, wherein the nucleic acid construct encodes a fusion protein having from N-terminus to C-terminus the structure of formula (I):

CAR-(L1)-E1-(L2)-E2 (I)

wherein the content of the first and second substances,

CAR denotes a chimeric antigen receptor polypeptide which is,

l1 and L2 each independently represent a linker peptide comprising a self-splicing site,

e1 and E2 are different from each other and are independently selected from IL-15 and optimized IL-15Ra,

8. The nucleic acid combination according to claim 7, wherein E1 represents IL-15 encoded by the second nucleic acid molecule and E2 represents optimized IL-15Ra encoded by the third nucleic acid molecule.

9. The nucleic acid combination according to claims 7-8, wherein L1 and L2 are different from each other and independently of each other comprise an self-splice site selected from P2A, T2A, E2A or F2A; preferably, L1 comprises the P2A site (preferably, comprises the amino acid sequence of SEQ ID NO: 17); and L2 comprises a T2A site (preferably, comprising the amino acid sequence of SEQ ID NO: 18).

10. The nucleic acid combination according to claims 1-9, wherein said nucleic acid combination, when introduced into an immune effector cell, such as a T cell, results in a reduced amount of IL-15 released in the extracellular environment, and thereby a reduced toxicity induced by IL-15, compared to a control immune effector cell into which only the first nucleic acid molecule encoding said CAR and the second nucleic acid molecule encoding said IL-15 are introduced.

11. Polypeptide encoded by the nucleic acid combination of claims 1-10, preferably said polypeptide is a single fusion polypeptide comprising functionally linked:

(i) A Chimeric Antigen Receptor (CAR) polypeptide;

(ii) An IL-15 polypeptide; and

(iii) Optimizing the IL-15Ra polypeptide.

More preferably, the fusion polypeptide has a structure according to formula (I) as described in claims 7-9.

12. A vector, such as a lentiviral vector, comprising the combination of nucleic acids of claims 1-11.

13. The vector of claim 12, wherein said first, second and third nucleic acid molecules are present on said vector in polycistronic form.

14. An armored CAR-T cell, wherein the armored CAR-T cell comprises a combination of nucleic acid molecules according to claims 1-10, or incorporates the vector of claim 13.

15. Armored CAR-T cell according to claim 14, comprising the fusion polypeptides of formula (I) described in claims 7 to 9.

16. An armored CAR-T cell according to claims 14-15 comprising the CAR polypeptide, IL-15 polypeptide and optimized IL-15Ra polypeptide isolated from each other.

17. Armored CAR-T cell according to claims 14-16, which has at least one of the following properties:

in vivo antitumor efficacy and reduced adverse events associated with IL-15 in CAR-T treatment.

18. A pharmaceutical composition comprising an armored CAR-T cell according to claims 14-17.

19. Use of an armored CAR-T cell according to claims 14-17, preferably the tumor is a hematological or solid tumor, more preferably the CAR-T cell is administered systemically (e.g. intravenously) or locally (e.g. intratumorally) for the preparation of a medicament for the prevention or treatment of cancer or for providing anti-tumor immunity.

20. An optimized IL15Ra polypeptide comprising double mutations S202R and D203E at amino acid positions 201 and 202, wherein the amino acid positions are determined according to SEQ ID No. 6, and preferably further comprising the amino acid sequence of SEQ ID No. 6; or an amino acid sequence having at least 90%, 92%, 95%,96%,97%,98%, 99% or 99.5% identity thereto.

21. A polynucleotide encoding an optimized IL15Ra according to claim 20, preferably comprising the sequence depicted in SEQ ID No. 3 or a polynucleotide having at least 90%, 92%, 95%,96%,97%,98%, 99% or 99.5% identity thereto.

22. A method for increasing persistence and reducing toxicity of a CAR-T cell comprising introducing and expressing in said CAR-T cell a combination of nucleic acids according to claims 1-10.

23. The method of claim 22, wherein the nucleic acid molecule encoding the optimized IL-15Ra polypeptide and the nucleic acid molecule encoding the CAR polypeptide and the nucleic acid molecule encoding the IL-15 polypeptide are functionally linked to each other in a single nucleic acid construct and expressed to produce a fusion polypeptide having the structure of formula (I) as described in claims 7-9.