WO2018103734A1

WO2018103734A1 - Chimeric antigen receptor and use thereof and preparation method therefor

Info

Publication number: WO2018103734A1
Application number: PCT/CN2017/115184
Authority: WO
Inventors: 路国秋; 任克勇
Original assignee: 北京中捭生物科技有限公司
Priority date: 2016-12-09
Filing date: 2017-12-08
Publication date: 2018-06-14

Abstract

Disclosed are a chimeric antigen receptor (CAR) and the use thereof and a preparation method therefor, a T-cell containing the chimeric antigen receptor, a nucleic acid sequence encoding the chimeric antigen receptor, a vector comprising the nucleic acid sequence and a pharmaceutical composition comprising the vector or T-cell.

Description

Chimeric antigen receptor, use thereof and preparation method

Technical field

The present invention relates to a novel chimeric antigen receptor (CAR), its use and a preparation method. The chimeric antigen receptor can specifically bind to the ligand PD-L1 and/or PD-L2 of the immunosuppressive receptor PD-1; T cells (CAR T cells) expressing the chimeric antigen receptor can be specific Killing target cells expressing PD-L1 and/or PD-L2 ligands.

Background technique

The regulation of T cell function by the human immune system includes a positive activation pathway and a negative inhibition pathway. In the forward activation pathway, activation of T cells requires two signals, including a first signal and a second signal. The first signal is triggered by the binding of a major histocompatibility complex (MHC)-antigen polypeptide complex on the surface of the antigen presenting cell (APC) membrane to the T cell receptor (TCR) on the surface of the T cell membrane, and the second signal is composed of the antigen. The costimulatory protein presented on the surface of the cell membrane is triggered by binding to the corresponding co-receptor protein on the surface of the T cell. Co-receptor proteins include CD28, OX40, and CD137. In the activation pathway, the T cell receptor transmits a signal triggered by the TCR/CD3 transmembrane protein complex, and CD3ζ plays an important role in this process. After T cells are activated by the forward pathway, they can be abundant. Amplifies and releases cytokines that promote immune response and kills target cells.

In addition to the positive activation pathway, there is also a negative inhibition pathway. The negative inhibition pathway can inhibit the activation of T cells and deplete their functions, thereby negatively regulating the immune response. In the negative inhibition pathway, receptor PD-1 is the most important co-inhibitory protein in the body. Receptor PD-1 is a type I transmembrane protein expressed in activated T cells, B cells, activated NK cells, monocytes, and immature Langerhans cells. In humans, receptor PD-1 has two ligands, PD-L1 and PD-L2. Under physiological conditions, binding of the receptor PD-1 expressed by T cells to the ligand PD-L1 or PD-L2 negatively regulates T cell proliferation and cytokine production.

A variety of tumor cells utilize this negative regulatory mechanism, which directly upregulates the expression of ligand PD-L1 or PD-L2 through transcription, post-transcriptional regulation, and epigenetic mechanisms. The ligand PD-L1 or PD-L2 on the surface of tumor cells binds to receptor PD-1 on T cells, inhibiting the activation of T cells, thereby avoiding attack by immune cells. The ligand PD-L1 or PD-L2 is highly expressed in various tumor tissues, including non-small cell lung cancer, melanoma, renal cell carcinoma, prostate cancer, breast cancer, glioma, etc., ligand PD-L1 or PD-L2. Binding to the receptor PD-1 on T cells depletes T cell function, loses proliferation and kills tumor cells.

Blocking the receptor PD-1 binding to the ligand PD-L1 or PD-L2 can be used as a tumor immunotherapy, ie, checkpoint therapy, by blocking PD-1 mediated negative regulatory signals, T The cells restore activity, thereby enhancing the immune response to cancer cells. Currently, drugs that block PD-1/PD-L1 or PD-L2 binding are mainly monoclonal antibodies. The drugs that block the PD-1 signaling pathway are Opdivo (Nivolumab) from Bristol-Myers Squibb (BMS) and Keytruda (Pembrolizumab) from Merck Sharp & Dohme. Other drugs at the stage of research include Pidilizumab (CT-011). , CureTech), Avetumab (Merck, BMS-936559 (MDX-1105, BMS), Atezolizumab (MPDL3280A, Roche) and MED14736 (Astrazeneca). However, antibody treatment methods have a half-life limitation, require continuous administration, are expensive, and the whole body The use of side effects is large, and there are disadvantages such as the inefficient use of single drugs.

Adoptive cellular immunotherapy is also a method of tumor immunotherapy, and the use of chimeric antigen receptor T cells (CAR T cells) to kill cancer cells is one of the adoptive cell immunotherapy methods. The chimeric antigen receptor (CAR) comprises an scFv fragment (or antigen recognition domain) which specifically recognizes a tumor antigen, and the hinge region, the transmembrane region and the intracellular signal domain are sequentially connected to each other. When the antigen on the target cell binds to the antigenic domain of the chimeric antigen receptor (CAR), the signal is transmitted to the cell through the hinge region and the transmembrane region, and the intracellular signal domain converts the signal into an activation signal, activating the effector cell. Proliferation, production of cytokines to kill target cells. Recently, it has been reported in the literature that CD19ZCAR and PD-1CD28 proteins are co-expressed in T cells to kill target cancer cells, that is, the chimeric antigen receptor of CD19Z on the surface of T cells binds to CD19 on the surface of tumor cells, thereby recognizing and killing CD19 antigen. Tumor cells, at the same time, the PD-1CD28 protein expressed on the surface of T cells competes with endogenous PD-1 on the surface of T cells to bind to PD-L1 or PD-L2 molecules on the surface of tumor cells, and converts the inhibition signal into a stimulation signal. In turn, the PD-1 pathway-mediated negative inhibition of T cells is abolished (Xiaojun Liu, et al, Cancer Research 2016, 76(6): 1578-1590). CD19ZCAR and PD-1CD28 synergistically achieve the killing effect on tumor cells. However, this protocol cannot kill tumor cells or immunosuppressive cells expressing the ligand PD-L1 or PD-L2, and the microenvironment of tumor immunosuppression still exists, so there is still a need for a more efficient method for killing cancer cells by immunotherapy.

Summary of the invention

The present invention provides a chimeric antigen receptor (CAR) which binds to PD-L1 and/or PD-L2 on the surface of a target cell and activates a signaling structure through it to enable a T expressing CAR The cells activate, massively expand, release cytokines that promote immune responses and kill target cells. This reverses the negative inhibition pathway produced by the binding of T cells that do not express this CAR to PD-L1 and/or PD-L2 on the surface of target cells.

A chimeric antigen receptor of the invention comprising: (i) a domain having a binding to a ligand PD-L1 or PD-L2; (ii) Hinge region; (iii) transmembrane domain; (iv) costimulatory signaling domain; (v) CD3ζ signaling domain.

In one embodiment, wherein the domain (i) has a domain that binds to an immunosuppressive receptor PD-1 ligand PD-L1 and/or PD-L2 molecule.

The chimeric antigen receptor domain (i) of the present invention may be a domain that binds to a ligand in a PD-1 molecule, or an scFv of an antibody of PD-L1 or PD-L2. The antibody can be from a human or animal source, including a murine source.

In one embodiment, wherein domain (i) has a domain that binds to ligand PD-L1.

In one embodiment, the chimeric antigen receptor domain (i) of the invention is part of a PD-1 protein and has at least 90%, 92%, 95%, 96%, 97% of the sequence of SEQ ID No: 1. , 98%, 99% or 100% identity, more preferably the sequence shown in SEQ ID No: 1. The chimeric antigen receptor is capable of binding to cells expressing the ligand PD-L1 or PD-L2, or cells expressing the PD-L1 and PD-L2 ligands.

In one embodiment, the domain (i) on the PD-1 molecule has the sequence set forth in SEQ ID No: 2 or 3.

In one embodiment, the hinge region comprises at least one of: a hinge region of CTLA4, a hinge region of CD28, a hinge region of CD7, a hinge region of IgG1, a hinge region of IgG4, a hinge region of IgD, a hinge region of CD7, CD8α The hinge area or the hinge area of PD-1. Preferably, the hinge region is derived from a human; more preferably, the hinge region is the hinge region SEQ ID No: 4 of hPD-1 (human PD-1).

In one embodiment, the hinge region comprises a hinge region of PD-1 and a hinge region of CD8α having the sequence set forth in SEQ ID No: 5. In another embodiment, the hinge region has the sequence set forth in SEQ ID No: 6.

In one embodiment, wherein the transmembrane domain comprises at least one of: a transmembrane domain of CD4, a transmembrane domain of CD7, a transmembrane domain of CD8α, a transmembrane domain of CD28, a transmembrane domain of CD134, a span of CD137 Membrane domain, transmembrane domain of FcεRIγ or transmembrane domain of H2-Kb. Preferably, the transmembrane region is derived from a human; more preferably, the transmembrane domain is the transmembrane domain of hCD28 (human CD28) SEQ ID No: 7.

In one embodiment, the costimulatory signaling domain comprises at least one of the following: CD28, 4-1BB, ICOS, OX40, CD244, FcεRIγ, CD8α, BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2 Costimulatory signaling domains of NKG2C, LIGHT, and DAP12. Preferably, the costimulatory signaling domain is derived from a human; more preferably, the costimulatory signaling domain is the costimulatory signaling region of hCD28 SEQ ID No: 8.

In one embodiment, the costimulatory signaling domain comprises the costimulatory signaling domain of 4-1BB SEQ ID No: 9. In another embodiment, the costimulatory signaling domain comprises a co-stimulatory signaling domain of CD28 and a costimulatory signaling domain of 4-1BB having the sequence set forth in SEQ ID No: 10.

In one embodiment, wherein the CD3ζ signaling domain has at least 95% of the sequence of SEQ ID No: 11 96%, 97%, 98%, 99% or 100% identity. Preferably, the CD3ζ signaling domain is the protein represented by the sequence of SEQ ID No: 11.

In one embodiment, the chimeric antigen receptor of the invention has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID No:12. protein. Preferably, the chimeric antigen receptor of the present invention is the sequence of SEQ ID No: 12, each of which is of human origin.

In one embodiment, the chimeric antigen receptor of the invention has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID No: protein. Preferably, the chimeric antigen receptor of the present invention is the sequence of SEQ ID No: 13, each of which is of human origin.

In one embodiment, the chimeric antigen receptor of the invention has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID No: 14. protein. Preferably, the chimeric antigen receptor of the present invention is the sequence of SEQ ID No: 14, each of which is of human origin.

In one embodiment, the chimeric antigen receptor of the invention has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID No: 15. protein. Preferably, the chimeric antigen receptor of the present invention is the sequence shown in SEQ ID No: 15, each of which is of human origin.

In one embodiment, a chimeric antigen receptor of the invention has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID No: protein. Preferably, the chimeric antigen receptor of the present invention is the sequence of SEQ ID No: 16, each of which is of human origin.

In one embodiment, the chimeric antigen receptor of the invention has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID No:17. protein. Preferably, the chimeric antigen receptor of the present invention is the sequence of SEQ ID No: 17, each of which is of human origin.

In another aspect, the invention provides a nucleic acid sequence encoding a chimeric antigen receptor of the invention. Preferably, the invention provides a nucleic acid sequence encoding a chimeric antigen receptor having any one of SEQ ID Nos: 12-17. Preferably, the nucleic acid is the sequence set forth in any one of SEQ ID Nos: 18-23.

In another aspect, the invention provides a vector comprising the aforementioned nucleic acid sequence.

In another aspect, the invention provides a T cell expressing the aforementioned chimeric antigen receptor.

In another aspect, the present invention provides a method of producing the aforementioned T cell, which comprises the step of introducing the aforementioned nucleic acid sequence into a T cell.

In another aspect, the invention provides a pharmaceutical composition comprising the aforementioned carrier or T cell together with a pharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the invention provides a method of treating a disease associated with PD-L1 or PD-L2 expression, comprising An effective amount of a T cell of the present invention expressing the aforementioned chimeric antigen receptor is administered to an animal.

In another aspect, the present invention provides the use of the T cell for the preparation of a medicament for treating a PD-L1 or PD-L2 expression-related disease.

In another aspect, the invention provides the use of the T cell described in the manufacture of a medicament for modulating the immune system.

In another aspect, the present invention provides the use of the T cell for inhibiting PD-L1 or PD-L2 expression-related cells and treating a related disease. Related cells include various tumor cells, such as hematological tumor cells including, but not limited to, leukemia, lymphoma, and/or myeloma, and solid tumors including, but not limited to, lung cancer, gastric cancer, esophageal cancer, colon cancer, breast cancer, ovary Cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck cancer, glioma, and soft tissue sarcoma; also included in the microenvironment of the tumor, expressing PD-L1 and PD-L2 ligands Other cell types include immunosuppressive cells such as macrophages, dendritic cells, and T cells, as well as stromal cells.

The invention has the advantages that the traditional CAR T cell kills the target cell and has the advantage of the test point drug, that is, the negative negative inhibition pathway is released by the receptor PD-1. Moreover, the existing target of CAR T structure design is a tumor antigen, and the target is to directly kill tumor cells. Whereas the targeting cells of the present invention include not only tumor cells expressing PD-L1 and/or PD-L2 ligands, but also immunosuppressive cells expressing PD-L1 and/or PD-L2 ligand molecules in the tumor microenvironment and Stromal cells. The effect achieved by the technical scheme of the present invention is not only effective killing tumor cells but also anti-tumor immunity of the tumor microenvironment by killing cells expressing PD-L1 and/or PD-L2 ligand in the tumor microenvironment. The inhibition of the reaction is beneficial to the anti-tumor effect of the human immune system.

DRAWINGS

Figure 1 is a plasmid map of the pCDH-EF1-MCS-T2A-copGFP vector.

Figure 2 shows the release of cytokine IFN-γ after co-culture of hPD-1-CAR-A T cells and target cells.

Figure 3 shows the release of cytokine IL-2 after co-culture of hPD-1-CAR-A T cells and target cells.

Figure 4 shows the killing effect of hPD-1-CAR-A T cells on K562-PD-L1 target cells.

Figure 5 shows the killing effect of hPD-1-CAR-A T cells on K562-PD-L2 target cells.

Figure 6 shows inhibition of tumor growth in mice by hPD-1-CAR-A T cells.

Figure 7 is a plasmid map of the pLVX-EF1α-IRES-mCherry vector.

Figure 8 shows the release of cytokine IL-2 after co-culture of hPD-1-CARs T cells with K562 series target cells, respectively.

Figure 9 shows the release of cytokine IL-2 after co-culture of hPD-1-CARs T cells with Raji series target cells, respectively. put.

Figure 10 shows the release of cytokine IFN-γ after co-culture of hPD-1-CARs T cells with K562 series target cells, respectively.

Figure 11 shows the release of cytokine IFN-γ after co-culture of hPD-1-CARs T cells with Raji series target cells, respectively.

Figure 12 shows the killing effect of hPD-1-CARs T cells on K562-PDL1 target cells.

Figure 13 shows the killing effect of hPD-1-CARs T cells on K562-PDL2 target cells.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning

Chimeric antigen receptor (CAR)

Chimeric antigen receptors (also known as chimeric T cell receptors, artificial T cell receptors, and chimeric immunoreceptors) are engineered receptors that implant any specific receptor into immune effector cells. In a classical CAR, a scFv fragment of a monoclonal antibody that specifically recognizes a tumor antigen is implanted into a T cell, an NK cell, or an NKT cell. A nucleic acid encoding a CAR can be introduced into a T cell, an NK cell, or an NKT cell using, for example, a retroviral vector. In this way, a large number of tumor-specific T cells, NK cells or NKT cells can be generated for adoptive cell transfer. Early clinical studies of this approach have shown efficacy in some tumors, particularly malignant tumors that treat B cells with the B cell antigen CD19. When the target antigen binding domain of the CAR binds to the target antigen, the activation signal is transmitted into the T cells expressing the CAR through the hinge region and the transmembrane domain, thereby stimulating the immune response of the CAR T cells.

PD-1

PD-1, a programmed cell death factor 1, is a costimulatory molecule belonging to the CD28 family and is expressed inducibly on the surface of activated T cells, B cells and NK cells. Its interaction with its ligand is autoimmune, It plays an important role in transplantation immunity, tumor immunity, and chronic viral infection. The extramembranous region of PD-1 used in the present invention is selected from human PD-1, which is 921 bp in length and encodes 288 amino acids, which comprises a conserved IgV domain, and its sequence size starts from amino acid to 125. Amino acid. Mammalian-derived PD-1 protein molecules are highly homologous.

PD-1 has two ligands that specifically bind to it, PD-L1 and PD-L2. PD-L2 and PD- at the genetic level L1 has 37.4% homology. PD-L1 is expressed in T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, and some non-hematopoietic cells (including cardiovascular endothelial cells, renal tubular epithelial cells, glial cells, pancreatic beta cells). , hepatocytes, etc.), PD-L2 is mainly expressed in dendritic cells, monocytes, bone marrow-derived mast cells, and B cells in germinal centers. In humans, PD-L2 is also found in vascular endothelium and T cells. A small amount of expression. When PD-1 binds to PD-L1/PD-L2, it can inhibit the activation of primary T cells and the function of effector T cells, induce the regulation of T cell production and maintain the regulatory function of T cells. In addition, PD-L1 can also bind to CD80 on APC to suppress the immune response.

scFv

A single-chain antibody variable fragment (or scFv), ie, a single-chain antibody, is linked by an antibody heavy chain variable region (VH) and a light chain variable region (VL). The peptide (Linker) is ligated with a molecular weight of 27-30 kDa, which is the smallest functional structural unit of the antigen binding specificity of the parent antibody. The DNA sequence of a single chain antibody can be transformed into a mammalian cell by a viral vector or a specific mammalian expression vector. The single-chain antibody gene is fused with other effector gene genes by recombinant DNA technology, and after expression, a single-chain antibody fusion protein having single-chain antibody properties and fused effector protein activity can be obtained.

Hinge area

The CAR of the present invention may comprise a hinge region, a "domain that binds to the ligand PD-L1 or PD-L2" and a transmembrane domain. The hinge region sequence may be, for example, the hinge region of CTLA4, the hinge region of CD28, the hinge region of IgG1, the hinge region of IgG4, the hinge region of IgD, the hinge region of CD7, the hinge region of CD8α, the hinge region of PD-1, or the hinge region above. Mutant. Preferably, the hinge region is the hinge region of PD-1 SEQ ID No: 4. The spacer may be a short spacer, such as a spacer comprising less than 100, less than 80, less than 60, or less than 45 amino acids. The spacer may be or include a hinge region of CTLA4, a hinge region of CD28, a hinge region of IgG1, a hinge region of IgG4, a hinge region of IgD, a hinge region of CD7, a hinge region of CD8α, or a hinge region of PD-1 or the above hinge Mutant of the region.

Transmembrane domain

The transmembrane domain is a CAR sequence spanning the membrane that may comprise a hydrophobic alpha helix. The transmembrane domain can be derived from CD28 with good receptor stability. The transmembrane domain can be derived from any type I transmembrane protein. The transmembrane domain can be a synthetic sequence that is predicted to form a hydrophobic helix. Preferably, the transmembrane domain of the invention may be a mutant derived from the transmembrane domain of CD4, CD7, CD8α, CD28, CD134, CD137, FcεRIγ, H2-Kb or the above transmembrane domain.

Intracellular costimulatory signaling region (intracellular domain)

The intracellular domain is the signal transmission part of the CAR. After antigen recognition, the receptors cluster and the signal is transmitted to the cells. The most commonly used intracellular domain component is the intracellular domain of CD3ζ containing 3 ITAMs. This transmits an activation signal to the T cells after antigen binding. CD3ζ may not provide a sufficient activation signal and may require additional costimulatory signals. For example, chimeric CD28 and OX40 can be used with CD3ζ to transmit proliferation/survival signals, or all three can be used together (Pule et al, Molecular therapy, 2005: Volume 12; Issue 5; Pages 933-41).

The costimulatory signaling region comprises one or more of the following: CD28, 4-1BB, ICOS, OX40, CD244, FcεRIγ, CD8α, BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, and DAP12 Co-stimulation signal transduction zone. Preferably, the co-stimulatory signaling region of CD28.

CD28

CD28 is a membrane protein molecule that mediates co-stimulatory signals on the surface of T cells and belongs to the CD28 family. CD 28 molecules are constitutively expressed in T cells, and CD 28 plays a leading role in various cascades of co-stimulatory signaling molecules on the surface of T cells, playing the role of "master switch", and is the earliest involved in the activation process of T cells. The conjugated co-stimulatory signal plays an important role in the activation of the initial T cells after antigen stimulation, and induces the expression of other co-stimulatory molecules such as ICOS, OX 40, CD 154, and the expression of co-stimulatory molecules. The co-stimulatory signal function is exerted in different activation or differentiation stages of T cells and in different micro-environments, thereby initiating a T cell costimulatory signal network to ensure complete and sustained activation of T cells. In human close relatives, such as murine and primate, CD28 molecules also play similar roles and functions.

4-1BB

4-1BB (CD137, receptor-induced by lymphocyte ac-tivation) is a newly discovered T cell costimulatory molecule other than CD28/CTLA-4, belonging to the tumor necrosis factor receptor (TNF-R) family. As a type I transmembrane protein, the extracellular domain is rich in cysteine, and the intracellular region contains a potential phosphorylation site. It is not expressed on the surface of resting T cells, but only on activated T cells. In addition to expression on activated CD4+ T cells, CD8+ T cells and NK cells, 4-1BB is also expressed on CD4+, CD25+ regulatory T cells, and 4-1BB plays an important role in lymphocyte activation regulation.

CD3ζ signaling domain

The term ""ζ" or "ζ chain", "CD3-ζ" or "TCR-ζ" means the protein provided by GenBank ID: NM_000734.3, or an equivalent residue to a human close relative; the ζ chain is a Receptor-activated protein tyrosine kinase substrate, when the receptor binds to the ligand, the ζ chain rapidly undergoes tyrosine phosphorylation and participates in the transduction of lymphocyte activation signals.

Identity

The correlation between two amino acid sequences is described by the parameter "identity".

For the purposes of the present invention, the degree of identity between two amino acid sequences uses the Needle program as in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends in Genetics 16: 276-277). It is preferably determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) performed in version 3.0.0 or higher. The optional parameters used were a gap penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). Use the output of the Needle tag as "longest identity" (obtained with the -nobrief option) as a percentage identity and calculate as follows:

(same residue x 100) / (alignment length - total number of gaps in the alignment).

Chimeric antigen receptor of the present invention

In the embodiment, the chimeric antigen receptor (CAR) of the present invention replaces the specificity of the monoclonal antibody targeting the tumor antigen of the classical CAR with a similar structure of the extramembranous region of PD-1 or the extramembranous region of PD-1. In the sexual region, the chimeric antigen receptor of the present invention is capable of specifically binding to a PD-L1 and/or PD-L2 ligand of a target cell.

In the chimeric antigen receptor of the present invention, the domain (i) which binds to the ligand PD-L1 or PD-L2 may be a domain which binds to a ligand in the PD-1 molecule, or PD-L1 or PD- An scFv of an antibody of L2, such as an antibody of human or animal origin.

In a specific embodiment, the domain of the chimeric antigen receptor (CAR) of the present invention that binds to the ligand PD-L1 or PD-L2 is the N-terminus of the PD-1 protein represented by the sequence of SEQ ID No: 1. a domain of an immunoglobulin variable region (IgV), or at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% of the protein sequence of SEQ ID No: 1. Variants. The variant of the protein of SEQ ID No: 1 in the present invention may be an amino acid change performed outside the IgV domain (positions 25-101 of the amino acid sequence of SEQ ID No: 1), and the change does not affect the domain and the match. The ability of the ligand PD-L1 or PD-L2 to bind.

In one embodiment, in the chimeric antigen receptor of the present invention, the domain that binds to the ligand in the PD-1 molecule has the sequence shown in SEQ ID No: 2 or 3.

In a specific embodiment, the chimeric antigen receptor of the invention is PD-1 of the sequence set forth in SEQ ID No: The N-terminus of the protein comprises a domain sequence of an immunoglobulin variable region (IgV) or a variant thereof, which binds to a hinge molecule, a transmembrane domain, a costimulatory signaling region, and a CD3ζ signaling domain. Preferably, the chimeric antigen receptor of the present invention is a domain having the above sequence of SEQ ID No: 1 or a variant thereof, a hinge region of PD-1 and/or a hinge region of CD8α, a transmembrane domain of CD28, CD28 A co-stimulatory signaling region and/or a costimulatory signaling region of 4-1BB, and a receptor molecule composed of a CD3ζ signaling domain. More preferably, the chimeric antigen receptor of the present invention has a sequence as shown in any one of SEQ ID Nos: 12-17 or at least 90%, 95 with the sequence shown in any one of SEQ ID Nos: 12-17. %, 96%, 97%, 98%, 99% or 100% identity protein molecules.

Variant refers to the inclusion of a change, ie, a substitution, insertion and/or deletion, at one or more (eg, several) positions with the combination of PD-L1 and/or PD-L2 as described herein. A polypeptide of a functional chimeric antigen receptor.

In another embodiment, the invention relates to a variant of the chimeric antigen receptor SEQ ID No: 12-17, a variant of domain (i) of SEQ ID No: 1, SEQ ID No: 11 Variants of the CD3ζ signaling domain are shown, which include a substitution, deletion, and/or insertion at one or more (eg, several) positions. In a specific embodiment, the CD3ζ signaling structure represented by SEQ ID No: 12-17 of the chimeric antigen receptor, or the domain (i) represented by SEQ ID No: 1 or SEQ ID No: 11 is introduced. The number of amino acid substitutions, deletions, and/or insertions in the domain is no more than ten, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. These amino acids may have minor changes in properties, ie, conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; typically smaller deletions of 1-10 amino acids; smaller amino or carboxy terminal extensions , such as an amino terminal methionine residue; a smaller linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or another function, such as a polyhistidine sequence , antigenic epitope or binding domain. In one embodiment, the variant of domain (i) set forth in SEQ ID No: 1 is the domain (i) of SEQ ID No: 2, and the mutation sites are V64H, L65V, N66V, Y68H, M70E , N74G, K78T, L122V, A125V. In another embodiment, the variant of domain (i) of SEQ ID No: 1 is the domain (i) of SEQ ID No: 3, and the mutation sites are V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93A, L122V, A125V.

Examples of conservative substitutions are within the scope of the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and day) Asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine) , threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are, for example, H. Neurath and RL Hill, 1979, The Proteins, New York Academic Publishing (Academic Press, New York). Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/ Gly.

Alternatively, these amino acid changes have a property that changes the physicochemical properties of the protein. For example, amino acid changes can improve the thermal stability of a protein, its ability to bind to a ligand molecule, and the like.

The essential amino acids in the protein can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244). :1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the glucoamylase activity of the resulting mutant molecule is tested to identify amino acids that are critical to the activity of the molecule. Residues. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interactions can also be determined by structural physical analysis (as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling) in combination with putative amino acid mutations at the contact site. . See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al. , 1992, "FEBS Lett." 309: 59-64. The identity of the essential amino acids can also be inferred by comparison with a related protein.

Single or multiple amino acid substitutions, deletions, and/or insertions can be performed using known methods of mutagenesis, recombination, and/or shuffling followed by relevant screening procedures such as Reidhaar-Olson and Sol ( Sauer), 1988, Science 241: 53-57; Bowie and Sol, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625 to produce and test. Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and local temptation Change (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Nucleic acid sequence

A second aspect of the invention relates to a nucleic acid molecule encoding the CAR of the first aspect of the invention.

Due to the degeneracy of the genetic code, the nucleic acid sequence may encode an amino acid sequence corresponding to any one of SEQ ID No: 18-23, but with a different nucleic acid sequence. The nucleotide sequence of 1-72 in SEQ ID Nos: 18-23 respectively encodes a signal peptide, and the 73-final nucleotide sequence encodes a mature protein molecule, i.e., a protein molecule represented by SEQ ID Nos: 12-17.

Carrier

The invention also provides a vector comprising a nucleic acid sequence according to the invention. Such vectors can be used to sequence nucleic acids The column is introduced into a host cell such that it expresses and produces a chimeric antigen receptor (CAR) molecule according to the invention.

The vector may be, for example, a plasmid or a synthetic mRNA or viral vector, such as a retroviral vector or a lentiviral vector. The vector may be capable of transfecting or transducing effector cells.

Host cell

The invention also provides a host cell comprising a nucleic acid of the invention. The host cell may be capable of expressing a CAR according to the first aspect of the invention. The host cell can be a human T cell, a human NK cell, or a human NKT cell. T cells capable of expressing a CAR according to the present invention can be produced by transducing or transfecting T cells with a nucleic acid encoding CAR. The T cell can be an ex vivo T cell, for example, the T cell can be a sample from a peripheral blood mononuclear cell (PBMC). T cells can be activated and/or expanded prior to transduction with a nucleic acid encoding a CAR, for example by treatment with an anti-CD3 monoclonal antibody.

Pharmaceutical composition

The invention further relates to a pharmaceutical composition comprising a vector of the invention or a T cell expressing CAR and a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more additional pharmaceutically Active polypeptides and/or compounds. Such formulations may be, for example, in a form suitable for intravenous infusion.

treatment method

T cells expressing the CAR molecule of the present invention are capable of killing cancer cells such as blood cancer and solid tumors. Expression can be prepared ex vivo from the patient's own peripheral blood (first party), or from a hematopoietic stem cell transplant from a donor peripheral blood (second party), or from peripheral blood (third party) from an unrelated donor. CAR's T cells. Alternatively, the CART cells can be derived from ex vivo differentiated cells that can induce stem cells or embryonic stem cells to T cells. In these cases, CAR T cells are generated by introducing DNA or RNA encoding CAR into one of a number of means, including transduction with a viral vector, transfection using DNA or RNA.

T cells (CAR T cells) expressing the CAR molecule of the present invention can kill PD-L1/PD-L2 target cells (tumor cells or other immunosuppressive cells) which are immunosuppressive in the tumor microenvironment, and release the tumor microenvironment The immunosuppressive effect of the immune system or exogenous immunotherapy program in the body can fully exert anti-tumor immunity, so it can be used for tumor treatment related to the increased expression of PD-L1/PD-L2, due to chronic The infection also has an increased expression of PD-L1/PD-L2, and thus the present invention can also be used for the treatment of chronic infectious diseases. Tumors associated with elevated PD-L1/PD-L2 expression, such as hematological tumors, including but not limited to leukemia, lymphoma, and/or myeloma, as well as solid tumors including, but not limited to, lung cancer, gastric cancer, esophageal cancer , colon cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, Prostate cancer, melanoma, head and neck cancer, glioma and soft tissue sarcoma.

detailed description

Example 1. Determination of PD-1-CD28-CD3ζ sequence

According to the extracellular domain of human PD-1 (GenBank ID: L27440.1) gene in GenBank (including PD-1 signal peptide, N-terminal immunoglobulin variable region domain (IgV), and PD-1 hinge region , the transmembrane and intracellular regions of the human CD28 (GenBank ID: AF222341.1) gene, and the ζ chain of the human CD3 (GenBank ID: NM_000734.3) gene to construct the PD-1-CD28-CD3ζ of the present invention The entire sequence (SEQ ID No: 18), which is the coding sequence of the chimeric antigen receptor represented by SEQ ID No: 12 of the present invention.

The complete nucleic acid sequence of PD-1-CD28-CD3ζ was synthesized (Jin Weizhi Biotechnology Co., Ltd. artificial synthesis), followed by the extramembranous region of human PD-1 gene, the transmembrane region and intracellular region of human CD28 gene, and The intracellular region of the human CD3 gene ζ chain. The kozak sequence (GCCACC) was introduced at the 5' end, and the EcoR I and BamH I restriction sites were introduced at the 5' and 3' ends, respectively, to form a human PD-1-CD28-CD3ζ nucleic acid with an enzyme cleavage site. The full sequence (hereinafter referred to as "hPD-1-CAR-A").

Example 2. Construction of hPD-1-CAR-A expression plasmid

The full nucleic acid sequence of hPD-1-CAR-A was cloned into pCDH-EF1-MCS-T2A-copGFP lentiviral expression vector by Eco- and BamHI (purchased from SBI (system biosciences) company, hereinafter referred to as In the "pCDH vector" (shown in FIG. 1), an expression plasmid of hPD-1-CAR-A (hereinafter referred to as "pCDH-CAR-A plasmid") was obtained, and the obtained pCDH-CAR-A plasmid was transformed into DH5α strain (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

Example 3. Large-scale extraction of the desired plasmid and packaging plasmid

The pCDH-CAR-A plasmid constructed in Example 2, the pCDH vector plasmid, and the psPAX2 packaging plasmid (purchased from Wuhan Qiling Biotechnology Co., Ltd.) and the pMD2.G packaging plasmid (purchased from Wuhan Yuling Biotechnology Co., Ltd.) will be respectively contained. The strains were cultured in large amounts in LB medium, and the pCDH-CAR-A plasmid, pCDH vector plasmid, psPAX2 packaging plasmid and pMD2.G packaging plasmid (using endotoxin-free plasmid) were purchased in large quantities by alkaline lysis. Since Beijing Tiangen Biochemical Technology Co., Ltd.). The obtained plasmid was used for cell transfection.

Example 4. Packaging, concentration and titer determination of lentivirus

Lentiviral packaging

(1) Cell treatment: 24 hours before transfection, 4th to 12th generation Lenti-X 293T cells (purchased from Clontech) in logarithmic growth phase were digested with trypsin. The collected Lenti-X 293T cells were seeded at a density of 6×10 ⁶ /dish in a 10 cm cell culture dish and cultured in a 10% FBS (purchased from Hyclone), 1% sodium pyruvate, 1% glutamine Amide, 1% penicillin and 1% streptomycin (all percentages above are volume percent) in DMEM medium (purchased from Gibco). The culture conditions were 37 ° C, 5% CO ₂ incubator. The cells were cultured for 24 hours, and transfection was performed when the confluence rate reached 70-90%. Prior to cell transfection, the medium was changed to 10 ml of fresh virus packaging medium Opti-MEM (available from Gibco), and 5% FBS, 0.2% sodium pyruvate and 1% glutamine were added.

(2) Preparation of transfection system

The amount of reagents and steps required to transfect cells in a 10 cm dish are as follows:

1) Dilute the plasmid with 1.5 ml Opti-MEM:

psPAX2 5μg

pMD2.G 5μg

pCDH-CAR-A plasmid or pCDH vector plasmid 10μg

2) 60 μl of liposome transfection reagent lipfiter (purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.) was diluted with 1.5 ml of Opti-MEM and vortexed and mixed.

3) The transfection reagent obtained in the step 2) was added dropwise to the plasmid mixture of the step 1), and blown to thoroughly mix, and allowed to stand at room temperature for 20 minutes. The obtained liposome and plasmid mixture was added dropwise to Lenti-X 293T cells, and shaken and mixed. Incubate at 37 ° C under 5% CO ₂ .

4) After 6 hours of transfection, the solution was completely changed, and 10 ml of virus packaging medium (purchased from Gibco) was added to each dish. The cultivation was continued at 37 ° C under 5% CO ₂ .

5) 24 hours after the transfusion, the transfected Lenti-X 293T cells were observed under a fluorescence microscope. The results showed that the percentage of Lenti-X 293T cells expressing green fluorescent protein (GFP) was over 90% after transfection. , indicating that the transfection efficiency is very high.

6) The culture supernatants of Lenti-X 293T cells collected at 48 hours and 72 hours after the liquid exchange were placed in a 50 ml centrifuge tube, centrifuged at 2000 rpm for 5 minutes at room temperature, and the supernatant was collected.

7) The supernatant was filtered through a 0.45 μm filter to remove cell debris, and a pCDH lentivirus stock solution and a pCDH-CAR-A lentivirus stock solution were obtained, respectively.

Virus concentration

The lentiviral stock solution and the concentrate (purchased from Clontech) were mixed at a ratio of 1:3, and the resulting mixture was precipitated overnight. The mixture obtained by precipitating overnight was centrifuged at 1500 g for 45 minutes. After the supernatant was removed, the precipitate was resuspended in a stock volume of X-VIVO (purchased from Lonza) to obtain a concentrated lentivirus. The concentrated lentivirus was divided into 100 ul each frozen in a -80 ° C refrigerator.

Virus titer determination

Lenti-X 293T cells were infected with the concentrated lentivirus, and after 48 hours of infection, the ratio of expressing GFP cells was measured by flow cytometry. The titers of the concentrated pCDH lentivirus and pCDH-CAR-A lentivirus were calculated to be about 1 x 10 ⁸ TU/ml and 1 x 10 ⁸ TU/ml, and the TU was a transducing unit.

Example 5. In vitro culture, infection and expansion of T cells

(1) Take umbilical cord blood from healthy donors, and obtain umbilical cord blood mononuclear cells (UBMC cells) with Ficoll separation solution (purchased from GE company), and adjust the cell concentration to 1×10 with X-VIVO medium containing 10% FBS. ⁶ / ml. The cells were seeded at a rate of 1 ml/well into a 24-well plate, and magnetic beads conjugated to anti-CD3/CD28 antibody (purchased from Nearshore Protein Company) were added in an amount of 1 μl/10 ⁶ cells, and 40 IU/ml recombinant person was added. Interleukin 2 (rhIL-2) (purchased from Beijing Yiqiao Shenzhou Company). The virus was infected 48 hours after cell stimulation.

(2) A new 24-well plate was taken and previously coated with human fibronectin (Retronectin, available from TaKaRa). UBMC cells cultured in the step (1) for 48 hours were added to the coated 24-well plate at a cell volume of 1 × 10 ⁶ /well. Subsequently, 100 μl of concentrated pCDH lentivirus or concentrated pCDH-CAR-A lentivirus, and polybrene (purchased from Sigma) were added to each well to a final concentration of 8 μg/ml, and a final concentration of 40 IU/. Ml rhIL-2, to a total volume of 0.8 ml, mix. The 24-well plate was centrifuged at 1500 g for 90 minutes at 32 ° C, then placed in a 37 ° C, 5% CO ₂ incubator, and 1 ml of medium was added overnight.

(3) The UBMC cells were infected a second time after the lentivirus infected the UBMC cells for 24 hours. The first infected UBMC cells were collected, centrifuged at 1200 rpm for 10 minutes, and the supernatant was carefully aspirated. The first infected UBMC cells were resuspended by adding fresh X-VIVO medium containing 10% FBS, and the cell concentration was adjusted to 2 × 10 ⁶ /ml. The obtained UBMC cells were seeded at 0.5 ml/well into a new 24-well plate previously coated with Retronectin, followed by addition of lentivirus concentrate, polybrene and rhIL-2 in the same manner as in step (2). The 24-well plate was centrifuged at 1500 g for 90 minutes at 32 ° C, and then placed in an incubator to continue the culture.

(4) After the second infected UBMC cells were cultured for 24 hours, the cells were resuspended and centrifuged at 1200 rpm for 10 minutes, and then seeded in a 24-well plate at a density of 0.3 × 10 ⁶ /ml. 1ml. The cells were expanded by adding 300 IU/ml of rhIL-2, and then changed every 2-3 days, the cell growth density was adjusted to 0.3 × 10 ⁶ /ml, and the amount of rhIL-2 was the same as before until use.

(5) After 72 hours of the first infection, GFP expression of UBMC cells after infection was observed by fluorescence microscopy and photographed.

After 48 hours of the first infection and 24 hours after the second infection (i.e., 72 hours after the first infection), it was observed clearly that the infected T cells expressed GFP under a fluorescence microscope, indicating that the above-mentioned pCDH lentivirus and pCDH were observed. -CAR-A lentivirus has an ideal effect on T cell infection.

(6) Collect the second infected T cells, centrifuge at 1000 rpm for 10 minutes, wash once with PBS buffer, resuspend the cells in a flow tube with an appropriate amount of PBS buffer, and detect GFP positive by flow cytometry. The ratio of cells.

The results of the flow cytometry showed that the infection rate of pCDH lentivirus was 53.3%, and the infection rate of pCDH-CAR-A lentivirus was 37.8%.

Example 6. In vitro co-culture of T cells and target cells, and determination of cytokine release

Co-culture of T cells with target cells in vitro

(1) Infecting T cells with concentrated pCDH lentivirus and pCDH-CAR-A lentivirus, respectively, as shown in Example 5, obtaining T cells infected with pCDH lentivirus (hereinafter referred to as "control T cells") and T cells infected with pCDH-CAR-A lentivirus (hereinafter referred to as "hPD-1-CAR-A T cells"). Flow cytometry by staining with anti-hPD-1 antibody (purchased from BioLengend) showed that the positive expression rate of hPD-1-CAR-A T cells was 35%, and the ratio detected by GFP was basically the same, ie T The cells successfully expressed the chimeric antigen receptor represented by SEQ ID No: 12 of the present invention and were successfully anchored to the surface of T cells.

(2) K562 (ATCC-CCL-243 ^TM ), K562-PD-L1 and K562-PD-L2 target cell lines were established, respectively. The K562-PD-L1 and K562-PD-L2 cell lines were infected with pCDH-hPD-L1 (hPD-L1 GenBank ID: NM_014143.3) and pCDH-hPD-L2 (hPD-L2 GenBank ID: NM_025239) by K562 cell line. .3) Lentivirus, a cell line stably expressing was obtained (methods are the same as in Example 4 and Example 5).

(3) The above T cells and target cells were separately mixed, and a small amount of cells were centrifuged, resuspended in fresh X-VIVO medium, and counted.

(4) A 96-well V-well plate was plated with T cells and target cells in an amount of 1 × 10 ⁴ /100 μl/well, respectively. The cells were incubated overnight at 37 ° C, 5% CO ₂ .

(5) The culture supernatant was collected after 24 hours, and the obtained supernatant was subjected to ELISA (ELISA kit, purchased from Beijing Dakco is Biotech Co., Ltd.).

Detection of cytokines IFN-γ and IL-2 by ELISA

Table 1 Determination of the concentration of cytokine IFN-γ (pg/ml)

	hPD-1-CAR-A T细胞hPD-1-CAR-A T cell	对照T细胞Control T cell
T细胞+K562-PD-L1T cell + K562-PD-L1	760.06760.06	23.9923.99
T细胞+K562-PD-L2T cell + K562-PD-L2	101.56101.56	23.9823.98
T细胞+K562T cell + K562	23.6423.64	23.4223.42

As shown in Table 1 and Figure 2, the results showed that the concentration of IFN-γ released by hPD-1-CAR-A T cells after co-culture with K562-PD-L1 or K562-PD-L2 was much higher than that of control T. cell. The hPD-1-CAR-A T cells of the invention can effectively increase the killing ability of T cells to tumor cells.

Table 2 Determination of cytokine IL-2 concentration (pg/ml)

	hPD-1-CAR-A T细胞hPD-1-CAR-A T cell	对照T细胞Control T cell
T细胞+K562-PD-L1T cell + K562-PD-L1	1064.931064.93	20.2520.25
T细胞+K562-PD-L2T cell + K562-PD-L2	344.83344.83	20.0620.06
T细胞+K562T cell + K562	20.2820.28	20.1420.14

As shown in Table 2 and Figure 3, the results showed that the concentration of IL-2 released by hPD-1-CAR-A T cells co-cultured with K562-PD-L1 or K562-PD-L2 was much higher than that of control T. cell.

Example 7. Killing effect of hPD-1-CAR-A T cells on K562-PD-L1 target cells

Co-culture of T cells with target cells in vitro

(1) T cells were infected with the concentrated pCDH lentivirus and pCDH-CAR lentivirus, respectively, and specific steps were as shown in Example 5 to obtain control T cells and hPD-1-CAR-A T cells.

(2) A target cell K562-PD-L1 cell line was obtained. The K562-PD-L1 cell line is a cell line stably infected with a lentivirus in which the K562 cell line infects pCDH-hPD-L1.

(3) Blown the T cells and the target cells separately, and take a small number of cells.

(4) T cells and target cells were co-cultured in 48-well plates according to the target cells 5×10 ⁵ /well, T cells: target cells = 1:1, 5:1 and 10:1. The volume of the liquid is 400 ul.

(5) The 48-well plate was placed in an incubator and cultured overnight.

Cell staining and detection:

Cells were stained using the Annexin V Apoptosis Detection Kit (available from BIOLEGEND). Follow the kit instructions.

Table 3 Killing rate of hPD-1-CAR-A T cells to K562-PD-L1 target cells (%)

As shown in Table 3 and Figure 4, hPD-1-CAR-A T cells showed significant specific killing of K562-PD-L1 cells, and with the increase of the ratio of T cells to target cells, the killing rate also followed. A significant increase. When the ratio of T cells to target cells was increased to 10:1, after overnight co-culture, the killing rate reached 65.7%, and the killing effect was very obvious.

Example 8. Killing effect of hPD-1-CAR-A T cells on K562-PD-L2 target cells

Co-culture of T cells with target cells in vitro

(1) T cells were infected with concentrated pCDH lentivirus and pCDH-CAR-A lentivirus, respectively, and specific steps were as shown in Example 5 to obtain control T cells and hPD-1-CAR T cells.

(2) A target cell K562-PD-L2 cell line was obtained. The K562-PD-L1 cell line is a cell line stably expressed by a lentivirus in which p562H-PD-L2 is infected by K562 cells.

(4) T cells and target cells were co-cultured in 48-well plates according to the target cells 5 × 10 ⁵ /well, T cells: target cells = 2.5:1, 5:1 and 10:1. The volume of the liquid is 400 ul.

(5) The 48-well plate was placed in an incubator for 2.5 hours.

Cell staining and detection:

Cells were stained using the Annexin V Apoptosis Detection Kit.

Table 4 Killing rate of hPD-1-CAR-A T cells to K562-PD-L2 target cells (%)

The results showed that, as shown in Table 4 and Figure 5, hPD-1-CAR-A T cells showed significant specific killing of K562-PD-L2 cells, and the kill rate was also increased with the increase of the ratio of T cells to target cells. It has increased significantly. When the ratio of T cells to target cells increased to 10:1, after overnight co-culture, the killing rate reached 45.1%, and the killing effect was very obvious.

Example 9. Inhibition of tumor growth in mice by hPD-1-CAR-A T cells

15 mice were ordered, including 1 group of experimental groups (injected hPD-1-CAR-A T cells, as shown in Example 5), 1 group of control groups (injected with control T cells, as shown in Example 5) and 1 Group blanks (with X-VIVO medium as control), 5 mice per group. The mouse was a highly immunodeficient mouse (NPG) ordered from Beijing Weitongda Biotechnology Co., Ltd.

Mouse tumor formation

First, a Raji-PD-L1-luciferase cell line was constructed. On Raji cells (ATCC-CCL-86 ^TM) PD-L1 expression vector transiently transfected, together with a long-term screening stable puromycin PD-L1 expression in PD-L1-Raji cell line. On this basis, the Raji-PD-L1 cell line was infected with Lenti-luciferase lentivirus, and then Raji-PD-L1-luciferase with dual expression of PD-L1 and luciferase was obtained by flow sorting. Cell line.

Each mouse was injected with 2 x 10 ⁵ Raji-PD-L1-luciferase cells. The above cells were resuspended in 50 ul of PBS, mixed with 50 ul of Matrigel (purchased from BD), and injected into the peritoneal cavity of the mice.

Living imaging

In vivo imaging was performed after injection of Raji-PD-L1-luciferase cells for 48 hours.

Injection of T cells

Mice were grouped by mean fluorescence values, with the average fluorescence values of each group being as close as possible. For the experimental group, 1 x 107 h of PD-1-CAR-A T cells were injected per mouse. The cells were washed once with PBS, resuspended in 200 ul of X-VIVO, and intraperitoneally injected into the tumor of the mouse.

For the control group, an equivalent volume of control T cells was injected.

For the blank group, the same volume of X-VIVO was injected.

Post-treatment imaging

After T cells were injected, in vivo imaging was performed every 7 days to detect changes in tumor size in the peritoneal cavity of the mice.

The results showed that, as shown in Fig. 6, injection of hPD-1-CAR-A T cells significantly inhibited tumor growth compared to X-VIVO and control T cells.

Example 10. Construction of expression plasmid of human PD-1 chimeric antigen receptor series

(1) Structural design of human PD-1 chimeric antigen receptor series and determination of nucleic acid sequence

According to the extracellular domain of human PD-1 (GenBank ID: L27440.1) gene in GenBank (including PD-1 signal peptide, N-terminal immunoglobulin variable region domain (IgV), and PD-1 hinge region And its sequence containing partial site mutations (see WO2016/022994A2), the hinge region of human CD8α (GenBank ID: NM_001768.6), the transmembrane region and the cell of human CD28 (GenBank ID: AF222341.1) gene The inner region, the human 4-1BB (GenBank ID: U03397.1) intracellular region and the human CD3 (GenBank ID: NM_000734.3) gene ζ chain designed the human PD-1 chimeric antigen receptor series Nucleic acid sequence.

The sequence and nucleotide positions of the corresponding domains of the human PD-1 chimeric antigen receptor series are shown in Table 5.

hPD-1-CAR-A has the sequence shown in SEQ ID No: 12. Its corresponding nucleic acid sequence is SEQ ID No: 18.

hPD-1-CAR-B has the sequence shown in SEQ ID No: 13. Its corresponding nucleic acid sequence is SEQ ID No: 19. Its costimulatory signaling domain comprises the costimulatory signaling domain of CD28 and the costimulatory signaling domain of 4-1BB.

hPD-1-CAR-C has the sequence shown in SEQ ID No: 14. Its corresponding nucleic acid sequence is SEQ ID No: 20. Its costimulatory signaling domain is a costimulatory signaling domain of 4-1BB.

hPD-1-CAR-D has the sequence shown in SEQ ID No: 15. Its corresponding nucleic acid sequence is SEQ ID No:21. Its hinge region contains the hinge region of PD1 and the hinge region of CD8α.

hPD-1-CAR-E has the sequence shown in SEQ ID No: 16. Its corresponding nucleic acid sequence is SEQ ID No: 22. Based on hPD-1-CAR-A (SEQ ID No: 12), the binding sites of the binding domain were: V64H, L65V, N66V, Y68H, M70E, N74G, K78T, L122V, A125V. The mutation site of the hinge region is: A131I.

hPD-1-CAR-F has the sequence shown in SEQ ID No: 17. Its corresponding nucleic acid sequence is SEQ ID No: 23. Based on hPD-1-CAR-A (SEQ ID No: 12), the binding sites of the binding domain were: V64H, L65V, N66V, Y68H, M70E, N74G, K78T, C93A, L122V, A125V. The mutation site of the hinge region is: A131I.

Taking V64H as an example, it is indicated that the 64th amino acid is mutated from proline to histidine.

Table 5: Structure and sequence of six human PD-1 chimeric antigen receptors

(2) Construction of expression plasmid of human PD-1 chimeric antigen receptor series

The human PD-1-CD28-CD3 ζ nucleic acid full sequence (hereinafter referred to as "hPD-1-CAR-A", that is, the full nucleic acid sequence of A in Table 5) having the restriction enzyme cleavage site of Example 1 was cloned into pLVX. -EF1a-IRES-mCherry lentiviral expression vector (purchased from SBI (System Biosciences), hereinafter referred to as "pLVX vector", as shown in Figure 7), the expression plasmid of hPD-1-CAR-A was obtained (hereinafter referred to as "pLVX-CAR-A plasmid"), and the obtained pLVX-CAR-A plasmid was transformed into Stbl3 strain (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

The complete nucleic acid sequence of PD-1-CD28-4-1BB-CD3ζ (ie, B in Table 5) was synthesized (Jin Weizhi Biotechnology Co., Ltd.), and the sequence was the extramembranous region of human PD-1 gene, human origin. The transmembrane and intracellular regions of the CD28 gene, the intracellular region of human 4-1BB, and the intracellular region of the human CD3 gene ζ chain. The kozak sequence (GCCACC) was introduced at the 5' end, and the EcoRI and MluI restriction sites were introduced at the 5' and 3' ends, respectively, to form the human PD-1-CD28-4-1BB- with the restriction site. The full sequence of the CD3 ζ nucleic acid was cloned into the pLVX-EF1a-IRES-mCherry lentiviral expression vector, and the expression plasmid of hPD-1-CAR-B (hereinafter referred to as "pLVX-CAR-B plasmid") was obtained, and the obtained pLVX was obtained. The -CAR-B plasmid was transformed into the Stbl3 strain (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

PD1 upstream primer (CGGAATTCGCCACCATGCAGATCCCACAGGCG, SEQ ID No: 24) and 3' downstream primer (GCCACTGTTACTAGCAAGCTAT, SEQ ID No: 25) with EcoR I restriction site at the 5' end, and pLVX-CAR-B expression plasmid as template , PCR amplification, cut into pieces to recover about 600bp. The 5'-end upstream primer (ATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAA, SEQ ID No:26) and the 3'-end downstream primer (CGACGCGTTTAGCGAGGGGGCAGGGCCTGC, SEQ ID No:27) were used, and the pLVX-CAR-B expression plasmid was used as a template for PCR amplification and gel extraction. A fragment of about 500 bp. The two fragments recovered by the above-mentioned gelatin were used as templates, 100 ng each, without primers, and PCR amplification for 5 cycles. Adding 5' upstream primer (CGGAATTCGCCACCATGCAGATCCCACAGGCG, SEQ ID No: 24) and 3' downstream primer (CGACGCGTTTAGCGAGGGGGCAGGGCCTGC, SEQ ID No: 27) to the obtained PCR product, and adding 1 ul of KOD-PLUS enzyme for TOUCHDOWN PCR amplification. The PCR product was detected by electrophoresis, and the gel was recovered to a band of about 1100 bp. The cut-and-recovered band and the pLVX-EF1a-IRES-mCherry lentiviral expression vector were digested with EcoRI and MluI, and then the digested products were ligated to obtain the hPD-1-CAR-C expression plasmid (hereinafter referred to as " pLVX-CAR-C plasmid"), and the obtained pLVX-CAR-C plasmid was transformed into Stbl3 strain (purchased from Beijing Huayueyang) Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

The Touchdown PCR reaction conditions are as follows:

1. Pre-denaturation: 94 ° C 5 min

2. 3 cycles:

Denaturation: 98 ° C 15s

Annealing: 67 ° C 30 s

Extension: 68 ° C 2 min

3. 3 cycles

Denaturation: 98 ° C 15s

Annealing: 65 ° C 30 s

Extension: 68 ° C 2 min

4. 3 cycles

Denaturation: 98 ° C 15s

Annealing: 63 ° C 30 s

Extension: 68 ° C 2 min

5. 3 cycles

Denaturation: 98 ° C 15s

Annealing: 60 ° C 30 s

Extension: 68 ° C 2 min

6. 3 cycles

Denaturation: 98 ° C 15s

Annealing: 57 ° C 30 s

Extension: 68 ° C 2 min

7. 3 cycles

Denaturation: 98 ° C 15s

Annealing: 55 ° C 30 s

Extension: 68 ° C 2 min

8. Final extension:

68°C 10min

10°C Forever

The PD1 upstream primer (CGGAATTCGCCACCATGCAGATCCCACAGGCG, SEQ ID No: 24) and the 3'-end PD1 downstream primer (GTGGGCGCCGGTGTTGGTGGTCGCGGCGCTGGCGTCGTGGTGTGGGCTGTGGGCACTTC, SEQ ID No: 28) with the EcoR I restriction site at the 5' end, and the pLVX-CAR-A plasmid as a template , PCR amplification, gel recovery of about 500bp fragment 1. The 5' upstream primer (CATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTGTCCAAGTCCCCTATTT, SEQ ID No: 29) and the 3' downstream primer (CGACGCGTTTAGCGAGGGGGCAGGGCCTGC, SEQ ID No: 27) were used for PCR amplification using the pLVX-CAR-A plasmid as a template to recover a fragment 2 of about 700 bp. Fragment 1 was subjected to EcoR I single digestion, and fragment 2 was subjected to MluI single digestion, followed by column recovery of fragment 1 and fragment 2, respectively. The pLVX-EF1a-IRES-mCherry vector was double digested with EcoR I and MluI, and the digested vector was recovered. Subsequently, the digested vector was ligated with the digested fragments 1 and 2 recovered from the column to obtain an expression plasmid of hPD-1-CAR-D (hereinafter referred to as "pLVX-CAR-D plasmid"), and The obtained pLVX-CAR-D plasmid was transformed into Stbl3 strain (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

The extracellular sequence of the PD-1 mutant of the sequence E in Table 5 was artificially synthesized, 1-465 bp (Jin Weizhi Biotechnology Co., Ltd. artificial synthesis), and the PD1 upstream primer with the EcoR I restriction site at the 5' end (CGGAATTCGCCACCATGCAGATCCCACAGGCG, SEQ) ID No: 24) and the 3'-end downstream primer (GATCTTGGGGGCCAGGGAGATC, SEQ ID No: 30) were subjected to PCR amplification using the synthesized mutant sequence as a template to obtain a fragment 1 having a size of about 400 bp. Using the pLVX-CAR-B plasmid as a template, the 5'-end upstream primer (CTGGCCCCCAAGATCCAGATCAAAGAGAGCCTG, SEQ ID No: 31) and the 3' end The primer (CGACGCGTTTAGCGAGGGGGCAGGGCCTGC, SEQ ID No: 27) was subjected to PCR amplification to obtain a fragment 2 of about 700 bp in size. The two fragments recovered by the gelatinization were used as templates, 100 ng each, without primers, and PCR amplification for 5 cycles. Add 5' upstream primer (CGGAATTCGCCACCATGCAGATCCCACAGGCG, SEQ ID No: 24) and 3' downstream primer (CGACGCGTTTAGCGAGGGGGCAGGGCCTGC, SEQ ID No: 27) to the obtained PCR product, and add 1 ul of KOD-PLUS enzyme for TOUCHDOWN PCR amplification. The PCR product was detected by electrophoresis, and the gel was recovered to a band of about 1100 bp. The recovered band and the pLVX-EF1a-IRES-mCherry lentiviral expression vector were digested with EcoR I and MluI, and then the digested products were ligated to obtain an expression plasmid of hPD-1-CAR-E (hereinafter referred to as "pLVX". -CAR-E plasmid"), and the obtained pLVX-CAR-E plasmid was transformed into Stbl3 strain (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

The sequence F in Table 1 is a C93A amino acid mutation based on E, so the construction of the pLVX-CAR-F plasmid was carried out on the basis of the correctly sequenced pLVX-CAR-E plasmid. First, a 5'-end upstream primer (CAGCCCGGCCAGGACGCCCGCTTCCGTGTCACA, SEQ ID No: 32) and a 3'-end downstream primer (TGTGACACGGAAGCGGGCGTCCTGGCCGGGCTG, SEQ ID No: 33) were synthesized, and PCR amplification was carried out using the pLVX-CAR-E plasmid as a template to obtain about 10 kbp. The product was digested with DPNI enzyme (purchased from NEB). The product after digestion of the template was transformed into Stbl3 strain (purchased from Beijing Huayueyang Biotechnology Co., Ltd.), and the sequence mutation was confirmed to be correct by sequencing. The plasmid with the correct sequencing (Tiangen Small Plasmid Kit, purchased from Beijing Kainabo Technology Co., Ltd.) was extracted in small amounts, and the mini-extracted plasmid and pLVX-EF1a- were double-digested with EcoR I and Mlu I. IRES-mCherry lentiviral expression vector. The obtained digested product was ligated to obtain an expression plasmid of hPD-1-CAR-F (hereinafter referred to as "pLVX-CAR-F plasmid"), and the obtained pLVX-CAR-F plasmid was transformed into Stbl3 strain (purchased From Beijing Huayueyang Biotechnology Co., Ltd.). The sequence was confirmed to be correct by sequencing.

Through the above steps, the expression plasmids pLVX-CAR-A, pLVX-CAR-B, pLVX-CAR-C, pLVX-CAR-D, pLVX-CAR-E of the human PD-1 chimeric antigen receptor of the present invention are obtained. , pLVX-CAR-F (hereinafter referred to as "pLVX-CARs plasmid")

Example 11. Large extraction of the desired plasmid and packaging plasmid

The pLVX-CARs plasmid constructed in Example 10, the pLVX vector plasmid, and the psPAX2 packaging plasmid (purchased from Wuhan Qiling Biotechnology Co., Ltd.) and the pMD2.G packaging plasmid (purchased from Wuhan Qiling Biotechnology Co., Ltd.) will be respectively included. The strain was cultured in large amount in LB medium, and pLVX-CARs plasmid, pLVX vector plasmid, psPAX2 packaging plasmid and pMD2.G packaging plasmid (using endotoxin-free plasmid) were extracted by alkaline lysis method. Purchased from Beijing Tiangen Biochemical Technology Co., Ltd.). The obtained plasmid was used for cell transfection.

Example 12. Packaging, Concentration and Titer Determination of Lentivirus

Lentiviral packaging

(1) Cell treatment, same as the cell treatment method in Example 4: 24 hours before transfection, trypsinization and collection of 4th to 12th generation Lenti-X 293T cells in logarithmic growth phase (purchased from Clontech) ). The collected Lenti-X 293T cells were seeded at a density of 6×10 ⁶ /dish in a 10 cm cell culture dish and cultured in a 10% FBS (purchased from Hyclone), 1% sodium pyruvate, 1% glutamine Amide, 1% penicillin and 1% streptomycin (all percentages above are volume percent) in DMEM medium (purchased from Gibco). The culture conditions were 37 ° C, 5% CO ₂ incubator. The cells were cultured for 24 hours, and transfection was performed when the confluence rate reached 70-90%. Prior to cell transfection, the medium was changed to 10 ml of fresh virus packaging medium Opti-MEM (available from Gibco) containing 5% FBS, 0.2% sodium pyruvate and 1% glutamine.

(2) Preparation of transfection system

1) Dilute the plasmid with 1.5 ml Opti-MEM:

psPAX2 5μg

pMD2.G 5μg

pLVX-CARs plasmid or pLVX vector plasmid 10μg

2) 60 μl of liposome transfection reagent lipofectamine 3000 (purchased from Thermo Fisher) was diluted with 1.5 ml of Opti-MEM and vortexed and mixed.

5) The culture supernatants of Lenti-X 293T cells collected at 48 hours and 72 hours after the liquid exchange were placed in a 50 ml centrifuge tube, centrifuged at 2000 rpm for 5 minutes at room temperature, and the supernatant was collected.

6) The supernatant was filtered through a 0.45 μm filter to remove cell debris, and a pLVX lentiviral stock solution and a pLVX-CARs lentivirus stock solution were obtained, respectively.

Virus concentration (the same method as in Example 4)

The lentiviral stock solution and the concentrate (purchased from Clontech) were mixed at a ratio of 1:3, and the resulting mixture was precipitated at 4 ° C overnight. The mixture obtained by overnight precipitation was centrifuged at 1500 g for 45 minutes. After the supernatant was removed, the precipitate was resuspended in a stock solution volume of X-VIVO 15 (purchased from Lonza) to obtain a concentrated lentivirus. The concentrated lentivirus was divided into 100 ul each and stored in a -80 ° C refrigerator.

Virus titer determination

Lenti-X 293T cells were infected with the concentrated lentivirus, and 48 hours after infection, infected Lenti-X293T cells were collected. Lenti-X 293T cells infected with pLVX lentivirus and Lenti-X 293T cells infected with pLVX-CARs lentivirus were immunofluorescently labeled with monoclonal antibody to PD-1 (purchased from BD), followed by flow cytometry The instrument detects the ratio of cells expressing the chimeric antigen receptor. Calculate the titer of the concentrated pLVX lentivirus (refer to the lipofectamine 3000 transfection reagent lentiviral titer assay) and pLVX-CARs lentivirus. As shown in Table 6, TU is the transducing unit, TU /ml refers to the number of biologically active virus particles contained per milliliter. The titer assay showed that the plasmid-packaged lentivirus could be used for further experiments.

Table 6 Titer results of six pLVX-CARs lentiviruses

质粒名称Plasmid name	滴度(TU/ml)Titer (TU/ml)
pLVX pLVX	1×10⁸ 1×10 ⁸
pLVX-CAR-ApLVX-CAR-A	1.4×10⁸ 1.4×10 ⁸
pLVX-CAR-BpLVX-CAR-B	2.5×10⁸ 2.5×10 ⁸
pLVX-CAR-CpLVX-CAR-C	0.5×10⁸ 0.5×10 ⁸
pLVX-CAR-DpLVX-CAR-D	0.7×10⁸ 0.7×10 ⁸
pLVX-CAR-EpLVX-CAR-E	2.92×10⁷ 2.92×10 ⁷
pLVX-CAR-FpLVX-CAR-F	2.53×10⁷ 2.53×10 ⁷

Example 13. In vitro culture, infection and expansion of T cells

(2) A new 24-well plate was taken and previously coated with human fibronectin (Retronectin, available from TaKaRa). UBMC cells cultured in the step (1) for 48 hours were added to the coated 24-well plate at a cell volume of 1 × 10 ⁶ /well. Subsequently, 100 μl of concentrated pLVX lentivirus or concentrated pLVX-CARs lentivirus, and polybrene (purchased from Sigma) were added to each well to a final concentration of 8 μg/ml, and a final concentration of 40 IU/ml rhIL. -2, to a total volume of 0.8 ml, mix. The 24-well plate was centrifuged at 1500 g for 90 minutes at 32 ° C, then placed in a 37 ° C, 5% CO ₂ incubator, and 1 ml of medium was added overnight.

(3) The UBMC cells were infected a second time after the lentivirus infected the UBMC cells for 24 hours. The first infected UBMC cells were collected, centrifuged at 1200 rpm for 10 minutes, and the supernatant was carefully aspirated. The first infected UBMC cells were resuspended by adding fresh X-VIVO 15 medium containing 10% FBS, and the cell concentration was adjusted to 2 × 10 ⁶ /ml. The obtained UBMC cells were seeded at 0.5 ml/well into a new 24-well plate previously coated with Retronectin, followed by addition of lentivirus concentrate, polybrene and rhIL-2 in the same manner as in step (2). The 24-well plate was centrifuged at 1500 g for 90 minutes at 32 ° C, and then placed in an incubator to continue the culture.

(4) After the second infected UBMC cells were cultured for 24 hours, the cells were resuspended and centrifuged at 1200 rpm for 10 minutes, and then inoculated into a T75 flask at a density of 0.3 × 10 ⁶ /ml, and 300 IU was added. /ml of rhIL-2 to expand the cells, and then change every 2-3 days, the cell growth density was adjusted to 0.3 × 10 ⁶ /ml, the amount of rhIL-2 was the same as before.

(5) Collect the second infected UBMC cells (72h after infection), centrifuge at 1000rpm for 10 minutes, wash once with PBS buffer, UBMC cells after pLVX lentivirus infection and UBMC cells after pLVX-CARs lentivirus infection. Immunofluorescent labeling was performed using a monoclonal antibody of PD-1 (purchased from BD). The labeled cells were centrifuged at 1000 rpm for 5 min and resuspended in an appropriate amount of PBS. The ratio of cells expressing the chimeric antigen receptor was detected by flow cytometry.

The results of flow cytometry showed that the infection rates of pLVX-CARs lentivirus were:

UBMC cells (hereinafter referred to as "hPD-1-CAR-A T cells") after pLVX-CAR-A lentivirus infection were immunofluorescently labeled with a monoclonal antibody of PD-1 (purchased from BD), and the results showed that The infection rate was 74.01%, and the MOI (multiplicity of infection, the number of viruses infected per cell) was 12.6.

UBMC cells (hereinafter referred to as "hPD-1-CAR-B T cells") after pLVX-CAR-B lentivirus infection were immunofluorescently labeled with a monoclonal antibody of PD-1 (purchased from BD), and the results showed that The infection rate was 81.84% and the MOI was 47.5.

UBMC cells (hereinafter referred to as "hPD-1-CAR-C T cells") after pLVX-CAR-C lentivirus infection were immunofluorescently labeled with a monoclonal antibody of PD-1 (purchased from BD), and the results showed that The infection rate was 83.04% and the MOI was 0.72.

UBMC cells (hereinafter referred to as "hPD-1-CAR-D T cells") after pLVX-CAR-D lentivirus infection were immunofluorescently labeled with a monoclonal antibody of PD-1 (purchased from BD), and the results showed that The infection rate was 56.10% and the MOI was 13.3.

UBMC cells (hereinafter referred to as "hPD-1-CAR-E T cells") after pLVX-CAR-E lentivirus infection were immunofluorescently labeled with a monoclonal antibody of PD-1 (purchased from BD), and the results showed that The infection rate was 8.73% and the MOI was 4.38.

UBMC cells (hereinafter referred to as "hPD-1-CAR-F T cells") after pLVX-CAR-F lentivirus infection were immunofluorescently labeled with a monoclonal antibody of PD-1 (purchased from BD), and the results showed that The infection rate was 9.06% and the MOI was 3.8.

The control T cells are UBMC cells (hereinafter referred to as "control T cells") after pLVX lentivirus infection. Immunofluorescence labeling with a monoclonal antibody of PD-1 (purchased from BD) revealed that the control T cells did not express the chimeric antigen receptor.

Example 14. In vitro co-culture of T cells and target cells, and determination of cytokine release

Construction of target cell lines

In ^{K562 (ATCC-CCL-243 TM} ) cell lines were constructed based on K562- luciferase (luciferase GenBank ID: EU581860.1), K562-PD-L1- luciferase (hPD-L1 GenBank ID: NM_014143 .3), K562-PD-L2-luciferase (hPD-L2 GenBank ID: NM_025239.3) target cell line. The construction method is that the K562 cell line is infected with pLVX-EF1α-luciferase, pLVX-EF1α-PD-L1-luciferase, and K562-PD-L2-luciferase, respectively, to obtain stably expressing cells. (Methods are the same as in Example 4 and Example 5).

A Raji-PD-L1-luciferase cell line was constructed. On Raji cells (ATCC-CCL-86 ^TM) infection pLVX-EF1α-PD-L1- luciferase lentivirus stably expressing PD-L1 of Raji-PD-L1- luciferase cell lines. On Raji cells (ATCC-CCL-86 ^TM) infection pLVX-EF1α-PD-L2- luciferase lentivirus stably expressing PD-L2 to give the Raji-PD-L2- luciferase cell lines.

The target cell K562-luciferase cell line does not express PD-L1 or PD-L2 on the surface, so hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR -C T cells, hPD-1-CAR-D T cells, hPD-1-CAR-E T cells, hPD-1-CAR-F T cells, hPD-1-CAR-G T cells, hPD-1-CAR -I T cells There is no specific killing effect on the K562-luciferase cell line. The K562-PD-L1-luciferase cell line expresses only PD-L1 but not PD-L2, so CAR T cells containing the domain that binds to PD-L1 are K562-PD-L1-luciferase cells. It has a specific killing effect. The K562-PD-L2-luciferase cell line does not express PD-L1 on the surface and expresses PD-L2, so CAR T cells containing the domain that binds to PD-L2 are K562-PD-L2-luciferase cells. It has a specific killing effect. The Raji-PD-L1-luciferase, Raji-PD-L2-luciferase target cell lines were consistent with K562-PD-L1-luciferase and K562-PD-L2-luciferase cells.

Pre-recovery of the established target cell lines: K562-luciferase, K562-PD-L1-luciferase, K562-PD-L2-luciferase, Raji-PD-L1-luciferase, Raji-PD-L2-luciferase, cultured for use.

Co-culture of T cells with target cells in vitro

(1) The T cells and target cells obtained in Example 13 were separately mixed, and a small number of cells were counted, resuspended in fresh X-VIVO 15 medium, and counted.

(2) Set the ratio of target cells to T cells to 1:1, and the total culture system was 200 ul/well. A 96-well V-well plate was plated with T cells and target cells in an amount of 1 × 10 ⁴ /100 μl/well, respectively. The cells were incubated overnight at 37 ° C, 5% CO ₂ .

(3) The culture supernatant was collected 24 hours later, and the obtained supernatant was subjected to ELISA (ELISA kit, purchased from Beijing Dakco as Biotechnology Co., Ltd.).

ELISA for detection of cytokine IL-2

Table 7 Determination of concentration of cytokine IL-2 co-cultured with K562 series target cells (pg/ml)

	无靶细胞Targetless cell	K562K562	K562-PD-L1K562-PD-L1	K562-PD-L2K562-PD-L2
对照T细胞+靶细胞Control T cell + target cell	39.8039.80	44.6644.66	44.6644.66	44.6644.66
hPD1-CAR-A T细胞+靶细胞hPD1-CAR-A T cell + target cell	41.0141.01	64.9164.91	1120.911120.91	3703.073703.07
hPD1-CAR-B T细胞+靶细胞hPD1-CAR-B T cell + target cell	41.0141.01	73.0973.09	1175.271175.27	3962.283962.28
hPD1-CAR-C T细胞+靶细胞hPD1-CAR-C T cell + target cell	41.0141.01	70.5970.59	1475.241475.24	4595.874,595.87
hPD1-CAR-D T细胞+靶细胞hPD1-CAR-D T cell + target cell	41.0141.01	45.2745.27	1228.541228.54	6229.176229.17
hPD1-CAR-E T细胞+靶细胞hPD1-CAR-E T cell + target cell	41.0141.01	37.4037.40	1487.651487.65	39.8839.88
hPD1-CAR-F T细胞+靶细胞hPD1-CAR-F T cell + target cell	41.0141.01	47.8447.84	1199.531199.53	48.9948.99
无T细胞+靶细胞No T cells + target cells	N/AN/A	44.0544.05	44.0544.05	44.8644.86

As shown in Table 7 and Figure 8, the results showed that the concentration of IL-2 released by hPD-1-CARs T cells co-cultured with K562 series target cells was significantly different from that of control T cells or target cells. Specifically, hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR-C T cells, hPD-1-CAR-D T cells to target cells K562-PD-L1 Or the concentration of cytokine IL-2 after K562-PD-L2 co-culture was significantly higher than the background value of control T cells or target cells. This indicates that hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR-C T cells, hPD-1-CAR-D T cells successfully expressed the corresponding PD-1 The chimeric antigen receptors can specifically recognize and bind to the PD-L1 and PD-L2 antigens on the target cells, respectively, and activate the downstream signaling pathway of the CAR to promote the release of the cytokine IL-2. In addition, the data indicated that hPD-1-CAR-E T cells and hPD-1-CAR-F T cells successfully expressed chimeric antigen receptors containing the corresponding PD-1, but only specifically recognized and bound to target cells. The PD-L1 antigen has no effect on the PD-L2 antigen.

Table 8 Determination of concentration of cytokine IL-2 co-cultured with Raji series target cells (pg/ml)

	无靶细胞Targetless cell	RajiRaji	Raji-PD-L1Raji-PD-L1	Raji-PD-L2Raji-PD-L2
对照T细胞+靶细胞Control T cell + target cell	63.2963.29	67.8267.82	60.7360.73	62.0162.01
hPD1-CAR-A T细胞+靶细胞hPD1-CAR-A T cell + target cell	55.0755.07	60.1060.10	8365.388365.38	7210.687210.68
hPD1-CAR-D T细胞+靶细胞hPD1-CAR-D T cell + target cell	62.6562.65	65.8765.87	1546.891546.89	1257.211257.21
hPD1-CAR-E T细胞+靶细胞hPD1-CAR-E T cell + target cell	62.6562.65	238.62238.62	13136.9213136.92	103.69103.69
无T细胞+靶细胞No T cells + target cells	N/AN/A	59.4659.46	56.3256.32	59.4659.46

As shown in Table 8 and Figure 9, the results showed that the concentration of IL-2 released by hPD-1-CARs T cells co-cultured with Raji series target cells was significantly different from that of control T cells or target cells. Specifically, hPD-1-CAR-A T cells and hPD-1-CAR-D T cells were significantly higher in concentration of cytokine IL-2 after co-culture of target cells Raji-PD-L1 or Raji-PD-L2. The background value of the control T cell or target cell. This indicates that hPD-1-CAR-A T cells and hPD-1-CAR-D T cells successfully expressed chimeric antigen receptors containing the corresponding PD-1, and thus each specifically recognizes and binds to target cells. PD-L1 and PD-L2 antigens cause activation of the downstream signaling pathway of CAR and promote the release of cytokine IL-2. In addition, the data indicate that hPD-1-CAR-E T cells successfully express the chimeric antigen receptor containing the corresponding PD-1, but can only specifically recognize and bind the PD-L1 antigen on the target cell to PD-L2. The antigen has no effect.

ELISA for detection of cytokine IFN-γ

Table 9 Concentration determination of cytokine IFN-γ co-cultured with K562 series target cells (pg/ml)

	无靶细胞Targetless cell	K562K562	K562-PD-L1K562-PD-L1	K562-PD-L2K562-PD-L2
对照T细胞+靶细胞Control T cell + target cell	22.8122.81	29.7229.72	30.0830.08	36.2336.23
hPD1-CAR-A T细胞+靶细胞hPD1-CAR-A T cell + target cell	21.4521.45	33.5733.57	727.57727.57	212.73212.73
hPD1-CAR-B T细胞+靶细胞hPD1-CAR-B T cell + target cell	20.1020.10	31.5431.54	501.94501.94	203.09203.09
hPD1-CAR-C T细胞+靶细胞hPD1-CAR-C T cell + target cell	27.6327.63	32.5532.55	596.81596.81	216.94216.94
hPD1-CAR-D T细胞+靶细胞hPD1-CAR-D T cell + target cell	22.1322.13	30.7830.78	466.33466.33	463.36463.36
无T细胞+靶细胞No T cells + target cells	N/AN/A	30.0830.08	29.0329.03	35.5935.59

As shown in Table 9 and Figure 10, the results showed that the concentration of IFN-γ released by hPD-1-CARs T cells co-cultured with K562 series target cells was significantly different from that of control T cells or target cells. Specifically, hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR-C T cells, hPD-1-CAR-D T cells to target cells K562-PD-L1 Or the concentration of cytokine IFN-γ after K562-PD-L2 co-culture was significantly higher than the background value of control T cells or target cells. This indicates that hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR-C T cells, hPD-1-CAR-D T cells successfully expressed the corresponding PD-1 The chimeric antigen receptors can specifically recognize and bind to the PD-L1 and PD-L2 antigens on the target cells, respectively, and activate the downstream signaling pathway of the CAR to promote the release of the cytokine IFN-γ.

Table 10 Concentration determination of cytokine IFN-γ co-cultured with Raji series target cells (pg/ml)

	无靶细胞Targetless cell	RajiRaji	Raji-PD-L1Raji-PD-L1	Raji-PD-L2Raji-PD-L2
对照T细胞+靶细胞Control T cell + target cell	24.8324.83	24.6924.69	24.3224.32	25.5125.51
hPD1-CAR-A T细胞+靶细胞hPD1-CAR-A T cell + target cell	36.3436.34	35.0135.01	2662.602662.60	2765.952765.95
hPD1-CAR-D T细胞+靶细胞hPD1-CAR-D T cell + target cell	23.9623.96	24.1024.10	347.96347.96	290.32290.32
hPD1-CAR-E T细胞+靶细胞hPD1-CAR-E T cell + target cell	25.9625.96	471.36471.36	3331.353,331.35	368.24368.24
无T细胞+靶细胞No T cells + target cells	N/AN/A	23.1823.18	23.7523.75	24.3224.32

As shown in Table 10 and Figure 11, the results showed that the concentration of IFN-γ released by co-culture of hPD-1-CARs T cells and Raji series target cells was significantly different from that of control T cells or target cells. Specifically, hPD-1-CAR-A T cells, hPD-1-CAR-D T cells co-cultured with target cells Raji-PD-L1 or Raji-PD-L2 The concentration of IFN-γ was significantly higher than that of the control T cells or target cells. This indicates that hPD-1-CAR-A T cells and hPD-1-CAR-D T cells successfully expressed chimeric antigen receptors containing the corresponding PD-1, and thus each specifically recognizes and binds to target cells. PD-L1 and PD-L2 antigens cause activation of the downstream signaling pathway of CAR and promote the release of cytokine IFN-γ. In addition, the data indicate that hPD-1-CAR-E T cells successfully express the chimeric antigen receptor containing the corresponding PD-1, but can only specifically recognize and bind the PD-L1 antigen on the target cell to PD-L2. The antigen has no effect.

In summary, the six PD-1 chimeric antigen receptors differ in function, hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR-C T cells. hPD-1-CAR-D T cells can specifically recognize and bind PD-L1 and PD-L2 antigens on target cells, respectively, and promote the release of cytokines IL-2 and IFN-γ. hPD-1-CAR-E T cells and hPD-1-CAR-F T cells can specifically recognize and bind to PD-L1 antigen on target cells, and promote the release of cytokines IL-2IL-2 and IFN-γ.

Example 15. Killing effect of hPD-1-CARs T cells on target cells

Co-culture of T cells with target cells in vitro

(1) The hPD-1-CARs T cells were separately mixed with the target cells, and a small number of cells were counted.

(2) Co-culture of T cells and target cells in 96-well plates in a ratio of 1×10 ⁴ /50 μl/well, T cells: target cells = 1:1, 5:1, 10:1. The volume of the culture solution was 100 ul.

(3) The 96-well plate was placed in an incubator for 6 hours, after which the 96-well plate was taken out from the incubator, and 100 ul of room temperature-balanced steady-Glo luciferase substrate (purchased from Promega) was added to each well. The 96-well plate was gently lysed by shaking on a shaker for 15 minutes, and then the fluorescence value was measured using a Victor X Light chemiluminescence detector (available from PerkinElmer Co., Ltd.).

Table 11 Killing rate of target cells by hPD-1-CARs T cells (%)

As shown in Table 11 and Figures 12 and 13, only low levels of non-specific killing were observed after co-culture of control T cells and target cells. hPD-1-CAR-A T cells, hPD-1-CAR-B T cells, hPD-1-CAR-C T cells, hPD-1-CAR-D T cells, and four hPD-1-CARs T cells After the target cells were co-cultured, a significantly enhanced killing effect was observed, and as the dose of T cells increased, the killing efficiency of the target cells increased sharply. When the target ratio was 10:1, the highest killing efficiency was close to 70. %, which demonstrates that the four hPD-1-CARs T cells have a dose-dependent effect on the killing of target cells, and also demonstrates a specific killing effect on target cells containing the PD-L1 and PD-L2 antigens, respectively. In addition, the data also showed that hPD-1-CAR-E T cells and hPD-1-CAR-F T cells have stronger specific killing effect on target cells containing PD-L1 antigen, and the highest killing efficiency is close to 100%, but No specific killing of target cells containing the PD-L2 antigen.

Claims

A chimeric antigen receptor comprising:

(i) having a domain that binds to the ligand PD-L1 or PD-L2;

(ii) a hinge area;

(iii) a transmembrane domain;

(iv) a costimulatory signaling domain;

(v) CD3ζ signaling domain.
The chimeric antigen receptor of claim 1, wherein said domain (i) is a domain that binds to a ligand in a PD-1 molecule, or an scFv of an antibody of PD-L1 or PD-L2.
The chimeric antigen receptor of claim 1, wherein said domain (i) is a domain having binding to a ligand PD-L1.
The chimeric antigen receptor of claim 2, wherein the antibody is an antibody of human or animal origin.
The chimeric antigen receptor according to claim 2, wherein the domain (i) on the PD-1 molecule has the sequence shown in SEQ ID No: 1, or has at least the sequence shown in SEQ ID No: 1. 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical protein.
The chimeric antigen receptor according to claim 5, wherein the domain (i) on the PD-1 molecule has the sequence of SEQ ID No: 2 or 3.
A chimeric antigen receptor according to any of the preceding claims, wherein said hinge region comprises at least one of: a hinge region of CTLA4, a hinge region of CD28, a hinge region of CD7, a hinge region of IgG1, a hinge of IgG4 The region, the hinge region of IgD, the hinge region of CD8α, or the hinge region of PD-1.
The chimeric antigen receptor of claim 7, wherein said hinge region comprises the hinge region of PD-1 SEQ ID No: 4.
The chimeric antigen receptor of claim 7, wherein said hinge region comprises a hinge region of PD-1 and a hinge region of CD8α, having the sequence of SEQ ID No: 5.
The chimeric antigen receptor of claim 7, wherein said hinge region has the sequence of SEQ ID No: 6.
A chimeric antigen receptor according to any of the preceding claims, wherein said transmembrane domain comprises at least one of: a transmembrane domain of CD4, a transmembrane domain of CD7, a transmembrane domain of CD8[alpha], a transmembrane domain of CD28 Domain, transmembrane domain of CD134, transmembrane domain of CD137, transmembrane domain of FcεRIγ or transmembrane domain of H2-Kb.
The chimeric antigen receptor of claim 11, wherein said transmembrane domain comprises a transmembrane domain of CD28 SEQ ID No: 7.
A chimeric antigen receptor according to any of the preceding claims, wherein said costimulatory signaling domain comprises at least one of the following: CD28, 4-1BB, ICOS, OX40, CD244, FcεRIγ, CD8α, BTLA, CD27 Co-stimulatory signaling regions in CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, and DAP12.
The chimeric antigen receptor of claim 13, wherein said costimulatory signaling domain comprises a co-stimulatory signaling domain of CD28 SEQ ID No: 8.
The chimeric antigen receptor of claim 13, wherein said costimulatory signaling domain comprises a costimulatory signaling domain of 4-1BB SEQ ID No: 9.
The chimeric antigen receptor of claim 13, wherein said costimulatory signaling domain comprises a co-stimulatory signaling domain of CD28 and a costimulatory signaling domain of 4-1BB having SEQ ID No: 10 The sequence shown.
A chimeric antigen receptor according to any of the preceding claims, wherein said CD3ζ signaling domain has the sequence set forth in SEQ ID No: 11 or at least 95% identical to the sequence set forth in SEQ ID No: , 96%, 97%, 98%, 99% or 100% identity.
The chimeric antigen receptor of claim 1, which has the sequence set forth in SEQ ID No: 12 or at least 90%, 95%, 96%, 97% of the sequence set forth in SEQ ID No: 12. , 98%, 99% or 100% identical protein.
The chimeric antigen receptor of claim 1, which has the sequence set forth in SEQ ID No: 13 or at least 90%, 95%, 96%, 97% of the sequence set forth in SEQ ID No: 13. , 98%, 99% or 100% identical protein.
The chimeric antigen receptor of claim 1, which has the sequence set forth in SEQ ID No: 14, or at least 90%, 95%, 96%, 97% of the sequence set forth in SEQ ID No: 14. , 98%, 99% or 100% identical protein.
The chimeric antigen receptor of claim 1, which has the sequence of SEQ ID No: 15 or at least 90%, 95%, 96%, 97% of the sequence set forth in SEQ ID No: 15. , 98%, 99% or 100% identical protein.
The chimeric antigen receptor of claim 1, which has the sequence of SEQ ID No: 16 or at least 90%, 95%, 96%, 97% of the sequence set forth in SEQ ID No: , 98%, 99% or 100% identical protein.
The chimeric antigen receptor of claim 1, which has the sequence set forth in SEQ ID No: 17 or at least 90%, 95%, 96%, 97% of the sequence set forth in SEQ ID No: 17. , 98%, 99% or 100% identical protein.
A nucleic acid sequence encoding the chimeric antigen receptor of any of the preceding claims 1-23.
The nucleic acid sequence of claim 24, which encodes the chimeric antigen receptor of any one of claims 18-23.
The nucleic acid sequence of claim 25 having the sequence set forth in any one of SEQ ID Nos: 18-23.
A vector comprising the nucleic acid sequence set forth in any one of claims 24-26.
A T cell expressing the chimeric antigen receptor of any one of claims 1-23.
A method of producing the T cell of claim 28, which comprises the step of introducing the nucleic acid sequence of any one of claims 24-26 into a T cell.
A pharmaceutical composition comprising the vector of claim 27 or the T cell of claim 28 together with a pharmaceutically acceptable carrier, diluent or excipient.
A method of treating a disease associated with PD-L1 or PD-L2 expression comprising administering to an animal an effective amount of the T cell of claim 28.
Use of a T cell according to claim 28 for the preparation of a medicament for the treatment of a disease associated with PD-L1 or PD-L2 expression.
Use of a T cell of claim 28 for inhibiting PD-L1 or PD-L2 expression-associated cells.
The PD-L1 or PD-L2 expression-related cells of claim 33 include tumor cells, macrophages, dendritic cells, T cells, and intratumoral stromal cells.
The tumor cells of claim 34 include tumor cells of various human or animal blood system tumors and solid tumors.
The hematological tumors of claim 35 include leukemia, lymphoma, and myeloma.
The solid tumor of claim 35 includes lung cancer, gastric cancer, esophageal cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck tumor, glioma, and soft tissue sarcoma.