CD24 car-t cells for treatment of hematopoietic and solid tumors

Abstract

Chimeric antigen receptors that bind to CD24, optionally further including CXCR3 or IL-15, and optionally further including EGFRt, polynucleotides encoding the receptors, cells comprising the polynucleotides, and methods of treating cancers using said chimeric antigen receptors or cells are disclosed.

Description

CD24 CAR-T CELLS FOR TREATMENT OF HEMATOPOIETIC AND SOLID TUMORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 659,256 filed on June 12, 2024. The content of which is incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (18250600050. xml; Size: 71,844 bytes; and Date of Creation: June 1 , 2025) is incorporated by reference in its entirety.BACKGROUND

[0003] Recent breakthroughs in comprehending the immune tumor microenvironment's significance have spurred the creation of innovative immunotherapeutic approaches, bolstering the battle against cancer. Notably, the advent of T cells engineered to express chimeric antigen receptors (CAR-T) aimed at distinct tumor targets has emerged as a highly promising therapeutic avenue.

[0004] Mantle cell lymphoma (MCL) is an incurable and aggressive subtype of B-cell lymphoma. A vast majority of MCL patients will die of their disease despite high initial rates of response to recently introduced anti-MCL agents. The majority of MCL patients experience disease progression after frontline therapy with a poor overall survival after relapse. The most significant advancement in MCL treatment is evident by the recent FDA approval of CD 19 CAR T therapy. Chimeric antigen receptor (CAR) T-cells against CD19 (CD19 CAR-T) represent a potentially transformative therapy for MCL, emerging as a curative option for relapsed or refractory (R / R) MCL. However, despite high initial complete remission rates, relapses occur within the first year of therapy in approximately 50% of patients. Relapses can be classified into two patterns: CD 19- positive relapse related to CAR T-cell exhaustion and exclusion due to immunosuppressive tumor microenvironment (TME), or CD 19- negative relapse related to down-regulation or loss of CD 19 surface expression. Patients who relapse after CAR19 T-cell therapy have dismal outcome; hence, there is an urgent need to develop the next-generation of engineered CAR or immunotherapy that utilize alternative targets to circumvent resistance mechanisms and provide a therapeutic option for relapsed patients as well as improve the efficacy and persistence of CAR T cells.

[0005] Additionally, while CAR-T therapies have achieved notable success in certain blood cancers by targeting CD 19 and BCMA, their application in solid tumors has been hindered by the limited infiltration of these cells into the tumor, confining their effectiveness primarily to the tumor periphery. Therefore, novel CAR-T therapies that effectively target hematopoietic cancers, as well as solid tumors, are needed.SUMMARY

[0006] In an aspect, provided herein is an engineered polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular CD24 scFv, wherein the CD24 scFv comprises a sequence having at least 90% identity to SEQ ID NO: 4. The CAR may further comprise CXCR3 or IL-15. The CXCR3 may comprise a sequence having at least 90% identity to SEQ ID NO: 5. The IL- 15 may comprise a sequence having at least 90% identity to SEQ ID NO: 6. The engineered CAR may further comprise EGFRt. The EGFRt may comprise a sequence having at least 90% identity to SEQ ID NO: 11. The CAR may further comprise at least one of a 4-1BB domain, and a CD3(^ signaling domain. The CAR may comprise a sequence having at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0007] In another aspect, provided herein is a construct comprising the engineered polynucleotide described herein operably linked to a promoter.

[0008] In another aspect, provided herein is a CAR encoded by the engineered polynucleotide described herein.

[0009] In another aspect, provided herein is a cell comprising the engineered polynucleotide, the construct, or the CAR described herein. The cell may be an immune cell. The cell may be a T cell, a B cell, a natural killer (NK) cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell. In embodiments, the cell is a T cell.

[0010] In another aspect, provided herein is a pharmaceutical composition comprising the engineered polynucleotide, the construct, the CAR, or the cell described herein; and a pharmaceutically acceptable carrier.

[0011] In another aspect, provided herein is a method for treating a cancer in a subject, the method comprising administering to the subject the pharmaceutical composition described herein. The cancer may be a B-cell lymphoma. The B-cell lymphoma may be mantle cell lymphoma. The cancer may comprise a solid tumor. The cancer may be a colon cancer, liver cancer, prostatecancer, breast cancer, pancreatic cancer, ovarian cancer, or glioblastoma.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or patent application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0013] FIGS. 1 A-1C. (A) Surface CD24 expression in different MCL cell lines measured by Flow- Cytometry. (B) Violin plot showing CD24 RNA expression measured by qRT-PCR in sub-types of B-cell malignancies. (C) Overall survival of patients with MCL (B, n = 71) with high or low CD24 expression. Patients at risk in the CD24 high-expression group (red) compared with the CD24 low-expression group (blue). High expression of CD24 indicates a poor prognosis in MCL.

[0014] FIG. 2. Schematic diagrams of three CD24 CAR constructs.

[0015] FIG. 3A-3C. CD24 CAR-T cytotoxicity. (A) Luciferase-based cytotoxicity assay of DHL- 16 (upper) and Z 138(1 ower) cocultured with different CD24 CAR-T cells, showing E:T ratios for 48h and 72h. (B) Cytotoxicity assay of MCL cocultured with CD24 CAR-T cells, E:T ratios for 48h. For each cell line, the left panel shows results from Mock T cells, while the right panel shows results from CD24 CAR-T cells. (C) Comparison of the cytotoxicity of CD24 and CD19 CAR-T in Z138-Nluc, different E:T ratios for 48h.

[0016] FIGS. 4A-4E. (A-C) illustrate CD24 CAR-T shows superior cytotoxicity to CD 19 CAR-T cells against MCL over time. E:T ratio (effector-to-target) (D) CD24 CAR-T cells enhanced macrophage-mediated phagocytosis. Green, MCL cells and Red, macrophage. (E) illustrate a transwell migration assay.

[0017] FIG. 5 illustrates the CD24-EGFR CAR construct.

[0018] FIG. 6. illustrates the CD24-EGFR-IL-15 CAR construct.

[0019] FIG. 7. Illustrates the CD24-EGFR-CXCR3 CAR construct.

[0020] FIGS. 8A-8C. (A) Dose dependent cytotoxicity of CD24-CXCR3 CAR-T cells against Al 72 (glioblastoma). (B) CD24 CAR-T cells used to treat solid tumors. CD24-CXCR3 CAR-T cells attaching to glioblastoma cell lines, proliferating, and eliminating the cancer cells after coculture. (C) CD24-CXCR3 CAR-T construct shows cytotoxicity against different solid tumor cell lines.DETAILED DESCRIPTION

[0021] The inventors have developed CD24 CAR-T cells as novel therapeutic approaches for treating MCL and other cancers.

[0022] In a first aspect, provided herein is an engineered polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular CD24 scFv, wherein the CD24 scFv comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4.

[0023] CD24 (cluster of differentiation 24) is a protein expressed at the surface of most B lymphocytes and differentiating neuroblasts.

[0024] In a second aspect, provided herein is a CAR encoded by the engineered polynucleotides described herein. The CAR may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0025] As used herein, “a polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term “nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, including DNA, RNA, ORFs, analogs and fragments thereof. The polynucleotides disclosed herein may be optimized, for example codon optimized or host cell optimized. For example, the polynucleotide may encode a fusion protein comprising a chimeric antigen receptor. As used herein a “fusion protein” refers to proteins created through the joining of two or more genes that originally coded for separate proteins.

[0026] The terms “engineered polynucleotide”, “recombinant polynucleotide”, “genetically engineered polynucleotide”, and “genetically modified polynucleotide” refer to any manipulation of a polynucleotide that results in a detectable change in a naturally occurring polynucleotide, wherein the manipulation includes, but is not limited to, changes in the sequence of the polynucleotide or inclusion of non-naturally occurring nucleotides or nucleosides.

[0027] The terms “chimeric antigen receptor”, “chimeric receptor”, and “CAR” refer to a polypeptide having a binding specificity to a desired target and operably connected to (e.g., as a fusion or as separate chains linked by one or more disulfide bonds, etc.) the intracellular part of an immune cell activation domain. When expressed, a CAR is present at the plasma membrane of an immune cell. More particularly, CARs are engineered receptors which, when expressed, graft an antigen specificity onto a cytotoxic cell, such as a T cell, a natural killer (NK) cell, a macrophage, etc. For example, CARs are engineered to give T cells the new ability to target a specific protein. CARs comprise an extracellular domain having at least one antigen specific targeting region that binds to a hapten, a transmembrane domain (TM), and an intracellular domain (ID) including a T cell activation domain, the T cell activation domain comprising one or more co-stimulatory domains (CSD), in a combination that is not naturally found together on a single protein. The antigen specific targeting region may be a single chain antibody (scFv). CARs known in the art typically bind to antigens expressed on tumor cells. The CARs of the present disclosure bind to CD24.

[0028] As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues connected to by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein” and “polypeptide” refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to an encoded gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0029] An extracellular domain is external to the cell or organelle and functions to recognize and respond to a ligand. A transmembrane domain spans the membrane of a cell. An intracellular domain is situated inside a cell. Intracellular co-stimulatory domains provide secondary signals to the cell. They can recruit signaling molecules, cytoskeletal mobilization or induce cell proliferation, differentiation or survival. In the present disclosure a CAR may include an antigen specific extracellular domain, a transmembrane domain and one or more intracellular domains with one or more co-stimulatory domains.

[0030] The extracellular domain antigen binding region of the present disclosure comprises a single chain variable fragment (scFv) which is comprised of six complementarity determining regions (CDRs). CDRs are hypervariable domains that determine specific antibody binding. scFv are polypeptides that contain the variable light chain and variable heavy chain of an antibody connected by a flexible linker peptide. The scFv of the present disclosure may comprise the scFv of CD24 (scFv). The scFv may be encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity SEQ ID NO: 18. The scFv may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 4.

[0031] The engineered polynucleotide may further comprise a cytokine such as CXCR3 or IL-15. CXC3 is a chemokine receptor expressed primarily on activated T lymphocytes, natural killer cells, and some epithelial cells. The CXCR3 may be encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity SEQ ID NO: 19. The CXCR3 (chemokine receptor CXCR3) may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 5. IL-15 (interleukin-15) is an inflammatory cytokine that binds to and signals through a complex composed of IL-2 and IL-15 receptor beta chain (CD122) and the common gamma chain (CD132). IL-15 induces the proliferation of natural killer cells. The IL-15 may be encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity SEQ IDNO: 20. The IL-15 may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6.

[0032] The engineered polynucleotide may further comprise EGFRt (truncated epidermal growth factor receptor) is a transduction marker and safety switch for clinical T cell therapy. It is a functionally inactive protein. EGFRt may be targeted by an antibody that binds it and trigger an antibody-dependent cellular cytotoxicity (ADCC) response. When bound by an antibody (e.g. cetumixab), an immune cell can recognize the EGFRt and eliminate the CAR T cell. As such, EGFRt may serve as a safety switch for modulating potential systemic toxicity of CAR T cell therapy. The EGFRt may be encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity SEQ ID NO: 25. The EGFRt may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11.

[0033] The CAR of the present disclosure may comprise a transmembrane domain and a hinge sequence. A hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be positioned between the antigen recognition moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. For example, the hinge sequence may be derived from a CD8a molecule or a CD28 molecule. In exemplary embodiments, the hinge domain is a CD8 domain.

[0034] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon , CD45, CD4, CD5, CD8 (e.g. , CD8 alpha, CD8 beta), CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD11 a, CD18) , ICOS (CD278) , 4-1 BB (CD137) , GITR, CD40, BAFFR, HVEM (LIGHTR) , SLAMF7, NKp80 (KLRF1), CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11 d, ITGAE, CD 103, ITGAL,ITGAM , CD1 1 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, ITGB7, TNFR2, DNAM 1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM 1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, and PAG / Cbp. In exemplary embodiments, the transmembrane region is derived from CD8. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A short oligo- or polypeptide linker, such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR. In some embodiments, the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain or can be different transmembrane domains.

[0035] The CAR of the present disclosure may comprise at least one intracellular signaling domain. The signal sequence plays a determinant role in protein distribution and can allow the CAR to be glycosylated and anchored in the cell membrane. The intracellular signaling domain may be a co- stimulatory domain. A costimulatory domain is required for an efficient antigen response in immune cells. The intracellular signaling domain may be derived from CD3 zeta (CD3(^ (TCR zeta, GenBank acc no. BAG36664.1). T-cell glycoprotein CD3 zeta (CD3^ chain, also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene. Other co-stimulatory domains include CD28, 4- 1BB, OX-40, ICOS and other members of the TNF receptor superfamily or immunoglobulin (Ig) superfamily. In exemplary embodiments, the CAR comprises a CD3(^ and a 4-1BB co-stimulatory domain. However, any co-stimulatory domains may be used.

[0029] The CD3(^ domain may be encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity SEQ ID NO: 23. The CD3(^ domain may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 9.

[0030] The 4- IBB domain may be encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity SEQ ID NO: 22. The 4-1BB domain may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 8.

[0031] “Percentage of sequence identity", “percent similarity”, or “percent identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (z. ., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0032] The term “substantial identity" or “substantial similarity” of polynucleotide or peptide sequences means that a polynucleotide or peptide comprises a sequence that has at least 75% sequence identity. Alternatively, percent identity can be any integer from 75% to 100%. Embodiments described herein have at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0033] In a third aspect, provided herein is a construct comprising any of the engineered polynucleotides described herein operably linked to a promoter.

[0034] As used herein, the term “construct” refers to a recombinant polynucleotide, i.e., a polynucleotide that was formed artificially by combining at least two polynucleotide components from different sources (natural or synthetic). For example, the constructs may comprise a portion of the coding region of a transgene of interest operably linked to a promoter that (1) is associated with another gene found within the same genome, (2) is from the genome of a different species, or (3) is synthetic. The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, apromoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0035] As used herein, the terms “heterologous promoter,” “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5’ or 3’ side of a polynucleotides described herein, or within the coding region of said polynucleotides. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. The typical 5’ promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Heterologous promoters useful in the practice of the present disclosure include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters. The heterologous promoter may be a plant, animal, bacterial, fungal, or synthetic promoter. Suitable promoters are known and described in the art. Suitable promoters include the T3, T7 and SP6 promoter sequences, which are often used for in vitro transcription of RNA. In mammalian cells, typical promoters include, without limitation, promoters for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, as well as the translational elongation factor EFla promoter or ubiquitin promoter. In exemplary embodiments, the promoter is an EF-la promoter.

[0036] The CAR construct may be comprised in a vector. The term “vector” refers to a nucleic acid molecule capable of propagating a nucleic acid segment within it, e.g. the engineered CAR polynucleotide described herein. The term includes the vector as a self-replicating nucleic acid structure as well as a vector incorporated into the genome of a host cell into which it has been introduced. The term vector encompasses “plasmids”, the most commonly used form of vector. Plasmids are circular double-stranded DNA loops into which additional DNA segments (e.g., those encoding peptides) may be ligated. In some embodiments, the vector is a mini-circle DNA (mcDNA) vector. Mini-circle DNA vectors are episomal DNA vectors that are produced ascircular expression cassettes devoid of any bacterial plasmid DNA backbone. See, for example, System Biosciences, Mountain View CA, MN501A-1. Their smaller molecular size enables more efficient transfections and offers sustained expression over a period of weeks as compared to standard plasmid vectors that only work for a few days. The vectors may further a comprise heterologous nucleic acid backbone sequence. As used herein, “heterologous nucleic acid sequence” refers to a non-human nucleic acid sequence, for example, a bacterial, viral, or other non-human nucleic acid sequence that is not naturally found in a human. Heterologous backbone sequences may be necessary for propagation of the nucleic acid segment and / or expression of encoded peptides.

[0037] In a fourth aspect, provided herein is a cell comprising any of the engineered polynucleotides, constructs, or CARs described herein. As used herein, a “cell” or “engineered cell” is a cell in which the engineered polynucleotide is expressed or a cell comprising the CAR. The cell may be a mammalian cell. The cell may be a human cell. In exemplary embodiments, the cell is an immune cell. The cell may comprise a T cell, a natural killer (NK) cell, a B cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell. Accordingly, the cell comprising the CAR protein and / or the engineered polynucleotide may be a CAR-T cell, CAR- NK cell, CAR-B cell, a CAR-macrophage, or a CAR-iNKT cell. In exemplary embodiments, the cell is a T cell. The cell may be derived from a subject, engineered to express the CAR described herein, then used to treat the subject. Any suitable means for delivering the CAR constructs and vectors to the host cell may be used including, but not limited to, transfection, transduction, transformation, and nanoparticle delivery.

[0038] In a fifth aspect, provided herein is a pharmaceutical composition comprising any of the engineered polynucleotides, CARs, and cells described herein. As used herein, the term “pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammal. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Examples of compositions appropriate for such therapeutic applications include preparations for parenteral, subcutaneous, transdermal, intradermal, intramuscular, intracoronarial,intramyocardial, intraperitoneal, intravenous or intraarterial (e.g., injectable), or intratracheal administration, such as sterile suspensions, emulsions, and aerosols. In some cases, pharmaceutical compositions appropriate for therapeutic applications may be in admixture with one or more pharmaceutically acceptable excipients, diluents, or carriers such as sterile water, physiological saline, glucose or the like.

[0039] In a sixth aspect, provided herein is a method for treating a cancer in a subject, the method comprising administering to the subject any of the engineered polynucleotides, constructs, CARs, or pharmaceutical compositions described herein.

[0040] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. For example, treating cancer in a subject includes the reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. The term “treatment” can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; (d) reducing or ameliorating at least one symptom of cancer; and (e) extending the survival of the subject.

[0041] In embodiments, the cancer is a B cell lymphoma. The B cell lymphoma may be a mantle cell lymphoma. In embodiments, the cancer comprises a solid tumor. The cancer comprising a solid tumor may be a colon cancer, a liver cancer, a prostate cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, or a glioblastoma.

[0042] The method may further comprise administering to the subject an additional cancer treatment. Additional cancer treatments include, but are not limited to chemotherapy, radiation, bone marrow transplant, surgery and immunotherapy.

[0043] The method may further comprise administering an antibody against EGFRt. The antibody may be a monoclonal antibody. The antibody may be cetumixab.

[0044] As used herein, the term “administering” an agent, such as a therapeutic entity to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. In terms of the therapeutic agent, the term “administering” is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitableroute for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, topical administration, and administration by the intranasal or respiratory tract route.

[0045] The term “subject” or “patient” are used herein interchangeably to refer to a mammal, preferably a human, to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. The subject may be a human. The subject may be a mammal in need of treatment for a cancer.

[0046] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results. The amount of the pharmaceutical composition that is therapeutically effective may vary depending on the particular pathogen or the condition of the subject. Appropriate dosages may be determined, for example, by extrapolation from cell culture assays, animal studies, or human clinical trials taking into account body weight of the patient, absorption rate, half-life, disease severity and the like. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0047] Miscellaneous

[0048] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0049] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus orminus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0050] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0051] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0052] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in formingmeasurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0053] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or£B or “A and B.”

[0054] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0055] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0056] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0057] The present disclosure will be more fully understood upon consideration of the following non-limiting examples.EXAMPLES

[0058] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.

[0059] Example 1

[0060] Introduction

[0061] MCL is an aggressive, disseminated B cell malignancy, and its treatment has incorporated many targeted therapies. However, many patients succumb to disease due to relapse following these novel therapies. In the past decade, immunotherapies such as CAR-T therapy [1] and immune checkpoint inhibitors targeting regulatory pathways of adaptive immune cells resulted in significant clinical advances[2]. Chimeric antigen receptor T (CAR-T) cells are an emerging and novel cell type that consist of an extracellular single-chain variable fragment (scFv) with a hinge region, a transmembrane domain, intracellular activation, and co-stimulation domains [3, 4], The widely studied CAR-T cell targeting CD 19 has shown excellent therapeutic efficacy against patients suffering from relapsed and refractory (R / R) B-cell malignancies [5-7], CAR-T cell therapy and bispecific antibody (BsAb) cells have indeed significantly shaped the treatment landscape for refractory hematologic malignancies including large B-cell lymphomas and MCL. While response rates have improved and have shown remarkable clinical efficacy in the treatment of R / R MCL, over half of patients eventually relapsed. Moreover, CAR-T cells are often overactivated with the emission of significantly increased levels of serum inflammatory cytokines, which causes cytokine-release syndrome and other severe toxicitiesfl]. Relapses can be classified into two patterns: CD 19-positive relapse or CD 19- negative relapse related to down-regulation or loss of CD19 surface expression. Patients who relapse after CAR19 T-cell therapy have dismal outcome; hence, there is an urgent need to develop the next-generation of engineered CAR thatutilize alternative targets to circumvent resistance mechanisms and improve the efficacy and safety of CAR T cells.

[0062] Recently, immune checkpoints in innate immune cells have attracted great interest as therapeutic targets[8] [9], Generally, healthy cells express antiphagocytic molecules to prevent clearance by phagocytes

[0010] , However, tumor cells can use this mechanism to evade immune clearancefl 1], Cluster of differentiation (CD)47 represents the first “don't eat me” signal

[0012] . Studies showed that CD47 signals expressed on tumor cells evade phagocytosis via binding to signal regulatory protein-a (SIRPa), an inhibitory immunoreceptor expressed on macrophages and dendritic cells[ 13 , 14], In recent years, cluster of differentiation 24 (CD24) has been reported as a novel antiphagocytic molecule

[0015] , CD24 is expressed by many cells of the immune system

[0016] and has been extensively studied in the context of cancer biology, with it being defined as a cancer stem cell marker in various malignancies, such as breast

[0017] , pancreas

[0018] , and ovarian carcinoma

[0019] , CD24-mediated signaling further promotes cell migration, invasion, and cell proliferation[20-22]. More recently, CD24 was also described as an innate immune checkpoint with apparent significance in several solid cancer types

[0015] . Specifically, CD24 relayed antiphagocytic signals to phagocytes through its interaction with Siglec-10, a lectin expressed on tumor-associated macrophages (TAMs). Accordingly, CD24 blockade induced macrophage- mediated phagocytosis of tumor cells and inhibited tumor growth in mouse model

[0015] . Notably, high expression of CD24 has been associated with poor prognosis in several cancers[23-26],

[0063] CD24 is physiologically expressed on human B cells

[0027] , In B-cell lymphomas, CD24 is reportedly overexpressed in various lymphomas in which CD24 levels are elevated compared to healthy subjects

[0028] , B-cell lymphomas such as mantle cell lymphoma (MCL) or follicular lymphoma (FL), retained higher CD24 expression in contrast to healthy counterparts

[0029] . Moreover, given CD24 is overexpressed and its expression is correlated with poor overall survival (OS) in MCL [30-31], we propose to target expressed CD24 on MCL cells which is minimally expressed in normal B-lymphocytes. Antibody-mediated targeting of CD24 was shown to robustly enhance the phagocytic uptake of MCL cells and phagocytic uptake upon CD24 mAb treatment was significantly greater than uptake upon CD47 mAb treatment in MCL cell lines and primary patient-derived MCL cells[3O-31],

[0064] Here, we develop CD24 CAR-T cells that will be a dual functional immunotherapeutic strategy of direct T cell mediated killing and enhanced phagocytosis for MCL. Successfulcompletion of these highly translational studies will lead to a new therapeutic paradigm for MCL patients and have immediate impact on MCL and other B-cell lymphoma patient care.

[0065] Results

[0066] CD24 surface expression and significance in MCL. We examined the CD24 expression in a set of MCL cell lines and primary MCL cells. Using flow cytometry and quantitative real time PCR (qRT-PCR), as shown in FIGS. 1A-1B, variable CD24 expression was detected in MCL lines as well as higher CD24 was detected in primary MCL samples. In addition, we and others also observed that CD24 expression were correlated with MCL patient clinical prognosis and high CD24 is indicative of poor prognosis (FIG. 1C).

[0067] Generation of different CD24 CAR T cells and CD24 CAR T cells cytotoxicity against MCL. CAR-T therapy has shown great potential in lymphoma treatment. However, many factors impair the efficacy of CAR-T therapy, such as antigenic loss, limited potency and persistence, poor infiltration capacity of CAR-T cells, and a suppressive TME. To overcome these obstacles, we designed and constructed three third-generation CD24 CARs by using the sequence of CD24 antibodies. The CD24-CAR was constructed by successively linking single-chain variable fragment (scFv) binder, hinge and transmembrane region of CD8a, costimulatory domain of 4-1BB, and signal transduction domain of CD3(^ (CD24 CAR-T). As a reference, we used a clinically tested vector and implemented industrial manufacturing standard. In addition, by capitalizing novel CAR as a backbone, we generated additional two CD24 CAR-T : 1) CD24 CAR- T armed with CXCR3 (to increase CAR-T cell activity and infiltration), 2) CD24 CAR-T cells armed with IL 15 (to increase T and NK cell activity and infiltration) to enhance their anti -tumor capacity, FIG. 2). FIG. 5 shows the CD24 EGFR CAR designed to target CD24 tumors. This CAR includes expression of human EGFR receptor which serves as an off switch. FIG. 6 shows the CD24 EGFR CAR designed to target CD24 tumors in combination with co-expression of interleukin 15 which activates NK (natural killer) cells inside the tumor. This CAR includes coexpression of human EGFR receptor which serves as an off switch. FIG. 7 shows the CD24 EGFR CAR designed to target CD24 and also promote co-expression of the T cell specific homing and activation growth factor CXCR3. This allows CAR-T cells to overcome the tumor suppressive microenvironment and allows activated cells to migrate into the tumor tissue. This CAR also includes co-expression of human EGFR receptor which serves as an off switch.

[0068] To this end, we tested cytotoxicity of all three CD24 CAR-T cells against MCL lines exvivo. We examined the cytotoxic activity of the T cells transduced with the different CD24-CARs (FIG. 3). As tumor target cells, our established luciferase stably expressed target cells (B cell lymphoma cell lines Z 138, REC-1, HBL-2) were co-cultured with CD24 CARs or control T cells from 12 hours to 72 h. Then, the luciferase-activity based-cytotoxicity was measured by bioluminescence assays and showed sustained cytotoxicity for all three novel CD24 CAR-T cells. As shown observed in FIG. 3A-B, all CAR constructs mediated significant cytotoxicity in a time (A) and dose (B) dependent manner (B) against the MCL cell lines, in comparison with control T cells ( O.OOOl). Intriguingly, we observed that, in contrast to CD19 CAR-T, CD24 CAR-T cells showed a slow acting but comparable cytotoxicity against MCL (FIGS. 3C, 4A-4E).

[0069] References1. Neelapu, S.S., et al., Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. Nat Rev Clin Oncol, 2018. 15(1): p. 47-62.2. He, M., et al., Immune Checkpoint Inhibitor-Based Strategies for Synergistic Cancer Therapy. Adv Healthc Mater, 2021. 10(9): p. e2002104.3. Fesnak, A., et al., CAR-T Cell Therapies From the Transfusion Medicine Perspective. Transfus Med Rev, 2016. 30(3): p. 139-45.4. Maus, M.V., et al., T Cells Expressing Chimeric Antigen Receptors Can Cause Anaphylaxis in Humans. Cancer Immunology Research, 2013. 1(1): p. 26-31.5. Kalos, M., et al., T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med, 2011. 3(95): p. 95ra73.6. Kochenderfer, J.N., et al., Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood, 2010. 116(19): p. 3875-86.7. Neelapu, S. S., et al., Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B- Cell Lymphoma. New England Journal of Medicine, 2017. 377(26): p. 2531-2544.8. Feng, M., et al., Phagocytosis checkpoints as new targets for cancer immunotherapy. Nat Rev Cancer, 2019. 19(10): p. 568-586.9. Place, D.E. and T.D. Kanneganti, The innate immune system and cell death in autoinflammatory and autoimmune disease. Curr Opin Immunol, 2020. 67: p. 95-105.10. Kelley, S.M. and K.S. Ravichandran, Putting the brakes on phagocytosis: “don't-eat-me ” signaling in physiology and disease. EMBO Rep, 2021. 22(6): p. e52564.a. Landry, M R., J.M Walker, and C. Sun, Exploiting Phagocytic Checkpoints in Nanomedicine: Applications in Imaging and Combination Therapies. Front Chem, 2021. 9: p. 642530. b. Majeti, R., et al., CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell, 2009. 138(2): p. 286-99.11. Cao, X., et al., Effect of cabazitaxel on macrophages improves CD47-targeted immunotherapy for triple-negative breast cancer. J Immunother Cancer, 2021. 9(3).12. Zhang, W ., et al., Advances in Anti-Tumor Treatments Targeting the CD47,'SIRPalpha Axis. Front Immunol, 2020. 11 : p. 18.13. Barkal, A. A., et al., CD24 signalling through macrophage Siglec-10 is a target fo a. Schlossman, S.F. Leucocyte typing V: white cell differentiation antigens: proceedings of the fifth international workshop and conference held in Boston, USA, 3-7 November, 1993. 1993. Oxford University Press, USA.14. Li, W ., et al., Unraveling the roles ofCD44 / CD24 andALDHl as cancer stem cell markers in tumorigenesis and metastasis. Sci Rep, 2017. 7(1): p. 13856.15. Salaria, S., et al., Expression of CD24, a Stem Cell Marker, in Pancreatic and Small Intestinal Neuroendocrine Tumors. Am J Clin Pathol, 2015. 144(4): p. 642-8.16. Yang, W., et al., Therapeutic Strategies for Targeting Ovarian Cancer Stem Cells. Int J Mol Sci, 2021. 22(10).17. Overdevest, J.B ., et al ., CD24 expression is important in male urothelial tumorigenesis and metastasis in mice and is androgen regulated. Proc Natl Acad Sci U S A, 2012. 109(51): p. E3588- 96.18. Altevogt, P., et al., Novel insights into the function of CD24: A driving force in cancer. Int J Cancer, 2021. 148(3): p. 546-559.19. Fang, X., et al., CD24: from A to Z. Cell Mol Immunol, 2010. 7(2): p. 100-3.20. Kwon, M. J., et al., CD24 Overexpression Is Associated with Poor Prognosis in Luminal A and Triple-Negative Breast Cancer. PLoS One, 2015. 10(10): p. e0139112.21. Zhang, P., P. Zheng, and Y. Liu, Amplification of the CD24 Gene Is an Independent Predictor for Poor Prognosis of Breast Cancer. Front Genet, 2019. 10: p. 560.22. Tarhriz, V., et al., Overview of CD24 as a new molecular marker in ovarian cancer. J Cell Physiol, 2019. 234(3): p. 2134-2142.23. Wang, J.L., et al., CD24 Overexpression Related to lymph Node Invasion and Poor Prognosis of Colorectal Cancer. Clin Lab, 2018. 64(4): p. 497-505.24. Mensah, F.F.K., et al., CD24 Expression and B Cell Maturation Shows a Novel Link With Energy Metabolism: Potential Implications for Patients With Myalgic Encephalomyelitis / Chronic Fatigue Syndrome. Front Immunol, 2018. 9: p. 2421.25. Shapira, S., et al., Feasibly of CD24 / CD1 lb as a Screening Test for Hematological Malignancies. J Pers Med, 2021. 11(8).26. Katrina Lancaster- Shorts, S.P. CD24 Expression in Follicular Lymphoma: An Alternative B-Cell Marker in Therapy Selected, Recurrent Lymphoma. 2020. Graduate Medical Education Research Journal.27. Freile, J.A., et al., CD24 Is a Potential Immunotherapeutic Target for Mantle Cell Lymphoma. Biomedicines, 2022. 10(5).28. Aroldi A, et al., Effects of blocking CD24 and CD47 'don't eat me' signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia. J Cell Mol Med. 2023 0ct;27(20):3053-3064

[0070] Example 2

[0071] Since CD24 is overexpressed in many solid tumors such as breast, pancreas, colon, liver, brain and ovarian carcinoma, CD24 CAR-T cells can be used to treat solid tumors. FIGS. 8A-8C show the efficacy of CD24-CXCR3 against glioblastoma and several solid tumor cell lines.

[0072] Table 1. Informal Sequence Listing

Claims

CLAIMSWhat is claimed:

1. An engineered polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular CD24 scFv, wherein the CD24 scFv comprises a sequence having at least 90% identity to SEQ ID NO: 4.

2. The engineered polynucleotide of claim 1, wherein the CAR further comprises CXCR3 or IL-15.

3. The engineered polynucleotide of claim 2, wherein the CXCR3 comprises a sequence having at least 90% identity to SEQ ID NO: 5.

4. The engineered polynucleotide of claim 2, wherein the IL-15 comprises a sequence having at least 90% identity to SEQ ID NO: 6.

5. The engineered polynucleotide of any one of claims 1-4, wherein the CAR further comprises EGFRt.

6. The engineered polynucleotide of claim 5, wherein the EGFRt comprises a sequence having at least 90% identity to SEQ ID NO: 11.

7. The engineered polynucleotide of any one of claims 1-6, wherein the CAR further comprises at least one of a 4-1BB domain, and a CD3(^ signaling domain.

8. The engineered polynucleotide of any one of claims 1-7, wherein the CAR comprises a sequence having at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

9. A construct comprising the engineered polynucleotide of any one of claims 1-8 operably linked to a promoter.

10. A CAR encoded by the engineered polynucleotide of any one of claims 1-8.

11. A cell comprising the engineered polynucleotide of any one of claims 1-8, the construct of claim 9, or the CAR of claim 10.

12. The cell of claim 11, wherein the cell is an immune cell.

13. The cell of claim 12, wherein the cell is a T cell, a B cell, a natural killer (NK) cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell.

14. The cell of claim 13, wherein the cell is a T cell.

15. A pharmaceutical composition comprising the engineered polynucleotide of any one of claims 1-8, the construct of claim 9, the CAR of claim 10, or the cell of any one of claims 11-14; and a pharmaceutically acceptable carrier.

16. A method for treating a cancer in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 15.

17. The method of claim 16, wherein the cancer is a B-cell lymphoma.

18. The method of claim 17, wherein the B-cell lymphoma is mantle cell lymphoma.

19. The method of claim 16, wherein the cancer comprises a solid tumor.

20. The method of claim 19, wherein the cancer is colon cancer, liver cancer, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, or glioblastoma.

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