Cell therapy

Abstract

The present application provides chimeric antigen receptor constructs capable of expression in dendritic cells (DCs), as well as DCs modified to express one or more chimeric antigen receptors (CARs), as well as compositions comprising these modified DCs and methods of stimulating an adaptive immune response in a subject. The intracellular domain of the CAR comprises a Toll-Interleukin Receptor (TIR) intracellular signaling domain and a co-stimulatory signaling domain selected from the group consisting of a CD3 signaling domain, a CD28 signaling domain, and a CD28 and CD3 combined signaling domain.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to Australian Provisional Patent Application AU2022903034, filed on October 17, 2022, the entire content of which is incorporated herein by reference.

[0003] Sequence listing citation

[0004] The entire content of the electronically submitted sequence listing is incorporated herein by reference for all purposes. Technical Field

[0005] Disclosed herein are dendritic cells (DCs) modified to express one or more chimeric antigen receptors (CARs), compositions comprising such cells, and methods of generating an adaptive immune response in a subject. Background Art

[0006] Chimeric antigen receptor (CAR) therapy has achieved great clinical success in the treatment of hematological malignancies. It is based on a synthetic receptor with both antigen recognition and signal transduction functions. The single-chain variable fragment (scFv) in a CAR retains the antigen recognition specificity of the variable regions of the monoclonal antibody heavy and light chains. Signal transduction of the CAR construct depends largely on the signaling domains of the native immune receptor. However, despite the great success of CAR-T therapy in treating blood cancers such as leukemia, its application in solid tumors still has many problems. For example, CAR-T cells show a significant clinical response (CR) of 80 - 100% in patients with end-stage relapsed acute lymphoblastic leukemia (ALL), but the CR in solid tumors is only 1%. This is because there is usually a physical barrier formed by tumor-associated stromal cells around solid tumors, which limits T cell infiltration, solid tumors show extensive antigen heterogeneity, and there is a highly immunosuppressive microenvironment around some solid tumors, so it is difficult to develop CAR-T cells that can enter solid tumors. In addition, CAR-T cells downregulate the expression of their cell surface adhesion molecules, thus limiting their adhesion properties to the already dysregulated tumor blood vessels. Besides these extrinsic factors, intrinsic factors inherent to CAR-T cells also weaken their efficacy, leading to their exhaustion. In addition, CAR-T cell therapy may cause toxic reactions, thus destroying healthy cells, including CAR-T cells themselves. In some trials, CAR-T therapy has also produced strong off-tumor toxicity to healthy tissues.

[0007] Given the above challenges, researchers have also turned their attention to harnessing myeloid cells, including monocytes, macrophages, and DCs, which can produce large amounts of pro-inflammatory cytokines to activate T cells and may cultivate the tumor microenvironment to promote anti-tumor immune responses. Some researchers have found that CAR carried by an adenovirus vector overcomes the inherent resistance of primary human macrophages to genetic manipulation and confers on them a sustained pro-inflammatory phenotype. CAR macrophages (CAR-M) exhibit antigen-specific phagocytosis and tumor clearance capacity in vitro (M Klichinsky et al., (2020) Nature Biotechnology Vol 38:947-953).

[0008] However, the ability of macrophages to present antigens to T cells is limited because their main function in the tumor environment is to phagocytose dead and dying tumor cells and produce pro-inflammatory or immunosuppressive cytokines depending on the environment in which they encounter dead or dying cells. This may be a key limitation of macrophages as a cell therapy because the initiation of effector T cell and memory T cell responses is crucial for tumor clearance and is also key to preventing tumor recurrence, which is the main cause of death in cancer patients. Due to the non-replicative nature of macrophages, expressing CAR in the cells has also been shown to be a challenge (Mukhopadhyay (2020) Nature Methods 17:561).

[0009] Dendritic cells (DCs) are professional antigen-presenting cells that bridge innate and adaptive immunity and are crucial for inducing protective immune responses against pathogens. Both T lymphocytes and B lymphocytes are key players in generating an adaptive immune response to pathogens or malignant cells, resulting in the generation of effector T cells and high-affinity antibodies produced by plasma cells. An important feature of the adaptive immune system is that antigen-specific T cells and B cells can differentiate into memory cell populations that can persist in the host for many years. When re-exposed to the same pathogen, these cells can respond quickly and robustly to the threat, resulting in a faster and more effective immune response.

[0010] DCs recognize, uptake, and present pathogen-derived antigens to T cells via major histocompatibility complex (MHC) molecules. DCs also provide the necessary co-stimulatory signals and pro-inflammatory cytokines to effectively initiate an adaptive immune response. Their activity is crucial for the ability to distinguish danger from benign and to promote the optimal type of immune response.

[0011] In addition, the expression of CAR in monocyte-derived dendritic cells (moDC) was also explored. Studies have shown that the expression of an anti-HER2 CAR with truncated intracellular domain in moDC can enhance the phagocytosis of exosomes derived from tumor cells, thereby increasing its antigen presentation. US20200247870 describes the use of monocytes-derived macrophages generated from CD14+ selected cells (apheresis products from normal donors) differentiated in GM-CSF conditioned medium for 7 days. To optimize the delivery of lentiviral-transduced CAR, anti-HER2 lentivirus was used to transduce macrophages at different stages of the differentiation process of monocytes into macrophages.

[0012] There is a need in the art for alternative and improved therapies to combat tumors, particularly semi-solid tumors and solid tumors, while generating an adaptive immune response by harnessing anti-tumor T cells. Summary of the Invention

[0013] The present disclosure is based on generating modified dendritic cells (DCs) comprising a chimeric antigen receptor (CAR), which exhibit enhanced tumor recognition ability, thereby having superior antigen presentation ability, and thus can generate a more effective adaptive immune response in vivo. Specifically, the modified DCs show enhanced ability to cross-present tumor-specific antigens to CD8+ T cells, thereby promoting T cell proliferation and cytokine secretion.

[0014] Specifically, the inventors have successfully generated functional CAR-DCs comprising a Toll-interleukin-1 receptor (TIR) domain (in particular, the TIR from Toll-like receptor 4, i.e., TIR4). In addition, the inventors have also found that a strong DC pro-inflammatory response can be obtained when the CD28 signaling domain and / or the CD3ζ signaling domain in the CAR binds to TIR4.

[0015] In a first aspect, there is provided a chimeric antigen receptor (CAR) construct comprising:

[0016] (i) an antigen-binding domain;

[0017] (ii) a transmembrane domain (TM); and

[0018] (iii) an intracellular domain (IC) comprising a Toll interleukin receptor (TIR) intracellular signaling domain and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain;

[0019] wherein the CAR construct is capable of being expressed or functioning in dendritic cells.

[0020] In a second aspect, there is provided a modified dendritic cell comprising a chimeric antigen receptor (CAR) construct, the CAR construct comprising:

[0021] (i) an antigen-binding domain;

[0022] (ii) a transmembrane domain (TM); and

[0023] (iii) an intracellular domain (IC) comprising a Toll-interleukin receptor (TIR) intracellular signaling domain; and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a CD28 and CD3 combined signaling domain.

[0024] In one embodiment, the TIR intracellular signaling domain is located after the co-stimulatory signaling domain, that is, it is located distally to the co-stimulatory signaling domain. In a specific embodiment, the TIR is located after the co-stimulatory domain such that the order of the domains from N to C is: transmembrane domain - co-stimulatory domain. In a specific embodiment, the TIR is located after each of the CD28 and CD3 co-stimulatory domains, or after the CD28 and CD3 combined co-stimulatory domain, such that the order of the domains from N to C and the arrangement of the domains are TM - CD3 - TIR, or TM - CD28 IC - TIR or TM - CD28 IC - CD3 - TIR or TM - CD3 - CD28 TM - TIR.

[0025] In one embodiment, the transmembrane domain is the CD28 transmembrane domain.

[0026] In a specific embodiment, the order of the domains from N to C and the arrangement of the domains are: CD28 TM - CD3 - TIR or CD28 TM - CD28 IC - TIR or CD28 TM - CD28 IC - CD3 - TIR or CD28 TM - CD3 - CD28 TM - TIR.

[0027] In another embodiment, the CD28 transmembrane domain comprises the following sequence or consists of the following sequence: FWVLVVGGVLACYSLLVTVAFIIFWVRS (SEQ ID NO:1).

[0028] In another embodiment, the transmembrane domain comprises the following sequence or consists of the following sequence: FWLTVALILGIFLGTFIAFWVVYLLWVRS (SEQ ID NO:17).

[0029] In one embodiment, the dendritic cell is a conventional type 1 cell (cDC1). In another embodiment, the cDC1 expresses one or more markers selected from the group consisting of BTLA, CADM1, CD8A, CLEC9A, ITGAE, ITGAX, LY75, THBD (CD141), XCR1, and CD26.

[0030] In a third aspect, there is provided a modified dendritic cell comprising a nucleic acid sequence encoding a CAR construct, the CAR construct comprising:

[0031] (i) an antigen-binding domain;

[0032] (ii) a transmembrane domain (TM); and

[0033] (iii) an intracellular domain (IC) comprising a Toll-interleukin receptor (TIR) intracellular signaling domain and a co-stimulatory signaling domain, the co-stimulatory signaling domain being selected from: CD3

[0034] signaling domain, CD28 signaling domain, and a combined CD28 and CD3 signaling domain.

[0035] In one embodiment, the transmembrane domain is the CD28 transmembrane domain. In another embodiment, the CD28 transmembrane domain comprises the following sequence or consists of the following sequence: FWVLVVGGVLACYSLLVTVAFIIFWVRS (SEQ ID NO:1).

[0036] In another embodiment, the transmembrane domain comprises the following sequence or consists of the following sequence: FWLTVALILGIFLGTFIAFWVVYLLWVRS (SEQ ID NO:17).

[0037] In some embodiments, the CAR further comprises a CD8 hinge sequence. In one embodiment, the CD8 hinge sequence comprises the following sequence or consists of the following sequence: NGVTVSSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL (SEQ ID NO:2). In one embodiment, the CD8 hinge is located before the CD28 transmembrane domain.

[0038] In some embodiments, the CAR further comprises a tag. In one embodiment, the tag is a Myc tag. In another embodiment, the tag is near the C-terminal sequence of the antigen-binding domain or within the C-terminal sequence of the antigen-binding domain. In another embodiment, the tag is at the N-terminal of the CD8 hinge sequence. In one embodiment, the tag comprises the following sequence or consists of the following sequence: EQKLISEEDL (SEQ ID NO:3).

[0039] In one embodiment, the co-stimulatory signaling domain is the CD28 intracellular domain, which comprises the following sequence or consists of the following sequence:

[0040]

[0041] In one embodiment, the co-stimulatory signaling domain is the CD3 signaling domain, preferably the CD3ζ domain. In one embodiment, the CD3ζ intracellular co-stimulatory signaling domain comprises the following sequence or consists of the following sequence:

[0042]

[0043] In one embodiment, the TIR intracellular signaling domain is selected from Toll-like receptor 4, Toll-like receptor 3, Toll-like receptor 9, Toll-like receptor 11, and Toll-like receptor 13. In another embodiment, the TIR domain is the TLR4 domain (named TIR4). In another embodiment, the TIR4 domain comprises the following sequence or consists of the following sequence:

[0044]

[0045] In one embodiment, the CAR construct comprises an intracellular domain, and the intracellular domain comprises the co-stimulatory signaling domains of CD28, CD3ζ, and TIR4 (the whole CAR construct is named CD28 TM -CD28 IC -CD3ζ-TIR4CAR). In another embodiment, the CD28 TM -CD28 IC -CD3ζ-TIR4CAR comprises the following sequence or consists of the following sequence:

[0046]

[0047] In one embodiment, the CAR construct comprises the co-stimulatory signaling domains of CD28 and TIR4 (the whole CAR construct is named CD28 TM -CD28 IC-TIR4CAR). In another embodiment, the CD28 TM -CD28 IC -TIR4CAR comprises the following sequence or consists of the following sequence:

[0048]

[0049] In one embodiment, the CAR construct comprises a co-stimulatory signaling domain of TIR4 and CD3ζ. In another embodiment, the CD28 TM -TIR4-CD3ζCAR comprises the following sequence or consists of the following sequence:

[0050]

[0051] In some embodiments, the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

[0052] In some embodiments, the antibody or an antigen-binding fragment thereof has a binding affinity for a tumor cell antigen. In another embodiment, the antibody or an antigen-binding fragment thereof binds to or specifically binds to the HER2 antigen. In one embodiment, the antigen-binding fragment is a scFv sequence, which comprises the sequence:

[0053]

[0054] Without being bound by theory, the applicant has determined that the continuity of the CD28 transmembrane domain (CD28 TM ) and the CD28 intracellular domain (CD28 IC ) co-stimulatory signaling domain is very important for the normal function of CD28. In addition, the integrity of the CD28 TM and CD28 IC co-stimulatory signaling domain seems to be very important for the optimal activation of TIR4. After adding the CD3ζ domain, the signal intensity is further enhanced. In addition, the applicant has also found that adding the TIR4 domain to the C-terminus of the CAR is the most effective position for optimizing T cell proliferation and function. Therefore, in a preferred embodiment, the optimal arrangement of the domains in the expressed protein is:

[0055] N-terminus: Antibody / antigen-binding fragment: CD8 hinge CD28 TM domain: CD28 IC domain: CD3ζ domain: TIR domain C-terminus.

[0056] In one embodiment, anti-HER2-CD28 TM -CD28 IC-CD3ζ-TIR4 CAR, which comprises the sequences:

[0057]

[0058] In another embodiment, an anti-HER2-CD28 TM -CD28 IC -TIR4 CAR is provided, which comprises the sequences:

[0059]

[0060] In another embodiment, an anti-HER2-CD28 TM -TIR4-CD3ζ CAR is provided, which comprises the sequences:

[0061]

[0062] In one embodiment, an anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR is provided, which comprises the sequences:

[0063]

[0064] In one embodiment, the CAR comprises the sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, wherein the Myc tag sequence EQKLISEEDL is not included or the Myc tag sequence is replaced with another tag sequence. Other tags suitable for use in the structures of the present disclosure are well known to those skilled in the art. Examples of tags include hemagglutinin (HA) tag (YPYDVPDYA), 6His tag (HIS), FLAG TM tag (DYKDDDDK), AU1 tag (DTYRYI), AU5 tag (TDFYLK), GluGlu tag (EYMPME), OLLAS tag (SGFANELGPRLMGK), T7 tag (MASMTGGQQMG), V5 tag (GKPIPNPLLGLDST), E-tag (GAPVPYPDPLEPR), and D-tag (KETAAAKFERQHMDS), and Avi tag (CGLNDIFEAQKIEWHE) or HSV tag (SQPELAPEDPED).

[0065] In a fifth aspect, one or more nucleic acid sequences encoding the CAR are provided, which comprise any one of the sequences of SEQ ID NO.7, 8, 9, 11, 12, 13, 14, 17, 18, 19 or 20.

[0066] In one embodiment, the antigen-binding domain is encoded by a first nucleic acid, and the transmembrane domain and the intracellular domain are encoded by a second nucleic acid.

[0067] In some embodiments, the nucleic acid sequence encoding the CAR further comprises a signal peptide.

[0068] In one embodiment, the dendritic cell is a conventional type 1 cell (cDC1). In some embodiments, the dendritic cell is genetically modified.

[0069] In one embodiment, the modified dendritic cell is capable of cross-presenting antigens to T cells, eliciting an adaptive immune response, or activating anti-tumor T cells.

[0070] In some embodiments, the modified dendritic cell is capable of selectively phagocytosing tumor cells (especially apoptotic bodies released by tumor cells), cross-presenting tumor antigens, and / or activating T cells to respond to tumor antigens.

[0071] In some embodiments, the modified dendritic cell is capable of cross-presenting tumor antigens (or having tumor antigen cross-presentation), wherein antigen cross-presentation refers to the ability of a cell to present internalized antigens to major histocompatibility complex class I molecules (MHC I), which is necessary for an efficient cytotoxic T cell response against tumor cells.

[0072] In a sixth aspect, there is provided a pharmaceutical composition comprising the modified dendritic cell described herein.

[0073] In a seventh aspect, there is provided a method of stimulating an adaptive immune response in a subject, the method comprising: administering to the subject a therapeutically effective amount of the modified dendritic cell according to any one of the first to fourth aspects, or the pharmaceutical composition according to the sixth aspect.

[0074] In one embodiment, a CAR comprising any one of the sequences set forth in SEQ ID NO: 7, 8, 9, 11, 12, 13, 17, 18, 19, or 20 is administered to a subject.

[0075] In another embodiment, a modified dendritic cell comprising a CAR having any one of the sequences set forth in SEQ ID NO: 7, 8, 9, 11, 12, 13, 17, 18, 19, or 20 is administered to a subject.

[0076] In an eighth aspect, there is provided a method of stimulating an adaptive immune response in a subject, which comprises administering to the subject a dendritic cell comprising a chimeric antigen receptor (CAR-DC), the CAR comprising:

[0077] (i) Antigen-binding domain;

[0078] (ii) Transmembrane domain (TM); and

[0079] (iii) Intracellular domain (IC), comprising an intracellular signaling domain of Toll-interleukin receptor (TIR) and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain.

[0080] In one embodiment, a CAR comprising any one of the sequences of SEQ ID NO:7, 8, 9, 11, 12, 13, 17, 18, 19, or 20 is administered to a subject.

[0081] In another embodiment, a modified dendritic cell comprising a CAR having any one of the sequences of SEQ ID NO:7, 8, 9, 11, 12, 13, 17, 18, 19, or 20 is administered to a subject.

[0082] In one embodiment of the seventh or eighth aspect, the subject has cancer. In one embodiment, the cancer is a malignant tumor, a solid tumor, or a liquid tumor. In another embodiment, the method can induce phagocytosis of cancer cells in a subject.

[0083] In one embodiment, the modified cell or composition induces an immune response that eliminates tumors.

[0084] In a ninth aspect, a method for preparing a population of dendritic cells containing a chimeric antigen receptor (CAR-DC) is provided, comprising:

[0085] (i) Providing a population of cells from a subject;

[0086] (ii) Culturing the population of cells in a medium containing an FMS-like tyrosine kinase 3 (Flt3) agonist for at least one day;

[0087] (iii) Introducing a chimeric antigen receptor (CAR) into the population of cells, the chimeric antigen receptor comprising an antibody or an antigen-binding fragment thereof, a CD28 transmembrane domain, and an intracellular domain comprising an intracellular signaling domain of Toll-interleukin receptor (TIR) and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain; and / or

[0088] (iv) Culturing the cells from (iii) in a medium comprising an FMS-like tyrosine kinase 3 (Flt3) agonist for a time sufficient to form modified dendritic cells.

[0089] In one embodiment, the time required to form modified dendritic cells is about 2 days to 15 days. In another embodiment, the time is about 7 days.

[0090] In one embodiment, the cell population is selected from monocytes or stem cells in circulation, umbilical cord or bone marrow.

[0091] In another embodiment, the dendritic cells are conventional type I dendritic cells (cDC1).

[0092] In some embodiments, introducing the CAR into dendritic cells comprises introducing a nucleic acid sequence encoding a protein product that comprises an antibody or an antigen-binding fragment thereof, a CD28 transmembrane domain, and an intracellular domain that comprises a Toll-interleukin receptor (TIR) intracellular signaling domain and a co-stimulatory signaling domain, and the co-stimulatory signaling domain is selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 Showing that deficiency of DC-SCRIPT (encoded by the Zfp366 gene), which results in reduced numbers and impaired function of cDC1, impairs antigen-specific T cell responses in tumors. (A) CD45.2+WT and Zfp366 - / - mice were inoculated with B16-OVA melanoma (5x10 5 ), and then adoptively transferred with 2x10 7 CD45.1+OT-1 cells / mouse on day 7. Tumor size was measured every 2 - 3 days (n = 5). (B) Counts of tumor-infiltrating cDC1 (PI-SiglecHCD11c+MHCII+XCR1+CD11b-), cDC2 (PI-SiglecH-CD11c+MHCII+XCR1-CD11b+), and pDC (PI-CD11c+MHCII+SiglecH+) from the mice in A. (C) Ratio of the number of NK cells to the number of cDC1 cells. The data shown are mean ± SEM. Each point represents one mouse. P values were calculated using unpaired Student's t-test, *P < 0.05.

[0094] Figure 2 shows that cDC1 is essential for anti-tumor immunity. (A) Wild-type (WT), Zfp366 - / -, CD11c cre Irf8 + / + and CD11c cre Irf8 fl / fl mice. Tumor size was measured every 2 - 3 days (n = 5). (B) Tumor weight on day 18 after tumor inoculation. (C) Statistical analysis of tumor-infiltrating cDC1 (PI-SiglecH-CD11c+MHCII+XCR1+CD11b-). (D) Ratio of tumor size to the number of cDC1 cells. (E) WT, CD11c cre Irf8 fl / fl , Rag1 - / - , Rag2 - / - gc - / - and Irf8 fl / fl for their ability to inhibit tumor growth.

[0095] Figure 3 Shows the structure and performance of a CAR construct lacking the TIR signaling domain. (A) Shows the constructs of anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR and empty CAR expressed in a retroviral vector. Anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR contains, from left to right: anti-HER2 scFv, myc tag, CD8 hinge, CD28 transmembrane domain (CD28 TM ), CD28 intracellular domain (CD28 IC ) and CD3ζ signaling domain, internal ribosome entry site (IRES) and mCherry tag. The empty CAR contains only mCherry. (B) Representative FACS plots of the expression of empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR in mutuDC. (C) Representative confocal images showing direct GFP (green, Mutu cells) and mCherry (red, retrovirally transduced) fluorescence against myc (yellow), overlaid with DAPI (blue). Cells were analyzed 5 days after transduction. Scale bar, 100 μm. (D) Representative FACS plots of myc expression in mutuDC transduced with empty vector (upper panel) or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR. (E, F) Representative confocal images showing anti-IRF8 (E, cyan) and anti-DEC205 (F, pink). n = 3 independent cell cultures for each condition.

[0096] Figure 4It is shown that CAR+cDC1 can exhibit direct anti-tumor phagocytic activity. (A) Empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR transduction of HER2 expression on E0771 tumor cells and mutuDC. (B) Representative FACS plots of HER2 expression on mutuDC transduced with empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR before and 2 hours after co-culture with irradiated HER2+ E0771 tumors. (C) Representative confocal images showing that anti-HER2-CD28TM-CD28IC-CD3ζ CAR-transduced mutuDC (cyan) uptake CTV-labeled apoptotic HER2+ E0771 tumor cells (red) after 2 hours. (D) Representative FACS plots of CTV signal on mutuDC transduced with empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR before and 2 hours after co-culture with CTV-labeled irradiated HER2 + or HER2 - E0771 tumors. The right bar graph shows the statistical results of the left flow cytometry data (n = 3). (E) Representative FACS plots of CTV signal on mutuDC transduced with empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR before and after co-culture with CTV-labeled irradiated HER2 + E0771 tumors for 15 minutes, 30 minutes, 45 minutes, 1 hour, and 2 hours, respectively. (F) Statistic of (E) (n = 3). The data shown are mean + SEM. Each point represents one mouse *P < 0.05; **P < 0.01; ***P < 0.001.

[0097] Figure 5 It shows the activation of CAR+cDC1 in response to tumor cell-associated antigens. (A) Stimulation of mutuDC transduced with empty vector or anti-HER2-CD28 + -CD28 TM -CD28 IC -CD3ζ CAR with LPS, CpG, or apoptotic HER2 - GFP + mCherry + MHC II +CD11c + )。(B - C) Empty vector or anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR - transduced mutuDCs were stimulated with apoptotic HER2 + E0771 tumor cells for 16 hours. mutuDCs (PI - GFP + mCherry + MHC II + CD11c + ) MFI of CD86 (B) and CD80 (C). (D) Anti - HER2 - CD28 + co - cultured with apoptotic HER2 TM -CD28 IC -CD3ζ CAR - transduced mutuDCs (mCherry+). (E - F) Analysis of empty vector or anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR - transduced mutuDCs (5x10 3 ) antigen cross - presentation ability to OT - I T cells (2.5x10 + ) in response to irradiated HER2 4 E0771 tumor cells loaded with OVA at the indicated concentrations. Data represent 3 independent experiments. (G) Counting of PI + tumor cells for each condition. Data shown are mean + SEM. P - values were calculated using unpaired Student’s t - test (A, B, and C) or two - way Anova (F and G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0098] Figure 6 Show that anti - HER2 CAR+ BM - derived cDC1s exhibit enhanced phagocytic ability in response to tumor - specific antigens. (A) WT BM progenitors were transduced with empty vector, intracellular domain - truncated anti - HER2 CAR, or anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR (all expressing mCherry). mCherry - myc - or mCherry + myc + pDCs (SiglecH+CD11c+), cDC1s (FVD - SiglecH -MHC II + CD11c + XCR1 + CD11b 低 ) and cDC2 (FVD - SiglecH - MHC II + CD11c + XCR1 - CD11b + ) The frequencies of (B) Histograms show anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-transduced pDC, cDC2, and cDC1 for myc expression. mCherry - Expression of Myc in pDC was used as a negative control. (C) Histograms show uninfected or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-transduced mCherry - myc - or mCherry + myc + Expression of the indicated cell surface markers in BM-derived cDC1 (selected area as in A). (D) BM-derived pDC, cDC2, and cDC1 (selected area as shown in A) transduced with anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR were separately incubated with CTV-labeled apoptotic HER2 + or HER2 - E0771 tumors for 2 h before and after co-culture. Representative FACS plots of CTV signals are shown. CAR-expressing cells were labeled with mCherry. The right bar graph shows the percentage of CTV + or mCherry - cells in the mCherry + population (n = 3). Data shown are mean + standard deviation. P values were calculated using two-way Anova. *P < 0.05; **P < 0.01.

[0099] Figure 7 The predicted protein structures of individual CARs are shown. (A) Structure of TIR4, (B) anti-HER2-CD28 TM -CD28 IC -CD3ζ structure, (C) structure of truncated CAR (intra trunc), (D) anti-HER2-CD28 TM -CD28 IC- Structure of TIR4, (E) anti-HER2-CD28 TM - Structure of TIR4-CD3ζ, (F) anti-HER2-CD28 TM - Structure of TIR4, and (G) anti-HER2-CD28 TM - CD28 IC - Structure of CD3ζ-TIR4. The CAR protein structure was predicted by AlphaFold2. The results are 3D models (below) and predicted alignment errors (above). Each amino acid in the protein chain is labeled relative to itself and other amino acids. In the 3D models (A, B) and the predicted alignment error heatmaps (A - G), different functional domains are color-coded and labeled.

[0100] Figure 8 Indicating that the expression of TIR4-CAR can activate cDC1. (A) Using empty vector, anti-HER2 intracellular domain truncated CAR, anti-HER2-CD28 TM - TIR4-CD3ζ, anti-HER2-CD28 TM - CD28 IC - TIR4, anti-HER2-CD28 TM - CD28 IC - CD3ζ-TIR4CAR, anti-HER2-CD28 TM - TIR4 or anti-HER2-CD28 TM - CD28 IC - The retroviruses encoding CD3ζCAR (all expressing mCherry) were used to transduce mutuDC. The frequency of mCherry+Myc+mutuDC was determined by flow cytometry. (B) Representative confocal images show the overlay images of α-tubulin (cyan) and DAPI (blue). Cells were stained 2 days after seeding on slides. Scale bar, 20 μm. (C) The MFI of CTV signal on the indicated CAR-transduced mutuDC before and 2 hours after co-culture with CTV-labeled apoptotic HER2 + or HER2 - E0771 tumors. The bar graphs show the statistical results of FACS data (n = 3). (D - E) As in C, the bar graphs show the MFI of HER2 (D) and MHCII (E) relative to resting cells in the indicated CAR-transduced mutuDC co-cultured with CTV-labeled apoptotic HER2 + or HER2 - E0771 tumors for 2 hours. (F - H), the bar graphs show the MFI of HER2 + or HER2 -MFI of CD86 (F), CD80 (G), and PD-L1 (H) in mutuDCs transduced with the indicated CARs and co-cultured with E0771 tumors or stimulated with LPS for 16 hours. Data shown are mean + standard deviation. P values were calculated using two-way Anova. *P < 0.05; **P < 0.01, ****P < 0.0001.

[0101] Figure 9 Shown is that autoactivation of CARs containing TIR4 is reduced by modifying the TM domain. (A) mutuDCs were transduced with empty vector, anti-HER2 intracellular domain truncated CAR, anti-HER2-CD28 TM -TIR4-CD3ζ, anti-HER2-CD28 TM -CD28 IC -TIR4, anti-HER2-CD28 TM -CD28 IC -CD3ζ-TIR4CAR, anti-HER2-CD28 TM -TIR4, anti-HER2-CD28 TM -CD28 IC -CD3ζCAR, anti-HER2-ProCAR1-CD28 IC -CD3ζ-TIR4CAR, anti-HER2-ProCAR2-CD28 IC -CD3ζ-TIR4CAR, anti-HER2-ProCAR3-CD28 IC -CD3ζ-TIR4CAR or anti-HER2-ProCAR4-CD28 IC -CD3ζ-TIR4CAR-encoding retroviruses. Expression of CD86 in MYC-mCherry-mutuDCs or MYC+mCherry+mutuDCs lacking HER2 + E0771 tumors was determined by flow cytometry. (B) Representative confocal images showing mCherry (red, retrovirally transduced) fluorescence. Cells were analyzed 7 days after transduction. Scale bar, 100 μm. (C-D) Analysis of apoptosis of HER2+ E0771 tumor cells expressing OVA in response to indicated concentrations by empty vector or anti-HER2 intracellular domain truncated CAR or ProCAR1 / 2 / 3 or 4-CD28IC-CD3ζ-TIR4 (labeled as Pro-CAR1 / 2 / 3 / 4-TIR4) CAR-transduced mutuDCs (5x10 3 ) and antigen cross-presentation to OT-I T cells (2.5x10 4) capacity. (E-F) The numbers of granzyme B+ OT-1 and IFNγ+ OT-1 in the co-culture system specified in C were analyzed by flow cytometry. (G) Empty or anti-HER2-ProCAR1-CD28IC-CD3ζ-TIR4CAR (labeled Pro-CAR2-TIR4CAR) was transduced into HOXB8 cells and then differentiated into cDC1 in the presence of OP9-DL1. On day 5, empty or anti-HER2-ProCAR1-CD28IC-CD3ζ-TIR4CAR (labeled Pro-CAR2-TIR4)-expressing cDC1 were injected into HER2+ E0771-challenged human HER2 transgenic mice (purple arrows). The tumor area (length x width) was measured every two to three days.

[0102] Key information of the sequence listing

[0103] SEQ ID NO:1: CD28 TM Domain sequence

[0104] SEQ ID NO:2: CD8 hinge region sequence

[0105] SEQ ID NO:3: Myc tag sequence

[0106] SEQ ID NO:4: CD28 IC Domain sequence

[0107] SEQ ID NO:5: CD3ζ signaling domain sequence

[0108] SEQ ID NO:6: TIR4 domain sequence

[0109] SEQ ID NO:7: CD28 lacking CD8 hinge TM -CD28 IC -CD3ζ-TIR4 CAR sequence

[0110] SEQ ID NO:8: CD28 lacking CD8 hinge TM -CD28 IC -TIR4 CAR sequence

[0111] SEQ ID NO:9: CD28 lacking CD8 hinge TM -TIR4-CD3ζCAR sequence

[0112] SEQ ID NO:10: HER2-binding scFv sequence

[0113] SEQ ID NO:11: anti-HER2-CD28 TM-CD28 IC -CD3ζ-TIR4 CAR sequence

[0114] SEQ ID NO:12: Anti-HER2-CD28 TM -CD28 IC -TIR4 CAR sequence

[0115] SEQ ID NO:13: Anti-HER2-CD28 TM -TIR4-CD3ζCAR sequence

[0116] SEQ ID NO:14: Anti-HER2-CD28-CD3 CAR sequence

[0117] SEQ ID NO:15: Truncated CAR sequence

[0118] SEQ ID NO:16: Anti-HER2-TIR4 CAR sequence

[0119] SEQ ID NO:17: Anti-HER2-Pro-CAR2-CD28 IC -CD3ζ-TIR4 CAR sequence

[0120] SEQ ID NO:18: Anti-HER2-Pro-CAR1-CD28 IC -CD3ζ-TIR4 CAR sequence

[0121] SEQ ID NO:19: Anti-HER2-Pro-CAR3-CD28 IC -CD3ζ-TIR4 CAR sequence

[0122] SEQ ID NO:20: Anti-HER2-Pro-CAR4-CD28 IC -CD3ζ-TIR4 CAR sequence

[0123] SEQ ID NO:21: HA tag sequence

[0124] SEQ ID NO:22: HIS tag sequence

[0125] SEQ ID NO:23: FLAG tag sequence

[0126] SEQ ID NO:24: AU1 tag sequence

[0127] SEQ ID NO:25: AU5 tag sequence

[0128] SEQ ID NO:26: GluGlu tag sequence

[0129] SEQ ID NO:27: OLLAS Tag Sequence

[0130] SEQ ID NO:28: T7 Tag Sequence

[0131] SEQ ID NO:29: V5 Tag Sequence

[0132] SEQ ID NO:30: E Tag Sequence

[0133] SEQ ID NO:31: D Tag Sequence

[0134] SEQ ID NO:32: Avi Tag Sequence

[0135] SEQ ID NO:33: HSV Tag Sequence Detailed Implementation Modes

[0136] Conventional techniques

[0137] In this specification, unless otherwise specifically stated or the context otherwise indicates, a single step, a composition of substances, a group of steps, or a group of compositions of substances mentioned shall be considered to include one and more (i.e., one or more) of these steps, compositions of substances, groups of steps, or groups of compositions of substances.

[0138] Those skilled in the art should understand that, in addition to the specifically described content, the present disclosure may have other variations and modifications. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds individually or jointly mentioned or specified in this specification, as well as any two or more or any and all combinations of said steps or features.

[0139] The scope of the present disclosure is not limited by the specific embodiments described herein, which are only for illustrative purposes. Products, compositions, and methods with equivalent functions clearly fall within the scope of the present disclosure.

[0140] Unless otherwise specifically stated, any embodiment of the present disclosure should be applicable to any other embodiment of the present disclosure after appropriate modification.

[0141] Unless otherwise clearly defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those of ordinary skill in the art (e.g., those in the fields of cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0142] Unless otherwise specified, the recombinant proteins, recombinant DNA techniques, molecular biology, microbiology, cell culture, and immunology techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in various documents, such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0143] In this specification, the word “comprise” or its variants, such as “comprises” or “comprising”, shall be understood to mean the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0144] Definitions

[0145] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein.

[0146] In addition, "and / or" as used herein shall be understood to specifically disclose each of the two specified features or components, whether or not any other features or components are present. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is also intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0147] The term "about" as used herein means approximately, roughly, around, or in this region. When the term "about" is used in connection with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. Generally, the term "about" as used herein means a numerical value modified up or down (higher or lower) by ten percent (%).

[0148] The term "conventional DC" as used herein refers to dendritic cells derived from common DC progenitors (CDP). Conventional dendritic cells (DC) are divided into two types, type I and type II. This term generally does not include plasmacytoid dendritic cells. They are present in tissues and are activated and migrate to draining lymph nodes after tissue infection or injury to promote an adaptive immune response.

[0149] The term "CAR-DC" as used herein refers to dendritic cells expressing a chimeric antigen receptor (CAR).

[0150] The term "chimeric antigen receptor (CAR)" as used herein refers to a recombinant fusion protein that comprises an extracellular ligand-binding domain (antigen recognition domain, antigen-binding domain), a transmembrane domain, and an intracellular signal transduction domain.

[0151] The term "cross-presentation" as used herein refers to the process by which CAR dendritic cells (CAR-DC) internalize, process, and present an antigen (such as a tumor cell antigen) in complex with MHC class I molecules on the cell surface. Then, the antigen is recognized by T cells.

[0152] The term "cross-priming" as used herein refers to the process by which T cell recognition of an antigen leads to T cell activation. The activated T cells are capable of enhancing proliferation, persistence, and / or targeting to enhance cytotoxicity against tumor cells expressing such antigen.

[0153] The term "allogeneic or xenogeneic" as used herein refers to a source that is different from the host.

[0154] The term "autologous" as used herein refers to a source obtained from the same individual.

[0155] The term "construct" as used herein is generally understood to mean any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, self-replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, from any source, capable of genomic integration or self-replication, and containing one or more operably linked nucleic acid molecules.

[0156] The term "composition" as used herein refers to a combination of an immunotherapeutic cell population and one or more pharmaceutically acceptable carriers.

[0157] The term "therapeutically effective amount" should be understood to mean an amount of an antibody or antigen-binding fragment sufficient to reduce or inhibit one or more symptoms of a cell proliferative disease below the level observed clinically and recognized as characteristic of the disease. Those skilled in the art will appreciate that the therapeutically effective amount will vary depending on the particular antibody, fragment, and / or the particular subject and / or the type or severity or level of the disease. Accordingly, the term should not be construed as limiting the invention to a specific quantity.

[0158] The terms "treat", "treating", "treatment" and their grammatical variants as used herein refer to the administration of a regimen, course, process or intervention to an individual patient with the expectation of obtaining a physiological response or prognosis in said patient. Since not every patient treated will respond to a particular treatment regimen, course, process or therapy, treatment does not require that every patient or patient population achieve the desired physiological response or prognosis. Thus, a particular patient or patient population may not respond or may respond inadequately to treatment.

[0159] The terms "tumor" or "cancer" are used interchangeably and refer to a cell or cell population having a growth, proliferation or survival rate that is higher than that of normal corresponding cells, e.g., having a disorder of cell proliferation or differentiation. Typically, such growth is uncontrolled.

[0160] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, and / or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0161] Dendritic cells

[0162] Previously, dendritic cells (DCs) were described as universal "omnipotent" antigen-presenting cells, but an important aspect of controlling the immune response is the existence of several different types of DCs, each of which specifically responds to a particular pathogen and interacts with a specific subset of T cells. The term includes DCs that initiate an immune response and / or present an antigen to T lymphocytes and / or provide any other activation signal required to stimulate an adaptive immune response to T cells.

[0163] As a cell type of different taxa, they are widely distributed throughout the human body. They have evolved into various cell subsets with different phenotypes and functions, which can be roughly divided into conventional dendritic cells (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs (moDCs). Conventional cDCs can be further divided into type 1 cDCs (cDC1) and type 2 cDCs (cDC2).

[0164] In addition to expressing CD11c and MHC-II, other cell surface markers can be used to distinguish DC subsets. cDC1 can be identified as CD141+ DC. In addition, some cDC1 markers are shown to be conservatively expressed in both mice and humans, such as CLEC9A, CD26, CADM1, and BTLA. In peripheral lymphoid organs and non-lymphoid organs (such as the lung, intestine, and lymph nodes), cDC1 can also be identified as CD103+CD11b- cDC. cDC1 in mice and humans has excellent antigen cross-presentation properties, which are crucial for the response of CD8+ T cells to antigens of viral or tumor origin.

[0165] The cDC2 subset is defined by the presence of CD11b, Sirpα (CD172a), and CD4 on the cell surface. cDC2 in peripheral lymphoid organs and non-lymphoid organs is CD103+CD11b+.

[0166] pDCs can be identified by the expression of Bst2, B220, and SiglecH, and they also express relatively low levels of CD11c and MHC-II. Under inflammatory conditions, moDCs can respond to pro-inflammatory chemokines such as CCL2 and CCL7 and upregulate the expression of cell surface MHC-II, CD11c, and CD11b, thus being easily confused with cDC2. Other markers such as CD64 and MAR-1 can be used to distinguish moDCs and cDC2.

[0167] In addition to directly or indirectly presenting antigens to T cells, DCs can also produce a large number of cytokines in response to environmental stimuli, ultimately fine-tuning T cells to promote an effective immune response.

[0168] Given the natural adjuvant properties of DCs and their unique attributes in promoting T cell priming and recruitment to solid tumors, DCs have long been the focus of cancer therapies.

[0169] This disclosure is based on the discovery that DCs genetically modified to express a chimeric antigen receptor (CAR) can uptake tumor cells through phagocytosis, micropinocytosis, or receptor-mediated endocytosis and promote cytotoxicity through T cell cross-priming. CAR dendritic cells (CAR-DCs) can be used to treat various cancers and malignancies, including solid tumors. Previously described CAR macrophages (CAR-Ms) do not successfully cross-prime T cells after phagocytosing or pinocytosing tumor cells and do not successfully eliminate solid tumors in clinical trials.

[0170] This disclosure describes a method for generating functional CAR-DCs that can selectively phagocytose tumor cells and cross-present endogenous tumor antigens in a manner that cross-primes tumor antigen-reactive T cells. Thus, CAR-DCs can generate an adaptive immune response for targeted killing of CAR-Ag+ and CAR-Ag- tumors or cancer cells.

[0171] CAR-DCs can be generated by exposing isolated DC progenitors such as stem cells (pluripotent, multipotent, hematopoietic, or other stem cells), multipotent progenitors, common myeloid progenitors (CMPs), myeloid dendritic cell progenitors (MDPs), common dendritic cell progenitors (CDPs), bone marrow monocytes, peripheral blood mononuclear cells (PBMCs), or spleen cells to a DC proliferation stimulant such as Flt3L. Then, the cells can be transduced with the CAR of interest and further exposed to the DC differentiation factor Flt3L for a sufficient period of time before treatment to generate dendritic cell-like cells (DC-like cells). For example, the cells can be exposed to Flt3L for about 2 to 15 days to promote differentiation.

[0172] The present disclosure provides modified DCs. Numerous studies have shown that DCs are often restricted in the tumor microenvironment and even in cancer patients. In addition, even if DCs are present, they may induce tolerance or rejection of antigens, or may not work at all, because they usually do not have a strong signal indicating that tumor cells are foreign or threatening and need to be eliminated.

[0173] The dendritic cell may be a subpopulation of dendritic cells. For example, the subpopulation of DC may be plasmacytoid DC (pDC), monocytic DC, myeloid / conventional / classical DC1 (cDC1), or myeloid / conventional / classical DC2 (cDC2).

[0174] The present disclosure provides modified conventional type I dendritic cells (cDC1) that can be generated by differentiating CAR-DC. Such cells can effectively perform antigen cross-priming. Antigen cross-priming refers to antigen presenting cells obtaining and cross-presenting extracellular antigens (in this case, the extracellular antigens are obtained from tumors) to stimulate antigen-specific naive cytotoxic CD8 T cells into activated cytotoxic CD8 T cells. Antigen cross-presentation refers to the ability of cells to present internalized antigens on type I major histocompatibility complex molecules (MHC-I). It is well known that antigen cross-presentation and cross-priming are necessary for effective adaptive immune responses against tumor cells.

[0175] cDC1 can be identified by flow cytometry based on specific surface protein expression markers described herein and confirmed by its T cell cross-priming function against phagocytic cell-associated antigens. In one embodiment, cDC1 can be identified based on NK1.1 - 、CD19 - 、TCRb - , SinglecH - 、MHC-II + 、CD11c + 、XCR1 - and CD172a - In another embodiment, cDC1 generated in vitro can be sorted according to the expression of one or more of the single cH - 、MHC-II + 、CD11c + 、XCR1 + and CD172a - The expression of one or more of the above proteins is sorted.

[0176] Chimeric antigen receptor (CAR)

[0177] Despite extensive research efforts to determine the optimal CAR design, no universal CAR structure has been found yet. Some studies have shown that minor alterations can have a significant impact on the treatment prognosis.

[0178] CARs are designed to contain an extracellular target-binding domain (such as an antigen-binding domain, a tumor-binding domain), a hinge region, a transmembrane domain that anchors the CAR to the cell membrane, and one or more intracellular domains that transmit activation signals. Traditionally, CARs have used intracellular signaling motifs derived from the T cell receptor (TCR), which have been optimized to promote T cell proliferation and function, but these motifs are not relevant to the use of CAR-cDC1 cells, which require cDC1-specific effector functions such as tumor cell phagocytosis, antigen processing and presentation, co-stimulatory receptor expression, and cytokine secretion. The CD28 and 4-1BB co-stimulatory signaling domains have been the most widely used in CAR-T cell clinical trials, but these two domains have shown very different outcomes in CAR-T cells. Clinical trials for B cell malignancies have shown that CD28-based CAR-T cells are usually undetectable after 3 months, while 4-1BB-based CAR-T cells can persist in patients for several years after treatment (Fraietta et al, 2018, Nat Med 24, 563-571). Some studies have shown that the transmembrane domains of certain co-stimulatory molecules can participate in synapse formation or T cell signaling.

[0179] The inventors surprisingly found that, in the case of DC cells, the arrangement of the intracellular signaling domain affects the activity of CAR-DC. In a preferred arrangement of the present application, connecting the proximal intracellular domain to its corresponding transmembrane domain results in better performance. In the present disclosure, the CAR comprises an intracellular domain that is responsible for intracellular signal transduction after extracellular ligand-binding domain binds to the target, thereby activating immune cells and immune responses. The signaling (transduction) domain is responsible for activating at least one normal effector function of the immune cells expressing the CAR. For example, the effector functions of DC can be increased survival rate, differentiation, cytokine secretion, phagocytosis, and / or antigen cross-presentation. Therefore, the "signaling domain" as used herein refers to the part of a protein that transmits the effector signal function signal and guides the cell to perform a specific function. According to the number of co-stimulatory signaling domains, CARs can be classified into first-generation (only CD3ζ), second-generation (one co-stimulatory domain + CD3ζ), or third-generation CARs (more than one co-stimulatory domain + CD3ζ). The co-stimulatory signaling domains used in the present CAR-DC can also be used to increase or decrease the function, persistence, or proliferation of cells. Introducing the CAR molecule into DCs successfully redirects the DCs to have additional antigen specificity and provides the necessary signals to drive overall DC activation and function.

[0180] One of the main physiological activation mechanisms of cDC1 is the recognition of pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs). cDC1 expresses high levels of TLR3, TLR4, TLR9, TLR11, and TLR13, which recognize different PAMPs usually derived from viruses and bacteria. The intracellular signaling molecule is preferably a motif derived from TLR4. The intracellular signaling molecule is preferably the Toll / interleukin-1 receptor (TIR) from TLR4.

[0181] TLR4 agonists have been successfully used to treat colorectal cancer and lung cancer, but other synthetic ligands have also been tested in the treatment of different tumors. For example, TLR7 and TLR8 ligands have been used to treat chronic lymphocytic leukemia and skin cancer. Similarly, TLR9 ligands have been used to treat lymphoma, kidney cancer, skin cancer, and brain cancer. Therefore, it is expected that different TLRs will initiate different immune responses through differences in cytokine production against different types of cancer.

[0182] The TIR domain adopts a dianthrin-like fold, which is characterized by a central five-stranded parallel β-sheet, with five α-helices surrounding both sides of the β-sheet and connected by loops. Its structure is similar to the commonly used CD28 IC and / or CD3ζ co-stimulatory signaling domains. Therefore, using and maintaining its dianthrin-like fold structure in the CAR may be the key to retaining its signal function.

[0183] In response to ligand binding, TLRs produce a strong pro-inflammatory response. The inventors noticed that in cDC1s, TLR4 stimulation promotes both the MyD88-dependent and TRIF-mediated pathways, leading to their maturation and promoting the production of Th1 cytokines by activating the NF-kB, AP-1, and IRF transcription complexes. TLR4 signaling is also necessary for DCs to process and cross-present dying tumor cell antigens by inducing the transfer of phagosomes from the endosomal recycling compartment to MHC-I. The selection of this TIR domain in the CAR is an important factor of the present invention. The presence of TLR signaling is positively correlated with the therapeutic prognosis of radiotherapy and chemotherapy.

[0184] The differentiation of conventional type 1 dendritic cells (cDC1s) from bone marrow progenitors depends on FLT3 (FMS-like tyrosine kinase 3) signaling. Previous studies have shown that integrating the intracellular domain of FLT3 into the intracellular domain of a chimeric antigen receptor (CAR) construct helps to generate a large number of cDC1 cells from DC progenitors (as described in WO2021 / 127024A1). However, the ability of cDC1 cells generated by this method is limited and fails to exhibit strong maturation signals when encountering tumor cells. The TLR intracellular domain selected in the present invention overcomes this limitation.

[0185] In some embodiments, the CAR construct domains may be operably linked by a linker. The linker can be any nucleotide sequence capable of linking the domains described herein. For example, the linker can be any amino acid sequence suitable for this purpose (e.g., 8-80 amino acids in length, depending on the target binding domain used).

[0186] The present disclosure provides a modified mutant cell comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular domain that specifically binds to a ligand expressed on the surface of a tumor cell, a transmembrane domain, and a signaling domain derived from a Toll-like receptor (TLR), and the signaling domain is combined with another different co-stimulatory signaling domain, for example, to generate a "third-generation" CAR comprising two or more signaling domains. In some embodiments, the CAR further comprises another co-stimulatory signaling domain. In some embodiments, the other co-stimulatory signaling domains can be selected from the group consisting of: the signaling domains of CD28, 4-1BB (CD137), OX40, CD27, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, the ligand specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2R-γ, IL7R-α, ITGA6 (VLA6, CD49f), CD49a, VLA-1, IGTA4 (CD49D), ITGAD, ITGAM (CD11b), ITGAE (CD103), ITGAL (CD11a), ITGA X (CD11c), ITGB1 (CD29), ITGB2 (CD18), LFA-1 (CD11a), ITGB7, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-a, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NCR2, NCR3, NCR1 or NKG2D.

[0187] Each co-stimulatory signaling domain may have unique properties. The affinity of the scFv, the intensity of antigen expression, the likelihood of off-target toxicity, or the differences in the disease to be treated may all affect the selection of this domain.

[0188] Typically, an extracellular antigen-binding domain is linked to a signaling domain of a chimeric antigen receptor (CAR) via a transmembrane domain. The transmembrane domain traverses the cell membrane, anchoring the CAR to the DC surface and connecting the extracellular ligand-binding domain to the signaling domain, thereby affecting CAR expression on the DC surface. In the present disclosure, a notable property of the transmembrane domain is its ability to be expressed on the DC surface, thus guiding immune cells to respond to pre-determined target cells. The transmembrane domain can be from natural or synthetic sources. Alternatively, the transmembrane domains of the present disclosure can be derived from any membrane-bound or transmembrane protein.

[0189] In some embodiments, the transmembrane domain can be selected from the group consisting of: TNFR2, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4 (CD49D), IA4, ITGA6 (VLA-6, CD49f), ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a, LFA-1), ITGAM (CDllb), ITGA X (CDllc), ITGB1 (CD29), ITGB2 (CD18), ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D or NKG2C transmembrane domains.

[0190] The inventors surprisingly found that, in some embodiments, the selection of the transmembrane domain is independent of its expression pattern. For example, although CD28 is mainly expressed on naive T cells (about 80% of human CD4 +T cells and 50% CD8 + is constitutively expressed on T cells), and it is not currently known whether it is expressed on DC cells, but the use of the CD28 transmembrane protein is effective in the present invention.

[0191] In some embodiments, the transmembrane domain is from a synthetic source, i.e., designed to modify certain properties of the CAR. In some embodiments, the transmembrane domain can be selected from one of the constructs described in WO2021229581, which is incorporated herein by reference in its entirety.

[0192] In some embodiments, the synthetic transmembrane domain is designed to enhance the following properties: oligomerization, specificity (by guiding the DC cell to respond to a predefined target cell), inhibition of constitutive signaling, or a combination of these properties.

[0193] The transmembrane domain may further comprise a hinge region between the extracellular antigen-binding domain and the transmembrane domain. The term "hinge region" generally refers to any oligopeptide or polypeptide that connects the transmembrane domain to the extracellular antigen-binding domain. The hinge region can be used to provide greater flexibility and accessibility to the extracellular antigen-binding domain. The hinge generally provides stability for the effective expression and activity of the CAR. The hinge (which also binds to the transmembrane domain) also ensures proper proximity to the target. The hinge region can contain up to 300 amino acids, preferably 5 to 100 amino acids, and most preferably 8 to 50 amino acids. The hinge region can be derived from all or part of a naturally occurring molecule, such as CD28, 4-1BB (CD137), OX-40 (CD134), CD3C, the alpha or beta chain of the T cell receptor, CD45, CD4, CD5, CD8β, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, ICOS, CD154, or all or part of an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or the hinge region can be a completely synthetic hinge sequence.

[0194] In some embodiments, the antigen-binding domain is linked to the transmembrane domain via a hinge region. In some embodiments, the hinge region comprises a CD8α hinge region. In another embodiment, the hinge region comprises a sequence that is at least 70% or at least 80% or at least 90% or at least 95% identical to SEQ ID NO:2. In another embodiment, the hinge region comprises the sequence SEQ ID NO:2 or consists of SEQ ID NO:2.

[0195] The CAR may comprise an antibody or an antigen-binding domain. The antigen-binding domain may comprise any domain or fragment thereof that binds to an antigen expressed by the targeted cell type, such as an antigen expressed by tumor cells (see, e.g., Saar Gill et al., U.S. Application No. US15 / 747,555, which is incorporated herein by reference in its entirety). For example, the antigen-binding domain may be an antibody (from humans, mice, or other animals), a humanized antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a camelid antibody, a native receptor or ligand, or a fragment thereof. For example, the antigen-binding domain may be a single-chain variable fragment (scFv) of an antibody. The antigen-binding domain may be directed to various tumor-associated proteins. The tumor antigen may be selected from the group consisting of: HER2, EphA2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1 antibody, CD19, CD20, CD123, CD22, CD30, SlamF7, CD33, epidermal growth factor receptor (EGFR), BCMA, GD2, CD38, PSMA, B7H3, EPCAM, IL-13Rα2, PSCA, mesothelin, LewisY, LewisA, CIAX, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), aberrant products of ras or p53, or other proteins found at higher levels on the surface of tumor cells than in key normal tissues. Any protein expressed on tumor cells may be used in the tumor-related embodiments described herein. Antigen sources include, but are not limited to, oncoproteins. The antigen may be expressed as a full-length protein or as a partial protein. The full-length protein or a portion thereof may be native or mutagenized.Non-limiting examples of tumor antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cytomegalovirus (CMV)-infected cells (such as cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinases erb-B2,3,4 (erb-B2,3,4-), folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), k light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, Survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, integrin beta7, ICAM-1, CD70, CLEC12A, and ERBB.

[0196] In some embodiments, the antigen-binding domain comprises an antibody, an antibody fragment, or a receptor ligand. In some embodiments, the antigen-binding domain comprises an scFv, Fv, Fab, 'Fab')2, single-domain antibody (SDAB), VH or VL domain, or camelid VHH domain.

[0197] In one embodiment, the antigen-binding domain is a scFv. ScFvs are well known in the art and can be used as the binding moiety in various constructs (see, for example, Sentman 2014 Cancer J. 2015 6 - 159; Guedan 2019 Mol Ther Methods Clin Dev. 12 145 - 156). Any scFv known in the art or generated against an antigen using methods known in the art can be used as the binding moiety. The form of the scFv is generally two variable domains linked by a flexible peptide sequence, and the orientation can be VFI - linker - VL or VL - linker - VFI. The orientation of the variable domains within the scFv, depending on the structure of the scFv, may affect whether the CAR will be expressed on the DC surface or whether the CAR - DC will target the antigen and signal. In addition, the length and / or composition of the variable domain linker also affect the stability or affinity of the scFv. In some embodiments, the antigen - binding domain comprises an amino acid sequence that is at least 70% or at least 80% or at least 90% or at least 95% identical to SEQ ID NO: 10. In another embodiment, the antigen - binding domain comprises or consists of the SEQ ID NO: 10 sequence.

[0198] The TLR intracellular domain described herein can be used in CARs having more than one antigen - binding domain. Thus, in some embodiments, the CAR is a multi - specific CAR comprising two or more antigen - binding domains. The two or more antigen - binding domains can bind the same or different targets.

[0199] Genetic modification of DC

[0200] Gene modification of DCs can be achieved by transducing substantially syngeneic cell components with a recombinant DNA construct. In some embodiments, a retroviral vector (γ - retrovirus or lentivirus) is used to introduce the DNA construct into the cells. For example, the polynucleotide encoding the CAR can be cloned into a retroviral vector and expressed under the control of its endogenous promoter, the retroviral long terminal repeat, or a promoter specific to the relevant target cell type. Other viral or non - viral vectors can also be used.

[0201] When genetically modifying DCs to include CARs, retroviral vectors are generally used for transduction, but any other suitable viral vector or non-viral delivery system (such as lipid nanoparticles) can also be used. The CAR can be constructed together with co-stimulatory molecules (such as cytokines) in a single polycistronic expression cassette, multiple expression cassettes on a single vector, or multiple vectors. Elements for creating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRESs, such as FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-III IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (such as 2A peptides, such as P2A, T2A, E2A, and F2A peptides). In other embodiments, any of the vectors or CARs disclosed herein can include a P2A peptide. The combination of a retroviral vector with an appropriate packaging line is also suitable, where the capsid protein will have the function of infecting human cells. There are various cell lines known to produce amphotropic viruses, including but not limited to PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Nat. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also applicable, such as VSVG, RD114, or GALV envelope pseudotyped particles, and any other particles known in the art.

[0202] Possible transduction methods also include co-culturing the cells directly with producer cells, such as by the method of (Bregni, et al. (1992) Blood 80:1418-1422), or culturing with only the viral supernatant or concentrated vector stock solution (with or without appropriate growth factors and polycations), such as by the methods of (Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J Clin. Invest. 89:1817).

[0203] The resulting cells can be grown under conditions similar to those of unmodified cells, enabling the modified cells to be expanded and used for various purposes.

[0204] Any targeted genome editing method can be used to implant the CARs of the present disclosure into one or more endogenous gene loci of the immune response cells of the present disclosure. In some embodiments, the CRISPR system is used to deliver the CARs of the present disclosure to one or more endogenous gene loci of the immune response cells of the present disclosure.

[0205] The present disclosure also encompasses nucleic acid sequences encoding the CARs described herein, as well as signal peptides. Signal peptides can direct the transport of secreted or transmembrane proteins to the cell membrane and / or cell surface, enabling the correct localization of the polypeptide. In particular, the signal peptides of the present disclosure can direct the attached polypeptide (i.e., the CAR receptor) to the cell membrane, where the extracellular antigen-binding domain of the attached polypeptide is presented on the cell surface, the transmembrane domain of the attached polypeptide spans the cell membrane, and the signaling domain of the attached polypeptide is located in the cytoplasm of the cell. In one embodiment, the signal peptide is the signal peptide of human CD8α. In another embodiment, the signal peptide is a fragment of at least 10 amino acids of the CD8α signal peptide that directs the attached polypeptide to the cell membrane and / or cell surface.

[0206] The CAR-DCs of the present disclosure can comprise one or more different CAR constructs. For example, dual CAR-DCs can be generated by cloning the protein-coding sequence of a first extracellular antigen-binding domain into a viral vector containing one or more co-stimulatory domains and a signaling domain, and cloning the second protein-coding sequence of a second extracellular antigen-binding domain into the same viral vector containing one or more additional co-stimulatory domains and a signaling domain, thereby obtaining a plasmid that expresses two CAR constructs from the same vector. Tandem CAR-DCs are DCs with a single chimeric antigen polypeptide that comprises two different extracellular antigen-binding domains capable of interacting with two different cell surface molecules, wherein the extracellular antigen-binding domains are linked together by a flexible linker and share one or more co-stimulatory domains, and wherein the binding of the first or second extracellular antigen-binding domain signals through one or more co-stimulatory domains and a signaling domain.

[0207] Adaptive T - cell response

[0208] The CAR-DCs described herein are capable of eliciting an adaptive immune response in a subject. An adaptive anti-tumor T cell response can be initiated or enhanced through antigen cross-presentation or cross-priming by the CAR-DCs. In one embodiment, the adaptive anti-tumor T cell response can include an enhancement of T cell function. For example, T cell function can be evaluated by a cytotoxic T lymphocyte assay (CTL), in which effector T cells are mixed with target tumor cells at increasing ratios for a period of time (typically 4 hours), and tumor cell killing is quantified by tumor luciferase activity. The adaptive anti-tumor T cell response also encompasses an increase in T cell activation or proliferation. For example, the proliferation or activation markers (such as cytokine release) of CD4 and CD8 T cells can be evaluated by FACS analysis to measure T cell activation or proliferation.

[0209] A successful adaptive anti-tumor T cell response can result in tumor cell cytotoxicity, further tumor cell uptake, and a reduction in tumor volume. The anti-tumor T cell response can directly eliminate antigen-positive (Ag + ) tumors targeted by the CAR, and indirectly eliminate CAR-Ag - tumor cells (which cannot be directly recognized by the CAR) through cross-presentation and epitope spreading. Epitope spreading refers to the expansion of the immune response such that the specificity of T cells and antibodies extends beyond the antigen that initially triggered the immune response. For example, epitope spreading can result in tumor cells that do not express the CAR-targeted antigen being targeted by T cells.

[0210] Accordingly, the present disclosure provides a method of stimulating an adaptive anti-tumor T cell response in a subject, wherein the method generally comprises administering to the subject an effective amount of the CAR-DCs described herein. The CAR-DCs target tumor or cancer cells (through phagocytosis, macropinocytosis, or receptor-mediated endocytosis) to uptake proteins associated with the tumor cells. The tumor or cancer cells and cross-presented tumor antigens are presented to the T cells of the subject. Thus, the CAR-DCs directly target antigen-positive (Ag + ) tumors or cancer cells for elimination, and / or indirectly target CAR antigen-negative (Ag - ) tumors or cancer cells for elimination through cross-presentation and epitope spreading.

[0211] Compositions

[0212] The present disclosure also provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a plurality of CAR-DCs and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a diluent, binder, filler, buffer, pH regulator, disintegrant, dispersant, preservative, lubricant, taste masking agent, flavoring agent, or coloring agent.

[0213] Compositions containing the CAR-DCs of the present disclosure can be provided in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to a selected pH value. The liquid or viscous compositions can contain a carrier, which can be a solvent or a dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0214] If desired, sterile injectable solutions can be prepared by adding the CAR-DCs to the required amount of a suitable solvent and varying amounts of other components. Such compositions can be used in admixture with suitable carriers, diluents or excipients, such as sterile water, physiological saline, dextrose, dextran or the like. The compositions can also be lyophilized. Other auxiliary substances can also be included, such as emulsifying agents, wetting agents, dispersing agents, pH buffering agents, gelling agents, preservatives or coloring agents. Various additives that enhance the stability and sterility of the compositions can be added, including antibacterial preservatives, antioxidants, chelating agents and buffering agents.

[0215] The number of cells administered varies depending on the subject being treated. In one embodiment, about 10 3 to about 10 10 , about 10 5 to about 10 9 or about 10 6 to about 10 8 of the CAR-DCs of the present disclosure are administered to human subjects. More effective cells can be administered in smaller numbers. In certain embodiments, at least about 1x10 8 , about 2x10 8 , about 3x10 8 , about 4x10 8 or about 5x10 8 of the CAR-DCs of the present disclosure are administered to human subjects. In certain embodiments, about 1x10 7 to 5x10 8 of the CAR-DCs of the present disclosure are administered to human subjects. How to accurately determine the effective dose may depend on specific factors for each subject, including their body size, age, gender, weight and the medical condition of the specific subject. Those skilled in the art can readily determine the dose based on the present disclosure and knowledge in the art.

[0216] One skilled in the art can easily determine the amounts of cells and optional additives, vehicles, and / or carriers in the composition, as well as the amounts administered in the methods described herein. Typically, any additive (other than the active cells and / or agents) is present in phosphate buffered saline at a level of from 0.001 to 50% (by weight), and the active ingredient is present at the microgram to milligram level, such as from about 0.0001 to about 5% by weight, from about 0.0001 to about 1% by weight, from about 0.0001 to about 0.05% by weight, or from about 0.001 to about 20% by weight, from about 0.01 to about 10% by weight, or from about 0.05 to about 5% by weight. For any composition to be administered to an animal or human, the following can be determined: toxicity, e.g., by determining the lethal dose (LD) and the median lethal dose (LD50) in a suitable animal model (such as a rodent, e.g., a mouse); the dose of the composition, the concentration of the components therein, and the time of administration of the composition, such that a suitable response is elicited. These determinations do not require undue experimentation, given the common general knowledge in the art, the present disclosure, and the documents cited herein. Moreover, the duration of continuous administration can be determined without undue experimentation.

[0217] The compositions described herein can be provided to a subject systemically or parenterally for inducing and / or enhancing an immune response against an antigen and / or treating and / or preventing a neoplasm, a pathogen infection, or an infectious disease.

[0218] In certain embodiments, the CAR-DCs or compositions comprising CAR-DCs of the present disclosure are directly injected into the relevant tumor or organ (e.g., the organ affected by the neoplasm). Alternatively, the CAR-DCs or compositions comprising CAR-DCs of the present disclosure can be provided indirectly to the relevant organ, e.g., by administration to the circulatory system (such as the tumor vasculature). Expansion agents and differentiation agents can be provided before, during, or after administration of the cells or composition to increase the production of T cells, NK cells, or CTL cells in vitro or in vivo.

[0219] The CAR-DCs of the present disclosure can be administered in any physiologically acceptable vehicle, typically by intravascular administration, but they can also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (such as lymphatic vessels). The CAR-DCs of the present disclosure can comprise a purified cell population. Those skilled in the art can readily determine the percentage of the CAR-DCs of the present disclosure in the population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable purity ranges for the population containing the CAR-DCs of the present disclosure are from about 50% to about 55%, from about 5% to about 60%, from about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Those skilled in the art can adjust the dosage at any time (for example, a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or similar means.

[0220] The administration of the composition can be autologous or allogeneic. For example, CAR-DCs or progenitor cells can be extracted or obtained from one subject and then administered to the same subject or a different compatible subject.

[0221] Treatment

[0222] The present disclosure also provides methods for treating cancer in a subject or reducing or preventing cancer recurrence in a subject, which comprise administering an effective amount of the modified dendritic cells or CAR-DCs described herein, which target antigens expressed by cancer cells. In one embodiment, a subject is administered modified dendritic cells according to any one of the first to fourth aspects. In one embodiment, a subject is administered the pharmaceutical composition according to the sixth aspect. In one embodiment, a subject is administered a CAR comprising any one of the sequences of SEQ ID NO:7, 8, 9, 11, 12 or 13, 17, 18, 19.

[0223] Cancer returning after primary treatment is recurrence. This can occur weeks, months, or even years after treating the primary or original cancer.

[0224] The methods described herein are particularly effective for treating semi-solid tumors or solid tumors. In clinical trials to date, the complete response rate of traditional chimeric antigen receptor (CAR) T cells to solid tumors is only 1%. Solid tumors cannot be recognized by CAR-Ts if not all cells express the target antigen. Therefore, if an adaptive immune response can be successfully generated in a patient, the failures of these two immunotherapies can be overcome.

[0225] In one embodiment, the cancer is selected from the group consisting of: lung cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, ovarian cancer, skin cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, brain cancer, glioblastoma, neuroblastoma, laryngeal cancer, esophageal cancer, bladder cancer, and head and neck cancer.

[0226] In one embodiment, the cancer is a hematological malignancy. Hematological malignancies include leukemia, lymphoma, multiple myeloma, and their subtypes. There are various classification methods for lymphoma, usually based on the basic type of malignant cells, including Hodgkin lymphoma (usually a cancer of Reed-Sternberg cells, but sometimes also originating from B cells; all other lymphomas are non-Hodgkin lymphoma), B-cell lymphoma, T-cell lymphoma, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, and other lymphomas defined herein and known in the art.

[0227] B-cell lymphoma includes, but is not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and other lymphomas defined herein and known in the art.

[0228] T-cell lymphoma includes T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and other diseases defined herein and known in the art.

[0229] Leukemia includes acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (sometimes classified as lymphoma), and other leukemias defined herein and known in the art. Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0230] Administering the CAR-DCs described herein to a subject can be by one or more of subcutaneous injection, intradermal injection, intratumoral injection, intranodular injection, intramedullary injection, intramuscular injection, intravenous injection, or intralymphatic injection or intraperitoneal injection. In one embodiment, it is administered intratumorally.

[0231] In another aspect, the present disclosure also provides the use of chimeric antigen receptor-expressing dendritic cells (CAR-DCs) in the preparation of a medicament for stimulating the adaptive immune response of a subject, the cells comprising:

[0232] (i) an antigen-binding domain;

[0233] (ii) Transmembrane domain (TM); and

[0234] (iii) Intracellular domain (IC), comprising an intracellular signaling domain of Toll-interleukin receptor (TIR) and a costimulatory signaling domain, said costimulatory signaling domain being selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain.

[0235] In one embodiment, the drug can be used to treat the semi-solid tumors, solid tumors or hematological malignancies described herein.

[0236] Cancers treatable according to the methods of the present disclosure include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma (childhood cerebellar or cerebral), basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumors (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor (childhood, gastrointestinal), carcinoma of unknown primary, central nervous system lymphoma (primary), cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma of the Ewing tumor family, extracranial germ cell tumor (childhood), extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, germ cell tumor (childhood extracranial, extragonadal, ovarian), gestational trophoblastic tumor, glioma (adult, childhood brainstem, childhood cerebral astrocytoma, childhood visual pathway and hypothalamus), gastric carcinoma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma (childhood), intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell), lip and oral cavity cancer, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma (AIDS-related, Burkitt, cutaneous T-cell, Hodgkin, non-Hodgkin, primary central nervous system), macroglobulinemia (Waldenstrom), malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma (childhood), melanoma, intraocular melanoma, Merkel cell carcinoma, mesothelioma (adult malignant, childhood), metastatic cervical squamous cell carcinoma with occult primary, oral cancer, multiple endocrine neoplasia syndrome (childhood), multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative diseases, myelocytic leukemia (chronic), myeloid leukemia (adult acute, childhood acute), multiple myeloma, myeloproliferative diseases (chronic), nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone,Ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumors), ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, pancreatic cancer (islet cell), cancer of the sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pinealoma and supratentorial primitive neuroectodermal tumors (childhood), pituitary tumor, plasmacytoma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma (childhood), salivary gland cancer, sarcoma (Ewing family tumors, Kaposi, soft tissue, uterine), Sezary syndrome, skin cancer (non-melanoma, melanoma), skin cancer (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck with occult primary (metastatic), gastric cancer, supratentorial primitive neuroectodermal tumors (childhood), T-cell lymphoma (cutaneous), T-lymphocyte leukemia and lymphoma, testicular cancer, laryngeal cancer, thymoma (childhood), thymoma and thymic carcinoma, thyroid cancer, thyroid cancer (childhood), transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumors (pregnancy), primary site unknown (adult, child), transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer (endometrial), uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma (childhood), vulvar cancer, Waldenstrom macroglobulinemia or Wilms tumor (childhood).

[0237] The methods described herein are generally performed on subjects in need thereof. Subjects in need of the treatment methods described herein can be subjects who have, are diagnosed with, suspected of having, or are at risk of developing cancer or a proliferative disease, disorder, or condition. Whether treatment is needed is typically evaluated by medical history, physical examination, or diagnostic tests consistent with the relevant disease or condition.

[0238] The administration of the CAR-DC therapy described herein can be a single administration or can be administered in a course. For example, DC-based therapies can be administered once daily, weekly, biweekly, or monthly. For chronic conditions, treatment may be extended from weeks to months or years.

[0239] In some embodiments, the CAR-DC therapy can be administered concurrently with, before, or after conventional cancer treatment methods. In one embodiment, the CAR-DC therapy can be administered concurrently with or sequentially with another agent (such as an anti-cancer agent). In some embodiments, the anti-cancer agent is a chemotherapeutic agent, a radiotherapeutic agent, or an immunotherapeutic agent.

[0240] The CAR-DCs described herein can be administered in combination with drugs that suppress immunosuppressive pathways, including but not limited to inhibitors of TGFβ, interleukin 10 (IL-10), adenosine, VEGF, indoleamine 2,3-dioxygenase 1 (IDO1), indoleamine 2,3-dioxygenase 2 (IDO2), tryptophan 2,3-dioxygenase (TDO), lactate, hypoxia, arginase, and prostaglandin E2. In another embodiment, the CAR-DCs or populations of CAR-DCs of the present disclosure can be used in combination with T cell checkpoint inhibitors, including but not limited to anti-CTLA4 (such as ipilimumab), anti-PD1 (such as pembrolizumab, nivolumab, cemiplimab), anti-PDL1 (such as atezolizumab, avelumab, durvalumab), anti-PDL2, anti-BTLA, anti-LAG3, anti-TIM3, anti-VISTA, anti-TIGIT, and anti-KIR.

[0241] Those skilled in the art will appreciate that various changes and / or modifications can be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Accordingly, the present embodiments are to be considered in all respects as illustrative and not restrictive. The present disclosure includes the following non-limiting examples.

[0242] Examples

[0243] Those skilled in the art will appreciate that various changes and / or modifications can be made to the above-described embodiments and the following examples without departing from the broad general scope of the present disclosure. Accordingly, the present embodiments are to be considered in all respects as illustrative and not restrictive. Those skilled in the art should understand, in light of the present disclosure, that many changes can be made to the specific embodiments disclosed, and similar results can still be obtained without departing from the spirit and scope of the invention.

[0244] Materials and methods

[0245] Mice

[0246] All experiments were performed using wild-type (WT) C57BL / 6 mice or C57BL / 6 CD45.2 Zfp366 mice + / + or CD45.2 Zfp366 mice - / - maintained under specific pathogen-free conditions. The mice used in the experiments were between 8 and 12 weeks of age, and all experiments had to be approved by the ethics committee.

[0247] Generation of retroviral particles

[0248] Retroviral particles were generated by transient transfection of three plasmids into 293T cells. 3.5 x 10 6293T cells were seeded in a 10-cm culture dish and 10 ml of DMEM containing 10% FCS was added. One day later, the DMEM medium was replaced with pre-warmed OptiMEM medium containing 10% FCS. A plasmid mixture was prepared for each dish, which contained the packaging Gag / pol plasmid (1.2 μg), the envelope plasmid (VSV-G, 0.6 mg), and the transfer retroviral plasmid (3.6 mg), and then added to 100 μl of serum-free OptiMEM medium. 30 μl of FuGENE-6 reagent was added to this system and incubated at room temperature for 15 minutes. The plasmid / FuGENE mixture was added to the 293T cells and cultured overnight at 37 °C and 5% CO2. The next day, the medium was replaced with pre-warmed DMEM containing 10% FCS. After 48 hours, the virus-containing supernatant was harvested for transduction and used immediately or stored at -80 °C.

[0249] Cell transduction

[0250] Six-well plates were coated with RetroNectin (Takara) overnight at 4 °C. The retroviral supernatant was centrifuged onto the coated plates at 2000 G for 2 hours at 32 °C. Then, it was centrifuged at 1700 rpm for 20 minutes at 32 °C. After 48 hours, the transduction efficiency was detected by flow cytometry.

[0251] Cell line and primary cell culture

[0252] MutuDCs were derived from the spleens of CD11c:SV40LgT transgenic C57BL / 6 mice. The cells were cultured in IMDM containing 10% FCS and 50 mM β-mercaptoethanol. Parental and human HER2-transduced E0771 cells were cultured in DMEM containing 10% FCS.

[0253] Immunofluorescence analysis

[0254] Two days before immunofluorescence staining, CAR-transduced cDC1s were seeded on 8-well glass slides (ibdi) at a concentration of 1x10 3 cells / well. After the cells were washed, fixed, and blocked, they were incubated overnight in a cold room with anti-tubulin-Alexa647 or anti-MYC APC. After staining, the slides were washed and then fixed with ProLong Gold antifade mounting medium containing DAPI (Invitrogen) for confocal microscopy, imaged with a confocal microscope (Zeiss LSM 780), and then processed with ImageJ.

[0255] Heat - shock and irradiation - induced tumor cell apoptosis

[0256] Cell line (HER2+ or HER2 - E0771) were cultured in 10 cm dishes until 80% confluence. Cells were then detached with trypsin-EDTA solution, resuspended in 1 ml of DMEM, and subjected to heat shock (44 °C, 50 minutes) or irradiation (3000 rad). To label tumor cells with CTV, cells were washed and resuspended in 5 nM of CTV at 37 °C for 15 minutes after heat shock.

[0257] In vitro phagocytosis assay

[0258] Label apoptotic HER2 with CTV + or HER2 - E0771 tumor cells were then co-cultured with CAR-transduced BMDC or mutuDC at a 1:1 ratio in a U-bottom 96-well plate for a specified time. After co-culture, the plates were immediately placed on ice, stained with an antibody cocktail, and subjected to FACS analysis.

[0259] In vitro generation of DC

[0260] The hips, femurs, and tibias of the mice were crushed and placed in FACS buffer (PBS + 0.5% BSA, Sigma-Aldrich), and red blood cells were removed with ACK lysis buffer. BM cells were cultured at a concentration of 1.5 x 10 6 cells / ml in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine (GIBCO), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 100 U / ml penicillin / streptomycin (GIBCO), and containing FLT3L (200 ng / ml, BioXCell) for 7 days to generate conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC).

[0261] Flow cytometry

[0262] Bone marrow single cell suspensions were resuspended in FACS buffer and stained at 4 °C. All analyses were performed on a BD FACSCanto or BD LSRFortessa (BD Biosciences).

[0263] Cell sorting and magnetic enrichment

[0264] Harvested spleens were digested with collagenase (Worthington) and DNase I (Sigma). Red blood cells were removed with ACK lysis buffer. Light density cells were collected by density centrifugation procedures. CD11c + DC were enriched by CD11c magnetic beads (Miltenyi). Enriched CD11c+ The purity of the DC preparation is >90%. cDC1 was sorted as PI - NK1.1 - CD19 - TCRb - SiglecH - MHC-II + CD11c + XCR1 + CD172a - ; while cDC2 was sorted as PI - NK1.1 - CD19 - TCRb - SiglecH - MHC-II + CD11c + XCR1 - CD172a + 。DCs generated in vitro were sorted as PI - SiglecH - MHC-II + CD11c + XCR1 + CD172a - (cDC1) and PI - SiglecH - MHC-II + CD11c + XCR1 - CD172a + (cDC2). To enrich OVA-specific OT-I or OT-II cells, lymph nodes (LNs) were harvested and passed through a 70 mm mesh. Using BioMag goat anti-rat IgG beads (Qiagen), LN cells were negatively selected with a lineage Lin mixture containing monoclonal antibodies against MHC-II, B220, MAC-1, Ly6G, CD44, and TER119, and LN cells were positively selected with CD4 (for OT-II enrichment) or CD8 (for OT-I enrichment). Cell purity (>99%) was detected by analytical flow cytometry using CD8 or CD4, TCRva2 antibodies.

[0265] Antigen presentation and cross - presentation assay

[0266] For in vitro antigen presentation assays, purified OVA-specific SIINFEKL-specific CD8 + (OT-I T cells) were labeled with CellTrace TM Violet (CTV). 5x10 4OT-I T cells were co-cultured with purified CAR-transduced cDC1s, with or without irradiated (3000 rad) OVA-loaded HER2 + E0771 tumor cells. At 60 - 72 hours after stimulation, the number of proliferating T cells was measured by flow cytometry.

[0267] Design and construction of retroviral vectors

[0268] The coding fragments of anti-HER2 scFv, CD8 hinge, CD28 TM and mCherry were obtained by PCR from the anti-HER2-CD28 TM -CD28 IC -CD3ζ-mCherry CAR plasmid in the pMSCV backbone, using the following primers with 20 bp overlaps with the insert fragment.

[0269] Forward primer: GTCTGTAGCGACCCTTTGCAGGCAGCGG

[0270] Reverse primer: CACCCAAAAGGGATCCAGCCCCCTCGTG

[0271] The designed DNA insert fragment encoding the intracellular signaling tail was ordered from IDT (Integrated DNA technology). It has 20 bp overlaps with the backbone vector at the 3'-end and 5'-end.

[0272] Then the PCR products were assembled using Gibson cloning (NEB). After Sanger sequencing, the correct clones containing the designed plasmids were selected.

[0273] Forward primer: GGCTGGATCCCTTTTGGGTG

[0274] Reverse primer: ACAGACCTTGCATTCCTTTGGC Cloned sequences The sequences used in this study are shown in Table 1 below.

[0275] Table 1 Potential CAR domain sequences.

[0276]

[0277]

[0278]

[0279] As a control group, the inventors made a truncated CAR consisting of anti-HER2 scFv, Myc tag, CD8 hinge, CD28 TMand a truncated CD28 consisting of the sequence KRSR IC domain.

[0280] The sequences of exemplary CARs are shown below:

[0281] anti-HER2-CD28 TM -CD28 IC -The complete amino acid sequence of CD3ζCAR is as follows:

[0282]

[0283] anti-HER2-CD28 TM -CD28 IC -The complete amino acid sequence of TIR4 CAR is as follows:

[0284]

[0285] anti-HER2-CD28 TM -The complete amino acid sequence of TIR4-CD3ζCAR is as follows:

[0286]

[0287] anti-HER2-CD28 TM -The complete amino acid sequence of TIR4 CAR is as follows:

[0288]

[0289] anti-HER2-CD28 TM -CD28 IC -The complete amino acid sequence of CD3ζ-TIR4 CAR is as follows:

[0290] The complete amino acid sequence of the intracellular truncated CAR is as follows:

[0291]

[0292] anti-HER2-Pro-CAR2-CD28 IC -The complete amino acid sequence of CD3ζ-TIR4-CAR is as follows:

[0293] anti-HER2-Pro-CAR1-CD28 IC -The complete amino acid sequence of CD3ζ-TIR4-CAR is as follows:

[0294] Anti-HER2-Pro-CAR3-CD28 IC -The complete amino acid sequence of CD3ζ-TIR4-CAR is as follows:

[0295]

[0296] Anti-HER2-Pro-CAR4-CD28 IC -The complete amino acid sequence of CD3ζ-TIR4-CAR is as follows:

[0297]

[0298] Experiment

[0299] A series of novel CARs were generated that contain the Toll-interleukin receptor (TIR) intracellular signaling domain of TLR4 and can promote the activation of cDC1s after antigen recognition in the absence of adjuvant. In addition, experiments with different combinations of other intracellular domains were also conducted. Control experiments without the TIR domain helped to understand the role of this domain in the present invention.

[0300] The research results showed that expressing anti-HER2 CAR on conventional type I (cDC1) containing the TIR domain could increase their targeting of HER2 + tumor cells, thereby increasing the acquisition of tumor proteins. Most importantly, it could present other tumor antigens to CD8+ T cells.

[0301] Example 1 cDC1 controls tumor growth by enhancing T - cell response

[0302] It has been reported previously that the lack of DC-SCRIPT (a protein encoded by an 8 kb mRNA that is preferentially expressed in DCs; V Triantis et al., (2006) J Immunol 15; 176(2):1081) impairs the development and antigen presentation of cDC (S Zhang et al., (2021) Sci Immunol 2; 6(58)). To determine whether the impaired antigen presentation function of cDC1 affects the efficiency of adoptive T cell therapy, B16-F10 melanoma cells expressing OVA antigen (B16-OVA) were used to attack Ly5.2+ WT and Zfp366 - / - mice. Subsequently, Ly5.1+ CD8+ OT-1 T cells were intravenously injected into the tumor-challenged mice on day 7. Compared with Zfp366 - / - mice, adoptive T cell transfer significantly reduced the tumor burden in WT mice ( Figure 1 A). Compared with WT mice, Zfp366 - / -The number of tumor-infiltrating cDC1s in mice was low, and the numbers of pDCs and cDC2s within tumors remained unchanged ( Figure 1 B). Zfp366 - / - The numbers of Ly5.1+ OT-1 cells in mouse tumors, spleens, and tumor-draining lymph nodes (LNs) were also reduced (data not shown), indicating that impaired cDC1s reduced the efficiency of adoptive T cell therapy. Intriguingly, tumor-infiltrating T cells expressed high levels of PD-1 regardless of adoptive T cell therapy, suggesting that T cell function may still be inhibited within tumors. The numbers of CD4+ T cells, Tregs, macrophages, monocytes, and granulocytes did not change compared with the WT group, but the number of tumor-infiltrating NK cells in the Zfp366 - / - group was reduced after OT-1 treatment. The number of NK cells was positively correlated with the number of cDC1s in the tumor microenvironment ( Figure 1 C).

[0303] Example 2 cDC1 is essential for anti - tumor immunity

[0304] To investigate the effect of cDC1 on tumor growth, MC38 colorectal cancer cells were injected into WT, Zfp366 - / - , CD11c cre Irf8 fl / fl and CD11c cre Irf8 + / + mice. Compared with the WT control group, tumors grew fastest in CD11c cre Irf8 fl / fl mice and Zfp366 - / - mice (Figure 2A, Figure 2B). The largest tumor burden was observed in CD11c cre Irf8 fl / fl mice, which had a complete absence of cDC1s (Figure 2C). The number of tumor-infiltrating cDC1s was representatively negatively correlated with tumor size (Figure 2D). These data indicate that cDC1 is crucial for controlling tumor burden. The number of tumor-infiltrating cDC1s was positively correlated with the numbers of tumor-infiltrating CD8+ T cells and NK cells but not with the number of CD4+ T cells, suggesting that tumor-infiltrating DCs exhibit cross-talk with CD8+ T cells compared with CD4+ T cells, which confirms that the antigen cross-presentation ability of cDCs mainly affects CD8+ T cells.

[0305] In addition, when mice were challenged with SM1WT1 melanoma, CD11c cre Irf8 fl / flMice could inhibit tumor growth at an early stage but could not control tumor growth in the long term, indicating that in this model, cDC1 is necessary for controlling tumor progression in both the early and late stages of melanoma (Figure 2E). This was compared with Rag1 - / - mice and Rag2 - / - γC - / - mice, as well as CD11c cre Irf8 + / + -deficient mice and WT mice that cleared tumors.

[0306] Example 3 CAR - engineered cDC1 shows enhanced phagocytic activity against targeted antigen - positive tumors

[0307] The inventors hypothesized that expressing CAR in cDC1 could enhance their antigen recognition, uptake, and presentation capabilities and thus be used as a targeted anti-tumor therapy. To investigate the potential of CAR-mediated redirection of cDC1 phagocytosis, the inventors transduced the murine cDC1 cell line (mutuDC) (SA Fuertes Marraco et al., (2012) Frontiers in Immunology 3:331) with a second-generation anti-HER2 CAR, which encodes an anti-human HER2 single-chain variable fragment (ScFv), a transmembrane domain, and the intracellular domains of CD28 and CD3ζ ( Figure 3 A).

[0308] The anti-HER2 CAR was engineered into the pMSCV-mCherry retroviral vector, which was efficiently transduced into mutuDC and expressed the CAR. Purified mCherry-positive cDC1 could maintain a high mCherry signal during two months of culture. Since the CAR construct encodes a Myc tag, the inventors confirmed that mCherry-transduced cDC1 had cell surface expression of anti-HER2 ScFv ( Figure 3 B). Subsequently, the inventors tested whether the expression of the CAR would impair the characteristics of cDC1 by measuring the transcription of interferon regulatory factor 8 (IRF8) or the expression of the cell surface marker DEC205 ( Figure 3 C, Figure 3 D). This analysis showed that the expression of these markers was normal compared with WT mutuDC, indicating that the transduced cells maintained their cDC1 characteristics ( Figure 3 E, Figure 3 F). These data also showed that cDC1 could be efficiently transduced and express the CAR.

[0309] To test the function of anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-cDC1, the inventors measured human HER2+ Uptake of apoptotic bodies released by the E0771 breast cancer cell line. Flow cytometry confirmed the expression of HER2 in E0771 ( Figure 4 A). Intriguingly, in contrast to cDC1 transduced with empty CAR, the inventors found that after co - culturing with apoptotic HER2 + E0771 tumor cells for 2 hours, approximately 28% of anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR - transduced cDC1 were HER2 - positive, while none of the cDC1 transduced with the empty vector were ( Figure 4 B), indicating that CAR expression promoted more efficient uptake of tumor - associated antigens. Apoptotic HER2+ E0771 tumor cells were labeled with CTV and co - cultured with anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR - transduced cDC1 as described herein. Confocal imaging confirmed that cDC1 captured apoptotic bodies released by HER2 + E0771 tumor cells ( Figure 4 C). Measurement of CTV density by flow cytometry confirmed that transduction with anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR promoted cDC1 capture of HER2 + tumor - associated antigens ( Figure 4 D). According to time - course analysis, expression of anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR in cDC1 appeared at least 15 minutes after co - culture ( Figure 4 E and Figure 4 F).

[0310] This indicates that CAR expression on the surface of DC cells may enable them to increase uptake of tumor - derived apoptotic bodies. In addition, anti - HER2 - CD28 TM -CD28 IC -CD3ζ CAR expression promoted cDC1 recognition of HER2 + tumors, ultimately leading to capture of a large amount of tumor - associated antigens.

[0311] Example 4 CAR + cDC1 promotes cDC1 activation upon encounter with tumor - cell - associated antigens

[0312] After receiving the stimulatory signal, cDC1 will be activated and express co-stimulatory factors, cytokines, chemokines, and chemokine receptors. The most obvious features are CD80 and CD86, which are necessary for presenting antigens to T cells. To evaluate whether CAR expression in cDC1 would initiate the activation of cDC1 upon encountering tumor-associated antigens, the inventors used apoptotic HER2 + E0771 tumor cells or TLR ligands (such as LPS or CpG) to stimulate empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-transduced cDC1 for 6 hours.

[0313] Empty vector or anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-transduced cDC1's response to TLR ligands (LPS or CpG) was to increase the expression of MHC-II on their surface ( Figure 5 A), and MHC-II is a typical marker of DC maturation. Then the stimulation was extended for 16 hours to determine the expression of co-stimulatory factors CD80 and CD86. The results showed that anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-transduced cDC1 could be activated upon encountering apoptotic HER2 + E0771 tumor cells, but the empty vector control cDC1 maintained low expression of CD80 / 86 ( Figure 5 B and Figure 5 C). Morphologically, anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR-transduced cDC1 tended to form clusters after being stimulated by apoptotic HER2 + E0771 tumor cells, which is a typical manifestation of activated cDC1 ( Figure 5 D). These data show that the expression of CAR in cDC1 can promote the activation of cDC1 upon encountering tumor-associated antigens.

[0314] Most tumor-specific antigens have not been identified, so the CAR-T strategy cannot be directly adopted. However, the ability of cDC1 to acquire many potential unrecognized tumor-associated antigens through phagocytosis and cross-present them to CD8+ T cells may overcome this limitation. To study whether the expression of CAR in cDC1 can help these cells acquire independent tumor antigens, the inventors loaded OVA onto HER2 +E0771 cells were incubated with mutuDCs expressing anti-HER2 CAR in the presence of OT-I cells (transgenic CD8+ T cells specific for OVA peptide). As previously described, expression of anti-HER2 CAR in cDC1 enhanced the uptake of HER2 + tumor-associated antigen and OVA by cDC1, enabling efficient presentation of OVA-derived peptides to OT-I T cells and their proliferation ( Figure 5 E). In this experimental setting, empty CAR mutuDCs had limited ability to stimulate CD8 T cells. Additionally, expression of anti-HER2 CAR in cDC1 increased their sensitivity across a wide range of tumor cell numbers, as measured by OT-I proliferation and tumor killing ( Figure 5 F, Figure 5 G).

[0315] Collectively, these data show that expression of CAR on cDC1 can generate a broader CD8+ T cell response through cross-presentation of a panel of tumor cell-associated antigens.

[0316] Example 5 Bone - marrow - derived CAR cDC1 improves its phagocytosis in response to tumor - associated antigens

[0317] To enhance the anti-tumor immune response in patients, autologous cDC1s are needed clinically. It is important to understand whether the anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR retrovirus can efficiently transduce bone marrow-derived dendritic cells (BMDCs), whether the signaling tails affect the in vitro development of cDC1, and whether the resulting BMDCs can appropriately express CAR on their cell surface. To study these questions, the inventors cultured murine BM cells in the presence of FLT3L and transduced these cells with a standard anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR or a CAR variant with an intracellular domain truncation (containing a truncated CD28 signaling domain) and used them on day 4. On day 7, the differentiation of DCs was examined by flow cytometry. In BMDCs, the Myc tag was co-expressed with mCherry, indicating that anti-HER2 CAR could be transduced into BMDCs and expressed on the cell surface ( Figure 6 A).

[0318] The proportions of pDC, cDC1, and cDC2 generated from mCherry+Myc+ and mCherry-Myc-BMDCs were similar, as measured by anti-MYC antibody staining ( Figure 6 A). The transduction and expression levels of the CAR were similar among DC subtypes ( Figure 6B). More importantly, compared with cDC1 expressing truncated CAR, the expression of typical DC markers in cDC1 expressing anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR was similar ( Figure 6 C). These data indicate that in the presence of functional CAR, cDC1 derived from BM can be normally formed.

[0319] To investigate whether the expression of anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR could enhance the uptake of tumor-associated antigens, the inventors co-cultured BMDC with CTV-labeled HER2 + or HER2-E0771 apoptotic tumor cells for 2 hours and then evaluated antigen uptake by flow cytometry. Consistent with earlier reports (S Zhang et al., (2021) Sci Immunol 2; 6(58) eabf4432. doi: 10.1126 / sciimmunol.abf4432), cDC1 has a stronger ability to phagocytose phagocyte-associated antigens compared with pDC or cDC2. The expression of anti-HER2-CD28TM-CD28IC-CD3ζ CAR in cDC1 enhanced their uptake of HER2+ tumor-associated antigens but not HER2- tumor-associated antigens ( Figure 6 D). This data further demonstrates that anti-HER2-CD28 TM -CD28 IC -CD3ζ CAR cDC1 can also enhance the phagocytic ability of BM-derived cDC1 in a target-specific manner.

[0320] Example 6 Transduction of a novel TIR4 - containing CAR generates overloaded cDC1

[0321] TLR4 is a family member of pattern recognition receptors that defends against microbial infections by interacting with lipopolysaccharide (LPS). TLR4 contains a cytoplasmic Toll / interleukin-1 receptor (TIR) domain, and signal transduction is initiated by dimerization of the TIR domain after binding to LPS, which leads to the recruitment of adaptor proteins, thereby triggering the activation of downstream inflammatory and anti-pathogen responses. TLR4 signal transduction promotes cDC1 maturation and favors the production of Th1 cytokines by activating NF-kB, AP-1, and interferon regulatory (IRF) signals in the MyD88-dependent and TRIF-mediated pathways, thus promoting the generation of cytotoxic T lymphocytes (CTL) and Th1 cells (T Ve et al., (2012) Curr Drugs Targets 13:1360-1374; NJ Gay et al., (2014) Nature reviews. Immunology 14:546-558). Therefore, the inventors propose that TLR4 signal transduction can enhance the anti-tumor efficiency of CAR-cDC1.

[0322] The inventors hypothesized that replacing the CD28 IC -CD3ζ intracellular domain of CAR with the TIR4 domain of TLR4 could initiate an inflammatory response after DC encounters the CAR-targeted tumor antigen. To test this hypothesis, the inventors designed four novel CARs that target human HER2 by replacing the CD28 and / or CD3ζ signaling domains with the intracellular signaling (TIR) domain of Toll-like receptor 4 (TLR4), or attaching it to the C-terminus of the CD3ζ domain. The novel protein structures of the new CARs were predicted by AlphaFold 2 with high confidence (>90%) for the functional domains. The TIR domain adopts a folding pattern similar to that of dioscin, characterized by a central five-stranded parallel β-sheet with five α-helices surrounding it on both sides of the sheet, and these sheets are linked together by loops ( Figure 7 A). The extracellular anti-HER2 ScFv domain forms multiple anti-parallel β-sheets and is linked to the α-helical CD28 TM domain through the random coil of the CD8 hinge ( Figure 7 B). The intracellular CD28 signaling domain forms a random coil and attaches to CD3ζ to form a triple helix. To evaluate the necessity of the anti-HER2 CAR intracellular domain for the normal function of cDC1, the intracellular domain of the control CAR was truncated.

[0323] After truncating the intracellular domain, the α-helical structure of the transmembrane domain was maintained. Figure 7C). This property is crucial for the cell surface expression of truncated CARs. It is speculated that replacing the CD28 and / or CD3ζ domains with the TIR domain can retain its luteolin-like folding structure ( Figure 7 D- Figure 7 G), which is essential for retaining its signal transduction function. In addition, after modifying the intracellular domain, the extracellular anti-HER2-ScFV structure is well preserved ( Figure 7 D- Figure 7 G), which is unlikely to affect the affinity of anti-HER2-ScFV for the human HER2 antigen.

[0324] To test whether four different TIR4 CARs can be expressed on the surface of cDC1 cells, the CAR constructs were transduced into mutuDCs. Anti-Myc staining of mutuDCs transduced with anti-HER2 CAR showed that the anti-HER2 CAR was efficiently and continuously expressed on the cell surface ( Figure 8 A). Morphologically, anti-HER2-CD28 TM -TIR4-CD3ζ CAR, anti-HER2-CD28 TM -CD28 IC -TIR4 CAR or anti-HER2-CD28 TM -CD28 IC -CD3ζ-TIR4 CAR-transduced cDC1s showed enlarged dendrites without any stimulatory signal ( Figure 8 B), but this feature was not observed in cDC1s transduced with empty vector, intracellular domain truncated CAR, TIR4 CAR only, or CD28 IC -CD3ζ CAR.

[0325] Phagocytosis experiments showed that anti-HER2 CAR-transduced cDC1s readily internalized HER2 + tumor-associated antigens, especially anti-HER2-CD28 TM -TIR4-CD3ζ CAR, anti-HER2-CD28 TM -CD28 IC -TIR4 CAR or anti-HER2-CD28 TM -CD28 IC -CD3ζ-TIR4 CAR-transduced cDC1s ( Figure 8 C). This transfer is specific because the HER2 antigen can only be transferred from HER2 + tumor cells to anti-HER2 CAR-transduced cDC1s, but not to uninfected or empty vector CAR-transduced cDC1s ( Figure 8 D). The inventors observed that in HER2 +On stimulated cDC1, the expression level of MHC-II only increased slightly ( Figure 8 E). The inventors investigated the responsiveness of CAR-expressing cDC1 to HER2 + tumor cells and TLR4 agonists. All cDC1 transduced with the intracellular domain of anti-HER2 CAR responded to HER2 + tumor-specific antigens, increasing the expression of CD80 and CD86 on their surface, which are typical markers of DC maturation ( Figure 8 F and Figure 8 G). The expression levels of CD80 and CD86 were comparable to those induced by lipopolysaccharide (LPS) stimulation. Consistent with the morphological changes of cDC1 transduced with anti-HER2-CD28 TM -TIR4-CD3ζ CAR, anti-HER2-CD28 TM -CD28 IC -TIR4 CAR or anti-HER2-CD28 TM -CD28 IC -CD3ζ-TIR4 CAR, CD80 and CD86 were also more highly expressed in unstimulated cells compared to other treatment groups. The expression of PD-L1 remained unchanged in anti-HER2 CAR-transduced cDC1 after stimulation with HER2 - or HER2 + tumor cells ( Figure 8 H).

[0326] These data indicate that the anti-HER2-CD28 TM -TIR4-CD3ζ CAR, anti-HER2-CD28 TM -CD28 IC -TIR4 CAR and anti-HER2-CD28 TM -CD28 IC -CD3ζ-TIR4 CAR of the present invention: 1) are expressed on the surface of cDC1 cells; 2) endow cDC1 with the ability to efficiently phagocytose HER2 + tumor-specific antigens; 3) promote TLR4-dependent cDC1 activation after homologous recognition of their targets.

[0327] Example 7 Activation of anti - HER2 - CD28TM - CD28IC - CD3ζ - TIR4 CAR requires the presence of co - stimulatory factors

[0328] In integrating the TIR4 domain into anti-HER2-CD28 TM -CD28 IC-After the distal part of the CD3ζ CAR signaling domain, the inventors observed that TIR4 was autonomously activated in an antigen-independent manner. Activation of TLR4 in vivo generally requires the formation of a homodimer after binding to LPS. Hinge - CD28 TM region contains a cysteine residue that drives disulfide-linked receptor homodimerization. In addition, CD28 TM region contains a highly conserved polar YxxxxT motif that is crucial for optimizing dimerization and recruiting complementary endogenous co-stimulatory signals. However, adding TIR4 alone after the CD28 TM domain does not result in the (auto)activation of the TIR4 domain ( Figure 8 B), indicating the importance of the CD28 / CD3ζ distal signaling domain in promoting TIR4 activation.

[0329] Therefore, the inventors explored the possibility of designing different motifs in the transmembrane domain to prevent oligomerization in the absence of ligand and thus prevent TLR4 autoactivation. To this end, the inventors mutated the cysteine residue in the CD8a hinge region to alanine (mutant - TIR4), and in further experiments, changed the hinge region and replaced the YSLLVT motif in the transmembrane domain with different motifs, aiming to promote the dimer state (labeled Pro - CAR2 - TIR4). The design of this synthetic transmembrane domain refers to previous studies published in WO2021 / 229581 and Elazar, et al. eLife 2022;11. Compared with earlier CARs, anti - HER2 - CD28 TM -CD28 IC -CD3ζ - TIR4 CAR, in addition to a single mutation in the hinge region, changing the transmembrane domain can also significantly reduce autoactivation, but only mutating the cysteine residue in the hinge region has no same effect on autoactivation ( Figure 9 A and Figure 9 B). These findings suggest that the activation of the TIR4 domain may be the result of the synergistic effect of the transmembrane domain and the CD28 intracellular signaling domain.

[0330] To explore whether using this synthetic, designed transmembrane domain (labeled Pro - CAR2 - TIR4) in cDC1 can activate these cells in a tumor antigen - dependent manner, the inventors loaded HER2 + E0771 cells with OVA and co - cultured them with cells expressing anti - HER2 - Pro - CAR2 - CD28 ICThe mutuDCs of -CD3ζ-TIR4 CAR were co-incubated with OT-I cells. OVA was used as the neoantigen, and the proliferation of OT-1 cells stimulated in response to the cognate recognition of HER2+ E0771 tumor cells in various embodiments of the present invention was measured, and ultimately the killing ability of OVA-loaded tumor cells was promoted, which is a measure of antigen cross-presentation and anti-tumor immunity.

[0331] Anti-HER2-Pro-CAR2-CD28 IC The expression of -CD3ζ-TIR4 CAR in cDC1 increased the uptake of HER2 + tumor-associated antigen and OVA, thereby effectively presenting OVA-derived peptides to OT-I T cells and subsequent proliferation ( Figure 9 C and Figure 9 D). In the presence of cDC1 expressing anti-HER2-Pro-CAR2-CD28 IC -CD3ζ-TIR4 CAR, the numbers of granzyme B+ cytotoxic T cells and IFN-γ-producing T cells increased significantly. The anti-HER2-Pro-CAR2-CD28 IC -CD3ζ-TIR4 CAR-expressing cDC1 was adoptively transferred into human HER2 transgenic mice attacked by HER2 + E0771 tumors, which could inhibit tumor progression, indicating that the CAR-DC1 approach has therapeutic potential.

[0332] In summary, DCs transduced with CARs whose intracellular domain contains only the TIR domain cannot generate a mature branched morphology, which is consistent with the findings in WO2021 / 1277024. Anti-HER2-CD28 TM -TIR4 CAR-transduced DCs can bind to HER2 + tumor-associated antigens, but the expression of the DC maturation markers CD86 and CD80 of anti-HER2-TIR4 CAR-transduced cDC1 did not change significantly compared with that of cDC1 transduced with empty CAR or CAR with truncated intracellular domain before and after stimulation with HER2 + tumor antigens.

[0333] In cDC1 transduced with CARs containing TIR4 and CD28 IC and CARs containing TIR4 and CD3ζ and CARs containing TIR4, CD28 IC and CD3ζ, a mature branched morphology and increased expression of maturation markers were observed. These experiments indicate that the most effective CAR sequences include the TIR4 domain and additional co-stimulatory activation domains (such as CD3ζ, CD28 ICor both). The inventors also examined the transcriptomic changes of anti-HER2-CD28 TM -TIR4 CAR-transduced cDC1 by RNA sequencing. The transcriptomic changes of anti-HER2-CD28 TM -TIR4 CAR-transduced cDC1 were very close to those of intracellular domain-truncated CAR-transduced cDC1 (data not shown). The number of differentially expressed genes between anti-HER2-CD28 TM -TIR4 CAR- and intracellular domain-truncated CAR-transduced cDC1 (401 upregulated, 37 downregulated) was much less than that between anti-HER2-CD28 TM -CD28 IC -TIR4 CAR- and intracellular domain-truncated CAR-transduced cDC1 (1344 upregulated, 592 downregulated) or between anti-HER2-CD28 TM -CD28 IC -CD3ζ-TIR4CAR-transduced cDC1 and intracellular domain-truncated CAR-transduced cDC1 (1817 upregulated, 855 downregulated). These data indicate that after HER2 + tumor antigen stimulation, the TIR domain alone cannot induce the activation of cDC1, and additional costimulatory domains such as CD3ζ, CD28 IC or both are required.

[0334] All documents cited or referenced herein, as well as all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product brochures of any products mentioned herein or incorporated herein by reference, are hereby incorporated herein by reference in their entirety.

Claims

1. A chimeric antigen receptor (CAR) construct, comprising: (i) an antigen-binding domain; (ii) a transmembrane domain (TM); and (iii) an intracellular domain (IC), comprising: a Toll-interleukin receptor (TIR) intracellular signaling domain; and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain, wherein the CAR construct is capable of being expressed or functioning in dendritic cells.

2. A modified dendritic cell, comprising a chimeric antigen receptor (CAR) construct, the CAR construct comprising: (i) an antigen-binding domain; (ii) a transmembrane domain (TM); and (iii) an intracellular domain (IC), comprising: a Toll-interleukin receptor (TIR) intracellular signaling domain; and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain.

3. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the TIR signaling domain is located distally to the transmembrane domain, and the co-stimulatory signaling domain is located at the N-terminus of the transmembrane domain.

4. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the transmembrane domain is the CD28 transmembrane domain.

5. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the transmembrane domain is a synthetic transmembrane domain.

6. The modified dendritic cell according to claim 5, wherein the synthetic transmembrane domain is designed to promote oligomerization, enhance specificity, inhibit constitutive signaling, or a combination of these properties.

7. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the dendritic cell is a conventional type I (cDC1) cell.

8. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the CAR further comprises a CD8 hinge sequence.

9. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the co-stimulatory domain is the CD3 signaling domain.

10. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the co-stimulatory domain is the CD28 signaling domain.

11. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the co-stimulatory domain is the combined CD28 and CD3 signaling domain.

12. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the TIR intracellular signaling domain is selected from Toll-like receptor 4, Toll-like receptor 3, Toll-like receptor 9, Toll-like receptor 11, and Toll-like receptor 13.

13. The CAR construct or modified dendritic cell according to claim 12, wherein the TIR4 domain comprises or consists of the following sequence: AGCKYSRGESIYDAFVIYSSQNEDWRVNELKNLEEGVPRFHLCLHYRDFIPGVAIAANIIQEGFHKSRKVIVVSRHFIQSRWCIFEYEIAQTWQFLSSRSGIIFIVLEKVELLKSRQQVELYLLSRNTYLEWEDNPLGRHIFWRRLKNALLDGKASNPEQTAEEEQETWT (SEQ ID NO:6).

14. The CAR construct according to claim 1 or the modified dendritic cell according to claim 2, wherein the CAR construct comprises the following sequence or consists of the following sequence: SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

15. The CAR construct or modified dendritic cell according to any one of claims 1 to 14, wherein the antigen-binding domain comprises an antibody, an antibody fragment thereof, or an antigen-binding fragment.

16. The CAR construct or modified dendritic cell according to claim 15, wherein the antibody, the antibody fragment thereof, or the antigen-binding fragment has binding affinity for a tumor cell antigen.

17. The CAR construct or modified dendritic cell according to any one of claims 1 to 16, wherein the antigen-binding domain binds to the HER2 antigen.

18. The CAR construct or modified dendritic cell according to claim 17, wherein the antigen-binding domain is a scFv comprising the sequence of SEQ ID NO:

10.

19. The CAR construct or modified dendritic cell according to any one of claims 1 to 18, wherein the CAR comprises the following sequence or consists of the following sequence: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:

20.

20. A modified dendritic cell comprising one or more nucleic acid sequences encoding a CAR construct, the CAR construct comprising: (i) an antigen-binding domain; (ii) a transmembrane domain (TM); and (iii) an intracellular domain (IC) comprising: a Toll-interleukin receptor (TIR) intracellular signaling domain; and a co-stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a CD28 and CD3 combined signaling domain.

21. A nucleic acid sequence encoding a CAR, wherein the CAR comprises any one of the sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:

20.

22. The modified dendritic cell according to any one of claims 1 to 20, wherein the modified dendritic cell is capable of cross - presenting antigens to T cells, initiating an adaptive immune response, or activating anti - tumor T cells.

23. A pharmaceutical composition comprising the modified dendritic cell according to claim 2.

24. A method of stimulating an adaptive immune response in a subject, comprising: Administering to the subject a therapeutically effective amount of the modified dendritic cell according to claim 2 or the pharmaceutical composition according to claim 23.

25. The method according to claim 24, wherein the modified dendritic cell is administered to the subject, and the modified dendritic cell comprises a CAR having any one of the sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:

20.

26. The method according to claim 24 or 25, wherein the subject has cancer.

27. The method according to any one of claims 24 to 26, wherein the method induces phagocytosis of apoptotic bodies released by cancer cells in the subject.

28. The method according to any one of claims 24 to 27, wherein the modified dendritic cell or the pharmaceutical composition according to claim 23 induces an immune response that eliminates tumors.

29. A method for preparing a population of dendritic cells containing a chimeric antigen receptor (CAR - DC), comprising: (i) providing a population of cells from a subject; (ii) culturing the population of cells in a medium containing an FMS - like tyrosine kinase 3 (Flt3) agonist for at least one day; (iii) introducing into the population of cells a chimeric antigen receptor (CAR) that comprises an antibody or an antigen - binding fragment thereof, a transmembrane domain (TM), and an intracellular domain (IC), wherein the intracellular domain comprises a Toll - interleukin receptor (TIR) intracellular signaling domain and a co - stimulatory signaling domain selected from a CD3 signaling domain, a CD28 signaling domain, and a combined CD28 and CD3 signaling domain; and (iv) culturing the cells from (iii) in a medium containing an FMS - like tyrosine kinase 3 (Flt3) agonist for a time sufficient to form modified dendritic cells.

30. The method according to claim 29, wherein the cell population is selected from monocytes or stem cells in circulation, umbilical cord or bone marrow.

31. The method according to claim 29 or 30, wherein the dendritic cells are autologous or allogeneic conventional type I dendritic cells (cDC1).

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