New chimeric antigen receptors for monocytes / macrophage and uses thereof

Abstract

The present invention discloses new chimeric antigen receptors for monocytes / macrophage and uses thereof.The Chimeric Antigen Receptor (CAR) construct comprising an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain connected in sequence; wherein the intracellular domain comprises at least one costimulatory signaling domain, comprising a TLR4 TIR signaling domain and a 4-1BB signaling domain connected in sequence; wherein the TLR4 TIR signaling domain is constructed to be positioned adjacent to the transmembrane domain.

Description

DESCRIPTIONTITLE OF INVENTION: NEW CHIMERIC ANTIGEN RECEPTORS FOR MONOCYTES / MACROPHAGE AND USES THEREOFREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority to Singapore patent application No. 10202500509T, filed on 26 February 2026, the contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates generally to new chimeric antigen receptors, particularly for monocytes / macrophage.BACKGROUND

[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.

[0004] Cancer is one of the leading causes of mortality worldwide and continues to represent a major public health challenge. Despite significant advances in early detection and treatment, many cancers, particularly solid tumours, remain difficult to treat effectively. Conventional therapeutic approaches, including surgical resection, chemotherapy, radiotherapy, or combinations thereof, are associated with various limitations. Surgical intervention is often unsuitable for patients with advanced or metastatic disease, while chemotherapy and radiotherapy frequently suffer from a lack of tumour specificity, resulting in damage to healthy tissues and undesirable systemic side effects. Consequently, there remains an ongoing need for therapeutic strategies that can selectively target tumour tissue, improve treatment efficacy, and reduce treatment-associated toxicity, particularly in patients with advanced solid tumours.

[0005] In recent years, adoptive cell-based immunotherapies have attracted increasing interest as alternative approaches for cancer treatment. Among these, chimeric antigen receptor (CAR)-modified immune cells are engineered to recognise tumour-associated antigens in an antigen-specific and major histocompatibility complex (MHC)-independent manner. CAR-T cell therapies have demonstrated clinical efficacy in certain hematological malignancies; however, their application to solid tumours has been considerably more limited. In particular, CAR-T cells often exhibit poor infiltration into solid tumour tissue, and their activity can be suppressed byimmunoregulatory factors present within the tumour microenvironment, thereby reducing therapeutic effectiveness (see, e.g., Ma et al., 2023, PMID: 37998743).

[0006] Monocytes and macrophages possess intrinsic capabilities to migrate into solid tumours, respond to local inflammatory cues, and actively modulate the tumour microenvironment. On this basis, CAR-engineered monocytes or macrophages (CAR-M) have been proposed as a potential therapeutic platform for the treatment of solid tumours (see, e.g., Zhang et al., 2023, PMID: 37388769). Preclinical studies have shown that CAR-M cells can reduce tumour growth and influence the tumour microenvironment in animal models (see, e.g., Lei et al., 2024, PMID: 38012418). Early-stage clinical investigations have further indicated that CAR-M cells are capable of trafficking to solid tumours and modulating tumour-associated immune responses, while exhibiting a generally favourable safety profile, with reported adverse effects primarily limited to low-grade cytokine release syndrome or transient flu-like symptoms (see, e.g., Reiss et al., 2022; Annunziata et al., 2020; Sloas et al., 2021, PMID: 34899748). Nevertheless, the therapeutic efficacy achieved with existing CAR-M constructs remains limited, and durable tumour regression has not yet been consistently demonstrated.

[0007] Accordingly, there exists a need to develop an improved CAR construct for monocytes and / or macrophages that alleviates at least one of the aforementioned problems. SUMMARY

[0008] Accordingly, an aspect of the invention refers to a Chimeric Antigen Receptor (CAR) construct comprising an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain connected in sequence; wherein the intracellular domain comprises at least one costimulatory signaling domain, comprising a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence; wherein the TLR4 TIR signaling domain is constructed to be positioned adjacent to the transmembrane domain.

[0009] In various embodiments, the antigen binding domain comprises an scFV targeting an antigen.

[0010] In various embodiments, the antigen is a tumour-specific antigen or a tumour-associated antigen.

[0011] In various embodiments, the antigen is selected from a group consisting of PD-L1, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, PD1, CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, and EGFR.

[0012] In various embodiments, the hinge region comprises a CD8a, CD28, or C6 hinge region.

[0013] In various embodiments, the transmembrane domain comprises a CD8a, CD28, or TLR4 transmembrane domain.

[0014] In various embodiments, the intracellular domain comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0015] In various embodiments, the intracellular domain comprises an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.

[0016] In various embodiments, the CAR construct comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6; or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6.

[0017] In various embodiments, the CAR construct comprises a nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5; or a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0018] According to another aspect there is a mononuclear phagocyte cell expressing the CAR construct described above, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.

[0019] In various embodiments, the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.

[0020] In various embodiments, the monocyte is THP-1 cell.

[0021] According to another aspect there is use of the mononuclear phagocyte cell described above in the manufacture of a medicament for treatment of a target tumour.

[0022] In various embodiments, the target tumour is a solid tumour.

[0023] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0024] In various embodiments, the treatment comprises reducing a burden of the target tumour.

[0025] In various embodiments, the burden of the target tumour comprises a volume of the solid tumour.

[0026] According to another aspect there is a mononuclear phagocyte cell for use in the treatment of target tumour, wherein the mononuclear phagocyte cell described above.

[0027] In various embodiments, the target tumour is a solid tumour.

[0028] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0029] According to another aspect there is a method of treatment comprising administrating the mononuclear phagocyte cell described above to a subject with a tumour.

[0030] In various embodiments, the tumour is a solid tumour.

[0031] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0032] In various embodiments, the treatment comprises reducing a load of the target tumour.

[0033] In various embodiments, the load of the target tumour is a volume of the solid tumour.

[0034] According to another aspect there is use of the CAR construct described above for inducing a mononuclear phagocyte cell towards an M1 phenotype, wherein the mononuclear phagocyte cell is engineered to express the CAR construct, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.

[0035] In various embodiments, the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.

[0036] According to another aspect there is a method of inducing a monocyte or a macrophage towards an M1 phenotype, wherein a monocyte or a macrophage is engineered to express the CAR construct described above, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.

[0037] In various embodiments, the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) IPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.

[0038] According to another aspect there is use of the mononuclear phagocyte cell described above in the manufacture of a medicament for mediating activities of T cells.

[0039] According to another aspect there is use of the mononuclear phagocyte cell described above, wherein the T cells are tumour-infiltrating T cells.

[0040] According to another aspect there is the use of the mononuclear phagocyte cell described above, wherein the T cells include CD4+ T cells, CD8+ T cells, or both.

[0041] According to another aspect there is the use of the mononuclear phagocyte cell described above, wherein the mononuclear phagocyte cell reduces exhaustion of the T cells.

[0042] According to another aspect there is use of the mononuclear phagocyte cell described above in the manufacture of a medicament for modulating a microenvironment of a solid tumour.

[0043] In various embodiments, the use of the mononuclear phagocyte cell described above, wherein the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0044] According to another aspect there is an intracellular domain of a Chimeric Antigen Receptor (CAR) construct,wherein the intracellular domain comprises at least one costimulatory signaling domain, comprising a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence;wherein the TLR4 TIR signaling domain is constructed to be positioned adjacent to a transmembrane domain to be connected to the intracellular domain.

[0045] In various embodiments, the intracellular domain comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0046] In various embodiments, the intracellular domain comprises an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.

[0047] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] [Fig. 1]: illustrates that CAR-THP-1 cells reduce lung tumour burden in humanized mice. Immunodeficient NOD / SCID IL2Ry- / - (NIKO) mice were engrafted with human CD34+ pluripotent stem cells to generate humanized mice. (A) At week 12, human cell chimerism was assessed by flow cytometry, determined as the percentage of human CD45+ cells within the total lymphocyte population (human CD45+ and mouse CD45.1+). The percentages of human T cells (CD3+), B cells (CD19+), and monocytes (CD14+) were identified within the human CD45+ cell population, while human CD4+ and CD8+ cells were determined as the percentage of positive cells within the CD3+ subset. (B) One million A549 lung tumour cells were engrafted into the flank of 12-week-old humanized mice. After one week, the mice were divided into three groups: no treatment (Ctrl), intravenously injected with 10 million wild-type THP-1 cells (THP-1), and injected with 10 million CAR-THP-1 cells (CAR-THP-1). Tumour dimensions were monitored weekly for 28 days. (C-D) After 28 days, tumours were retrieved from the indicated groups (C) and weighed (D).Each dot represents one individual mouse, with bar plots showing the mean ± SD. **: p<0.01; ****:p<0.001.

[0049] [Fig. 2]: illustrates that B4 Cells reduce lung tumour burden in humanized mice. One million A549 lung tumour cells were engrafted into the flank of 12-week-old humanized mice. After one week, the mice were divided into two groups: no treatment (Control), or intravenously injected with 5 million B4 cells (B4). (A) Tumour dimensions were monitored weekly. (B) Survival of mice were tracked on a survival plot. (C-D) After 60 days, tumours were retrieved and weighed (C). Weight difference to the mean of control tumors was computed (D). Each dot represents one individual mouse, with bar plots showing the mean ± SD. *p<0.001.

[0050] [Fig. 3]: illustrates that CAR-THP-1 cells require other human immune cells for antitumour activity. (A) Twelve-week-old immunodeficient NOD / SCID IL2Ry-Z- (NIKO) mice were engrafted with one million A549 lung tumour cells. After one week, the mice were divided into two groups: untreated controls (Ctrl) and those injected with 10 million CAR-THP-1 cells (CAR-THP-1). Tumour dimensions were tracked weekly for 28 days. (B-C) At the end of the 28-day period, tumours were harvested from the respective groups (B) and weighed (C). Each dot represents an individual mouse, with bar plots indicating the mean ± SD.

[0051] [Fig. 4]: CAR-THP-1 cells work synergistically with human T cells to reduce tumour burden. Twelve weeks old humanized mice were engrafted with one million A549 lung tumourcells. After one week, the mice were divided into four groups: no treatment (Ctrl), intravenously injected with 10 million CAR-THP-1 cells (CAR-THP-1), injected with 10 million CAR-THP-1 and a CD4 depleting antibody (CAR-THP-1 +aCD4), and injected with 10 million CAR-THP-1 and a CD8 depleting antibody (CAR-THP-1 + aCD8). (A-B) After 28 days, tumours were retrieved from the indicated groups (A) and weighed (B). Each dot represents one individual mouse, with bar plots showing the mean ± SD. ****: p<0.001.

[0052] [Fig. 5]: CAR-THP-1 Induces M1 polarization of macrophages within the tumour Microenvironment. Tumour-infiltrating lymphocytes (TILs) were isolated from tumours retrieved from humanized mice engrafted with A549 cells, either untreated or treated with CAR-THP-1 cells for one month. The extracted TILs were analyzed by flow cytometry for the expression of species markers (huCD45, mouse CD45.1), macrophage markers (CD14, CD16, CD68), an M0 marker (CD11b), an M1 marker (CD80), and an M2 marker (CD206). (A) t-SNE analysis of these markers, followed by FlowSOM clustering, highlighting clusters with high contributions from CD11b, CD80, and CD206. (B) t-SNE plots displaying events from untreated or CAR-THP-1 treated groups. (C-E) Bar plots showing the geometric mean fluorescent intensity (GMFI) of CD11b (C), CD80 (D), and CD206 (E) for individual mice from the indicated groups. For t-SNE plot, each dot represents a single cell. For bar plots, mean ± SD is shown, with each dot representing an individual mouse **: p<0.01. Thus, the results of Figure. 5 demonstrate that CAR-M therapy could polarize tumor infiltrating macrophages towards a pro-inflammatory M1 phenotype.

[0053] [Fig. 6]: B4 Induces M1 Polarization of Macrophages Within the Tumour Microenvironment. TILs were isolated from tumours retrieved from humanized mice engrafted with A549 cells, either untreated or treated with B4 cells for 60 days. TILs were analyzed by flow cytometry. (A-C) Bar plots showing the percentage of macrophage cells positive for CD11b (A), CD80 (B), and CD206 (C). Mean ± SD is shown, with each dot representing an individual mouse *: p<0.05.

[0054] [Fig. 7]: CAR-THP-1 Reduces PD-1 Expression on T Cells in the Tumour Microenvironment. (A-B) TILs were isolated from tumours retrieved from humanized mice engrafted with A549 cells, either untreated or treated with CAR-THP-1 cells for one month. The extracted TILs were analyzed by flow cytometry for the expression of species markers (huCD45, mouse CD45.1) and T cell markers (CD3, CD4, CDS, CD45RA, CD197, PD-1, and CD25). T cell subsets were identified as follows: central memory T cells (Tern: CD45RA-CD197+), effector memory T cells (Tern: CD45RA-CD197-), naive T cells (Tn: CD45RA+CD197+), and terminal effector T cells (Ttem: CD45RA+CD197-). Bar plots show the percentage of the indicated populations within CD4+ (A) and CD8+ (B) cells. Data are presented as mean ± SD,with each dot representing an individual mouse. ***: p<0.005. Thus, the results of Figure 7 demonstrate that CAR-M therapy could reduce exhaustion of tumor infiltrating T cells.

[0055] [Fig. 8]: CAR-THP-1 cells are activated after infiltrating tumour-derived organoids. Tumour organoids generated from A549 (lung adenocarcinoma), HT-29 (colorectal adenocarcinoma), Huh-7 (hepatocellular carcinoma), and NCC-LC59 (lung cancer) cell lines were incubated with CAR-THP-1 cells or control THP-1 cells expressing a CAR construct lacking intracellular signaling domains (NC). CAR-THP-1 or NC-THP-1 cells were overlaid onto organoids and allowed to infiltrate for 24 hours, after which non-infiltrated cells were washed off. Organoids were maintained for an additional 48 hours, dissociated on day 3, and analyzed by flow cytometry. Bar plots show percentage of CD80 positive cells for each organoid model. Each dot represents one independent biological experiment (each performed with three technical replicates). Data represent mean ± SEM.

[0056] [Fig. 9]: Pathway-level remodeling of tumour organoids following CAR-THP-1 infiltration. Tumour organoids generated from four human solid tumour cell lines (HUH7 (hepatocellular carcinoma), HT29 (colorectal adenocarcinoma), A549 (lung adenocarcinoma), and NCC59 (also spelled as NCC-LC59) (lung cancer)) were infiltrated with CAR-THP-1 macrophages or NC-THP-1 controls and dissociated on day 3 for bulk RNA sequencing. Gene set enrichment analysis (GSEA) was performed using the Hallmark pathway collection, and Normalized Enrichment Scores (NES) from CAR-THP-1 versus NC-THP-1 comparisons were visualized as a clustered heatmap. Each column represents one tumour type (averaged across three biological replicates), and each row represents a Hallmark biological pathway. Scale bar shows NES magnitude.

[0057] [Fig. 10]: illustrates schematic representations of chimeric antigen receptor (CAR) constructs according to the present invention. (A) shows a general CAR construct comprising an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain connected in sequence, wherein the intracellular domain comprises a costimulatory signaling domain including a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence. (B) shows a CAR-THP-1 construct, in which the antigen binding domain is an scFv targeting PD-L1, the hinge region comprises a CD8a hinge region, the transmembrane domain comprises a CD8a transmembrane domain, and the intracellular domain comprises a costimulatory signaling domain including a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence. (C) shows a B4 construct, in which the antigen binding domain is an scFv targeting B7-H3, the hinge region comprises a CD8a hinge region, the transmembrane domain comprises a CD8a transmembrane domain, and the intracellular domain comprises a costimulatory signaling domain including a TLR4 TIR signalingdomain and a 4-1 BB signaling domain connected in sequence.DETAILED DESCRIPTION

[0058] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.

[0059] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0060] As used herein, the term “monocyte” refers to a circulating mononuclear phagocyte lineage cell that is capable of migrating into tissues and differentiating into macrophages or other myeloid cells in response to environmental cues. As used herein, the term “macrophage” refers to a differentiated mononuclear phagocyte lineage cell that resides in tissues and exhibits immune effector functions, including phagocytosis and modulation of the local microenvironment. For the avoidance of doubt, the CAR constructs disclosed herein are applicable to mononuclear phagocyte lineage cells at different stages of differentiation. In particular, the CAR constructs may be expressed in monocytes prior to differentiation, such that the CAR-expressing monocytes subsequently differentiate in vivo or ex vivo into macrophages while retaining functional CAR expression. Alternatively, the CAR constructs may be expressed in macrophages after differentiation from monocytes or other precursor cells. Accordingly, references herein to CAR-expressing monocytes and / or macrophages are intended to encompass CAR expression before differentiation, after differentiation, or across the differentiation process, and are not limited to a specific developmental stage of the mononuclear phagocyte cell.

[0061] As used herein, the term “TLR4 TIR signaling domain” refers to an intracellular signaling domain derived from Toll-like receptor 4 (TLR4), comprising a Toll / lnterleukin-1 receptor (TIR) domain that is capable of initiating downstream innate immune signaling in mononuclear phagocyte lineage cells. When incorporated into a chimeric antigen receptor, the TLR4 TIR signaling domain functions as a costimulatory signaling module that mediates signal transduction upon receptor engagement, including activation of pathways associated with macrophage activation and polarization.

[0062] Unless defined otherwise, all other technical and scientific terms used herein have the same meaning as is commonly understood by a skilled person to which the subject matter herein belongs.

[0063] EMBODIMENT 1

[0064] Monocytes were engineered to express chimeric antigen receptors (CAR-M) targeting tumour surface proteins and were evaluated for their ability to reduce human tumour burden in an in vivo humanized mouse model. Schematic representations of the CAR constructs are shown in Figure 10, wherein TM denotes a transmembrane domain and TIR denotes a Toll / lnterleukin-1 receptor domain.

[0065] Specifically, the present invention discloses a chimeric antigen receptor (CAR) construct comprising an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain connected in sequence, wherein the intracellular domain comprises at least one costimulatory signaling domain including a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence, and wherein the TLR4 TIR signaling domain is positioned adjacent to the transmembrane domain.

[0066] Two CAR constructs were generated, each comprising a different antigen binding domain in the form of a single-chain variable fragment (scFv). A first CAR construct comprised an scFv targeting PD-L1 and an intracellular signaling domain including a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence. This CAR construct was expressed in THP-1 cells, a human monocytic leukemia cell line commonly used as an in vitro model for studying monocyte and macrophage functions, thereby generating CAR-THP-1 cells. A second CAR construct comprised an scFv targeting B7-H3, while the remaining structural and intracellular components were identical to those of the first CAR construct. This second CAR construct was designated B4 and was similarly expressed in THP-1 cells to generate B4 CAR- THP-1 cells (i.e., “B4 cells”), to distinguish them from THP-1 cells expressing the first CAR construct (i.e., “CAR-THP-1 cells”).

[0067] Table.1 Sequences of the CAR constructs of the present invention

[0068] As described below, the intracellular domain of the CAR construct in the present invention combines TLR4 TIR and 4-1 BB in a specific sequence (i.e., TLR4 TIR closer to the transmembrane domain, while 4-1 BB closer to the C-terminus of the CAR protein construct), which enables effective recruitment of both MyD88-NF-KB and IRF3 via efficient homodimerization of TLR4 TIR. Simultaneously, the CAR construct maintains robust activation of 4-1BB. These synergistic mechanisms promote M1 polarization of macrophages, significantly enhancing anti-tumor efficacy, particularly against solid tumors.

[0069] In various embodiments, the present invention provides a chimeric antigen receptor (CAR) construct comprising an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain connected in sequence, wherein the intracellular domain comprises at least one costimulatory signaling module including a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence, with the TLR4 TIR signaling domain positioned adjacent to the transmembrane domain.

[0070] In some embodiments, the antigen binding domain comprises a single-chain variable fragment (scFv) configured to recognize a target antigen expressed on tumour cells.

[0071] In various embodiments, the antigen is a tumour-specific antigen or a tumour- associated antigen.

[0072] In various embodiments, the antigen is selected from a group consisting of PD-L1, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, PD1, CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, and EGFR.

[0073] In various embodiments, the hinge region comprises a hinge region derived from CD8a, CD28, or C6.

[0074] In various embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8a, CD28, or TLR4.

[0075] In certain embodiments, the intracellular domain comprises an amino acid sequence corresponding to SEQ ID NO: 1, or a variant thereof having at least 95% sequence identity while retaining innate immune signaling functionality.

[0076] In some embodiments, the intracellular domain is encoded by a nucleic acid sequence corresponding to SEQ ID NO: 3, or a variant thereof having at least 95% sequence identity.

[0077] In certain embodiments, the CAR construct comprises an amino acid sequence corresponding to SEQ ID NO: 2 or SEQ ID NO: 6, or a variant thereof having at least 95% sequence identity.

[0078] In further embodiments, the CAR construct is encoded by a nucleic acid sequence corresponding to SEQ ID NO: 4 or SEQ ID NO: 5, or a variant thereof having at least 95% sequence identity.

[0079] In various embodiments, the CAR construct described herein is expressed in a mononuclear phagocyte lineage cell.

[0080] In various embodiments, the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.

[0081] In various embodiments, the monocyte is THP-1 cell or a derivative thereof.

[0082] According to various embodiment there is use of the mononuclear phagocyte cell described above in the manufacture of a medicament for treatment of a target tumour.

[0083] In various embodiments, the target tumour is a solid tumour.

[0084] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0085] In various embodiments, treatment comprises reducing tumour burden, including reduction in tumour volume.

[0086] According to various embodiment there is a mononuclear phagocyte cell for use in the treatment of target tumour, wherein the mononuclear phagocyte cell described above.

[0087] In various embodiments, the target tumour is a solid tumour.

[0088] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0089] According to various embodiment there is a method of treatment comprising administrating the mononuclear phagocyte cell described above to a subject with a tumour.

[0090] In various embodiments, the tumour is a solid tumour.

[0091] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0092] In various embodiments, treatment comprises reducing tumour burden, including reduction in tumour volume.

[0093] According to various embodiment, the use of the CAR construct described above for inducing a mononuclear phagocyte cell towards an M1 phenotype, wherein the mononuclear phagocyte cell is engineered to express the CAR construct, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.

[0094] In various embodiments, the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.

[0095] According to various embodiment, the method of inducing a monocyte or a macrophage towards an M1 phenotype, wherein the monocyte or a macrophage is engineered to express the CAR construct described above, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.

[0096] In various embodiments, the mononuclear phagocyte cell is (I) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.

[0097] According to various embodiment, the use of the mononuclear phagocyte cell described above in the manufacture of a medicament for mediating activities of T cells.

[0098] According to various embodiment, the use of the mononuclear phagocyte cell described above, wherein the T cells are tumour-infiltrating T cells.

[0099] According to various embodiment, the use of the mononuclear phagocyte cell described above, wherein the T cells include CD4+ T cells, CD8+ T cells, or both.

[0100] According to various embodiment, the use of the mononuclear phagocyte cell described above, wherein the mononuclear phagocyte cell reduces exhaustion of the T cells.

[0101] According to various embodiment, the use of the mononuclear phagocyte cell described above in the manufacture of a medicament for modulating a microenvironment of a solid tumour.

[0102] In various embodiments, the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.

[0103] According to various embodiment, an intracellular domain of a Chimeric Antigen Receptor (CAR) construct, wherein the intracellular domain comprises at least one costimulatory signaling domain, comprising a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence; wherein the TLR4 TIR signaling domain is constructed to be positioned adjacent to a transmembrane domain to be connected to the intracellular domain.

[0104] In various embodiments, the intracellular domain comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0105] In various embodiments, the intracellular domain comprises an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.

[0106] EMBODIMENT 2

[0107] CAR-THP-1 cells are able to control tumor growth in vivo in humanized mice.

[0108] In one exemplary embodiment, a CAR construct comprising a single-chain variable fragment (scFv) targeting PD-L1 and an intracellular signaling domain associated with macrophage M1 polarization pathways was generated. The CAR construct was expressed in THP-1 cells, a human monocytic leukemia cell line, to generate CAR-THP-1 cells. The antitumour activity of the CAR-THP-1 cells was evaluated in a human immune system humanized mouse model. The humanized mice were generated by engrafting immunodeficient NOD / SCID IL2RY" / “ (NIKO) mice with human CD34+pluripotent stem cells (Costa, Ye et al., 2017, PMID: 28765218), resulting in human immune cell chimerism of approximately 50-60% at 12 weekspost-engraftment. The reconstituted human immune compartment comprised approximately 50% CD3+T cells, 30% CD19+B cells, and 10% CD14+monocytes, as shown in Figure 1A.

[0109] As an illustrative example of therapeutic application in solid tumours, a human lung adenocarcinoma cell line, A549, which expresses PD-L1, was subcutaneously engrafted into the right flank of the humanized mice. One week following tumour implantation, the mice were allocated to one of three treatment groups: no treatment (Ctrl), administration of wild-type THP-1 cells (THP-1), or administration of CAR-THP-1 cells (CAR-THP-1). Tumour dimensions were monitored longitudinally for a period of one month. As shown in Figure 1B, mice treated with CAR-THP-1 cells exhibited reduced tumour growth compared with the control group, with a statistically significant difference in tumour volume observed from day 21 onwards (90.4 ± 45.6 mm3, n = 12 versus 188.6 ± 62.2 mm3, n = 8; multiple t-tests with Benjamini-corrected p-value < 0.05).

[0110] At day 28, the mice were sacrificed and tumours were excised for further analysis, as illustrated in Figure 1C. Tumour weights were measured and are shown in Figure 1D. Consistent with the tumour volume measurements, tumours retrieved from mice receiving CAR-THP-1 cells exhibited lower weights (0.6 ± 0.3 g, n = 12) compared with tumours from mice receiving wild-type THP-1 cells (1.3 ± 0.6 g, n = 15; one-way ANOVA with Tukey’s post hoc test, p = 0.006) and tumours from untreated control mice (1.8 ± 0.5 g, n = 8; one-way ANOVA with Tukey’s post hoc test, p < 0.0001).

[0111] B4 cells are able to control tumor growth in vivo in humanized mice.

[0112] Using the same intracellular signaling architecture, a second CAR construct Incorporating an scFv targeting B7-H3 was generated. This construct was designated B4 and was expressed in THP-1 cells to generate B4 CAR-THP-1 cells. The in vivo anti-tumour activity of the B4 cells was evaluated using the same human immune system humanized mouse model, in which A549 lung carcinoma cells were subcutaneously engrafted into the right flank of the mice.

[0113] One week following tumour implantation, the mice were assigned to receive either no treatment (Control) or B4 cells. Tumour dimensions were monitored longitudinally for up to 60 days, as shown in Figure 2A. Control-treated mice exhibited progressive tumour growth over the study period, whereas mice receiving B4 cells displayed attenuated tumour growth, with reduced tumour volumes observed at multiple time points.

[0114] Survival outcomes corresponding to the tumour growth analysis are shown in Figure 2B. Control-treated mice began to succumb to tumour burden from approximately day 40 onwards. In contrast, all mice treated with B4 cells survived through the 60-day study endpoint.

[0115] At the conclusion of the study, tumours were excised and weighed, as illustrated in Figure 2C. Relative to control-treated mice, B4-treated mice exhibited an approximately 55% reduction in tumour mass, consistent with the longitudinal tumour growth measurements. These results demonstrate that incorporation of a B7-H3-targeting scFv into the disclosed CAR intracellular signaling framework supports effective tumour control in a humanized mouse model of solid cancer.

[0116] Taken together, the results shown in Figures 1 and 2 demonstrate that CAR constructs incorporating different antigen binding domains, including scFvs targeting PD-L1 and B7-H3 respectively, are each capable of mediating effective anti-tumour activity in vivo when combined with the disclosed intracellular signaling architecture. In both cases, CAR-engineered mononuclear phagocyte cells exhibited attenuated tumour growth and reduced tumour burden in a human immune system humanized mouse model of solid cancer. These findings indicate that the observed anti-tumour effects are not limited to a particular target antigen, but are attributable to the intracellular signaling domain comprising the ordered combination of the TLR4 TIR signaling domain and the 4-1 BB signaling domain, which confers enhanced macrophage-mediated anti-tumour immune functionality.

[0117] CAR-THP-1 work synergistically with human T cells.

[0118] Having demonstrated that CAR-THP-1 cells targeting PD-L1 reduced tumour burden in humanized mice engrafted with the lung tumour cell line A549 by approximately 67% (Fig.1D), the anti-tumour activity of CAR-THP-1 cells was further evaluated in the absence of additional human immune cells using immunodeficient NOD / SCID IL2Ry7" (NIKO) mice engrafted with A549 cells. The mice were divided into two groups: no treatment (Ctrl) and treatment with CAR-THP-1 cells (CAR-THP-1). Tumour dimensions were measured over the course of one month. As shown in Figure 3A, no significant difference in tumour volume was observed between the two groups. Similarly, no significant difference in tumour weight was observed at day 28, when the mice were sacrificed (Figures 3B and 3C). These results indicate that CAR-THP-1 cells alone are insufficient to control tumour growth and require the presence of additional human immune cells to be effective.

[0119] Given the established roles of T cells in tumour eradication, the contribution of CD4+and CD8+T cells to CAR-THP-1 -mediated anti-tumour activity was assessed. Humanized mice engrafted with A549 tumour cells were divided into four groups: no treatment (Ctrl), CAR-THP-1cells (CAR-THP-1), CAR-THP-1 cells in combination with an anti-CD4 depleting antibody (CAR-THP-1 + aCD4), and CAR-THP-1 cells in combination with an anti-CD8 depleting antibody (CAR-THP-1 + aCD8). Tumours were harvested and analysed after one month. As shown in Figures 4A and 4B, a significant reduction in tumour weight was observed only in mice treated with CAR-THP-1 cells without T cell depletion. These results indicate that the anti-tumour effects mediated by CAR-THP-1 cells depend on the presence of both CD4+and CD8+T cells, consistent with cooperative or synergistic interactions between CAR-THP-1 cells and these T cell subsets.

[0120] CAR-THP-1 shifted macrophage polarization towards M1 in the TME.

[0121] To evaluate macrophage polarization within the tumour microenvironment following CAR-THP-1 treatment, tumour-infiltrating leukocytes were isolated from tumours retrieved from humanized mice engrafted with A549 cells, either untreated or treated with CAR-THP-1 cells for one month. The isolated cells were analysed by flow cytometry for the expression of species markers (huCD45, mouse CD45.1), macrophage markers (CD14, CD16, CD68), a panmacrophage marker (CD11b), an M1-associated marker (CD80), and an M2-associated marker (CD206).

[0122] Dimensionality reduction analysis using t-distributed stochastic neighbor embedding (t-SNE), followed by FlowSOM clustering, revealed at least seven distinct cell populations. Clusters exhibiting high expression of CD11b, CD80, or CD206 were identified, as shown in Figure 5A. When analysed by treatment group, macrophages from untreated humanized mice predominantly localized to clusters characterized by high CD206 expression, whereas macrophages from CAR-THP-1 -treated mice were primarily associated with clusters exhibiting elevated CD11b and CD80 expression (Figure 5B). Quantitative analysis at the individual mouse level further showed increased expression of CD11b (1472 ± 1357, n = 9 versus 147 ± 102, n = 7; t-test p = 0.002; Figure 5C) and CD80 (586 ± 323, n = 9 versus 168 ± 124, n = 7; t-test p = 0.006; Figure 5D), together with decreased expression of CD206 (135 ± 106, n = 9 versus 370 ± 153, n = 7; t-test p = 0.003; Figure 5E), in the CAR-THP-1 -treated group compared with the untreated group.

[0123] These results indicate that treatment with CAR-THP-1 cells is associated with a shift in macrophage polarization toward an M1-like phenotype within the tumour microenvironment. Such polarization is consistent with a pro-inflammatory functional state of macrophages that is conducive to supporting T cell activity and reshaping the tumour microenvironment, thereby contributing to the anti-tumour effects observed following CAR-THP-1 treatment.

[0124] B4 shifted macrophage polarization towards M1 in the TME.

[0125] To assess macrophage polarization following B4 treatment, tumour-infiltrating leukocytes were isolated from tumours retrieved from humanized mice engrafted with A549 cells, either untreated or treated with B4 cells. The isolated cells were analysed by flow cytometry for the expression of species markers (huCD45, mouse CD45.1), macrophage markers (CD14, CD16, CD68), a pan-macrophage marker (CD11b), an M1-associated marker (CD80), and an M2-associated marker (CD206). As shown in Figure 6A, macrophages from B4-treated tumours exhibited increased CD11b expression (approximately 60%) compared with macrophages from untreated control tumours (approximately 40%). Consistently, expression of CD80, a marker associated with pro-inflammatory macrophage activation, was elevated in B4-treated tumours (approximately 20%) relative to control tumours (approximately 7.5%) (Figure 6B). In contrast, a modest but statistically significant reduction in CD206 expression, an M2-associated macrophage polarization marker, was observed in B4-treated tumours compared with controls (Figure 6C).

[0126] These results indicate that B4 CAR constructs promote reprogramming of tumourinfiltrating myeloid cells toward a pro-inflammatory, M1-like macrophage phenotype within the tumour microenvironment.

[0127] Taken together, the results obtained with both CAR-THP-1 cells targeting PD-L1 and B4 cells targeting B7-H3 demonstrate that CAR constructs incorporating the disclosed intracellular signaling architecture consistently promote polarization of tumour-infiltrating macrophages toward an M 1 -like phenotype within the tumour microenvironment. In both cases, increased expression of M1 -associated markers and reduced expression of M2-associated markers were observed, despite the use of different antigen binding domains. These findings indicate that the ability to reprogram macrophage polarization toward a pro-inflammatory, antitumour functional state is not dependent on the specific target antigen, but is attributable to the shared intracellular signaling domain comprising the ordered combination of the TLR4 TIR signaling domain and the 4-1 BB signaling domain.

[0128] CAR-THP-1 reduced PD-1 expression on CD4+and CD8+T cells in the TME.

[0129] To assess the phenotype of tumour-infiltrating T cells following CAR-THP-1 treatment, TILs were analysed for CD4+and CD8+T cell subsets. No substantial differences were observed between treatment groups in the proportions of effector memory, central memory, naive, or regulatory T cells within either the CD4+or CD8+T cell populations. In contrast, the proportion of CD4+T cells expressing PD-1 was significantly reduced in tumours retrieved from mice treated with CAR-THP-1 cells compared with control-treated mice (47.9 ± 8.3%, n = 9 versus 68.0 ± 7.6%, n = 7; t-test p = 0.003; Figure 7A). Similarly, the proportion of CD8+T cellsexpressing PD-1 was also significantly lower in the CAR-THP-1 -treated group (36.6 ± 11.4%, n = 9 versus 61.7 ± 11.2%, n = 7; t-test p = 0.007; Figure 7B).

[0130] These results indicate that treatment with CAR-THP-1 cells is associated with reduced PD-1 expression on tumour-infiltrating CD4+and CD8+T cells. Reduced PD-1 expression on T cells has been associated with improved clinical outcomes in lung cancer patients (see, e.g., Mazzaschi, Madeddu et al., 2018, PMID: 29074606). Accordingly, modulation of PD-1 expression on tumour-infiltrating T cells by CAR-THP-1 treatment may contribute to enhancement of anti-tumour immune responses and improved tumour control.

[0131] CAR-THP-1 Cells Increase CD80 Expression After Infiltrating Tumour Organoids.

[0132] The activation of CAR-THP-1 cells following infiltration into three-dimensional tumour organoids was evaluated using organoid models derived from multiple human solid tumour types. Tumour organoids were established from A549 (lung adenocarcinoma), HT-29 (colorectal adenocarcinoma), Huh-7 (hepatocellular carcinoma), and NCC-LC59 (lung cancer) cells. CAR-THP-1 cells were overlaid onto the tumour organoids and allowed to infiltrate for 24 hours, after which non-infiltrated cells were removed by gentle washing. The organoids were subsequently maintained for an additional 48 hours, dissociated on day 3, and analysed by flow cytometry.

[0133] THP-1 cells expressing a CAR construct lacking intracellular signaling domains (NC-THP-1) were used as a negative control to assess activation attributable to antigen binding alone. Across all tumour organoid models examined, CAR-THP-1 cells exhibited increased expression of the M1-associated activation marker CD80 relative to NC-THP-1 controls, as shown in Figure 8. In contrast, NC-THP-1 cells displayed minimal CD80 induction following organoid infiltration, whereas CAR-THP-1 cells showed consistent upregulation of CD80, indicating effective intracellular signaling and activation within the three-dimensional tumour microenvironment.

[0134] These results indicate that antigen binding alone is insufficient to induce activation of THP-1 monocytes, and that the presence of the disclosed intracellular signaling domains is required to drive tumour-associated macrophage activation. The consistent induction of CD80 observed across tumour organoids derived from multiple solid tumour types further indicates that the disclosed CAR-macrophage platform is broadly applicable for targeting diverse solid cancers.

[0135] Pathway-Level Remodeling of Tumour Organoids Following CAR-THP-1 Infiltration.

[0136] Bulk RNA sequencing was performed on dissociated tumour organoids following infiltration by CAR-THP-1 cells to assess pathway-level transcriptional responses. Tumour organoids were generated from four human solid tumour types, including A549 (lung), HT-29 (colorectal), Huh-7 (hepatocellular), and NCC-59 (lung), with three independent biological replicates for each tumour type. Gene set enrichment analysis (GSEA) was conducted using the Hallmark pathway collection to compute Normalized Enrichment Scores (NES), which were visualized as a clustered heatmap, as shown in Figure 9. Across multiple tumour types, CAR-THP-1 treatment was associated with reduced enrichment of epithelial and pro-tumour-associated pathways, including epithelial-mesenchymal transition, coagulation, and complement pathways, indicating disruption of tumour-associated structural and stromal-support programs. In HT-29 organoids, CAR-THP-1 treatment was further associated with increased enrichment of DNA repair and mTORCI signaling pathways together with reduced enrichment of the G2 / M checkpoint pathway, consistent with the induction of genotoxic and metabolic stress and impaired tumour proliferative capacity.

[0137] In A549, Huh-7, and NCC-59 organoids, CAR-THP-1 treatment was also associated with reduced enrichment of inflammatory response, allograft rejection, and interferon-y response pathways. As these organoid systems comprised tumour cells and CAR-THP-1 macrophages and lacked adaptive lymphocytes, which are a primary source of interferon-y, the observed pathway changes reflect modulation of tumour-intrinsic transcriptional programs rather than suppression of adaptive immune activity. Collectively, the pathway signatures observed across the four tumour types indicate that CAR-THP-1 cells impose a shared anti-tumour transcriptional influence characterized by suppression of epithelial-mesenchymal transition, coagulation, inflammatory signaling, and proliferative pathways, while permitting tumour-type-specific metabolic and stress-response adaptations. These results indicate that CAR-THP-1 cells are capable of reprogramming transcriptional programs across diverse solid tumour models.

[0138] Taken together, the results shown in Figures 8 and 9 demonstrate that infiltration of tumour organoids by CAR-THP-1 cells is associated with both activation of tumour-infiltrating macrophages, as evidenced by increased expression of the M1-associated marker CD80, and concomitant reprogramming of tumour cell transcriptional pathways. The observation of these effects across organoids derived from multiple distinct solid tumour types indicates that the disclosed CAR-macrophage platform, which incorporates an intracellular signaling domain comprising an ordered combination of a TLR4 TIR signaling domain and a 4-1 BB signaling domain, is not limited to a particular tumour origin or molecular subtype. Accordingly, these data support the potential applicability of the present invention to a broad range of tumours byenabling coordinated macrophage activation and tumour-intrinsic pathway modulation within diverse tumour microenvironments.

[0139] In summary, the experimental results described herein demonstrate that the present invention achieves a unique technical effect by combining antigen-specific recognition with an intracellular signaling architecture specifically configured to drive ctivation and functional programming of mononuclear phagocyte cells, including monocytes and macrophages, toward a pro-inflammatory, anti-tumour functional state, particularly in the context of solid tumours. In particular, the ordered arrangement of the TLR4 TIR signaling domain proximal to the transmembrane domain and the 4-1 BB signaling domain within the intracellular domain enables effective macrophage polarization, cooperative interaction with tumour-infiltrating T cells, modulation of immune checkpoint-associated exhaustion markers, and reprogramming of tumour-intrinsic transcriptional pathways. Unlike conventional CAR constructs designed primarily for lymphoid cells, or prior macrophage-based approaches that rely on antigen binding alone or nonspecific activation signals, the present invention functionally integrates innate immune signaling pathways in a manner that confers robust and reproducible anti-tumour activity across multiple solid tumour models. Accordingly, the disclosed CAR-macrophage platform provides a distinct and advantageous technical solution for overcoming limitations of existing immunotherapies in solid tumours.

[0140] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention.

[0141] As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments.

[0142] REFERENCES1. Arch, R. H. and C. B. Thompson (1998). "4-1 BB and 0x40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kappaB." Mol Cell Biol 18(1): 558-565.2. Costa, V. V., W. Ye, Q. Chen, M. M. Teixeira, P. Preiser, E. E. Ooi and J. Chen (2017)."Dengue Virus-Infected Dendritic Cells, but Not Monocytes, Activate Natural Killer Cells through a Contact-Dependent Mechanism Involving Adhesion Molecules." mBio 8(4): e00741-00717.Kienzle, G. and J. von Kempis (2000). "CD137 (ILA / 4-1BB), expressed by primary human monocytes, induces monocyte activation and apoptosis of B lymphocytes." International immunology 12(1): 73-82.Lei, A., H. Yu, S. Lu, H. Lu, X. Ding, T. Tan, H. Zhang, M. Zhu, L. Tian, X. Wang, S. Su, D. Xue, S. Zhang, W. Zhao, Y. Chen, W. Xie, L. Zhang, Y. Zhu, J. Zhao, W. Jiang, G. Church, F. K.-M. Chan, Z. Gao and J. Zhang (2024). "A second-generation M1 -polarized CAR macrophage with antitumor efficacy." Nature Immunology 25(1): 102-116.Mazzaschi, G., D. Madeddu, A. Falco, G. Bocchialini, M. Goldoni, F. Sogni, G. Armani, C. A. Lagrasta, B. Lorusso, C. Mangiaracina, R. Vilella, C. Frati, R. Altieri, L. Ampollini, M. Veneziani, E. M. Silini, A. Ardizzoni, K. Urbanek, F. Aversa, F. Quaini and M. Tiseo (2018). "Low PD-1 Expression in Cytotoxic CD8+ Tumor-Infiltrating Lymphocytes Confers an Immune-Privileged Tissue Microenvironment in NSCLC with a Prognostic and Predictive Value." Clinical Cancer Research 24(2): 407-419.Niu, Z., G. Chen, W. Chang, P. Sun, Z. Luo, H. Zhang, L. Zhi, C. Guo, H. Chen and M. Yin (2021). "Chimeric antigen receptor-modified macrophages trigger systemic anti-tumour immunity." The Journal of pathology 253(3): 247-257.Singh, R., Y.-H. Kim, S.-J. Lee, H.-S. Eom and B. K. Choi (2024). "4-1 BB immunotherapy: advances and hurdles." Experimental & Molecular Medicine 56(1): 32-39.Stoll, A., H. Bruns, M. Fuchs, S. Volkl, F. Nimmerjahn, M. Kunz, M. Peipp, A. Mackensen and D. Mougiakakos (2021). "CD137 (4-1 BB) stimulation leads to metabolic and functional reprogramming of human monocytes / macrophages enhancing their tumoricidal activity." Leukemia 35(12): 3482-3496.

Claims

CLAIMSClaim 1. A Chimeric Antigen Receptor (CAR) construct comprising an antigen binding domain, a hinge region, a transmembrane domain, and an intracellular domain connected in sequence; wherein the intracellular domain comprises at least one costimulatory signaling domain, comprising a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence;wherein the TLR4 TIR signaling domain is constructed to be positioned adjacent to the transmembrane domain.Claim 2. The CAR construct according to Claim 1, wherein the antigen binding domain comprises an scFV targeting an antigen.Claim 3. The CAR construct according to Claim 2, wherein the antigen is a tumour-specific antigen or a tumour-associated antigen.Claim 4. The CAR construct according to Claim 2 or 3, wherein the antigen is selected from a group consisting of PD-L1, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, PD1, CD19, CD123, CD70, Her2, mesothelin, claudin 6, BCMA, and EGFR.Claim 5. The CAR construct according to any one of Claim 1 - 4, wherein the hinge region comprises a CD8a, CD28, or C6 hinge region.Claim 6. The CAR construct according to any one of Claims 1 - 5, wherein the transmembrane domain comprises a CD8a, CD28, or TLR4 transmembrane domain.Claim 7. The CAR construct according to Claim 1, wherein the intracellular domain comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1.Claim 8. The CAR construct according to Claim 1, wherein the intracellular domain comprises a nucleic acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.Claim 9. The CAR construct according to Claim 1, wherein the CAR construct comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6; oran amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6.Claim 10. The CAR construct according to Claim 1, wherein the CAR construct comprises a nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5; ora nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.Claim 11. A mononuclear phagocyte cell expressing the CAR construct according any one of Claims 1 - 10, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.Claim 12. The mononuclear phagocyte cell according to Claim 11, wherein the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.Claim 13. The mononuclear phagocyte cell according to Claim 11 or 12, wherein the monocyte is THP-1 cell.Claim 14. Use of the mononuclear phagocyte cell according to any one of Claim 11 - 13 in the manufacture of a medicament for treatment of a target tumour.Claim 15. The use according to Claim 14, wherein the target tumour is a solid tumour.Claim 16. The use according to Claim 15, wherein the solid tumour is selected from a group consisting of lung, colon, liver, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.Claim 17. The use according to any one of Claims 14 - 16, wherein the treatment comprises reducing a burden of the target tumour.Claim 18. The use according to Claim 17, wherein the burden of the target tumour comprises a volume of the solid tumour.Claim 19. A mononuclear phagocyte cell for use in the treatment of target tumour, wherein the mononuclear phagocyte cell is according to any one of Claims 11 - 13.Claim 20. The mononuclear phagocyte cell for use in the treatment of tumour according to Claim 19, wherein the target tumour is a solid tumour.Claim 21. The mononuclear phagocyte cell for use in the treatment of tumour according to Claim 20, wherein the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.Claim 22. A method of treatment comprising administrating the mononuclear phagocyte cell according to any one of Claims 11 - 13 to a subject with a tumour.Claim 23. The method of treatment according to Claim 22, wherein the tumour is a solid tumour.Claim 24. The method of treatment according to Claim 23, wherein the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.Claim 25. The method of treatment according to any one of Claims 22 - 24, wherein the treatment comprises reducing a load of the target tumour.Claim 26. The method of treatment according to Claim 25, wherein the load of the target tumour is a volume of the solid tumour.Claim 27. Use of the CAR construct according to any one of Claims 1 - 10 for inducing a mononuclear phagocyte cell towards an M1 phenotype, wherein the mononuclear phagocyte cell is engineered to express the CAR construct, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.Claim 28. The use according to Claim 27, wherein the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.Claim 29. The method of inducing a monocyte or a macrophage towards an M1 phenotype, wherein the a monocyte or a macrophage is engineered to express the CAR construct according to any one of Claims 1 - 10, wherein the mononuclear phagocyte cell is a monocyte or a macrophage.Claim 30. The method according to Claim 29, wherein the mononuclear phagocyte cell is (i) primary mononuclear phagocyte cell directly isolated from a biological source, or (ii) iPSC-derived mononuclear phagocyte cell differentiated from an induced pluripotent stem cell.Claim 31. Use of the mononuclear phagocyte cell according to any one of Claims 11 - 13 in the manufacture of a medicament for mediating activities of T cells.Claim 32. Use of the mononuclear phagocyte cell according to Claim 30, wherein the T cells are tumour-infiltrating T cells.Claim 33. The use of the mononuclear phagocyte cell according to Claim 30, wherein the T cells include CD4+ T cells, CD8+ T cells, or both.Claim 34. The use of the mononuclear phagocyte cell according to Claim 30, wherein the mononuclear phagocyte cell reduces exhaustion of the T cells.Claim 35. Use of the mononuclear phagocyte cell according to any one of Claims 11 - 13 in the manufacture of a medicament for modulating a microenvironment of a solid tumour.Claim 36. The use of the mononuclear phagocyte cell according to Claim 35, wherein the solid tumour is selected from a group consisting of lung, colon, liver, breast, testicular, ovary, bladder, uterine, cervical, pancreatic, esophageal and stomach tumour.Claim 37. An intracellular domain of a Chimeric Antigen Receptor (CAR) construct, wherein the intracellular domain comprises at least one costimulatory signaling domain, comprising a TLR4 TIR signaling domain and a 4-1 BB signaling domain connected in sequence;wherein the TLR4 TIR signaling domain is constructed to be positioned adjacent to a transmembrane domain to be connected to the intracellular domain.Claim 38. The intracellular domain according to Claim 37, wherein the intracellular domain comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 1.Claim 39. The intracellular domain according to Claim 37, wherein the intracellular domain comprises a nucleic acid sequence encoded by a nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence at least 95% identical to the nucleic acid sequence of SEQ ID NO: 3.

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