CN117645672A - Chimeric immune cell co-receptor and uses thereof - Google Patents
Chimeric immune cell co-receptor and uses thereof Download PDFInfo
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Abstract
The present invention relates to chimeric immune cell-assisted receptors and uses thereof, and in particular provides an isolated fusion protein that is a chimeric immune cell-assisted receptor comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains a biological function of binding to KIR ligands, a transmembrane region, and an intracellular region. The accessory receptor can enhance the immune synapse core connection, simultaneously transmit signals combined by HLA and receptor extracellular region into cells, activate an activation signal path of a downstream co-stimulatory molecule, and improve the activation level of immune effector cells through double functions.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chimeric immune cell auxiliary receptor and application thereof.
Background
Adoptive cell therapy (Adoptive Cell Therapy, ACT) has been shown to be highly effective in cancer therapy initially. However, one major problem faced by ACT in cancer immunotherapy is still that immune cells are not activated to a sufficient extent and that the feedback immune cells are immunosuppressed in the tumor microenvironment in vivo. In the Tumor Microenvironment (TME), there are many factors that negatively regulate T cell immune responses, allowing tumor cells to escape from the monitoring and clearance of the immune system of the body and to continue malignant proliferation, invasion and metastasis. Positive regulatory molecules of immune effector cells include CD3/TCR complex, CD28, ICOS, CD134 (OX 40), CD137 (4-1 BB), etc., and negative regulatory molecules include PD-1 (PDCD 1, CD 279), CTLA-4 (CD 152), LAG3 (CD 223), TIM3 (HAVCR 2), BTLA, etc. Typically, T cells are activated by a combination of a first signal from the binding of CD3/TCR to the MHC-antigen peptide complex (pMHC) and a second signal from the binding of CD4 or CD8 to the constant region of the MHC molecule, and a number of other activating receptor binding ligands on the surface of the T cells are co-stimulated upon stimulation of the first signal with the second signal, e.g., CD28,4-1BB, OX40, etc. The structure formed by the receptor-ligand interaction between these T cells and the target cells (or antigen presenting cells) is called an immune synapse, where the first signal and the second signal form the core of the immune synapse.
Killer cell immunoglobulin-like receptor (KIR) is a group of germline-encoded receptors expressed on NK cells and a small part of the T cell surface with activating or inhibitory functions, including KIR3DL1,2,3, KIR3d 1, KIR2d 1,2,3,4,5a,5b and KIR2DS1,2,3,4,5, and in addition 2 pseudogenes (pseudogenese) 2DP1 and 3DP1. The function of KIR receptors is determined by their transmembrane and cytoplasmic domains, with the intracellular domain of inhibitory KIR receptors being longer and comprising the ITIM motif. With the exception of KIR2DL4, which has a longer intracellular domain comprising the ITIM motif, is produced by binding to an activating linker Fc e R1γ conducts an activation signal into the cell. The ligands for which many inhibitory KIRs are studied are mainly various class I HLA molecules. In normal cells and tissues, the expression level of the type I HLA is normal, and the KIR combined with the surfaces of immune effector cells such as NK cells and T cells starts a downstream inhibition signal path, so that the effect of other activation signal paths is counteracted, and the immune effector cells cannot be activated to attack themselves, so that tolerance to the immune effector cells is maintained. In abnormal cells such as virus-infected cells or tumor cells, the expression level of HLA class I is reduced or not, and at this time, the absence of a "do not eat me" signal leads to the reduction or disappearance of the level of the inhibition signal pathway, and the signal pathway activating immune effector cells is dominant, so that the immune effector cells are brought into an activated and proliferated state, and the target cells are unfolded and attacked to remove the abnormal cells.
The introduction of chimeric activating receptors into immune effector cells in tumor immune cell therapy is one of the important technological means for relieving immunosuppression and activating immune effector cells. The vast majority of chimeric activated receptors reported presently, except CARs, are designed based on depletion markers, such as chimeric activated receptors based on PD-1 and BTLA (CN 104114233B), and still lack chimeric immune cell co-receptors based on KIR receptor interactions with HLA type I.
Disclosure of Invention
The present invention provides an isolated fusion protein that is a chimeric immune cell co-receptor comprising an extracellular domain of KIR or a functional fragment or variant thereof that retains a biological function of binding KIR ligands, a transmembrane region, and an intracellular region comprising a signaling domain and/or a costimulatory domain.
In one or more embodiments, the chimeric immune cell co-receptor further comprises a signal peptide.
In one or more embodiments, the chimeric immune cell co-receptor further comprises a hinge region. Optionally, the hinge region comprises a membrane proximal fragment of a native extracellular domain of the costimulatory signaling molecule. In some embodiments, the hinge region is located N-terminal to the transmembrane region.
In one or more embodiments, the KIR is an activated KIR or an inhibited KIR. In some embodiments, the activating KIR is KIR2DL4; the inhibition type KIR is any one or more selected from KIR2DL3, KIR3DL1 and KIR3DL 2.
In one or more embodiments, the costimulatory domain is an intracellular domain of a costimulatory signaling molecule or a functional fragment or mutant thereof that retains the costimulatory signaling molecule to deliver a costimulatory signal, activate a biological function of an immune cell.
In one or more embodiments, the chimeric immune cell co-receptor further comprises a membrane surface tag. In one or more embodiments, the membrane surface tag comprises a BCMA extracellular domain or variant thereof, or a claudin protein extracellular domain or fragment or variant thereof. Preferably, the membrane surface tag is located C-terminal to the extracellular domain of KIR.
In one or more embodiments, the chimeric immune cell co-receptor further comprises a linker between the KIR extracellular domain or a functional fragment or variant thereof and a membrane surface tag; preferably, the joint is a rigid joint or a flexible joint.
In one or more embodiments, the membrane surface tag further comprises a linker or hinge located at the N-terminus or C-terminus of the BCMA extracellular domain or variant thereof, or the claudin protein extracellular domain or fragment or variant thereof.
In one or more embodiments, the claudin protein is claudin18, preferably claudin18.2. In one or more embodiments, the fragment of the extracellular domain of claudin protein is an extracellular epitope of claudin protein. In one or more embodiments, a fragment of the extracellular domain of claudin18.2 protein comprises the sequence shown in SEQ ID NO. 30; the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 29.
In one or more embodiments, the BCMA extracellular domain comprises a sequence shown in SEQ ID No. 32; the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 31.
In one or more embodiments, the linker located N-terminal or C-terminal to the BCMA extracellular domain or variant thereof, or claudin protein extracellular domain or fragment or variant thereof, is a rigid linker or a flexible linker. In one or more embodiments, the linker is a rigid linker. In one or more embodiments, the sequence of the rigid linker comprises the sequence set forth in SEQ ID NO. 60; the coding nucleic acid sequence comprises the sequence shown in SEQ ID NO. 59.
In one or more embodiments, the transmembrane region includes, but is not limited to, any one or more of a transmembrane region selected from CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27, or mutants thereof that retain transmembrane function. Preferably, the transmembrane region is a CD28 transmembrane region, an IL7 ra transmembrane region, or a mutant thereof that retains transmembrane function.
In one or more embodiments, the co-stimulatory signaling molecule intracellular domain includes, but is not limited to, an intracellular domain selected from any one or more of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD 27. Preferably, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28 and/or the intracellular domain of OX 40.
In one or more embodiments, the hinge region includes, but is not limited to, a membrane proximal fragment of a natural extracellular domain selected from the group consisting of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD 27. Preferably, the hinge region is an extracellular hinge region of CD28 and/or an extracellular hinge region of il7rα.
In one or more embodiments, the chimeric immune-cell co-receptor comprises a KIR2DL3 extracellular region, a linker, a membrane surface tag, a transmembrane region, and one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7rα intracellular domain, the transmembrane region being a CD28 transmembrane region or an IL-7rα transmembrane region, or a mutant thereof. Wherein the membrane surface tag comprises a claudin18.2 epitope or BCMA extracellular domain; the chimeric immune cell co-receptor further comprises a CD28 hinge region between the membrane surface tag and the transmembrane region. In one or more embodiments, the chimeric immune cell co-receptor comprises a KIR2DL3 extracellular region, a claudin18.2 epitope, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain. In one or more embodiments, the chimeric immune cell co-receptor comprises a KIR2DL3 extracellular region, a BCMA extracellular domain, an IL7 ra transmembrane region or any one of its mutants 1-4, an IL-7 ra intracellular domain. In one or more embodiments, the chimeric immune cell co-receptor comprises a KIR2DL3 extracellular region, a linker, a BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.
In one or more embodiments, the chimeric immune-cell co-receptor comprises a KIR2DL4 extracellular region, a linker, a membrane surface tag, a transmembrane region, which is a CD28 transmembrane region or an il7rα transmembrane region or a mutant thereof (e.g., mutant 4), and one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7rα intracellular domain. Wherein the membrane surface tag comprises a claudin18.2 epitope or BCMA extracellular domain; the chimeric immune cell co-receptor further comprises a CD28 hinge region or an IL7 ra extracellular hinge region between the membrane surface tag and the transmembrane region. In one or more embodiments, the chimeric immune-cell co-receptor comprises a KIR2DL4 extracellular region, a claudin18.2 epitope, a CD28 extracellular hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain, or the chimeric immune-cell co-receptor comprises a KIR2DL4 extracellular region, a BCMA extracellular domain, an IL7 ra extracellular hinge region, an IL7 ra transmembrane region, and an IL-7 ra intracellular domain, or the chimeric immune-cell co-receptor comprises a KIR2DL4 extracellular region, a claudin18.2 epitope, an IL7 ra extracellular hinge region, an IL7 ra transmembrane region mutant 4, and an IL-7 ra intracellular domain. In one or more embodiments, the chimeric immune cell co-receptor comprises a KIR2DL4 extracellular region, a linker, a BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.
In one or more embodiments, the chimeric immune-cell co-receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a membrane surface tag, a transmembrane region, and one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain, and an IL-7rα intracellular domain, the transmembrane region being a CD28 transmembrane region. In one or more embodiments, the chimeric immune cell co-receptor comprises a KIR3DL1 extracellular region KIR3 or DL2 extracellular region, a claudin18.2 epitope, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.
In one or more embodiments, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO. 4.
In one or more embodiments, the amino acid sequence of the IL7Rα transmembrane region is shown in SEQ ID NO. 6.
In one or more embodiments, the IL7Rα transmembrane region mutants 1-4 have the amino acid sequences shown in SEQ ID NOS.8, 10, 12 and 14.
In one or more embodiments, the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO. 16.
In one or more embodiments, the amino acid sequence of the OX40 intracellular domain is shown in SEQ ID NO. 18.
In one or more embodiments, the amino acid sequence of the IL-7Rα intracellular domain is shown in SEQ ID NO. 20.
In one or more embodiments, the amino acid sequence of the extracellular region of KIR2DL3 is shown in SEQ ID NO. 22.
In one or more embodiments, the amino acid sequence of the extracellular region of KIR2DL4 is shown in SEQ ID NO. 24.
In one or more embodiments, the amino acid sequence of the extracellular region of KIR3DL1 is shown in SEQ ID NO. 26.
In one or more embodiments, the amino acid sequence of the extracellular region of KIR3DL2 is shown in SEQ ID NO. 28.
In one or more embodiments, the amino acid sequence of the Claudin 18.2 epitope is shown as SEQ ID NO. 30.
In one or more embodiments, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID No. 32.
In one or more embodiments, the CD28 extracellular hinge region has an amino acid sequence as set forth in SEQ ID NO. 34.
In one or more embodiments, the IL7Rα extracellular hinge region has an amino acid sequence as set forth in SEQ ID NO: 36.
In one or more embodiments, the amino acid sequence of the chimeric immune cell co-receptor is as set forth in any one of SEQ ID NOs 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62 and 64.
The present invention also provides a polynucleotide molecule having: a nucleic acid sequence encoding a chimeric immune cell co-receptor according to any one of the embodiments of the invention or a complement thereof.
In one or more embodiments, the transmembrane region is a CD28 transmembrane region having a coding sequence as set forth in SEQ ID NO. 3.
In one or more embodiments, the transmembrane region is an IL7Rα transmembrane region having a coding sequence as set forth in SEQ ID NO. 5.
In one or more embodiments, the transmembrane region is IL7Rα transmembrane region mutant 1-4, which has the coding sequence shown in SEQ ID NOS 7, 9, 11 and 13, respectively.
In one or more embodiments, the intracellular domain comprises a CD28 intracellular domain having a coding sequence as set forth in SEQ ID NO. 15.
In one or more embodiments, the intracellular domain comprises an OX40 intracellular domain having a coding sequence as set forth in SEQ ID NO. 17.
In one or more embodiments, the intracellular domain comprises an IL-7Rα intracellular domain, the coding sequence of which is shown in SEQ ID NO 19.
In one or more embodiments, the extracellular domain comprises a KIR2DL3 extracellular region, the coding sequence of which is shown in SEQ ID NO. 21.
In one or more embodiments, the extracellular domain comprises a KIR2DL4 extracellular region, the coding sequence of which is shown in SEQ ID NO. 23.
In one or more embodiments, the extracellular domain comprises a KIR3DL1 extracellular region, the coding sequence of which is set forth in SEQ ID NO. 25.
In one or more embodiments, the extracellular domain comprises a KIR3DL2 extracellular region, the coding sequence of which is set forth in SEQ ID NO 27.
In one or more embodiments, the chimeric immune cell co-receptor comprises a membrane surface tag comprising a Claudin 18.2 epitope as set forth in SEQ ID No. 29 or a BCMA extracellular domain as set forth in SEQ ID No. 31.
In one or more embodiments, the hinge region is a CD28 extracellular hinge region having a coding sequence as set forth in SEQ ID NO. 33.
In one or more embodiments, the hinge region is an IL7Rα extracellular hinge region having a coding sequence set forth in SEQ ID NO. 35.
In one or more embodiments, the polynucleotide molecule comprises a nucleic acid sequence selected from any one of SEQ ID NOs 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61 and 63, or is a complement of any one of the nucleic acid sequences shown.
The invention also provides a nucleic acid construct comprising a polynucleotide molecule according to any one of the embodiments of the invention.
In one or more embodiments, the nucleic acid construct is a vector.
In one or more embodiments, the vector is an expression vector or an integration vector.
In one or more embodiments, the vector is a viral vector or a non-viral vector, preferably a non-viral vector. In one or more embodiments, the non-viral vector is an integrated non-viral vector, preferably an integrated non-viral vector based on a transposon system.
The invention also provides a genetically engineered cell expressing the chimeric immune cell co-receptor of any embodiment of the invention and/or carrying the coding sequence of the chimeric immune cell co-receptor.
In one or more embodiments, the cell is an immune effector cell.
In one or more embodiments, the immune effector cells include T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T, and TIL.
In one or more embodiments, the cell also expresses a CAR, or carries a coding sequence for a CAR.
In one or more embodiments, the cell also expresses an exogenous TCR, or a coding sequence carrying an exogenous TCR.
The invention also provides a pharmaceutical composition comprising pharmaceutically acceptable excipients and any one or more of the chimeric immune cell accessory receptor, the polynucleotide molecule, the nucleic acid construct and the genetically engineered cell according to any one of the embodiments of the invention. The pharmaceutical composition is used for treating or preventing cancer.
In one or more embodiments, the cancer is melanoma.
The invention also provides the use of the chimeric immune cell accessory receptor, the polynucleotide molecule, the nucleic acid construct and the genetically engineered cell according to any embodiment of the invention in the preparation of a medicament for treating or preventing cancer.
The invention has the advantages that:
the chimeric immune cell accessory receptor of the invention plays an immune activating role by the following modes: 1) When the receptor is expressed in T cells, the first signal and the second signal which are already existing between the T cells and antigen presenting cells are enhanced through binding with HLA type I, the most core component of T cell immune synapse is further enhanced from physical structure, so that the interaction between the T cells and the antigen presenting cells is enhanced, and favorable conditions are created for antigen recognition and activation of the T cells; 2) After expressing the receptor in immune effector cells including T cells and NK cells, the KIR receptor extracellular domain in its extracellular domain binds to the corresponding HLA class I on the surface of antigen presenting cells, which in turn activates the co-stimulatory molecule intracellular domain downstream thereof, thereby increasing the level of activation and proliferation of immune effector cells, and by dual action, the level of activation of immune effector cells.
Drawings
Fig. 1: RTCA killing curves of TIL-CTRL, TIL-K23-7 and TIL-K24-4 against cognate pairing tumor primary target cells.
Fig. 2: TIL-CTRL, TIL-K23-7 and TIL-K24-4 killing effect curves on homologously paired PDX tumor tissue.
Detailed Description
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.
KIR receptors on the surface of immune effector cells are activated or inhibited by binding to HLA class I on the surface of antigen presenting cells. The invention fuses the extracellular region of KIR and the transmembrane region of classical co-stimulatory signal molecule with the intracellular domain to form a novel receptor, can bind different class I HLAs, and is a novel immune cell auxiliary receptor. The receptor promotes immune effector cell function by: 1) When the receptor is expressed in T cells, the first signal and the second signal which are already existing between the T cells and antigen presenting cells are enhanced through binding with HLA type I, the most core component of T cell immune synapse is further enhanced from physical structure, so that the interaction between the T cells and the antigen presenting cells is enhanced, and favorable conditions are created for antigen recognition and activation of the T cells; 2) After expression of the receptor in immune effector cells, including T cells and NK cells, the KIR receptor extracellular domain in its extracellular domain binds to the corresponding HLA class I on the surface of antigen presenting cells, which in turn activates the co-stimulatory molecule intracellular domain downstream thereof, thereby increasing the activation and proliferation levels of immune effector cells.
In the present invention, immune cells have a meaning well known in the art and refer to cells involved in or associated with an immune response, including various lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, and the like. Lymphocytes include, for example, T lymphocytes, tumor-infiltrating lymphocytes (TILs), B lymphocytes, NK lymphocytes, and DN T cells. Immune cells suitable for use in the present invention include, inter alia, those typically used in adoptive cell therapy of tumors.
Definition of the definition
The present invention uses the following terminology. For terms not specifically defined herein, they have meanings well known in the art.
The term "expression cassette" refers to the complete elements required for expression of a gene, including promoters, gene coding sequences, and PolyA tailing signal sequences.
The term "coding sequence" is defined herein as that portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., immune cell co-receptor, CAR). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame at the 5 'end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame at the 3' end of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
The term "costimulatory signaling molecule" refers to a molecule that is present on the surface of an antigen-presenting cell and that is capable of binding to a costimulatory signaling molecule receptor on a Th cell to produce a costimulatory signal. It can activate the second signal of immune cell, strengthen the proliferation capacity of immune cell and the secretion function of cell factor, and prolong the survival time of activated immune cell. Proliferation of lymphocytes requires not only antigen binding but also signal of the co-stimulatory molecule. The co-stimulatory signal is transmitted to the T cell primarily through the co-stimulatory molecule CD80, CD86 expressed on the surface of the antigen presenting cell binding to the CD28 molecule on the surface of the T cell. B cells receive costimulatory signals through common pathogen components such as LPS, or through complement components, or through activated antigen-specific CD40L on Th cell surfaces.
The term "linker" or "hinge" is a polypeptide fragment that connects between different proteins or polypeptides in order to maintain the connected proteins or polypeptides in their respective spatial conformations in order to maintain the function or activity of the protein or polypeptide. Exemplary linkers include G and/or S containing linkers, rigid linkers or flexible linkers, and e.g., furin 2A peptides.
The term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present invention in a subject.
The term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.
The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., receiving a pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate a disease or disorder described herein.
The term "extracellular region" refers to the region of a membrane protein that is located outside of a cell.
The term "domain" refers to a region of a protein having a specific structure and independent function, the number of amino acid residues of a common domain being between 100 and 400, the smallest domain being only 40 to 50 amino acid residues, and the large domain being more than 400 amino acid residues.
Immune cell accessory receptor
The immune cell-assisted receptor of the present invention is a fusion protein comprising an extracellular region (extracellular domain), a transmembrane region (transmembrane domain) and an intracellular region (or intracellular domain) of a KIR extracellular domain. The KIR extracellular domain may be itself or a fragment, provided that the fragment retains the biological function of binding to the KIR ligand. The intracellular region comprises a signal transduction domain and/or a co-stimulatory domain.
In the fusion proteins herein, KIR extracellular domains are used to interact with KIR ligands (mainly HLA class I molecules), thereby down-regulating "do-it-yourself" signaling pathway levels, activating the signaling pathway predominance of immune effector cells, and thus bringing immune effector cells into an activated and proliferative state, and deploying an attack on target cells, thereby clearing abnormal cells. The extracellular domain of an activating KIR or an inhibitory KIR, or a fragment thereof that retains KIR ligand binding capacity, may be used. An exemplary activated KIR is KIR2DL4; exemplary inhibitory KIRs are selected from KIR2DL3, KIR3DL1, and KIR3DL2. Preferably, the amino acid sequence of the extracellular region of KIR2DL3 is shown as SEQ ID NO. 22, and the coding sequence is shown as SEQ ID NO. 21; the amino acid sequence of the extracellular region of KIR2DL4 is shown as SEQ ID NO. 24, and the coding sequence is shown as SEQ ID NO. 23; the amino acid sequence of the extracellular region of KIR3DL1 is shown as SEQ ID NO. 26, and the coding sequence is shown as SEQ ID NO. 25; the amino acid sequence of the extracellular region of KIR3DL2 is shown as SEQ ID NO. 28, and the coding sequence is shown as SEQ ID NO. 27.
Co-stimulatory signaling molecules in the present invention include CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27. One or more of these intracellular domains (intracellular regions) or functional fragments thereof of costimulatory signaling molecules or mutants that retain the costimulatory signaling molecules to deliver costimulatory signaling, activate the biological function of immune cells can be used to construct chimeric immune cell-assisted receptors of the invention. The amino acid sequence of the intracellular region of exemplary CD28 and the corresponding coding sequence can be shown in SEQ ID NOS.16 and 15, respectively. The amino acid sequence and corresponding coding sequence of the intracellular region of exemplary OX40 can be as shown in SEQ ID NOS.18 and 17, respectively. An exemplary IL-7R may be IL-7Rα, and exemplary amino acid sequences of its intracellular region and corresponding coding sequences may be shown in SEQ ID NOS 20 and 19, respectively. The intracellular domain of the costimulatory signaling molecule may also be that described in WO2021244486, which is incorporated herein by reference in its entirety.
Herein, the transmembrane region includes, but is not limited to, any one or more of a transmembrane region selected from the group consisting of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD27, or mutants thereof that retain transmembrane function. Illustratively, the amino acid sequence and nucleotide sequence of the CD28 transmembrane region are shown in SEQ ID NOS.4 and 3, respectively; the amino acid sequence and the nucleotide sequence of the IL7 Ralpha transmembrane region are respectively shown in SEQ ID NO. 6 and SEQ ID NO. 5; the amino acid sequence and the nucleotide sequence of the IL7 Ralpha transmembrane region mutant 1 are respectively shown in SEQ ID NO. 8 and SEQ ID NO. 7; the amino acid sequence and the nucleotide sequence of the IL7Rα transmembrane region mutant 2 are respectively shown in SEQ ID NO 10 and SEQ ID NO 9; the amino acid sequence and the nucleotide sequence of the IL7Rα transmembrane region mutant 3 are respectively shown in SEQ ID NO. 12 and 11; the amino acid sequence and the nucleotide sequence of the IL7Rα transmembrane region mutant 4 are shown in SEQ ID NO 14 and SEQ ID NO 13 respectively.
In the present invention, the extracellular region comprising the KIR extracellular domain may be linked to the transmembrane region by a hinge region. The hinge region includes, but is not limited to, a membrane proximal fragment of a natural extracellular domain selected from the group consisting of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM, and CD 27. Preferably, the hinge region is an extracellular hinge region of CD28 and/or an extracellular hinge region of il7rα.
It is understood that "functional fragment" as used herein refers to a fragment that retains the desired biological function. For example, a functional fragment of an intracellular domain as described herein refers to a fragment that retains the biological function of the costimulatory signaling molecule to deliver a costimulatory signal, activating an immune cell. Functional fragments of each extracellular domain suitable for use in the present invention can be readily determined by one of skill in the art in combination with prior art means in the art.
The immune cell-assisted receptor of the present invention may also have a membrane surface tag extracellular. Thus, in some embodiments, the immune cell co-receptor described herein further comprises a membrane surface tag at the C-terminus of the KIR extracellular domain. The membrane surface tag may function as an immune braking element, recognition element, linker, element that induces ADCC, ADCP and/or CDC effects. The membrane surface tag comprises a membrane surface functional domain.
The membrane surface functional domain may be an extracellular domain of claudin protein or a fragment thereof. The claudin protein is preferably claudin18, e.g. claudin18.2. Fragments of the extracellular domain are primarily referred to as extracellular epitopes of the corresponding protein. Thus, a fragment of the extracellular domain of claudin protein is an extracellular epitope of claudin protein. Preferably, the fragment of the extracellular domain of claudin18.2 protein (i.e. the extracellular epitope of claudin18.2 protein) comprises the sequence shown in SEQ ID NO. 30; the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 29.
The membrane surface functional domain may also be a BCMA extracellular domain or a fragment thereof. Preferably, the BCMA extracellular domain comprises the sequence shown as SEQ ID NO. 32; the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 31.
The membrane surface tag may also have a linker fragment at the N-terminus or C-terminus of the membrane surface functional domain (BCMA extracellular domain or claudin protein extracellular domain) for linking to other polypeptides or polypeptide portions. The connecting segments are typically hinges or joints. The hinge comprises one or more selected from the group consisting of: an extracellular hinge region of CD8, an IgG1Fc CH2CH3 hinge region, an IgD hinge region, a CD28 extracellular hinge region, an IgG4 Fc CH2CH3 hinge region, and an extracellular hinge region of CD 4.
As used herein, "mutant" includes mutants of each domain, provided that the mutant retains the respective biological functions of the KIR extracellular domain, membrane surface tag, transmembrane region, and intracellular domain. For example, mutants of KIR extracellular domains suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the KIR extracellular domain as a comparison; mutants of the membrane surface tags suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the membrane surface tag as a comparison; mutants suitable for use in the transmembrane region of the invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the transmembrane region as a comparison; mutants of the intracellular domains suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the intracellular domain to be compared. Alternatively, the mutants of the present invention have one or more (e.g., 20 or less, 15 or less, 10 or less, 8 or less, 5 or less, or 3 or less, e.g., 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted as compared to the sequences used as a comparison. For example, conservative substitutions with amino acids that are similar or analogous in nature typically do not alter the function of the protein or polypeptide. "similar or analogous amino acids" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
The invention also includes mutants of the immune cell-assisted receptor described previously, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the immune cell-assisted receptor. More specifically, the present invention includes mutants having one or more (e.g., 20 or less, 15 or less, 10 or less, 8 or less, 5 or less, or 3 or less, e.g., 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted as compared to the immunocyte auxiliary receptor described above. Such mutants retain the biological functions of the immune cell accessory receptors of the present invention, including but not limited to those that will recognize KIR ligands and activate immune effector cells into an activated and proliferative state. Mutations can occur in any, any two, or all three of the extracellular domains, transmembrane regions, and intracellular domains described herein.
The polypeptides described herein may be modified polypeptides. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.
Exemplary chimeric immune cell co-receptors of the invention include, but are not limited to, chimeric immune cell co-receptors comprising or consisting of the extracellular domains, hinge regions, transmembrane regions, and intracellular regions shown in each row of table 1 below:
TABLE 1 chimeric immunocyte auxiliary receptor (N-terminal to C-terminal)
In some embodiments, the chimeric immune cell co-receptor described herein further comprises a signal peptide. Preferably, the signal peptide is located at the N-terminus of the chimeric immune cell co-receptor. The signal peptide may be any signal peptide capable of directing the nucleation of a polypeptide conventional in the art, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII and antibody light chain signal peptides. In some embodiments, the signal peptide is a CD8 signal peptide, and the CD8 signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 2, and the coding sequence is set forth in SEQ ID NO. 1.
It will be appreciated that the extracellular domains and transmembrane regions, and/or the transmembrane regions and intracellular domains described herein may be linked by a linker sequence, as desired. Linker sequences known in the art, such as those containing G and S, such as (GSSS) n or (GSSSs) n, where n is an integer from 1 to 8, may be used. The joint may also be a rigid joint or a flexible joint. For example, the linker is a rigid linker as shown in SEQ ID NO. 60.
Preferably, the amino acid sequence of the chimeric immune cell co-receptor of the present invention is as shown in any one of SEQ ID NOs 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62 and 64.
CAR-T
The immune cells of the invention may further express a CAR, or contain a coding sequence for a CAR. The CARs of the present invention may be a variety of CARs well known in the art.
The CAR may in turn comprise a polypeptide that binds to a tumor cell membrane antigen (e.g., scFv), a hinge region, a transmembrane region, and an intracellular signaling region. The CARs of the invention can be constructed using hinge, transmembrane and intracellular signal regions well known in the art for constructing CARs. In general, polypeptides that bind tumor cell membrane antigens are capable of binding with moderate affinity to membrane antigens that are widely expressed by tumor cells, and are typically inserted with an epitope at a position selected from any 1, 2 or 3 of the following 3 positions: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and within the polypeptide. The polypeptide combined with the tumor cell membrane antigen is a natural polypeptide or an artificial synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or Fab fragment.
The chimeric antigen receptor of the invention may be directed against one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT 1), FR (also known as Flavin reductase), PSMA (also known as prostate specific membrane antigen), PMEL pre-melanosome protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as mycin 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3 (also known as SIA 1), PSCA (prostate stem cell antigen), KLA (PSA 9), GAGE (also known as GRGE protein) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as MUC 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, GD3 (also known as FBST 8SIA 1), PSCA (prostate stem cell antigen), PSCA (PSA 9), PSA 3, GAGE 3, GRBB 3 (Grave 3, GRCA 3, and GLP 3 (Grave 3) type 3, GRCA 1).
A single cell may express multiple CARs, including CARs targeting different tumor antigens.
T Cell Receptor (TCR) -T
The immune cells of the invention may further express an exogenous TCR or contain a coding sequence that expresses an exogenous TCR gene. The TCRs of the present invention may be any known in the art, for example, HLA-matched TCRs, known in sequence and structure, and known in combination with antigen peptide sequences.
The exogenous TCRs described herein include αβ double chains that can form complete TCR complexes with double-stranded structures of γε, δε, and ζζ expressed endogenously by immune effector cells such as T cells. The exogenous gene encoding the exogenous TCR of the invention includes an alpha beta double-stranded gene, and the alpha chain and beta chain coding sequences are covalently linked by a linker sequence that can be cleaved in vivo, such as the coding DNA sequence of P2A, T A or F2A sequences, or by a DNA fragment encoding an IRES sequence. In addition to the αβ duplex encoding the exogenous TCR, the gene encoding the exogenous TCR of the present invention may comprise a tag protein gene, such as EGFP, RFP, YFP gene, or the like, expressed in fusion with the αβ gene. The tag protein gene may be covalently linked to the αβ double stranded gene by a linker sequence that can be cleaved in vivo, such as a 2A sequence, e.g., a DNA sequence encoding P2A, T2A or F2A, or by a DNA sequence encoding an IRES sequence. The tag protein, such as EGFP, RFP, YFP gene, which is expressed together with TCR alpha beta double chain, can be used as identification index for detecting exogenous TCR expression.
The TCR-T of the invention may be directed against one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT 1), FR (also known as Flavin reductase), PSMA (also known as prostate specific membrane antigen), PMEL pre-melanosome protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as mycin 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3 (also known as GD 8SIA 1), PSCA (prostate stem cell antigen), KLA (FSK 9), GAGE (also known as GAGE) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as MUC 1), CD22, CD23, CD30, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGFR2, EGP-40, FBP, GD3 (also known as GL 8SIA 1), PSCA (precursor 1), KLA (F3), GRCA 3 (Grave 3), GRCA 3, GRCA 1) and GRCA 1 (Grave 3, GRCA 1).
A single cell may express multiple exogenous TCRs, including exogenous TCRs targeting different tumor antigens.
Polynucleotide molecules
The present invention provides polynucleotide molecules encoding the chimeric immune cell co-receptors described herein. The invention also provides the complementary sequence of the coding sequence of the chimeric immune cell auxiliary receptor. The polynucleotide molecule may be a recombinant nucleic acid molecule or may be synthetic; it may comprise DNA, RNA and PNA (peptide nucleic acid) and may be a hybrid thereof. Illustratively, the polynucleotide molecules of the invention have a sequence set forth in any one of SEQ ID NOs 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61 and 63.
Also provided is an expression cassette for the chimeric immune cell co-receptor of the invention, which is a nucleic acid construct comprising a promoter, a chimeric immune cell co-receptor coding sequence and a PolyA tailing signal sequence. Other elements required for expression may also be included in the nucleic acid construct, including but not limited to enhancers and the like.
Also provided is a vector comprising a polynucleotide molecule, expression cassette or nucleic acid construct described herein. Vectors may be plasmids, cosmids, viruses, and phages. The vector may be a viral vector or a non-viral vector. The vector may be a cloning vector, an integration vector, or an expression vector. The expression vector may be a transposon vector. In certain embodiments, the expression vector is one or more selected from the group consisting of: piggybac, sleep reliability, frog priority, tn5 and Ty. In addition to the polynucleotide molecules of the invention, the expression vectors will typically contain other elements typically contained in vectors, such as multiple cloning sites, resistance genes, replication initiation sites, and the like. In certain embodiments, the recombinant expression vector employs pUC18, pUC19, pMD18-T, pMD19-T, pGM-T, pUC57, pMAX or pDC315 series vectors as the backbone. In other embodiments, the recombinant expression vector employs a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector, or a pDC315 series vector as a backbone. In certain embodiments, the invention uses the pNB vector constructed by CN105154473 a. In certain embodiments, the invention uses the pKB20 vector described in WO2022078310 A1.
The CARs of the invention may also be expressed in the immune cells of the invention by conventional vectors. The vector may be a conventional CAR-expressing vector, including but not limited to the various transposon vectors and recombinant expression vectors described previously.
In some embodiments, the same vector encodes both the chimeric immune cell co-receptor and CAR of the invention. The vector may be a bicistronic. The coding sequence of the CAR may be disposed 5 'or 3' to the chimeric immune cell co-receptor coding sequence. Expression of the CAR and chimeric immune cell co-receptor may be under the direction of the same or different regulatory sequences.
Where the polynucleotide sequence is known, each polynucleotide molecule may be prepared by methods conventional in the art and the corresponding vector constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory), ausubel et al (1989,Short Protocols in Molecular Biology,Wiley), or other techniques described in standard textbooks. Alternatively, the nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the invention may be transferred into host cells by well known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electrotransfection may be used for other cellular hosts, see Sambrook et al (supra).
Host cells
Herein, when expressing a heterologous nucleic acid sequence, "host cell" refers to a eukaryotic cell that is capable of replicating the vector and/or expressing the heterologous gene encoded by the vector. Host cells can be used as acceptors for vectors. The host cell may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid is transfected or transduced into the host cell. Transformed cells include primary subject cells and their progeny. The terms "engineered" and "recombinant" cells or host cells as used herein often refer to cells into which exogenous nucleic acid sequences, such as vectors, have been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.
Herein, host cells include cells carrying the polynucleotide molecules and/or polypeptides described herein. In particular, the invention provides cells carrying the chimeric immune cell accessory receptor and/or the coding sequence thereof. The cells of the invention are preferably immune cells and can be used for adoptive cell therapy of tumors. Such cells of the invention are also referred to as chimeric immune cell accessory receptor modified cells of the invention.
More specifically, the cells of the invention are preferably immune effector cells, including T cells, such as cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, cd8+ T cells, or killer T cells), NK cells, NKT cells, CAR-T, CAR-NK, TCR-T, CIK, TIL, DN T cells; and other immune cells that can elicit effector functions.
Herein, cells may be autologous cells, syngeneic cells, allogeneic cells, and even in some cases xenograft cells, relative to the individual receiving them.
The nucleic acid construct/recombinant expression vector of the invention may be transferred into a cell of interest. Methods of transfer are conventional in the art and include, but are not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, electrotransformation, and the like. In certain embodiments, the nucleic acid construct or recombinant expression vector is electrotransferred.
In addition to carrying the chimeric immune cell accessory receptor and/or the coding sequence thereof, the cells of the invention may also have one or more other properties useful in cellular immunotherapy (e.g., adoptive cell therapy for tumors). Such other properties may be inherent to the cell or may be part of the cell after genetic manipulation by a human. For example, the cells of the invention may carry chimeric antigen receptors, αβ T cell receptors, and/or antigen-specific receptors, such as tumor-specific receptors, or coding sequences thereof.
Pharmaceutical composition
Herein, "pharmaceutical composition" refers to a composition for administration to an individual and encompasses a composition of cells for immunotherapy. The pharmaceutical compositions of the invention may also comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to a subject in a suitable dosage.
The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on a variety of factors including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs administered simultaneously.
The compositions of the present invention may be administered locally or systemically. In certain embodiments, the compositions provided herein (e.g., cells expressing the chimeric immune cell accessory receptor of the present invention) can be administered parenterally, e.g., intravenously, intraarterially, intrathecally, subdermally, or intramuscularly. In certain other embodiments, DNA encoding the constructs provided herein may be administered directly to a target site, for example, delivered to an internal or external target site by a gene gun or to an intra-arterial site by a catheter. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, intravenously. Parenteral formulations include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, lin Geyou dextrose, dextrose and sodium chloride, ringer's lactate solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on Yu Linge dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise a proteinaceous carrier, such as serum albumin or an immunoglobulin, preferably of human origin. In addition to the proteinaceous chimeric cytokine receptor construct or nucleic acid molecule or vector encoding the same, it is contemplated that the pharmaceutical composition of the invention may also comprise a biologically active agent, depending on the intended use of the pharmaceutical composition.
Compositions for parenteral (e.g., intravenous) administration of the cells described herein may also be stored in lyophilized form or in solution (e.g., lyophilized formulations). The lyophilized formulation may be stored in a ready-to-use form or in a form that is further formulated prior to administration. The cryopreservation formulation can withstand long distance transport without damaging the cells. In addition to the cells themselves, cryopreservation formulations typically include components such as cell cryopreservation solution, human Serum Albumin (HSA), and the like. Prior to administration (e.g., intravenous infusion), the cryopreserved pharmaceutical composition is stored (e.g., in liquid nitrogen). The frozen preparation can be directly infused into a patient or formulated as an infusion composition after thawing. The composition and concentration of conventional frozen stock solutions are known to those skilled in the art. For example, the frozen stock solution or infusion composition may further comprise dimethylsulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, magnesium chloride, or the like, the concentration of which may be determined by one of skill in the art (e.g., an experienced physician) depending on the condition of the cell, disease, patient, or the like.
Method and application
The chimeric immune cell accessory receptor, the polynucleotide molecule, the vector, the host cell and the pharmaceutical composition containing the same can be used for preventing, treating or relieving cancers, especially cancers with corresponding tumor antigens expressed on the surfaces of cancer cells, or used for preparing medicines for preventing, treating or relieving cancers.
As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or lesions of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition under treatment (e.g., cancer). Treatment may optionally include a reduction or alleviation of symptoms of the disease or disorder, or a delay in the progression of the disease or disorder. "treating" does not necessarily mean complete eradication or cure of a disease or disorder or associated symptoms thereof.
"preventing" as used herein refers to a method for preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or disorder (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or disorder or delaying the onset or recurrence of symptoms of a disease or disorder. As used herein, "preventing" also includes reducing the intensity, impact, symptoms and/or burden of a disease or disorder before it occurs or recurs.
The invention includes the administration of cells, polynucleotide molecules and vectors, alone or in any combination, using standard vectors and/or gene delivery systems, optionally together with pharmaceutically acceptable carriers or excipients. In certain embodiments, the polynucleotide molecule or vector may be stably integrated into the genome of the subject following administration.
In particular embodiments, viral vectors that are specific for certain cells or tissues and persist in the cells may be used. Suitable pharmaceutical carriers and excipients are well known in the art. The compositions prepared according to the invention may be used to prevent or treat or delay the diseases identified above.
Furthermore, the present invention provides a method for preventing, treating or alleviating cancer, comprising the steps of: administering to a subject in need thereof an effective amount of cells carrying the chimeric immune cell accessory receptor, polynucleotide molecule and/or vector described herein and/or produced by the methods described herein.
The methods herein can be used to prevent, treat, or ameliorate a variety of cancers, including various solid and hematological tumors, including but not limited to lung cancer (e.g., non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, and glioma, among others. More specifically, cancers herein include, but are not limited to, breast, prostate, lung, and colon cancer or epithelial cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genital-urinary tract cancers, such as ovarian cancer, endometrial cancer, cervical cancer; renal cancer, lung cancer, gastric cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. Administration of the compositions of the invention may be useful for all stages and types of cancer, including for example, minimal residual disease, early stage cancer, advanced cancer, and/or metastatic cancer, and/or cancer that is refractory to treatment.
By way of example, a cancer patient or a patient susceptible to cancer or a patient suspected of having cancer is treated as follows. The modified cells as described herein may be administered to an individual and left for an extended period of time. The individual may receive one or more administrations of cells, and the interval between administrations may be days, weeks, months or years. In particular embodiments, multiple administrations may occur over weeks or months, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks or months. In some embodiments, the genetically modified cells are encapsulated to inhibit immune recognition and are located at a tumor site. In the case where cells are provided to an individual after tumor recurrence following initial treatment with cells of the invention, the cells may be altered to recognize different target tumor antigens. For example, where an initial round includes cells carrying a chimeric immune cell accessory receptor of the invention and another receptor specific for a particular antigen, the receptor for a different particular antigen may be used after a subsequent round (including after tumor recurrence).
In some embodiments, an effective amount of therapeutic cells carrying or expressing the chimeric immune cell co-receptor of any embodiment of the invention and optionally a CAR or exogenous transgenic TCR is provided to an individual in need thereof. These cells may be delivered simultaneously or non-simultaneously with one or more other cancer treatments. These cells and other cancer therapeutic agents may be delivered in the same or separate formulations. Cells and other cancer therapeutic agents may be provided to an individual by separate delivery routes. Cells and/or other cancer therapeutic agents may be delivered by injection or intravenous or oral administration, for example, at a tumor site. Conventional delivery routes for such compositions are known in the art.
The number of cells employed will depend on a variety of circumstances, such as the purpose of the introduction, the lifetime of the cells, the regimen to be used, the number of administrations, the ability of the cells to reproduce, the stability of the recombinant construct, etc.
Cells may be administered as desired. In some embodiments, a variety of schemes may be used to adjust the scheme parameters. In particular embodiments, the route or number or timing of administration, the lifetime of the cells, and/or the number of cells present may vary. The number of administrations may depend, for example, at least in part, on the factors described above.
Kit for detecting a substance in a sample
Any of the compositions described herein may be included in a kit. In one non-limiting example, cells expressing the chimeric immune cell accessory receptor of any of the embodiments of the invention and/or agents that produce one or more cells for use in cell therapy, the therapy comprising a recombinant expression vector, can be included in a kit. The kit components are provided in a suitable container format.
Some of the components of these kits may be packaged in an aqueous matrix or in lyophilized form. The container means of these kits typically comprise at least one vial, test tube, flask, bottle, syringe or other container means in which the component may be placed and preferably appropriately dispensed. In the case where more than one component is present in the kit, the kit will typically also contain a second, third or other container in which the other components may be separately placed. However, various combinations of components may be included in the vial. The kits of the invention will typically also comprise means for containing the components in a commercially available closed constraint format. Such containers may include injection molded or blow molded plastic containers, wherein the desired vials are retained.
When the components of the kit are provided in one and/or more liquid solutions, the liquid solutions are aqueous solutions, particularly preferably sterile aqueous solutions. In some cases, the container means may itself be a syringe, pipette, and/or other such device.
The components of the kit may also be provided in dry powder form. When the reagents and/or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. Thus, the kit may further comprise a second container means comprising a sterile, pharmaceutically acceptable buffer and/or other diluent.
The components of the kit may also be provided in the form of a cryopreservation formulation (e.g., a cryopreservation solution). The frozen preparation can be directly infused into a patient or formulated as an infusion composition after thawing. Thus, the kit may also comprise a cell cryopreservation bag, a cell cryopreservation tube, a temperature holding means (e.g. a container comprising liquid nitrogen), a thawing means, etc.
In a specific embodiment of the invention, the cells to be used in the cell therapies described herein are provided in a kit. In some embodiments, the cell is essentially the only component of the kit. The kit may contain reagents and materials for preparing the desired cells. In particular embodiments, the reagents and materials comprise primers, nucleotides, suitable buffers or buffer reagents, salts, and the like for amplifying the desired sequence, and in some cases, the reagents comprise DNA and/or vectors encoding the chimeric immune cell co-receptor and/or regulatory elements thereof described in any of the embodiments herein.
Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, guidelines for molecular cloning experiments, third edition, scientific Press, et al), corresponding references, or according to the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Examples
Example 1: construction of a Co-receptor expression vector
The pKB20 vector was constructed according to the method described in example 1 on page 21 of the specification of PCT application WO2022078310A 1. According to the method for constructing pKB20-EGFP described in this example, a pKB20 vector containing an expression cassette for an exogenous gene was constructed. Specifically, the sequences shown in SEQ ID NOs 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61 and 63 of Table 1 were obtained by the synthesis of the designated company, and the 2-terminal of SEQ ID NOs 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61 and 63 were ligated with a linker containing the corresponding cleavage site by using a ligase, and cloned into the prepared pKB20 vector according to the method described in example 1 of page 21 of WO2022078310A1, designated pKB20-K23-1, pKB20-K23-2, pKB20-K23-3, pKB20-K23-4, pKB20-K23-5, pKB20-K23-6, pKB20-K24-1, pKB20-K24-2, pKB20-K31-1, pKB20-K32-1, pKB20-K23-7 and pKB 20-24-4, respectively. The recombinant plasmids obtained above were transformed into E.coli (DH 5 c), and after sequencing was correct, plasmids were extracted and purified using the plasmid purification kit from Qiagen, to obtain high-quality plasmids for each recombinant expression vector. The names and sequences of the co-receptors are shown in Table 2.
TABLE 2 Structure and sequence of accessory receptors
Example 2: isolated culture of melanoma tissue-derived TIL cells
Freshly excised melanoma specimens were collected and immediately treated under sterile conditions. The melanoma tissue of this example was treated and cultured to obtain TIL according to the medium described in example 1 of WO2022111571A1 and the tumor sample treatment method and TIL culture method described in example 2. WO2022111571A1 is incorporated by reference in its entirety into the present application.
The method comprises the following steps:
1) Preparing physiological saline containing 100U/mL penicillin, 100 mug/mL streptomycin and 50 mug/mL gentamicin for later use;
2) Placing the obtained tumor tissue sample of the freshly isolated tumor patient in a 10cm culture dish added with 30mL of the physiological saline prepared in the step 1) in a sterile environment in a secondary biosafety cabinet for washing, transferring the tumor tissue sample to a new 10cm dish added with 30mL of the physiological saline prepared in the step 1) for washing, and repeating the washing for 3 times;
3) Removing fat tissue and necrotic tissue with a sterile scalpel, cutting tumor tissue becomes a diameter of 3X 3mm 3 2G-REX 100 culture tanks (available from Wilsonwolf), 42 randomly selected tumor tissue pieces were placed in each G-REX100 culture tank, seed cell culture medium was added to the culture tank, and the seed culture medium had the following composition: 3000IU/mL IL-2, 20ng/mL IL-7, 20ng/mL IL-15, 500U/mL GM-CSF, 1000IU/mL IFN-gamma, 3 μg/mL anti-CD 137 mAb, 3 μg/mL anti-CD 28mAb, 3 μg/mL anti-PD-1 mAb, 10ng/mL TNF-alpha, 5% v/v human AB serum, 1 XPS diabody, and a final volume of X-VIVO 15 basal medium; the redundant tumor tissue blocks are frozen and stored by a cryo-Stor 10 (purchased from BioLifeSolons) frozen solution through a program cooling instrument liquid nitrogen;
4) 3) adding 1L of the seed cell culture medium into a G-REX100 culture tank containing tumor tissue blocks, and adding 5% CO at 37 ℃ to the tumor tissue blocks 2 Culturing, removing half of old seed cell culture medium every 4 days, supplementing half of fresh seed cell culture medium, centrifuging at 12 th day, and counting total number and activity rate of cells after harvesting TIL seed cells;
5) Taking the seed cells harvested in 4), re-suspending to 5.0X10 s with an expanding medium containing 500IU/mL IL-2, 7ng/mL IL-7, 30ng/mL IL-15, 5% v/v human AB serum, 1 XPS diabody and a final volume of X-VIVO 15 basal medium 5 Per mL, in a cell culture vessel pretreated with anti-CD 3 mAb, anti-CD 28 mAb and anti-CD 137 mAb coating, 37℃5% CO 2 Activating for 2 days, centrifugally collecting activated cells, inoculating into G-REX500M culture tank containing preheated expansion medium, and expanding the expansion medium in G-REX500M culture tankThe large media is identical. The volume of the expansion medium in each G-REX500M was 5L. Activated seed cells were grown according to 2.5X10 5 /cm 2 Inoculation density inoculation of (C) 37℃ 5% CO 2 Culturing, removing half volume of old expansion medium after cell count every 4 days, and supplementing half volume of fresh expansion medium until total cell count in each G-REX500M tank reaches 1.0X10 10 Afterwards, the flasks were separated at a ratio of 1:2, and each flask was supplemented with fresh expansion medium to 5L and then continued to culture. Cells were harvested after a total of 12 days of culture in an enlarged medium of a G-REX500M culture tank before and after culturing to obtain TIL.
Example 3: genetic modification and proliferation of TIL
1) The X-VIVO 15 medium was previously added to a 12-well plate for a total of 14 wells, 2mL per well, and then transferred to a cell incubator at 37℃with 5% CO 2 Preheating for 1 hour;
2) The ratio of the electrotransport liquid with single dosage per hole is carried out according to the following table:
100μL Nucleocuvette TM Strip(μL) | |
Nucleofector TM volume of solution | 82 |
Electrolysis supplementary solution | 18 |
Plasmids pKB20-K23-1, pKB20-K23-2, pKB20-K23-3, pKB20-K23-4, pKB20-K23-5, pKB20-K23-6, pKB20-K24-1, pKB20-K24-2, pKB20-K24-3, pKB20-K31-1, pKB20-K32-1, pKB20-K23-7, pKB20-K24-4 and control empty plasmid pKB20 were tested as required, and groups of the experimental and control electrotransfer systems 13 and 1 were prepared;
3) The TIL obtained in example 2 was taken into 14 EP pipes, each of which was filled with 5X 10 6 Centrifuging at 1200rpm for 5min, discarding supernatant, subsequently re-suspending cells with 500 μl physiological saline, and repeating the centrifugation step to wash cell pellet;
4) Adding plasmids pKB20-K23-1, pKB20-K23-2, pKB20-K23-3, pKB20-K23-4, pKB20-K23-5, pKB20-K23-6, pKB20-K24-1, pKB20-K24-2, pKB20-K24-3, pKB20-K31-1, pKB20-K32-1, pKB20-K23-7, pKB20-K24-4 and control empty plasmid pKB 20-5 mug into the electrotransfer solution of each of the different experimental groups and control groups prepared in 2), and standing at room temperature for 30 min;
5) Resuspension of all tubes with plasmid-containing electrotransfer solution prepared in 4), 100. Mu.L of each tube, carefully pipetting the cell resuspension into a LONZA 100. Mu.L electrotransfer cup, placing the electrotransfer cup into LONZA Nucleofector TM 2b, starting an electric transfer program in the electric transfer groove, wherein the electric transfer program selects X001;
6) After completion of electrotransfer, carefully remove the electrotransfer cup, transfer the cell suspension to EP tube, add 200. Mu.L of pre-warmed X-VIVO 15 medium per tube, then transfer to 1) wells containing pre-warmed AIM-V medium in 12-well plates, 37℃C, 5% CO 2 Culturing; after 5 days of culture, TIL cells overexpressing the co-receptors K23-1, K23-2, K23-3, K23-4, K23-5, K23-6, K24-1, K24-2, K24-3, K31-1, K32-1, K23-7 and K24-4 were obtained, and TIL cells of the control group, designated TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K31-1, TIL-K32-1, TIL-K23-7, TIL-K24-4 and TIL-CTRL, respectively.
Example 4: phenotypic detection of TIL for electrotransport auxiliary receptors
1. Cell viability of TIL of electrotransport accessory receptor and accessory receptor expression positive rate
Cell viability was measured for each group by trypan blue staining and cell counter counting. The results showed that each of the helper receptor-expressing TIL prepared in example 3 and the control TIL had cell viability of 92% or more.
The BCMA extracellular domain or Claudin18.2 epitope is fused and expressed with the KIR extracellular domain in the extracellular domain of each auxiliary receptor, and can serve as a label of an exogenous transgene. The ratio of BCMA to Claudin18.2 epitope positive cells was detected using BCMA extracellular domain or Claudin18.2 epitope-targeting fluorescent antibody as a tag for TIL (TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-2, TIL-K23-7, TIL-K24-4) expressing a BCMA extracellular domain-containing co-receptor and TIL (TIL-K23-1, TIL-K24-3, TIL-K31-1, TIL-K32-1) expressing a Claudin18.2 epitope-containing co-receptor, respectively, as a tag, as follows:
1) Collecting each group of cells of TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K31-1, TIL-K32-1, TIL-K23-7, and TIL-K24-4, and collecting 1X 10 per group of cells 6 800g, centrifuging for 5min;
2) Discarding the supernatant, adding physiological saline to resuspend cells, and centrifuging for 5min at 800 g;
3) The supernatant was discarded, and 100. Mu.L of physiological saline was added to each sample to resuspend the cells. For each set of TILs electrotransferring the auxiliary receptor containing the BCMA extracellular domain, 2. Mu.L of BCMA flow antibody (Biolegend, cat#: 357504) was added to each tube and incubated for 30 minutes at room temperature; for electrotransformation of TIL containing auxiliary receptor of Claudin18.2 epitope, adding 2 mu L of primary antibody (Abcam, cat#: ab 222512) without fluorescent group of targeted Claudin18.2 epitope into each tube, incubating for 30 minutes at room temperature, 800g, centrifuging for 5min, re-suspending and washing with physiological saline, 800g again, centrifuging for 5min, repeating twice, re-suspending with 100 mu L of physiological saline, adding 2 mu L of primary-targeting fluorescent secondary antibody (Abcam, cat#: ab 72465), and incubating for 30 minutes at room temperature; TIL-CTRL detects background values with BCMA flow antibodies;
4) Centrifuging 800g of each group of cells marked by the antibody in the step 3) for 5min, adding a proper amount of physiological saline into each group of cells, centrifuging 800g for 5min, washing twice, and discarding the supernatant;
5) Resuspension with 400 μl of physiological saline, and detection by up-flow cytometry.
The positive rate of each group of cells is shown in table 3 below:
TABLE 3 TIL occupancy positive for accessory receptor expression
Sample name | Positive rate (%) |
TIL-K23-1 | 36.9 |
TIL-K23-2 | 35.6 |
TIL-K23-3 | 38.2 |
TIL-K23-4 | 34.4 |
TIL-K23-5 | 40.9 |
TIL-K23-6 | 39.4 |
TIL-K24-1 | 43.3 |
TIL-K24-2 | 47.2 |
TIL-K24-3 | 42.3 |
TIL-K31-1 | 55.7 |
TIL-K32-1 | 58.2 |
TIL-K23-7 | 46.9 |
TIL-K24-4 | 47.1 |
TIL-CTRL | 0.01 |
2. Lymphocyte phenotype and cytokine secretion levels of TIL expressing co-receptors
(1) Lymphocyte phenotype expressing the accessory receptor TIL
The associated lymphocyte phenotype of each group of cells is shown in Table 4
TABLE 4 TIL cell phenotype of electrotransport auxiliary receptors
(2) Secretion of the cytokine IFN-gamma for the electrotransport helper receptor TIL
a. Secretion levels of IFN-gamma without co-incubation with target cells
Directly taking the TIL and TIL-CTRL cell supernatants of each of the electric transfer auxiliary receptors prepared in example 3, and detecting the IFN-gamma concentration in the cell supernatants of each group according to the method described in the specification by using a CBA detection kit (Human IFN-gamma Flex Set, BDbiosciences, cat# 558269);
b. secretion levels of IFN-gamma following co-incubation with target cells
The fresh melanoma tissue of example 2 was cut into pieces of 3X 3mm size, and the pieces were mixed as homogeneously as possible and then mixed according to Robert Suriano et al.Ex Vivo Derived Primary Melanoma Cells: implications for Immunotherapeutic Vaccines J Cancer 2013;4 (5) culturing the primary melanoma cells by the method described in section 371-382.Materials and Methods, stably passaging for 2-3 passages, and then plating 14 wells of 12-well plate, and when the cell wall is stable and the confluency exceeds 80%, adding 5×10 cells prepared in example 3 to each well 5 Cell suspension of TIL and TIL-CTRL of each co-receptor was electrotransformed 1mL at 37℃with 5% CO at 2 The culture was incubated overnight in an incubator, centrifuged, and the supernatant of each sample was collected and assayed for IFN-. Gamma.concentration using a CBA assay kit (Human IFN-. Gamma.Flex Set, BDbiosciences, cat# 558269) according to the protocol described in the specification.
The results are shown in Table 5:
TABLE 5 IFN-gamma secretion levels before and after Co-incubation of the electrotransport auxiliary receptor TIL with target cells
IFN-gamma secretion levels of the groups of electrotransport auxiliary receptor TILs not co-incubated with primary melanoma cells were generally less different than IFN-gamma secretion levels of control cells TIL-CTRL; after co-incubation with target cells, IFN-gamma secretion levels of the electrotransport auxiliary receptor TILs of each group are significantly improved compared to IFN-gamma secretion levels of the respective groups when the target cells are not co-incubated. The IFN-gamma secretion level of TIL-CTRL after co-incubation with target cells is also significantly increased compared to that without co-incubation, but to a significantly lesser extent than the TIL of the electrotransport auxiliary receptors of each group.
The results show that the activation level is improved after the TIL is incubated with the target cells; the TIL of the electrotransport auxiliary receptor can further enhance the amplitude of the activation after being incubated with the target cells. This suggests that the presence of accessory receptors helps to further increase the level of activation of T cell production by target cells.
Example 5: killing effect of TIL of electric transfer auxiliary receptor on homologous tumor cells
The fresh melanoma tissue of example 2 was cut into pieces of 3X 3mm size, and the pieces were mixed as homogeneously as possible and then mixed according to Robert Suriano et al.Ex Vivo Derived Primary Melanoma Cells: implications for Immunotherapeutic Vaccines J Cancer 2013;4 (5) Primary melanoma cells were obtained by culturing according to the method described in section 371-382.Materials and Methods.
The TIL and TIL-CTRL cells of each of the electrotransport auxiliary receptors obtained in example 3 were tested for their in vitro killing activity against their cognate melanoma primary cells using a real-time label-free cell function analyzer (RTCA) from the company Eisen, and the specific procedure was as follows:
(1) Zeroing: adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;
(2) Target cell plating: the primary cells of melanoma obtained by culture were cultured at 10 per well 4 Spreading individual cells/50 mu L in a plate containing a detection electrode, standing for several minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;
(3) Adding effector cells: after the target cells are cultured for 18h to 24h, observing the cell index, when the cell index is 1, respectively adding effector cells TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K31-1, TIL-K32-1, TIL-K23-7, TIL-K24-4 and TIL-CTRL, wherein the effective target ratio is 2:1, starting step 3, and after the co-culture is over 90h, observing the cell proliferation curve, and calculating the target cell killing rate. The target cell killing rate was calculated as follows:
Where A is the cell index of the group to which no effector cells have been added and only target cells (i.e., tumor cells) are present, and B is the cell index of each group to which effector cells have been added.
The results are shown in Table 6 and FIG. 1. Compared with TIL-CTRL, TIL-K23-1, TIL-K23-2, TIL-K23-3, TIL-K23-4, TIL-K23-5, TIL-K23-6, TIL-K24-1, TIL-K24-2, TIL-K24-3, TIL-K31-1, TIL-K32-1, TIL-K23-7 and TIL-K24-4 have significantly stronger killing effect on homologous melanoma primary tumor cells.
TABLE 6 RTCA kill Rate of the electrotransport auxiliary receptor TIL on target cells
Sample name | Target cell killing Rate (%) |
TIL-K23-1 | 52.1 |
TIL-K23-2 | 54.6 |
TIL-K23-3 | 59.6 |
TIL-K23-4 | 57.8 |
TIL-K23-5 | 63.2 |
TIL-K23-6 | 60.1 |
TIL-K24-1 | 72.8 |
TIL-K24-2 | 69.6 |
TIL-K24-3 | 62.9 |
TIL-K31-1 | 71.7 |
TIL-K32-1 | 73.4 |
TIL-K23-7 | 72.7 |
TIL-K24-4 | 77.3 |
TIL-CTRL | 40.9 |
Example 6: killing of tumor graft (PDX) tumor tissue by TIL cells of an electric transfer helper receptor
Experimental animal
Immunodeficient B-NDG mice (purchased from Baioerskin) were used as PDX model to construct experimental animals. Experimental design and grouping: as shown in Table 7 below
Table 7 mice dosing regimen and groupings
The TIL used in groups 2 to 4 was TIL-CTRL, TIL among the TILs prepared in example 3-K23-7 and TIL-K24-4. Cells were resuspended in PBS by centrifugation prior to tail vein injection to a cell density of 1X 10 8 /mL PBS cell suspension.
Animal feeding
After purchasing the required amount of B-NDG mice, the mice are fed into SPF-class experimental animal houses for 7-10 days.
Environment: the mice will be housed in a clear resin plastic cage in an animal house. The mouse cage padding is the sawdust and corncob padding which are subjected to high-pressure sterilization and is replaced periodically. The animal room is equipped with a high efficiency air filter and the temperature will be maintained between 20-26 c (68-79F) with a relative humidity of 40-70%. Temperature and humidity were continuously observed and recorded. The lighting conditions were 12 hours of fluorescent light illumination and 12 hours of no illumination per day.
Food and drinking water: the experimental mice can obtain special mouse grains (sterilized by irradiation, shanghai Laike laboratory animal liability Co., ltd., china) in an unlimited amount, and can be used for approaching sterilized clean drinking water at any time without obstacle.
Construction of PDX model
1) Patient tumor tissue sample treatment: taking a part of melanoma tissue in example 2, removing necrotic part tissue, adipose tissue, connective tissue, etc. under aseptic conditions, after washing, the tissue is divided into a plurality of 5X 5mm by using a surgical knife 3 The tissue block is placed in a tumor-containing sample transportation preservation solution UW, and B-NDG mice are prepared to be inoculated with the tumor block;
2) Tumor tissue sample inoculation: several B-NDG mice were taken, the mice were fixed with a mouse subcutaneous tumor inoculation fixator after the shoulder blade portion was prepared, the iodophor was sterilized, and the tumor mass in 1) was inoculated to the right inguinal portion with a PDX model tumor mass inoculation trocar after local anesthesia of lidocaine. Inoculation when the astronomical is P0, tumor is measured 2 times per week, and the calculation formula of tumor volume is V=0.5×a×b 2 Wherein a and b are the long and short diameters of the tumor, respectively;
3) PDX tissue passaging: observing the growth condition of tumor tissue of each inoculated mouse until the tumor tissue volume is over 300mm 3 After the mice were anesthetized, the tumor mass was removed and cut into 5X 5mm pieces with a scalpel under aseptic conditions 3 Repeating step 2) after tissue blocks, inoculatingTo the right inguinal portion of the new mouse, waiting for the next generation growth of PDX tumor;
4) Repeating step 3), continuing to subculture for 2-3 generations, taking part of the in-vivo PDX tissue of the mice for histologic pathological analysis, determining that the PDX tissue is still human tissue (but not murine tissue), continuing to inoculate the mice with the PDX tissue (namely, inoculating 48 mice with a PDX tissue block) according to 1.5 times of the number of the mice used in the experimental design of the table 6, observing the tumor formation condition of the mice, measuring the tumor number 2 times per week, and waiting for the tumor formation.
Grouping and administration of animals
Tumor volume of equal PDX inoculated mice reaches 50mm 3 When selecting 32 animals with proper tumor volumes from 48 animals, randomly grouping the animals according to tumor volumes, wherein n=8, and ensuring that all groups have comparability on a base line. Grouping when the diary was D0, dosing was performed according to the protocol of table 7. Animal body weight and tumor volume were measured 3 times a week during the experiment, and animals were observed daily for clinical symptoms. Mm for tumor volume 3 The tumor measurement formula is the same as that described above.
Results
The experimental results are shown in FIG. 2. One-way analysis of variance (one-way ANOVA analysis) was performed on the tumor volume differences between the different groups, followed by post hoc examination (Bonferroni post hoc test) using the Bonferroni method to see if there were significant differences between the different groups. * : p <0.05; * P <0.01. Figure 2 shows that tumor volume increase was significantly inhibited in the TIL-CTRL dosed mice compared to PBS-injected control tumor-bearing mice by day 40 post-dosing. Compared with the TIL-CTRL administration group, the tumor volume of the tumor-bearing mice in the TIL-K23-7 and TIL-K24-4 administration groups is further remarkably reduced. The result shows that the unmodified TIL-CTRL has obvious inhibition effect on the homologous paired PDX tumor tissues, and the inhibition effect of the TIL on the homologous PDX tumor tissues is improved more obviously after the transgenic modification of the K23-7 or K24-4 auxiliary receptor, so that the auxiliary receptor can obviously improve the tumor killing capability of immune effector cells.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (14)
1. A chimeric immune cell co-receptor comprising an extracellular domain of a KIR or a functional fragment or variant thereof that retains the biological function of binding to a KIR ligand, a transmembrane region, and an intracellular region comprising a signaling domain and/or a costimulatory domain,
preferably, the method comprises the steps of,
the KIR is an activated KIR or an inhibited KIR,
more preferably, the activated KIR is KIR2DL4 and the inhibited KIR is any one or more selected from KIR2DL3, KIR3DL1 and KIR3DL 2.
2. The chimeric immune cell co-receptor of claim 1, further comprising a hinge region,
preferably, the hinge region comprises a membrane proximal fragment of the natural extracellular domain of the costimulatory signaling molecule, or, the hinge region comprises, but is not limited to, any one or more selected from the group consisting of: CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27,
More preferably, the hinge region is an extracellular hinge region of CD28 and/or an extracellular hinge region of il7rα.
3. The chimeric immune cell co-receptor according to claim 1 or 2, wherein the co-stimulatory domain is an intracellular domain of a co-stimulatory signaling molecule or a functional fragment or mutant thereof retaining the biological function of the co-stimulatory signaling molecule to transmit a co-stimulatory signal, activate an immune cell,
preferably, the co-stimulatory signaling molecule intracellular domain comprises any one or more selected from the group consisting of: CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27 or a mutant thereof,
more preferably, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28 and/or the intracellular domain of OX 40.
4. The chimeric immune cell co-receptor according to claim 1 or 2, wherein the chimeric immune cell co-receptor further comprises a membrane surface tag,
Preferably, the method comprises the steps of,
the membrane surface tag comprises a BCMA extracellular domain or variant thereof, or a claudin protein extracellular domain or fragment or variant thereof, and/or,
the membrane surface tag is located at the C-terminus of the extracellular domain of KIR,
more preferably, the claudin protein is claudin18 and the fragment of the claudin protein extracellular domain is a claudin protein extracellular epitope.
5. The chimeric immune cell co-receptor of claim 1 or 2, wherein the transmembrane region comprises one or more selected from the group consisting of: CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, CD40L, HVEM, CD5, CD2, CD46, CD8, CD97, GITR, CD30, SLAMF1-9, DAP10, CD64, CD69, CD16, CD89, myD88, KIR2DS, KIR3DS, NKp30, NKp44, NKp46, NKG2D, ICAM and CD27 or mutants thereof that retain transmembrane functions,
preferably, the transmembrane region is a CD28 transmembrane region, an IL7 ra transmembrane region, or a mutant thereof that retains transmembrane function.
6. The chimeric immune cell co-receptor of claim 1 or 2, wherein the chimeric immune cell co-receptor further comprises a signal peptide.
7. The chimeric immune cell co-receptor of claim 4,
the chimeric immune-cell co-receptor comprises a KIR2DL3 extracellular region, a membrane surface tag, a transmembrane region, and one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain and an IL-7Rα intracellular domain, the transmembrane region being a CD28 transmembrane region or an IL-7Rα transmembrane region or a mutant thereof,
preferably, the membrane surface tag comprises a Claudin18.2 epitope or a BCMA extracellular domain, and/or the chimeric immune-cell co-receptor further comprises a CD28 hinge region between the membrane surface tag and the transmembrane region,
preferably, the chimeric immune cell co-receptor further comprises a linker; preferably, the joint is a rigid joint or a flexible joint; preferably, the linker is located between the KIR2DL3 extracellular region and the membrane surface tag,
more preferably, the chimeric immune cell co-receptor comprises:
(1) KIR2DL3 extracellular region, claudin18.2 epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD28 intracellular domain and OX40 intracellular domain,
(2) Comprising a KIR2DL3 extracellular region, a BCMA extracellular domain, an IL7Rα transmembrane region or mutant thereof, an IL-7Rα intracellular domain, or
(3) Comprises a KIR2DL3 extracellular region, a linker, a BCMA extracellular domain, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain;
or,
the chimeric immune-cell co-receptor comprises a KIR2DL4 extracellular region, a membrane surface tag, a transmembrane region, and one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain and an IL-7Rα intracellular domain, the transmembrane region being a CD28 transmembrane region or an IL-7Rα transmembrane region or a mutant thereof,
preferably, the membrane surface tag comprises a claudin18.2 epitope or BCMA extracellular domain; and/or the chimeric immune cell co-receptor further comprises a CD28 hinge region or an IL7Rα extracellular hinge region between the membrane surface tag and the transmembrane region,
preferably, the chimeric immune cell co-receptor further comprises a linker; preferably, the linker is located between the KIR2DL3 extracellular region and the membrane surface tag,
more preferably, the chimeric immune cell co-receptor comprises:
(1) KIR2DL4 extracellular region, claudin18.2 epitope, CD28 extracellular hinge region, CD28 transmembrane region, CD28 intracellular domain and OX40 intracellular domain,
(2) KIR2DL4 extracellular region, BCMA extracellular domain, IL7 ra extracellular hinge region, IL7 ra transmembrane region and IL-7 ra intracellular domain,
(3) KIR2DL4 extracellular region, claudin18.2 epitope, IL7 ra extracellular hinge region, IL7 ra transmembrane region mutant and IL-7 ra intracellular domain, or
(4) KIR2DL4 extracellular region, linker, BCMA extracellular domain, CD28 transmembrane region, CD28 intracellular domain and OX40 intracellular domain
Or,
the chimeric immune-cell co-receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a membrane surface tag, a CD28 transmembrane region, one or more intracellular domains selected from the group consisting of a CD28 intracellular domain, an OX40 intracellular domain and an IL-7Rα intracellular domain,
preferably, the chimeric immune cell co-receptor comprises a KIR3DL1 extracellular region or a KIR3DL2 extracellular region, a claudin18.2 epitope, a CD28 transmembrane region, a CD28 intracellular domain, and an OX40 intracellular domain.
8. The chimeric immune cell co-receptor according to claim 1 or 2,
the amino acid sequence of the CD28 transmembrane region is shown as SEQ ID NO. 4,
the amino acid sequence of the IL7Rα transmembrane region is shown in SEQ ID NO. 6,
the amino acid sequences of the IL7Rα transmembrane region mutants are shown in SEQ ID NO. 8, 10, 12 and 14,
the amino acid sequence of the CD28 intracellular domain is shown as SEQ ID NO. 16,
the amino acid sequence of the OX40 intracellular domain is shown as SEQ ID NO. 18,
The amino acid sequence of the IL-7Rα intracellular domain is shown as SEQ ID NO. 20,
the amino acid sequence of the extracellular region of KIR2DL3 is shown as SEQ ID NO. 22,
the amino acid sequence of the extracellular region of KIR2DL4 is shown as SEQ ID NO. 24,
the amino acid sequence of the extracellular region of the KIR3DL1 is shown as SEQ ID NO. 26,
the amino acid sequence of the extracellular region of the KIR3DL2 is shown as SEQ ID NO. 28,
the amino acid sequence of the Claudin 18.2 epitope is shown as SEQ ID NO. 30,
the amino acid sequence of the BCMA extracellular domain is shown as SEQ ID NO. 32,
the amino acid sequence of the CD28 extracellular hinge region is shown as SEQ ID NO. 34,
the amino acid sequence of the IL7Rα extracellular hinge region is shown in SEQ ID NO. 36,
the linker is a rigid linker, the amino acid sequence of which is shown as SEQ ID NO. 60, or
The amino acid sequence of the chimeric immune cell auxiliary receptor is shown in any one of SEQ ID NO 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 62 and 64.
9. A polynucleotide molecule having: a nucleic acid sequence encoding the chimeric immune cell co-receptor according to any one of claims 1 to 8 or a complement thereof,
preferably, the method comprises the steps of,
the transmembrane region is a CD28 transmembrane region, the coding sequence of which is shown as SEQ ID NO. 3,
The transmembrane region is IL7 Ralpha transmembrane region, the coding sequence of which is shown as SEQ ID NO. 5,
the transmembrane region is an IL7 Ralpha transmembrane region mutant, the coding sequences of which are respectively shown as SEQ ID NO. 7, 9, 11 and 13,
the intracellular domain comprises a CD28 intracellular domain, the coding sequence of which is shown as SEQ ID NO. 15,
the intracellular domain comprises an OX40 intracellular domain having a coding sequence as shown in SEQ ID NO. 17,
the intracellular domain comprises an IL-7Rα intracellular domain, the coding sequence of which is shown in SEQ ID NO. 19,
the extracellular domain comprises an extracellular region of KIR2DL3, the coding sequence of which is shown as SEQ ID NO. 21,
the extracellular domain comprises an extracellular region of KIR2DL4, the coding sequence of which is shown as SEQ ID NO. 23,
the extracellular domain comprises an extracellular region of KIR3DL1, the coding sequence of which is shown as SEQ ID NO. 25,
the extracellular domain comprises a KIR3DL2 extracellular region, the coding sequence of which is shown as SEQ ID NO. 27,
the chimeric immune cell auxiliary receptor comprises a membrane surface tag, wherein the membrane surface tag comprises a Claudin 18.2 epitope with a coding sequence shown as SEQ ID NO. 29 or a BCMA extracellular domain with a coding sequence shown as SEQ ID NO. 31,
the hinge region is a CD28 extracellular hinge region, the coding sequence of which is shown as SEQ ID NO. 33,
The hinge region is an IL7Rα extracellular hinge region, the coding sequence of which is shown in SEQ ID NO. 35, or
The linker is a rigid linker, the coding sequence of which is shown as SEQ ID NO. 59,
more preferably, the process is carried out,
the polynucleotide molecule comprises a sequence selected from any one of SEQ ID NOs 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 61 and 63 or a complement thereof.
10. A nucleic acid construct comprising the polynucleotide molecule of claim 9;
preferably, the nucleic acid construct is a vector,
more preferably, the vector is a non-viral vector.
11. The nucleic acid construct of claim 10, wherein the non-viral vector is an integrated non-viral vector,
preferably, the non-viral vector is based on an integrated non-viral vector of a transposon system.
12. The invention also provides a genetically engineered cell expressing the chimeric immune cell co-receptor according to any one of claims 1 to 8 and/or carrying the coding sequence of said chimeric immune cell co-receptor,
preferably, the method comprises the steps of,
the cells are immune effector cells; more preferably, the immune effector cells include T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, DN T and TIL,
The cells also express a CAR, or carry a coding sequence for a CAR,
the cells also express an exogenous TCR, or a coding sequence carrying an exogenous TCR.
13. A pharmaceutical composition comprising a pharmaceutically acceptable adjuvant and any one or more of the chimeric immune cell co-receptor of any one of claims 1-8, the polynucleotide molecule of claim 9, the nucleic acid construct of claim 10, and the genetically engineered cell of claim 12.
14. Use of any one or more of the chimeric immune cell co-receptor of any one of claims 1-8, the polynucleotide molecule of claim 9, the nucleic acid construct of claim 10, and the genetically engineered cell of claim 12 in the preparation of a medicament for treating or preventing cancer.
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