Intracellular signal transduction region combination of HER2-targeting chimeric antigen receptor and application of intracellular signal transduction region combination

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an intracellular signal transduction region combination of a HER2-targeting chimeric antigen receptor and application of the intracellular signal transduction region combination. The chimeric antigen receptor containing the intracellular signal transduction region combination can keep the CAR positive rate, and can ensure the motility rate and the recovery amount of cells, so that the activity of CAR-immune cells is better ensured; immune cells of the chimeric antigen receptor containing the intracellular signal transduction region combination show significantly enhanced phagocytosis, killing and anti-depletion capabilities and T cell activation / activation level, and achieve stronger anti-tumor activity and stronger tumor wettability.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine technology, and specifically relates to a combination of intracellular signal transduction regions of a chimeric antigen receptor targeting HER2 and applications thereof. Background Art

[0002] Chimeric antigen receptor (CAR) technology has made significant progress in the treatment of hematological tumors, especially acute lymphoblastic leukemia (ALL), lymphoma and plasma cell myeloma (PCM). However, the effect of CAR-T cell therapy in the treatment of solid tumors is still unsatisfactory.

[0003] Macrophages are the main phagocytic cells in the body, which can engulf and digest pathogens such as bacteria, viruses and cancer cells. Macrophages not only have strong phagocytic ability, but also have antigen presentation function, which can activate adaptive immune response. Since the first engineering of carcinoembryonic antigen (CEA)-targeted CAR molecules into human monocytes in 2006, CAR-Macrophage / Monocyte (CAR-M) has become a powerful prospect because of its strong phagocytic ability, antigen presentation function, and ability to enter / regulate the tumor microenvironment and stimulate / trigger adaptive response.

[0004] CAR-M therapy is similar to CAR-T therapy, but its product is centered on macrophages. It is necessary to extract macrophages from the patient himself, introduce CAR molecules into macrophages through genetic engineering methods, and finally achieve tumor killing. As a key effector cell of innate immunity, macrophages have a powerful phagocytic effect. Among them, tumor-associated macrophages (TAMs) are the most important immune cells in the tumor microenvironment (TME). They have the dual role of killing tumors and promoting tumor growth. They play a very important role in the occurrence and development of malignant tumors, invasion and metastasis, immune escape, and angiogenesis and lymphangiogenesis.

[0005] CAR is an artificially designed transmembrane receptor, which usually includes four parts: single-chain variable region (scFv) that recognizes tumor antigens, hinge region, transmembrane region and intracellular signal transduction region (ICD). The ICD region of most CAR molecules used in CAR-T products is composed of CD3ζ and 41-BB or CD28 regions, which is responsible for transducing antigen recognition signals into T cells and then activating the immune response of cells.

[0006] In the design of CAR-M, the selection of different ICD regions will also give CAR-M different anti-tumor capabilities. At present, CAR-M is a method of introducing CAR molecules into macrophages through genetic engineering, enabling them to specifically recognize and kill cancer cells that express corresponding antigens. As a newcomer in the field of cell therapy, compared with the structural design of CAR-T, the structure of CAR-M is still in the stage of exploration and optimization and upgrading. Among them, the intracellular signal transduction region is an important part of the structural domain related to the function of CAR-M, and its optimization and screening are also the main directions for the optimization and upgrading of CAR-M. Summary of the invention

[0007] The present disclosure provides a CAR molecule targeting HER2, which includes two or more intracellular signaling domains (ICDs), modified immune cells containing the CAR molecule, and their use in preventing or treating tumors.

[0008] According to one aspect of the present disclosure, a chimeric antigen receptor (CAR) molecule targeting HER2 is provided, wherein the CAR molecule comprises: (a) an extracellular domain that specifically binds to HER2; (b) a transmembrane domain; and (c) two or more intracellular signaling regions, comprising: (i) an intracellular domain of FCE or CD3ζ or a portion thereof, and, (ii) an intracellular domain of any one or more of IFNgR, PI3Krecruit, TLR2, TLR4 and CD40 or a portion thereof.

[0009] In some embodiments, the CAR molecule may include two intracellular signaling regions, including: (i) the intracellular domain of FCE or a portion thereof; and, (ii) the intracellular domain of IFNgR, PI3K recruit, TLR2, TLR4 or CD40 or a portion thereof. In some embodiments, the CAR molecule may include: the intracellular domain of FCE or a portion thereof; and, the intracellular domain of IFNgR or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of FCE or a portion thereof, and the intracellular domain of PI3K recruit or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of FCE or a portion thereof, and the intracellular domain of TLR2 or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of FCE or a portion thereof, and the intracellular domain of TLR4 or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of FCE or a portion thereof, and the intracellular domain of CD40 or a portion thereof.

[0010] In some embodiments, the CAR molecule may include two intracellular signaling regions, which include from N-terminus to C-terminus: (i) the intracellular domain of CD3ζ or a portion thereof; and, (ii) the intracellular domain of IFNgR, PI3K recruit, TLR2, TLR4 or CD40 or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of IFNgR or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of PI3K recruit or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of TLR2 or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of TLR4 or a portion thereof. In some embodiments, the CAR molecule may include the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of CD40 or a portion thereof.

[0011] In some embodiments, the intracellular signaling region includes, from N-terminus to C-terminus: (1) the intracellular domain of FCE or a portion thereof, and the intracellular domain of IFNgR or a portion thereof; (2) the intracellular domain of FCE or a portion thereof, and the intracellular domain of PI3Krecruit or a portion thereof; (3) the intracellular domain of FCE or a portion thereof, and the intracellular domain of TLR2 or a portion thereof; (4) the intracellular domain of FCE or a portion thereof, and the intracellular domain of TLR4 or a portion thereof; (5) the intracellular domain of FCE or a portion thereof, and the intracellular domain of CD40 or a portion thereof; (6) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of IFNgR or a portion thereof; (7) the intracellular domain of CD3ζ or a portion thereof, and PI3K recruit or a portion thereof; (8) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of TLR2 or a portion thereof; (9) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of TLR4 or a portion thereof; or, (10) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of CD40 or a portion thereof.

[0012] In some embodiments, the intracellular domain of FCE may have an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 85% identity thereto.

[0013] In some embodiments, the intracellular domain of CD3ζ has an amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence that is at least 85% identical thereto.

[0014] In some embodiments, the intracellular domain of the IFNgR has an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence that is at least 85% identical thereto.

[0015] In some embodiments, the intracellular domain of PI3K recruit has an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 85% identity thereto.

[0016] In some embodiments, the intracellular domain of TLR2 has an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 85% identity thereto.

[0017] In some embodiments, the intracellular domain of TLR4 has an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 85% identity thereto.

[0018] In some embodiments, the intracellular domain of CD40 has an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 85% identity thereto.

[0019] In some embodiments, the two intracellular signaling regions described above can be directly connected or connected via a linker.

[0020] In some embodiments, the transmembrane domain is selected from the following transmembrane domains: CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, CD2, CD3D, CD3E, CD3G, CD3G, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150, CD166, CD200R, CD223, CD270, CD272, CD273, CD274, CD278, CD300, CD357, A2a R, ICAM-1, 2B4, BTLA, DAP10, FcR1, FcR1, Fyn, GAL9, IL7, IL12, IL15, KIR, KIR2DL4, KIR2DS1, LAG-3, Lck, LAT, LPA5, LRP, NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, SLP-76, SIRP7, pTR, T cell receptor polypeptide, TIM3, TRIM, ZAP70, or any combination thereof.

[0021] In some embodiments, the transmembrane domain may include a CD8 transmembrane domain. In a preferred embodiment, the CD8 transmembrane domain may have an amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least 85% identity thereto.

[0022] In some embodiments, the CAR molecule may further include an extracellular leader domain and / or an extracellular hinge domain.

[0023] In some embodiments, the extracellular leader domain may include a CD8 extracellular leader domain or a portion thereof. In some embodiments, the extracellular leader domain may have an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 85% identity thereto.

[0024] In some embodiments, the extracellular hinge domain is selected from the following hinge domains: CD8, CD28, CD4, IgG, PD-1, CTLA-4, CD2, LFA-1, CD5, CD27, CD70, 4-1BB, OX40, ICOS, IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgE Fc region, IgM Fc region, IgA Fc region or any combination thereof.

[0025] In some embodiments, the extracellular hinge domain may include a CD8 extracellular hinge domain or a portion thereof. In some embodiments, the extracellular hinge domain may have an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 85% identity thereto.

[0026] In some embodiments, the extracellular domain that specifically binds to HER2 includes a single-chain antibody (scFv), a heavy chain single domain (VHH) antibody, or an antigen-binding fragment thereof that can specifically bind to HER2. In some embodiments, the extracellular domain that specifically binds to HER2 includes a single-chain antibody (scFv) or an antigen-binding fragment thereof that can specifically bind to HER2. In some embodiments, the single-chain antibody (scFv) that can specifically bind to HER2 can have an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence that has at least 85% identity thereto.

[0027] In some embodiments, the CAR molecule may include, from N-terminus to C-terminus, an extracellular leader domain, an extracellular domain that specifically binds to HER2, an extracellular hinge domain, a transmembrane domain, and an intracellular signaling region.

[0028] In some embodiments, the CAR molecule can have an amino acid sequence as shown in any one of SEQ ID NOs: 13-17, 19-22, or an amino acid sequence having at least 85% identity thereto.

[0029] While maintaining the CAR positivity rate, the chimeric antigen receptor can ensure the cell viability and recovery rate, so that the CAR-cell activity is better guaranteed; cells containing the chimeric antigen receptor with this intracellular signal transduction region combination show significantly enhanced phagocytosis, killing, anti-exhaustion ability and T cell activation / activation level, achieving more powerful anti-tumor activity and stronger tumor infiltration.

[0030] According to another aspect of the present disclosure, a nucleic acid molecule encoding the above-mentioned CAR molecule of the present disclosure is provided.

[0031] In some embodiments, the nucleic acid molecule encoding the above-mentioned CAR molecule of the present disclosure may have a nucleotide sequence as shown in any one of SEQ ID NOs: 24-28, 30-33, or a nucleotide sequence having at least 85% identity thereto.

[0032] According to yet another aspect of the present disclosure, a vector is provided, which comprises the above-mentioned nucleic acid molecule of the present disclosure.

[0033] In some embodiments, the vector can be a non-viral vector or a viral vector, preferably a viral vector, such as, but not limited to, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector (such as a lentiviral vector or a gamma-retroviral vector). In some embodiments, the adeno-associated viral vector can include an Ad5 vector (such as an Adf35 viral vector).

[0034] According to another aspect of the present disclosure, a modified immune cell is provided, which includes the above-mentioned CAR molecule, nucleic acid molecule or vector of the present disclosure.

[0035] In some embodiments, the modified immune cells may include stem cells, macrophages, monocytes or dendritic cells. In a preferred embodiment, the modified immune cells may include macrophages and / or monocytes. In a preferred embodiment, the modified immune cells may include monocytes. In a preferred embodiment, the modified immune cells may be macrophages.

[0036] In some embodiments, the modified immune cells exhibit increased effector activity compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit increased tumor killing activity and / or phagocytic activity compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit increased depletion capacity compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit increased tumor infiltration capacity compared to unmodified immune cells.

[0037] In some embodiments, the modified immune cells exhibit increased polarization to the M1 phenotype compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit increased expression of one or more markers of the M1 phenotype compared to unmodified immune cells. In some embodiments, the modified immune cells exhibit decreased expression of one or more markers of the M2 phenotype compared to unmodified immune cells.

[0038] In some embodiments, the one or more markers of the M1 phenotype may include, but are not limited to, one or more of CD86, CD80, MHCII, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, CD68, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members, or ISG family members.

[0039] In some embodiments, the one or more markers of the M2 phenotype may include, but are not limited to, one or more of CD206, CD163, CD209, FIZZ1, and Ym1 / 2.

[0040] According to another aspect of the present disclosure, a method for preparing the modified immune cell of the present disclosure is provided, the method comprising the step of delivering a nucleic acid molecule or a vector encoding the CAR molecule of the present disclosure into the immune cell.

[0041] In some embodiments, the nucleic acid molecule can be an RNA molecule or a DNA molecule.

[0042] According to another aspect of the present disclosure, a pharmaceutical composition is provided, which includes the above-mentioned CAR molecules, nucleic acid molecules, vectors or immune cells disclosed herein. In some embodiments, the pharmaceutical composition may include a pharmaceutically acceptable carrier.

[0043] According to yet another aspect of the present disclosure, there is provided a method for preventing or treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of the present disclosure.

[0044] In some embodiments, the subject may include a mammal, such as a human or a non-human mammal. In some embodiments, the non-human mammal may include, but is not limited to, non-human primates (e.g., monkeys, gorillas), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cattle, pigs, etc.

[0045] According to another aspect of the present disclosure, provided is the use of the above-mentioned CAR molecules, nucleic acid molecules, vectors or immune cells of the present disclosure in the preparation of drugs for preventing or treating diseases or disorders.

[0046] In some embodiments, the disease or disorder may include a tumor / cancer, preferably a solid tumor.

[0047] In some embodiments, the tumor / cancer may include, but is not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, gastric cancer, esophageal cancer, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Schematic diagrams of the molecular structures of various HER2 CARs are shown.

[0049] Figure 2 Shown is the amount of macrophages recovered that were transfected with IVT RNA of each HER2 CAR molecule.

[0050] Figure 3 Shown is the viability of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0051] Figure 4 The results of characterization of macrophages transfected with IVT RNA of each HER2 CAR molecule are shown. Among them, the expression of CAR molecules on the surface of macrophage membranes is shown respectively ( Figure 4 A) CD80 molecule ( Figure 4 B) and CD163 molecules ( Figure 4 C) percentage.

[0052] Figure 5 Shown are the 2 h phagocytosis results of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0053] Figure 6 Shown are the 48h killing results of macrophages transfected with IVT RNA of each HER2 CAR molecule.

[0054] Figure 7 The viability of monocytes transfected with adenovirus expressing each HER2 CAR molecule is shown ( Figure 7A) and recovery volume ( Figure 7 B).

[0055] Figure 8 The percentage of monocytes transfected with adenovirus expressing each HER2 CAR molecule that expressed the CAR molecule is shown.

[0056] Fig. 9 The phenotypes of monocytes transfected with adenovirus expressing each HER2 CAR molecule are shown. Among them, the expression of CD80 molecules on the surface of macrophage membranes ( Fig. 9 A) and CD163 molecules ( Fig. 9 B) percentage.

[0057] Fig.10 The suppressive phenotype of monocytes transfected with adenovirus expressing each HER2 CAR molecule is shown. Among them, macrophages expressing PD-L1 ( Fig.10 A) and PD-L2 ( Fig.10 B) percentage.

[0058] Fig.11 Shown are the 48 h killing results of monocytes transfected with adenovirus expressing each HER2 CAR molecule.

[0059] Fig.12 The results show that after transfection with adenovirus expressing each HER2 CAR molecule, CAR molecules are expressed in monocytes ( Fig.12 A) and macrophages ( Fig.12 B) Expression levels on.

[0060] Fig.13 The results show that after transduction with adenovirus expressing each HER2 CAR molecule, monocytes ( Fig.13 A) and macrophages ( Fig.13 B) Phagocytosis level in vitro for 2 h.

[0061] Fig.14 Figure 2 shows the expression of HER2 CARs in monocytes after adenovirus transfection. Fig.14 A) and macrophages ( Fig.14 B) In vitro killing level at 48h.

[0062] Fig.15 Shown are the depletion levels of monocytes transfected with adenovirus expressing each HER2 CAR after 4 days of co-incubation with OE19 target cells.

[0063] Fig.16 The CAR expression of monocytes transfected with adenovirus expressing each HER2 CAR after 4 days of co-incubation with OE19 target cells is shown.

[0064] Fig.17 Shown are the phenotypes of monocytes transfected with adenovirus expressing each HER2 CAR after 4 days of co-incubation with OE19 target cells.

[0065] Fig.18 The long-term killing results of monocytes transfected with adenovirus expressing each HER2 CAR and OE19 target cells at E:T=5:1 are shown.

[0066] Fig.19 The long-term killing results of monocytes transfected with adenovirus expressing each HER2 CAR molecule and OE19 target cells at E:T=1:1 are shown.

[0067] Fig. 20 The long-term killing results of monocytes transfected with adenovirus expressing each HER2 CAR and OE19 target cells at E:T=1:5 are shown.

[0068] Fig.21 The graph shows the infiltration and killing results of monocytes transfected with adenovirus expressing each HER2 CAR against OE19-NLS mCherry monoclonal 3D tumor spheres at E:T=5:1 and 1:1.

[0069] Fig. 22 Shown are the 2 h phagocytosis results of macrophages transfected with adenovirus of each HER2 CAR molecule.

[0070] Fig.23 The plasmid map of pVAX1 is shown. DETAILED DESCRIPTION

[0071] Macrophages are immune cells specialized for detecting, engulfing, and destroying target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system, capable of performing at least three distinct anti-tumor functions: engulfing dead and dying cells, microorganisms, cancer cells, cell debris, or other foreign matter; cytotoxicity against tumor cells; and, presentation of tumor antigens to coordinate adaptive anti-tumor immune responses.

[0072] Although CAR-M therapy has shown potential in tumor treatment, its application in solid tumors still faces many challenges. The dense tissue structure and complex tumor microenvironment of solid tumors limit the infiltration and killing effect of CAR-M cells. Moreover, macrophages in the tumor microenvironment are easily induced into M2 macrophages that promote tumor growth, weakening their anti-tumor function.

[0073] The present invention discloses, through design and screening, a HER2 CAR molecule containing a combination of specific intracellular domains. Monocytes / macrophages containing the CAR molecule exhibit significantly enhanced phagocytosis, killing, and T cell activation / activation levels, thereby achieving more powerful anti-tumor activity and stronger tumor infiltration.

[0074] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0075] definition

[0076] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0077] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0078] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0079] Monocytes are a subset of leukocytes that play a key role in maintaining body homeostasis, pathogen recognition and clearance, and inflammation. After generation and development from myeloid progenitor cells, monocytes circulate in the vasculature, bone marrow, and spleen; they do not proliferate in steady state. Once monocytes migrate to peripheral tissues, either during normal development or due to pathogen attack, they differentiate into dendritic cells or macrophages.

[0080] Macrophages are specialized, long-lived, phagocytic cells of the innate immune system. Together with neutrophils, they are the first responders to infection. Macrophages are involved in the recognition, phagocytosis, and degradation of cell debris and pathogens. Macrophages also play a role in presenting antigens to T cells and inducing other antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. In addition, at the beginning of inflammation, macrophages play an important role by releasing cytokines and chemokines, which in turn recruit other immune cells to the site of inflammation. Macrophages are present in most tissues and therefore have different functions. In addition to initiating immune and inflammatory responses to pathogens, macrophages also play a role in maintaining tissue homeostasis and repair and remodeling of tissues. Macrophages also appear to play a key role in the tumor microenvironment, especially in matrix remodeling, angiogenesis, metastasis, and tumor progression. There are many sources of macrophages. Tissue-resident macrophages can differentiate from circulating monocytes or be generated during embryonic development in the fetal liver, yolk sac, or embryonic regions near the dorsal aorta and thus exist independently of monocytes in adulthood.

[0081] Different factors can lead to different phenotypes and activation states of macrophages. In general, macrophages can be divided into two subtypes according to their functions and activation effects: classically activated M1 macrophages and alternatively activated M2 macrophages. M1 macrophages mainly play a role in Th1 cell recruitment, pathogen resistance and tumor control through innate and adaptive immune responses. M1 macrophages have strong antigen presentation activity and secrete a large number of proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, TNF-α, nitric oxide (NO) and reactive oxygen species (ROS). M1 macrophages express high levels of major histocompatibility complex class II (MHC II), CD68, CD80, CD86, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, CD69, MHC I, CD64, CD32, CD16, IL1R, IFIT family members or ISG family members, and chemokines for Th1 cells, such as CXCL9 and CXCL12. M2 macrophages can be activated by parasitic or fungal infection, immune complexes, apoptotic cells, macrophage colony stimulating factor (M-CSF), IL-13, TGF-β, and type 2 helper T cell factors (Th2) IL-4, IL-33, and IL-25 through Th2 cell substitution. M2 macrophages express high levels of CD206, CD163, CD209, FIZZ1, Ym1 / 2, etc.

[0082] As used herein, the terms "effective amount" and "therapeutically effective amount" are interchangeable and refer to an amount of a compound, formulation, material or composition effective to achieve a specific biological result or provide a therapeutic or preventive benefit as described herein. Such results may include, but are not limited to, anti-tumor activity determined by any suitable means in the art.

[0083] The term "effector function" or "effector activity" as used herein refers to a specific activity performed by an immune cell in response to a stimulus to the immune cell. For example, the effector function of a macrophage is to engulf and digest cell debris, foreign matter, microorganisms, cancer cells, and other unhealthy cells by phagocytosis.

[0084] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0085] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs, and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. Mention herein that having "at least 85% identity" compared to a reference sequence can include at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0086] The term "single-chain antibody" or "scFv" as used herein refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region via amino acid engineering segments. Various methods for producing single-chain antibodies are known.

[0087] As used herein, the term "treatment" may refer to treatment and / or prevention, with a therapeutic effect being achieved by suppressing, alleviating or eradicating a disease state.

[0088] In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject. The immune cells may be autologous or derived from an allogeneic or universal donor. The cells may be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, tumors, and / or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells may be obtained from blood collected from a subject using any number of separation techniques known to the skilled artisan. In some embodiments, cells from circulating blood of a subject are obtained by apheresis or leukapheresis. In some embodiments, following enrichment, differentiation of immune cells (e.g., monocytes) comprises stimulation with GM-CSF. In some embodiments, the cells comprising blood cells (e.g., monocytes, lymphocytes, or dendritic cells) are isolated from the subject. In some embodiments, the selection of immune cells (e.g., monocytes, platelets, plasma and / or erythrocytes) includes selecting using CD14 positivity. Immune cell precursors can be differentiated into immune cells in vivo or in vitro. Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitor cells, myeloblasts, promonocytes, promonocytes, promonocytes or their intermediates. For example, induced pluripotent stem cells (iPSCs) can be used to generate monocytes, macrophages and / or dendritic cells. Induced pluripotent stem cells can be derived from normal human tissues, such as peripheral blood, fibroblasts, skin, keratinocytes or renal epithelial cells. Autologous, allogeneic or universal donor iPSCs can be differentiated into bone marrow lineages (e.g., monocytes, macrophages, dendritic cells or their precursors).

[0089] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention. The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods.

[0090] It should be noted that, if the specific conditions are not specified in the examples, the experimental conditions are carried out according to conventional conditions, manufacturer recommendations or publicly reported experimental conditions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. If the manufacturer of the reagents used is specified, similar products from other manufacturers are substitutable.

[0091] Example

[0092] Example 1. Construction of CAR-immunogenic cell death (ICD) in vitro transcription (IVT) plasmids with different combinations and IVT RNA preparation

[0093] Different CAR-ICD IVT vector plasmids were constructed, and the plasmid pVAX1 (purchased from Nanjing GenScript, Fig.23 ).according to Figure 1 The schematic diagrams of the structures of various CAR-ICDs are shown in the figure, and the sequences of various parts of CAR-ICD are shown in Table 1 below.

[0094] Table 1 Amino acid sequences of various parts

[0095]

[0096]

[0097] HER2 CAR-FCE (SEQ ID NO: 12), HER2 CAR-FCE-IFNR (SEQ ID NO: 13), HER2 CAR-FCE-PI3K (SEQ ID NO: 14), HER2 CAR-FCE-TLR2 (SEQ ID NO: 15), HER2 CAR-FCE-TLR4 (SEQ ID NO: 16), HER2 CAR-FCE-CD40 (SEQ ID NO:17), HER2 CAR-CD3ζ (SEQ ID NO:18), HER2CAR-CD3ζ-IFNR (SEQ ID NO:19), HER2 CAR-CD3ζ-PI3K (SEQ ID NO:20), HER2CAR-CD3ζ-TLR4

[0098] (SEQ ID NO:21) and HER2 CAR-CD3ζ-CD40 (SEQ ID NO:22) nucleic acid sequences (the corresponding nucleic acid sequences are shown in SEQ ID NO:23-33, respectively) were inserted into the pVAX1 plasmid to prepare the IVT plasmid.

[0099] The IVT plasmid was digested with BspQI restriction endonuclease (purchased from Takara), and the linearized DNA was purified and recovered using DNA magnetic beads (purchased from Shenji Biotechnology). The cap3011 RNA preparation kit (purchased from Shenji Biotechnology) was used to transcribe the linearized DNA into mRNA in vitro, and the IVT RNA was purified and recovered using RNA magnetic beads (purchased from Shenji Biotechnology).

[0100] Example 2. Detection of HER2 CAR molecule expression under IVT RNA transfection

[0101] The single blood sample obtained from a commercial company (which has passed relevant ethical approval) was centrifuged at 200g for 10 minutes to remove platelets and some red blood cells, and the supernatant was discarded. The cell pellet was mixed with PBS, added to Ficoll (lymphocyte separation fluid, purchased from Sigma), and centrifuged at 1200rpm for 30 minutes. After centrifugation, mononuclear cells (PBMC) were recovered and washed once with phosphate buffered saline (PBS) by centrifugation. PBMCs were incubated with CD14+ magnetic beads (purchased from Miltenyi Biopharmaceuticals) for 15 min, CD14+ cells were separated using a CD14 sorting column (purchased from Miltenyi Biopharmaceuticals), CD14+ monocytes were harvested, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of monocytes into macrophages (refer to the induction method in "MAS, et al. An Immune Atlas of Clear Cell Renal Cell Carcinoma. Cell. 2017 May 4; 169(4): 736-749.e18.").

[0102] After the monocytes were induced to differentiate into macrophages, they were divided into 12 experimental groups and different IVTRNA and Lipofectamine of different HER2 CAR-ICD molecules were added. TM MessengerMAX TM The negative control group (UTD group) consisted of macrophages that were not transfected with RNA. Cells were harvested 48 h after transfection, and at least 1×10 6 After mixing with PBS, centrifuge at 400g for 5 minutes, add 2μL FcR Blocking (Miltenyi Biotec, 130-059-901), incubate at room temperature in the dark for 10 minutes, add the antibody mixture in Table 2, incubate at 4°C in the dark for 30 minutes, centrifuge at 400g for 5 minutes, wash once with PBS and perform flow cytometry. The macrophage recovery rate of each group was analyzed by flow cytometry ( Figure 2 ) and survival rate ( Figure 3 ), and the percentage of CAR molecules targeting HER2 on the surface of macrophage membrane detected by flow cytometry ( Figure 4 A) CD80+ cell percentage ( Figure 4 B) and percentage of CD163+ cells ( Figure 4 C) Table 2 shows the antibodies used in the flow cytometry assay and their manufacturers.

[0103] Table 2. Antibodies for flow cytometry

[0104]

[0105]

[0106] The results showed that in terms of recovery rate and viability, the combined ICD-CAR was superior to either FCE or CD3ζ alone; at the same time, each combination had a good positive expression level of CAR molecules; phenotypic information showed that the groups after IVT RNA transfection tended to be polarized to M1 (CD80+), while showing resistance to M2 (CD163+).

[0107] Example 3. In vitro phagocytosis experiment of HER2 CAR-M transfected with IVT RNA

[0108] OE19 cells (human esophageal cancer cells, purchased from COBIOER BIOSCIENCES) were suspended in PBS. DiR dye (5 μM) was added to the cell suspension and incubated at 37°C for 20 minutes. The cells were centrifuged at 400 g for 5 minutes and the supernatant was aspirated. Pre-warmed X-VIVO15 medium (purchased from Lonza, hereinafter referred to as "complete medium") containing 10% FBS (purchased from Gibco) was added to resuspend the cells to a density of 1×10 6 cells / mL.

[0109] The IVT RNAs of HER2 CAR-FCE, HER2 CAR-FCE+IFNgR (IFNR), HER2 CAR-FCE+PI3K, HER2CAR-FCE+TLR2, HER2 CAR-FCE+TLR4, HER2 CAR-FCE+CD40, HER2 CAR-CD3ζ, HER2CAR-CD3ζ+IFNgR (IFNR), HER2 CAR-CD3ζ+PI3K, HER2 CAR-CD3ζ+TLR4 and HER2 CAR-CD3ζ+CD40 prepared in Example 1 were respectively transfected into the macrophages obtained according to the method of Example 2. After 2 days, the cells were suspended in PBS, centrifuged at 400 g for 5 minutes, the supernatant was aspirated, and preheated complete medium was added to the cell pellet to resuspend the cells to a density of 1×10 6 cells / mL.

[0110] The negative macrophage group (UTD group) consisted of macrophages not transfected with adenovirus. The cells were suspended in PBS and centrifuged at 400 g for 5 min. The supernatant was aspirated and pre-warmed complete medium was added to the cell pellet to resuspend the cells to a density of 1×10 6 cells / mL.

[0111] Transfected macrophages and negative macrophages are collectively referred to as effector cells, and OE19 cells are referred to as target cells. Effector cells and target cells were added to the culture dish at a ratio of 1:1. After culturing for 2 hours, the cells were collected, stained with anti-CD11b-FITC (purchased from eBioscience), and flow cytometry was used to analyze cells containing DiR markers in the CD11b+ population, which were macrophages that phagocytized OE19.

[0112] The results showed that the combined ICD-CAR was superior to the single HER2-FCE or HER2-CD3ζ in terms of phagocytic ability, and the phagocytic ability of ICD containing PI3K was significantly improved (see Figure 5 ).

[0113] Example 4. In vitro killing experiment of HER2 CAR-M transfected with IVT RNA

[0114] OE19 cells were plated in 6-well plates and cultured until the cell confluence was 60-80%. The target cells were transformed using commercial luciferase lentivirus (purchased from GeneCare Biotechnology Co., Ltd.) at an MOI of 10. Puromycin was used to screen the transformed cells, and the OE19 transformed cell line stably expressing luciferase (hereinafter referred to as OE19-luciferase cells) was obtained for in vitro killing detection.

[0115] Harvest OE19-luciferase cells and resuspend in pre-warmed complete medium to a cell density of approximately 1 × 10 5 cells / mL. 4 Cells were plated in 96-well plates (Corning / Cat: 3610) and incubated at 37°C for 2 hours.

[0116] Macrophages were transfected with IVTRNA of HER2 CAR-FCE, HER2 CAR-FCE+IFNgR, HER2 CAR-FCE+PI3K, HER2CAR-FCE+TLR2, HER2 CAR-FCE+TLR4, HER2 CAR-FCE+CD40, HER2 CAR-CD3ζ, HER2CAR-CD3ζ+IFNgR, HER2 CAR-CD3ζ+PI3K, HER2 CAR-CD3ζ+TLR4 and HER2 CAR-CD3ζ+CD40 prepared in Example 1. After 2 days, the cells were suspended in PBS, centrifuged at 400g for 5 minutes, the supernatant was aspirated, and pre-warmed complete medium was added to the cell pellet to resuspend the cells to a density of about 1×10 6 cells / mL.

[0117] The negative macrophage group (UTD group) consisted of macrophages not transfected with RNA. The cells were suspended in PBS and centrifuged at 400 g for 5 min. The supernatant was aspirated and pre-warmed complete medium was added to the cell pellet to resuspend the cells to a density of about 1 × 10 6 cells / mL.

[0118] Effector cells and OE19-luciferase cells were added according to the effector-target ratio of 5:1, 1:1, 1:5 and 1:10 (100 μL volume per well). After incubation at 37°C for 48 hours, centrifuge at 500 g for 5 minutes, discard 100 μL of the supernatant after the cells are precipitated. Add 50 μL of the prepared Luciferase Assay reagent (Promega / Cat: E2520). After 5 minutes of reaction, the microplate reader was used to read the results.

[0119] The results showed that based on OE19-luciferase target cells, the killing ability of HER2-CAR molecules containing ICD combinations was higher, among which the five ICD combinations of FCE-IFNgR, FCE-PI3K, FCE-TLR2, CD3ζ-IFNgR and CD3ζ-PI3K were the best. Especially under the condition of low efficiency target ratio, the killing ability of HER2-CAR molecules containing ICD combinations was better than that of HER2-FCE or HER2-CD3ζ CAR molecules containing single ICD (see Figure 6 ).

[0120] Example 5. Detection of HER2 CAR molecule expression in adenovirus-transfected monocytes

[0121] Single blood samples obtained from commercial companies (the single blood samples have passed relevant ethical approvals) were centrifuged at 200g for 10 minutes to remove platelets and some red blood cells, and the supernatant was discarded. The cell pellet was mixed with PBS, added to Ficoll (lymphocyte separation fluid, purchased from Sigma), and centrifuged at 1200rpm for 30 minutes. After centrifugation, mononuclear cells (PBMCs) were recovered and washed once with phosphate buffered saline (PBS). PBMCs were incubated with CD14+ magnetic beads (purchased from Miltenyi) for 15 minutes, CD14+ cells were separated using CD14 sorting columns (purchased from Miltenyi), and CD14+ monocytes were harvested.

[0122] The synthesized FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K six groups of CAR molecule encoding nucleic acid sequences were constructed into a plasmid system with a vector skeleton of AD5 / F35 (purchased from Yunzhou Biology), and the plasmid was linearized in vitro using Pac I endonuclease, and then 293A cells were infected and cultured until the cytopathic effect (plaques appeared between cells, some cells appeared in strings, etc.) reached 70-80%, that is, the P0 generation virus seed was harvested. The P0 generation virus seed was expanded and cultured, and the cells were harvested and lysed to collect the virus when the cytopathic effect reached ≥50%. After the lysis was completed, the virus was purified and divided for use.

[0123] FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K were divided into 6 experimental groups, and adenovirus with different HER2 CAR-ICD molecules was added respectively. The negative control group (UTD group) was macrophages not transfected with adenovirus. The cells were harvested on the second day after transfection, and at least 1×10 6 After mixing with PBS, centrifuge at 400g for 5 minutes. After mixing with PBS, the cell pellet was added with HER2 protein (purchased from Acro) (4μl / test), incubated at room temperature for 20 minutes, and centrifuged at 400g for 5 minutes to remove unbound HER2 protein. After washing, the cells were subjected to flow cytometry detection of CAR molecules targeting HER2 according to the method of Example 2, and the macrophage viability of each group was analyzed ( Figure 7 A) and recovery volume ( Figure 7 B).

[0124] Flow cytometry was used to detect the percentage of CAR molecules expressed on the surface of macrophage membranes ( Figure 8 ), percentage of CD80 molecule expression ( Fig. 9 A) The percentage of CD163 molecule expression ( Fig. 9 B), and inhibitory receptor molecules PD-L1 and PD-L2 ( Fig.10 A to Fig.10 B).

[0125] The results showed that in terms of viability, various ICD combinations of FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K were all on par with the negative control group; in terms of recovery rate, various ICD combinations were better than the negative control group; at the same time, each ICD combination had a good CAR positive expression level; phenotypic information showed that each group after adenovirus transfection tended to M1 (CD80+) polarization, while showing resistance to M2 (CD163+); in terms of inhibitory phenotype, the ICD combination showed an inhibitory effect with lower PD-L1 and PD-L2 expression.

[0126] Example 6. In vitro phagocytosis experiment of HER2 CAR-M in monocytes transfected with adenovirus

[0127] Resuspend OE19 cells in complete medium to a density of 1 × 10 6 cells / mL.

[0128] According to the method of Example 5, six groups of macrophages transfected with adenovirus derived from FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K for 1 day were suspended in PBS, DiR dye (5 μM) was added, and incubated at 37°C for 20 minutes. Centrifuge at 400 g for 5 minutes and aspirate the supernatant. Preheated complete medium was added to the cell pellet to resuspend the cells to a density of 1×10 6 cells / mL.

[0129] The negative macrophage group (UTD group) consisted of macrophages not transfected with adenovirus. The cells were suspended in PBS and centrifuged at 400 g for 5 min. The supernatant was aspirated and pre-warmed complete medium was added to the cell pellet to resuspend the cells to a density of 1×10 6 cells / mL.

[0130] Effector cells and target cells (OE19 cells) were added to the culture dish at a ratio of 1:1. After 2 hours of co-culture, the cells were collected and stained with anti-CD11b-FITC (purchased from eBioscience). The cells containing DiR markers in the CD11b+ group were analyzed by flow cytometry, and these cells were considered to be macrophages that phagocytized OE19 ( Fig. 22 ).

[0131] Example 7. In vitro killing experiment of HER2 CAR-M transfected with adenovirus into monocytes

[0132] OE19-luciferase cells were harvested and resuspended in pre-warmed complete medium at a cell density of 1 × 10 5 After mixing, add 100 μL (1×10 4 ), plated in 96-well plates (Corning / Cat: 3610), and incubated at 37°C for 2 hours.

[0133] A total of 6 groups of macrophages transfected with adenovirus derived from FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K for 1 day were suspended in PBS. Centrifuged at 400 g for 5 minutes, the supernatant was aspirated, and pre-warmed complete medium was added to the cell pellet to resuspend the cells to a density of 1×10 6cells / mL.

[0134] The negative macrophage group (UTD group) was composed of macrophages not transfected with adenovirus and suspended in PBS. Centrifuged at 400 g for 5 minutes and the supernatant was aspirated. Pre-warmed complete medium was added to the cell pellet to resuspend the cells to a density of 1×10 6 cells / mL.

[0135] Effector cells were added according to the effector-target ratio of 5:1, 1:1, 1:5 and 1:10 (100 μL volume per well). After incubation at 37°C for 48 hours, centrifuge at 500 g for 5 minutes. After the cells were precipitated, 100 μL of the supernatant was discarded and 50 μL of the prepared Luciferase Assay reagent (Promega / Cat: E2520), after 5 minutes of reaction, the results were read on a microplate reader.

[0136] The results showed that based on OE19-luciferase target cells, the killing ability of CAR molecules containing ICD combinations was higher, and the killing ability of ICD combinations FCE-PI3K and CD3ζ-PI3K2 was the best. Under low-efficiency target ratio conditions, CAR molecules containing ICD combinations were superior to CAR molecules containing single ICD (FCE or CD3ζ) (see Fig.11 )

[0137] Example 8. Comparison of the functions of monocytes transfected with adenovirus and induced macrophages

[0138] After plating the CD14+ monocytes obtained in Example 2, they were divided into 6 experimental groups. Adenoviruses expressing different HER2CAR-ICD molecules (prepared as in Example 5) were added respectively, containing different ICD combinations of FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K. There was one group in the negative control group (UTD group), which was monocytes not transfected with adenovirus. Cells were harvested on the 2nd day (Mono-D state) and the 7th day (macrophages) after transfection.

[0139] Detecting the percentage of CAR molecules expressed on the surface of monocytes and macrophages (see Example 5 for the method);

[0140] Detecting the phagocytic effect on OE19-luciferase target cells (method see Example 6);

[0141] According to the effector-target ratio of 5:1 and 1:1, the killing effect on OE19-luciferase target cells was detected (the method is shown in Example 7).

[0142] The results showed that each ICD combination had a good CAR positive expression level in monocytes and macrophages ( Fig.12 ); This cell is a monocyte (day 2) or macrophage (day 7) that phagocytizes OE19, and in terms of phagocytic ability, in the case of monocytes or macrophages, the combined ICD-CAR is superior to the single ICD-CAR ( Fig.13 ); in terms of killing ability, in the case of monocytes (day 2), the combined ICD-CAR was superior to the single ICD-CAR at high and low efficiency target ratios; in the case of macrophages (day 7), under the condition of high efficiency target ratio (5:1), the killing level tended to saturation (~100%); under the condition of low efficiency target ratio (1:1), the killing level was also significantly improved (>70%) ( Fig.14 ).

[0143] Example 9. Monocyte depletion levels after adenoviral transfection

[0144] After CD14+ monocytes were plated, IFNgR was divided into 6 experimental groups, and adenovirus expressing different HER2 CAR-ICD molecules was added, including different ICD combinations of FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K. There was one negative control group (UTD group), which was macrophages not transfected with adenovirus.

[0145] On the second day after transfection (day 2), CD14+ monocytes were harvested and co-incubated with OE19-luciferase target cells at an E:T=1:1 for 4 days (day 6). The harvested cells were then subjected to flow cytometry to detect the expression of HER2 CAR, CD11b, CD80, and CD86 molecules.

[0146] The results showed that the cell depletion level showed that the CAR-M in the ICD combination had lower depletion ability when co-incubated with tumor cells compared with the corresponding ICD group or UTD group ( Fig.15 ); and the CAR positive expression level of the ICD combination after co-incubation was significantly higher than that of the single ICD group ( Fig.16 ), while also maintaining the M1 macrophage phenotype (CD80 and CD86 expression) compared with the ICD group alone ( Fig.17 ).

[0147] Example 10. Long-term killing of monocytes after adenovirus transfection

[0148] After plating CD14+ monocytes, IFNgR were divided into 6 experimental groups, and adenoviruses with different HER2 CAR-ICD molecules were added, which contained different ICD combinations of FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K.

[0149] There was one negative control group (UTD group), which consisted of macrophages not transfected with adenovirus.

[0150] The cells were harvested on the second day after transfection (day0), and the effector cells were added to the OE19-luciferase target cells at an effector-target ratio of 5:1, 1:1, and 1:5, respectively (100 μL volume was added to each well, and parallel plates were laid). After 72 hours, the cells were centrifuged at 500g for 5 minutes, and 100 μL of the supernatant was discarded, and complete medium containing OE19-luciferase target cells was added (100 μL volume was added to each well). At the same time, luciferase substrate detection was performed on the second day (day2, 48h), the third day (day3, 72h before rehydration, 72-add after rehydration) and the sixth day (day6, 72-+72h) after plating.

[0151] Luciferase detection, kill 96-well white bottom transparent plate, centrifuge at 400g for 5 minutes. Prepare detection substrate reaction solution (Promega / Cat. E250), thaw at room temperature 100ml Luciferase Assay Buffer added 1 vial Luciferase Assay Substrate (lyophilized) was mixed; 50 μL of the prepared lyophilized Luciferase Assay Substrate was added to each well of the 96-well plate to be tested. Luciferase Assay reagent, react for 5 minutes, and read on the microplate reader.

[0152] The results showed that under the condition of E:T=5:1, compared with the UTD group, the luciferase values ​​of the ICD combination group and the corresponding ICD group showed a significant and sustained inhibition trend, among which the ICD combination group had a stronger inhibitory effect than the corresponding ICD group ( Fig.18 ); Under the condition of E:T=1:1, compared with the UTD group, the luciferase values ​​of the ICD combination group and the corresponding ICD group also showed an obvious continuous inhibition trend, among which the ICD combination group had a stronger inhibitory effect than the corresponding ICD group ( Fig.19); Under the condition of E:T=1:5, compared with the UTD group, the luciferase values ​​of the ICD combination group and the corresponding ICD group also showed a weaker inhibition trend, among which the ICD-PI3K combination group showed a significant inhibitory effect compared with the corresponding ICD group alone ( Fig. 20 ). This indicates that the ICD combination group has the ability to continuously kill and inhibit target cells, and under the condition of low efficiency target ratio, its killing and inhibition effect is significantly better than that of the single ICD group.

[0153] Example 11. Infiltration and killing of monocyte 3D tumor spheres after adenovirus transfection

[0154] The overexpression transformation scheme in Example 4 was used to transform the OE19 cell line with NLS-mCherry lentivirus (MOI of 10), and a single clone (hereinafter collectively referred to as OE19-NLS mCherry) was screened in a complete medium containing 300 μg / mL of hygromycin (purchased from MCE).

[0155] After CD14+ monocytes were plated, they were divided into 6 experimental groups, and different adenoviruses with HER2 CAR-ICD molecules were added, including different ICD combinations of FCE, FCE-IFNgR, FCE-PI3K, CD3ζ, CD3ζ-IFNgR and CD3ζ-PI3K. There was one negative control group (UTD group), which was macrophages not transfected with adenovirus.

[0156] Pre-recover OE19-NLS mCherry monoclonal cells, harvest cells when cell confluence reaches >50%, and resuspend in complete medium at a cell density of 5×10 4 / mL. Take a 96-well transparent round-bottom ultra-low adsorption microplate (purchased from Corning / #7007), add 100μL to each well, and place it in the Incucyte cell culture incubator for 4 days to form 3D tumor microspheres. The cells were harvested on the second day after transfection and added to the 96-well U-bottom plate of OE19-NLS mCherry monoclonal target cells that had formed 3D tumor spheres. According to the effector-target ratio of 5:1 and 1:1, the effector cells were added (100μL volume was added to each well, and parallel plates were laid), and placed in Incucyte for monitoring at an interval of 2h. After 96 hours, 50μL of complete medium was added (100μL volume was added to each well).

[0157] The results showed that compared with the UTD group, the ICD combination group and the ICD group alone had a certain killing and inhibitory effect on the OE19-NLS mCHerry (red) tumor spheres. At the same time, the ICD combination group had a better infiltration and lysis effect on the tumor spheres. Under the conditions of E:T=5:1 or 1:1, the ICD-PI3K group could achieve a near-clearing effect ( Fig.21 ).

[0158] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A chimeric antigen receptor (CAR) molecule targeting HER2, characterized in that: The CAR molecule comprises: (a) specifically binds to the extracellular domain of HER2; (b) a transmembrane domain; and (c) two or more intracellular signaling domains comprising: (i) the intracellular domain of FCE or CD3ζ or a portion thereof, and (ii) the intracellular domain of any one or more of IFNgR, PI3K recruit, TLR2, TLR4 and CD40, or a portion thereof.

2. The CAR molecule according to claim 1, characterized in that The CAR molecule comprises two intracellular signaling regions, preferably the intracellular signaling region comprises: (i) the intracellular domain of FCE or a portion thereof; and, (ii) the intracellular domain of IFNgR, PI3K recruit, TLR2, TLR4 or CD40 or a portion thereof, or (i) the intracellular domain of CD3ζ or a portion thereof; and, (ii) the intracellular domain of IFNgR, PI3K recruit, TLR2, TLR4 or CD40 or a portion thereof, Preferably, the intracellular signal transduction region comprises from the N-terminus to the C-terminus: (1) the intracellular domain of FCE or a portion thereof, and the intracellular domain of IFNgR or a portion thereof; (2) the intracellular domain of FCE or a portion thereof, and the intracellular domain of PI3K recruit or a portion thereof; (3) the intracellular domain of FCE or a portion thereof, and the intracellular domain of TLR2 or a portion thereof; (4) the intracellular domain of FCE or a portion thereof, and the intracellular domain of TLR4 or a portion thereof; (5) the intracellular domain of FCE or a portion thereof, and the intracellular domain of CD40 or a portion thereof; (6) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of IFNgR or a portion thereof; (7) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of PI3K recruit or a portion thereof; (8) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of TLR2 or a portion thereof; (9) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of TLR4 or a portion thereof; or (10) the intracellular domain of CD3ζ or a portion thereof, and the intracellular domain of CD40 or a portion thereof, Preferably, the intracellular domain of FCE has an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 85% identity thereto; Preferably, the intracellular domain of CD3ζ has an amino acid sequence as shown in SEQ ID NO: 6, or an amino acid sequence having at least 85% identity thereto; Preferably, the intracellular domain of the IFNgR has an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 85% identity thereto; Preferably, the intracellular domain of PI3K recruit has an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 85% identity thereto; Preferably, the intracellular domain of TLR2 has an amino acid sequence as shown in SEQ ID NO: 9, or an amino acid sequence having at least 85% identity thereto; Preferably, the intracellular domain of TLR4 has an amino acid sequence as shown in SEQ ID NO: 10, or an amino acid sequence having at least 85% identity thereto; Preferably, the intracellular domain of CD40 has an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 85% identity thereto.

3. The CAR molecule according to claim 1, characterized in that The transmembrane domain is selected from the following transmembrane domains: CD4, CD8, CD28, PD-1, OX40, 4-1BB, CTLA-4, CD2, CD3D, CD3E, CD3G, CD3ζ, CD8a, CD8b, CD16, CD25, CD27, CD40, CD79A, CD79B, CD80, CD84, CD86, CD95, CD150, CD166, CD200R, CD223, CD270, CD272, CD273, CD274, CD278, CD300, CD357, A2aR, ICA M-1, 2B4, BTLA, DAP10, FcRα, FcRβ, Fyn, GAL9, IL7, IL12, IL15, KIR, KIR2DL4, KIR2DS1, LAG-3, Lck, LAT, LPA5, LRP, NKp30, NKp44, NKp46, NKG2C, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, SLP-76, SIRPα, pTα, T cell receptor polypeptide, TIM3, TRIM, ZAP70, or any combination thereof; Preferably, the transmembrane domain comprises a CD8 transmembrane domain, preferably having an amino acid sequence as shown in SEQ ID NO: 4 or an amino acid sequence having at least 85% identity thereto.

4. The CAR molecule according to claim 1, characterized in that The CAR molecule further includes an extracellular leader domain and / or an extracellular hinge domain. Preferably, the extracellular leader domain comprises a CD8 extracellular leader domain or a portion thereof, preferably having an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 85% identity thereto, Preferably, the extracellular hinge domain is selected from the following hinge domains: CD8, CD28, CD4, IgG, PD-1, CTLA-4, CD2, LFA-1, CD5, CD27, CD70, 4-1BB, OX40, ICOS, IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgE Fc region, IgM Fc region, IgAFc region or any combination thereof, Preferably, the extracellular hinge domain comprises the CD8 extracellular hinge domain or a portion thereof, preferably having an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 85% identity thereto.

5. The CAR molecule according to claim 1, characterized in that The extracellular domain that specifically binds to HER2 includes a single-chain antibody (scFv), a heavy chain single-domain antibody or an antigen-binding fragment thereof that can specifically bind to HER2, preferably having an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence that has at least 85% identity thereto.

6. The CAR molecule according to claim 1, characterized in that The CAR molecule includes an extracellular leader domain, an extracellular domain specifically binding to HER2, an extracellular hinge domain, a transmembrane domain and an intracellular signal transduction region from the N-terminus to the C-terminus. Preferably, the CAR molecule has an amino acid sequence as shown in any one of SEQ ID NOs: 13-17, 19-22, or an amino acid sequence having at least 85% identity thereto.

7. A nucleic acid molecule encoding the CAR molecule according to any one of claims 1 to 6, wherein the nucleic acid molecule is a DNA or RNA molecule.

8. A vector comprising the nucleic acid molecule according to claim 7, Preferably, the vector is a viral vector or a non-viral vector, preferably a viral vector, more preferably an adenoviral vector, an adeno-associated viral vector or a retroviral vector.

9. A modified immune cell comprising the CAR molecule according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the vector according to claim 8, Preferably, the modified immune cells include stem cells, macrophages, monocytes or dendritic cells, more preferably macrophages and / or monocytes. Preferably, the modified immune cells exhibit increased effector activity, increased tumor killing activity and / or phagocytic activity, increased depletion capacity and / or increased tumor infiltration capacity compared to unmodified immune cells, Preferably, the modified immune cells exhibit increased polarization to an M1 phenotype compared to unmodified immune cells.

10. A pharmaceutical composition comprising: The CAR molecule of any one of claims 1 to 6, the nucleic acid molecule of claim 7, the vector of claim 8 or the immune cell of claim 9; and a pharmaceutically acceptable carrier.

11. Use of the CAR molecule according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the vector according to claim 8 or the immune cell according to claim 9 in the preparation of a drug for preventing or treating a disease or condition, Preferably, the disease or disorder comprises a tumor / cancer, preferably a solid tumor, Preferably, the tumor / cancer includes breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, gastric cancer and esophageal cancer.

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