WO2023030393A1

WO2023030393A1 - Chimeric antigen receptor and use thereof

Info

Publication number: WO2023030393A1
Application number: PCT/CN2022/116237
Authority: WO
Inventors: 阎锡蕴; 段红霞; 景林
Original assignee: 中国科学院生物物理研究所
Priority date: 2021-09-01
Filing date: 2022-08-31
Publication date: 2023-03-09
Also published as: CN117580863A

Abstract

Provided are a chimeric antigen receptor (CAR) and a use thereof. The CAR comprises a first co-stimulatory domain capable of binding to and activating lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation. Also provided are a nucleic acid sequence encoding the CAR, a nucleic acid construct comprising the nucleic acid sequence encoding the CAR, a vector comprising the nucleic acid sequence or nucleic acid construct, a cell expressing the CAR, and a method for preparing the cell.

Description

Chimeric antigen receptors and uses thereof

This application claims priority to Chinese Patent Application 202111019962.8 filed on September 1, 2021, the entire contents of which are incorporated herein by reference for all purposes.

field of invention

The present invention relates to a chimeric antigen receptor (CAR) and its use, especially in the treatment of cancer.

Background technique

In recent years, adoptive T cell therapy has achieved great success in tumor treatment, especially chimeric receptor T (CAR-T) cell therapy targeting CD19 antigen has shown significant and durable tumor suppression in the treatment of hematological malignancies. As a result, it has become an effective treatment method for hematological tumors in clinical practice.

Despite some success, the widespread application of CAR-T cells in cancer therapy still faces major challenges, especially in the treatment of solid tumors where the effect is not significant. At present, many clinical trials of CAR-T therapy for solid tumors that have been carried out at home and abroad have not achieved satisfactory results. Studies have shown that CAR-T cells are affected by the microenvironment of solid tumors, resulting in reduced killing ability and insufficient persistence, which are the key reasons for their poor therapeutic effect in solid tumors. Therefore, how to further improve the tumor killing ability of CAR-T cells is one of the key issues to solve the inefficiency of CAR-T therapy for solid tumors.

The core of CAR-T cells to achieve their tumor killing function is the CAR molecule, which is designed by simulating the TCR receptor of natural T cells. However, many studies have shown that compared with TCR molecules, CAR molecules have a significantly lower ability to activate T cells, which is only about 1/10-1/1000 of TCR activation ability. Therefore, improving the ability of CAR molecules to activate T cells is a key way to improve the tumor killing ability of CAR-T cells.

The activation of T cells by CAR molecules mainly depends on the CD3ζ domain of its intracellular segment. The ITAM motif in the CD3ζ domain can be activated by phosphorylation, and the activated CD3ζ activates a series of activation-related signaling pathways by activating downstream ZAP70 and other molecules, thereby activating T cells. How to increase the phosphorylation level of the CD3ζ domain of the CAR molecule is the key to increasing the activation level of CAR-T cells. Studies have shown that lymphocyte-specific protein tyrosine kinase (LCK) of the SRC family is a key molecule for phosphorylating the ITAM motif of CD3ζ in CAR-T cells. Therefore, if we can try to recruit more activated forms of LCK kinase to the vicinity of CD3ζ, we can improve the activation efficiency of CD3ζ, and then increase the activation level of CAR-T cells.

Therefore, there is a need to develop CAR molecules that increase the activation level of CAR-T cells.

Summary of the invention

The inventors unexpectedly found that the intracellular domain (cytoplasmic region) of the CD146 molecule in T cells can directly interact with the LCK molecule, and can promote the activation of LCK by promoting the autophosphorylation of LCK. This direct interaction is at least partially dependent on the KKGK motif at the membrane-proximal end of the CD146 cytoplasmic domain. Therefore, through the modification of the intracellular domain of the CAR molecule, the CD146 cytoplasmic region or its near-membrane fragment was integrated into the intracellular domain of the CAR molecule, and its ability to bind and activate LCK was used to integrate more activated forms of LCK molecules are recruited to the intracellular domain of the CAR molecule, thereby better activating the CD3ζ domain of the intracellular domain of the CAR molecule, so as to increase the activation level of CAR-T cells, thereby improving the tumor killing ability of CAR-T cells. The CAR molecule modified by the present invention can improve the activation of CAR-T cells, enhance the tumor killing ability of CAR-T cells, and thus be applied to the immunotherapy of various cancers including solid tumors.

Accordingly, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, an intracellular signaling domain and an intracellular co-stimulatory domain, wherein the The intracellular co-stimulatory domain includes a first co-stimulatory domain capable of binding and activating lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation.

In some embodiments of the CAR of the invention, the first co-stimulatory domain may comprise a sequence derived from the cytoplasmic region of the CD146 molecule. In some embodiments, the first co-stimulatory domain may comprise a KKGK motif at the membrane-proximal end of the cytoplasmic region of CD146.

In some embodiments, the first co-stimulatory domain may comprise the cytoplasmic region of CD146, or a membrane-proximal fragment thereof. In some embodiments, the length of the membrane-proximal fragment of the CD146 cytoplasmic region may be 4-55 amino acids, preferably 10-45 amino acids, more preferably 15-35 amino acids, and even more preferably 15-26 amino acids . In some embodiments, the membrane proximal fragment of the CD146 cytoplasmic region comprises amino acids 584-609 of the CD146 molecule.

In some embodiments, the intracellular co-stimulatory domain may further comprise a second co-stimulatory domain selected from the group consisting of 4-1BB (CD137), OX40 (CD134), ICOS (CD278) , 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), MyD88, CD2, Intracellular signaling regions of CD4, CD7, LIGHT, NKG2C and B7-H3 or any combination thereof. In some embodiments, the second co-stimulatory domain is the intracellular signaling region of 41BB.

In some embodiments, the C-terminus of the first costimulatory domain is linked to the N-terminus of the second costimulatory domain, optionally via a linker.

In some embodiments, the first co-stimulatory domain is the CD146 cytoplasmic region, the second co-stimulatory domain is the intracellular signaling region of 41BB, and the C-terminus of the first co-stimulatory domain is connected to the second co-stimulatory domain. N-terminal linkage of two co-stimulatory domains.

In some embodiments, the intracellular signaling domain may comprise a signaling region selected from the group consisting of TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, ICOS (CD278), and CD66d or any combination thereof. In some embodiments, the intracellular signaling domain comprises the signaling region of CD3ζ.

In some embodiments, the order of the first co-stimulatory domain, the second co-stimulatory domain, and the intracellular signaling domain from the N-terminus to the C-terminus is: first co-stimulatory domain-second Two co-stimulatory domains - intracellular signaling domains.

In some embodiments, the transmembrane domain may comprise an alpha chain selected from T cell receptor (TCR), beta chain of TCR, zeta chain of TCR, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16 , CD19, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (41BB), CD152, CD154 and the transmembrane domain of PD1 or any combination thereof.

In some embodiments, the antigen binding domain can bind one or more tumor-associated antigens (TAAs). The TAA is preferably selected from 5T4, alpha-fetoprotein, BCMA, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, ROR1, GPC3, Tyrp-1, TACI, ALK, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, ERBB2, FLT3, melanoma-associated antigen, mesothelin, MUC-1, VEGFR2. More preferably, the antigen binding domain specifically binds CD19.

In some embodiments, the antigen binding domain may be selected from an antigen binding domain derived from an antibody against the antigen and an antigen binding domain derived from a natural ligand of the antigen. Preferably, said antibody-derived antigen binding domain is in the form of a scFv, Fab or domain antibody (dAb).

In some embodiments, the CAR may also comprise a hinge region. Preferably, the hinge region may comprise a hinge selected from CD8, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE and IgM or a fragment thereof.

In another aspect, the invention provides a nucleic acid sequence encoding a CAR of the invention.

In yet another aspect, the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding the CAR of the present invention.

In some embodiments of the nucleic acid construct of the present invention, the nucleic acid construct has the following structure:

BD-hinge-TM-Costi-Singal;

in:

BD is the nucleotide sequence encoding the antigen binding domain;

hinge is the nucleotide sequence encoding the hinge region;

TM is a nucleotide sequence encoding a transmembrane domain;

Costi is a nucleotide sequence encoding an intracellular co-stimulatory domain;

Signal is a nucleotide sequence encoding an intracellular signaling domain.

In some embodiments, the nucleic acid construct has the following structure:

BD-hinge-TM-Costi1-Costi2-Singal;

in:

BD is the nucleotide sequence encoding the antigen binding domain;

hinge is the nucleotide sequence encoding the hinge region;

TM is a nucleotide sequence encoding a transmembrane domain;

Costi1 is a nucleotide sequence encoding the first co-stimulatory domain;

Costi2 is a nucleotide sequence encoding a second co-stimulatory domain;

Signal is a nucleotide sequence encoding an intracellular signaling domain.

In another aspect, the invention provides a vector comprising a nucleic acid sequence or nucleic acid construct of the invention.

In some embodiments, the vector is selected from lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.

In another aspect, the invention provides cells expressing a CAR of the invention.

In some embodiments, the cells are immune cells, preferably T cells, NK cells or macrophages. In some embodiments, the cells are stem cells, preferably pluripotent stem cells, induced pluripotent stem cells (iPSC), mesenchymal stem cells, hematopoietic stem cells or lymphoid progenitor cells.

In yet another aspect, the present invention provides a method of preparing the cells of the present invention, comprising the step of transducing or transfecting the cells with the vector of the present invention.

In some embodiments, the method may further comprise the step of expanding and/or activating cells before or after said transducing or transfecting.

In another aspect, the present invention provides a composition comprising the CAR, nucleic acid sequence, nucleic acid construct, vector, or cell of the present invention, and a pharmaceutically acceptable carrier or excipient.

In some embodiments of the composition of the present invention, the composition may further comprise a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

In yet another aspect, the invention provides a method of treating cancer in a subject comprising administering to said subject an effective amount of a cell of the invention.

In embodiments of the methods of the invention, said cells are autologous or allogeneic to said subject.

In some embodiments, the method comprises the steps of: (i) isolating a sample containing cells from the subject; (ii) transducing or transfecting the cells with a vector of the invention; and (iii) converting The cells obtained in step (ii) are administered to a subject.

In some embodiments, the method can also include administering a second therapeutic agent. Preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.

In some embodiments, the cancer may be selected from lymphoma, multiple myeloma, leukemia, and solid tumors.

In another aspect, the present invention provides the use of the CAR, nucleic acid sequence, nucleic acid construct, vector, cell or composition of the present invention in the manufacture of a medicament for treating cancer in a subject.

In yet another aspect, the invention provides a CAR, nucleic acid sequence, nucleic acid construct, vector, cell or composition of the invention for use in treating cancer in a subject.

In some embodiments of the uses of the present invention, the cancer may be selected from lymphoma, multiple myeloma, leukemia and solid tumors.

Description of drawings

Figure 1 shows the interaction assay between CD146 and LCK. (A) Schematic representation of recombinant CD146 cytoplasmic domain protein. (B) Pull-down assay of recombinant CD146 cytoplasmic domain protein and LCK-His protein.

Figure 2 shows the identification of CD146 interaction sites with LCK. (A) Schematic representation of CD146 intracellular mutants. (B) Immunoprecipitation experiments using anti-CD146 antibody AA1 to detect the interaction of CD146 intracellular mutants and LCK in 293T cells. (C) Histogram showing quantification of LCK and CD146 ratios (n=3).

Figure 3 shows the expression of the CAR molecule of the present invention in T cells. (A) Structural schematic diagrams of the CAR molecule of the present invention (CD146cyt-4-1BB) and the control CAR molecule (4-1BB). (B) Expression of the CAR molecule of the present invention (CD146cyt-4-1BB) and the control CAR molecule (4-1BB) in T cells.

Figure 4 shows the activation of T cells expressing the CAR of the present invention measured by flow cytometry. (A) Activation of T cells after co-culture with target cells. (B) Activation of T cells after co-culture with CD19 antigen. "CTR" indicates T cells not transfected with CAR, "4-1BB" indicates T cells transfected with 41BB-CD3ζCAR, and "CD146cyt-4-1BB" indicates T cells transfected with CD146cyt-41BB-CD3ζCAR. T cells not transfected with CAR served as a negative control.

Figure 5 shows the cytokine secretion of T cells expressing the CAR of the present invention measured by flow cytometry. "4-1BB" indicates T cells transfected with 41BB-CD3ζCAR, and "CD146cyt-4-1BB" indicates T cells transfected with CD146cyt-41BB-CD3ζCAR.

Fig. 6 shows the tumor killing activity of T cells expressing the CAR of the present invention measured by lactate dehydrogenase (LDH) method. "CTR" indicates T cells not transfected with CAR, "4-1BB" indicates T cells transfected with 41BB-CD3ζCAR, and "CD146cyt-4-1BB" indicates T cells transfected with CD146cyt-41BB-CD3ζCAR. T cells not transfected with CAR served as a negative control.

Figure 7 shows the in vivo tumor suppressive effect of the CAR T cells of the present invention on a mouse model of hematological tumor observed by in vivo imaging. (A) When the effect-to-target ratio is 1:1, the CAR T cells of the present invention have an in vivo tumor-inhibiting effect on a mouse model of hematological tumor. (B) When the effect-to-target ratio is 1:2, the in vivo tumor-suppressive effect of the CAR T cells of the present invention on a mouse model of hematological tumor. "CTR" indicates the injection of untransfected human primary T cells, "4-1BB" indicates the injection of 41BB-CD3ζCAR T cells, and "CD146cyt-4-1BB" indicates the injection of CD146cyt-41BB-CD3ζCAR T cells.

Figure 8 shows the in vivo tumor suppressive effect of the CAR T cells of the present invention on a solid tumor mouse model observed by in vivo imaging. "CTR" indicates the injection of untransfected human primary T cells, "4-1BB" indicates the injection of 41BB-CD3ζCAR T cells, and "CD146cyt-4-1BB" indicates the injection of CD146cyt-41BB-CD3ζCAR T cells.

Detailed description of the invention

the term

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, terms used herein such as "Immunobiology" (Immunobiology) by Janeway CA Jr, Travers P, Walport M, 5th edition, New York: Garland Science (2001) and "A multilingual glossary of biotechnological terms: (IUPAC Recommendations), Leuenberger, HGW, Nagel, B. and

Definitions described in H. ed. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the/said" include plural referents unless the context clearly dictates otherwise. Accordingly, the terms "a", "an", "one or more" and "at least one" may be used interchangeably. Similarly, the terms "comprising", "including" and "having" may be used interchangeably.

When the term "comprising" is used herein and in the appended claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising or comprising at least a certain number of embodiments, it is also to be understood that a group consisting preferably only of these embodiments is disclosed.

The term "and/or" as used herein is considered a specific disclosure that each of the two specified features or components is with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to encompass each of the following: A, B, and C; A, B, or C; A, or C A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

CD146 is also known as melanoma cell adhesion molecule, MCAM, MUC18. CD146 is a type I transmembrane glycoprotein consisting of 646 amino acids in humans. CD146 protein consists of extracellular region, transmembrane region and cytoplasmic region. Among them, the cytoplasmic region of CD146 consists of 63 amino acids (amino acids 584-646), with two putative PKC phosphorylation sites and a double leucine motif. The membrane-proximal end of the CD146 cytoplasmic region contains a conserved positively charged amino acid cluster KKGK motif, which is known to serve as a binding site for ERM proteins. The "membrane-proximal fragment of the CD146 cytoplasmic region" refers to the cytoplasmic region fragment of CD146 close to the transmembrane region, which means the fragment from the 584th amino acid to the 646th amino acid of the CD146 molecule.

Previous studies have shown that CD146 plays multiple roles in various cells (Chen J, Luo Y, Hui H, Cai T, Huang H, Yang F, et al. CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development.Proceedings of the National Academy of Sciences of the United States of America.2017;114(36):E7622-E31;Luo Y,Duan H,Qian Y,Feng L,Wu Z,Wang F,et al.Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis. Cell Res.2017; 27(3):352-72; Yan H, Zhang C, Wang Z, Tu T, Duan H, Luo Y, et al. CD146 is required for VEGF- C-induced lymphatic sprouting during lymphangiogenesis.Sci Rep.2017;7(1):7442;Wang Z,and Yan X.CD146,a multi-functional molecule beyond adhesion.Cancer Lett.2013;330(2):150-62 ).

LCK, also known as lymphocyte-specific protein tyrosine kinase, is a member of the Src kinase family and is critical in membrane signaling that activates intracellular signaling cascades. LCK deficiency abolishes proximal TCR signaling and blocks T cell development and activation. Accumulating evidence indicates that phosphorylation of LCK at Y394 is critical for its activation (Philipsen L, Reddycherla AV, Hartig R, Gumz J, Kastle M, Kritikos A, et al. De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling. Sci Signal.2017; 10(462)). Crystallization studies revealed that LCK forms asymmetric head-to-tail dimers in which the Y394 activation loop of one monomer is located in the active site of the other monomer. It has been reported that Transautophosphorylation is crucial for the activation of LCK (Eck MJ, Atwell SK, Shoelson SE, and Harrison SC.Structure of the regulatory domains of the Src-family tyrosine kinase Lck.Nature.1994; 368( 6473):764-9; Xu Q, Malecka KL, Fink L, Jordan EJ, Duffy E, Kolander S, et al. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases. Sci Signal. 2015; 8(405 ):rs13).

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an antigen-binding domain engineered to contain an antigen-binding domain that targets a specific antigen and activates immune cells (such as T cells or NK cells) upon binding to that antigen. cells, such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof) to attack and destroy molecules bearing the antigen. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

The classical chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain. The antigen-binding domain is usually a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms comprising an antibody-like antigen-binding site or an antigen-binding structure derived from the natural ligand of the antigen area. A hinge domain is generally required to separate the antigen-binding domain from the membrane and to allow its proper orientation. A common hinge domain used is the Fc of IgG1. Depending on the antigen, more compact spacers may suffice, such as the stem from CD8α, and even just the IgG1 hinge alone. The transmembrane domain anchors the protein in the cell membrane and connects the hinge domain to the intracellular domain (intracellular domain).

According to at least one non-limiting aspect, there have been at least three "generations" of CAR molecules. In the first generation of CARs, they were designed with intracellular domains derived from the gamma chain of FcεR1 or the intracellular portion of CD3ζ. Thus, these first-generation CARs transmit an immune signal1 that is sufficient to trigger T-cell killing of cognate target cells, but fails to fully activate T-cell proliferation and survival. To overcome this limitation, second-generation CARs have been constructed that possess a composite intracellular domain resulting from the fusion of the intracellular portion of a T-cell co-stimulatory molecule with that of CD3ζ, allowing simultaneous transmission of an activation signal following antigen recognition and costimulatory signals. The most commonly used co-stimulatory domain is that of CD28. This provides the most potent co-stimulatory signal - immune signal 2, which triggers T cell proliferation. Some CARs have also been described that include TNF receptor family intracellular domains such as the closely related OX40 and 41BB that transmit survival signals. Even more potent third-generation CARs have now been described with intracellular domains capable of transmitting activation, proliferation and survival signals.

Thus, a CAR typically comprises: (i) an antigen-binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more consensus domains. Stimulatory domain.

The term "antigen binding domain" as used herein refers to the portion of a chimeric antigen receptor that recognizes an antigen. In a classical CAR, the antigen-binding domain comprises: a single-chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been generated using domain antibodies (dAbs), VHH antigen-binding domains, or antigen-binding domains derived from the natural ligand of the antigen.

The term "antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Antigens may be expressed endogenously, ie from genomic DNA, or may be expressed recombinantly. An antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly. For example, the antigen is a tumor-associated antigen, such as all or a fragment of CD19 or CD20.

The term "hinge region" as used herein refers to the extracellular domain of the CAR molecule that is located between the antigen binding domain and the transmembrane domain and spatially separates the antigen binding domain from the intracellular domain. The hinge region may also be referred to as a "hinge domain" or a "spacer". A hinge may contribute to receptor expression, activity and/or stability. The hinge can also provide flexibility for binding the target antigen, allowing the antigen-binding domain to be oriented in different orientations to facilitate binding.

In the context of referring to a CAR, a "transmembrane domain" refers to a domain that has the property of being present in a membrane when present in a molecule at the cell surface or at the cell membrane (eg, spanning part or all of the cell membrane). A transmembrane domain can be any protein structure that is thermodynamically stable in a membrane. This is usually an alpha helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the chimeric receptor. The presence and span of transmembrane domains of proteins can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Alternatively, artificially designed TM domains can be used.

The intracellular domain (intracellular domain) is the signaling portion of the chimeric antigen receptor. It contains a signaling domain and one or more co-stimulatory domains. Following antigen recognition, the receptor cluster native CD45 and CD148 are expelled from the synapse and a signal is transmitted to the cell, thereby activating one or more immune cell effector functions (eg, native immune cell effector functions). The most commonly used intracellular domain component is that of CD3ζ containing 3 ITAMs. After antigen binding, it transmits an activation signal to T cells. CD3ζ may not provide a fully sufficient activation signal and additional co-stimulatory signaling may be required. Costimulatory signals promote T cell proliferation and survival. There are two main types of co-stimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).

The signaling domain of the intracellular domain mediates activation of at least one normal effector function of the immune cell. For example, effector functions of T cells may be cytolytic or helper activities involving cytokine secretion. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and/or other T cell functions. The intracellular domain may contain a signaling domain (intracellular signaling domain) as an activation domain. The intracellular domain may also comprise a signaling domain that is a costimulatory signaling domain (intracellular costimulatory domain).

The term "CD3ξ" as used herein is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from a non-human species such as mouse, rodent, monkey, ape, and the like. A "CD3 ξ intracellular signaling region" is defined as the amino acid residues from the cytoplasmic domain of the ξ chain that are sufficient to functionally transmit the initial signal required for T cell activation. For example, the cytoplasmic domain of CD3ξ comprises residues 52 to 164 of GenBan accession number BAG36664.1, or a functional ortholog thereof, i.e., from a non-human species such as mouse, rodent, monkey, ape, etc. equivalent residues.

The term "41BB" as used herein refers to a member of the TNFR superfamily having the amino acid sequence of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. base. The "41BB intracellular signal region" is defined as the amino acid sequence 214-255 of GenBank ACC. No. AAA62478.2, or equivalent residues from non-taxonomic species such as mouse, rodent, monkey, ape, and the like.

As used herein, the terms "nucleic acid sequence", "nucleic acid" and "polynucleotide" are intended to be synonymous with each other. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it should be understood that the skilled artisan can use routine techniques to make nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed. A nucleic acid according to the invention may comprise DNA or RNA. They can be single-stranded or double-stranded. They may also be polynucleotides which include synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones with the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For purposes of the uses described herein, it is understood that polynucleotides may be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or longevity of the polynucleotide of interest.

The term "sequence identity" as used herein means the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and Clustal W.

The term "vector" as used herein is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell where said nucleic acid molecule as a vector can eg be replicated and/or expressed.

The term "cell" as used herein refers to any type of cell, such as eukaryotic or prokaryotic cells, capable of expressing the CAR of the present invention.

The term "transfection" as used herein is the process of introducing nucleic acid molecules or polynucleotides, including vectors, into target cells. The term "transduction" is generally used to describe the virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to generate liposomes that fuse with cell membranes and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.

The term "treatment" as used herein includes therapeutic or prophylactic treatment in a subject in need thereof. "Therapeutic or prophylactic treatment" includes prophylactic treatment aimed at the complete prevention of clinical and/or pathological manifestations or therapeutic treatment aimed at amelioration or alleviation of clinical and/or pathological manifestations. Thus, the term "treating" also includes ameliorating or preventing a disease. For example, treatment may include: (i) preventing the disease, disorder and/or condition in a patient who may be predisposed to the disease, disorder and/or condition but has not been diagnosed with the disease, disorder and/or condition; (ii ) inhibiting said disease, disorder and/or condition, ie arresting its development; or (iii) alleviating said disease, disorder and/or condition, ie causing regression of said disease, disorder and/or condition.

The term "effective amount" as used herein means an amount of a therapeutic agent which, when administered to a subject for the treatment or prevention of a disease, is sufficient to effect such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. "Therapeutically effective amount" refers to an effective amount for therapeutic treatment. "Prophylactically effective amount" refers to an effective amount for prophylactic treatment.

The term "administration" as used herein refers to the physical introduction of an agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the active agents disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

As used herein, the terms "subject," "individual," and "patient" are art-recognized and are used interchangeably herein to refer to any subject, particularly a mammal, in need of treatment subject. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species. The terms individual, subject, and patient per se do not denote a specific age, sex, race, or the like.

The term "autologous" refers to any substance derived from the same individual into which it is later reintroduced. For example, the treatment methods herein involve collecting lymphocytes from a patient, then engineering them to express, for example, a CAR of the invention, and then administering them back to the same patient.

The term "allogeneic" refers to any substance derived from one individual and then introduced into another individual of the same species, such as an allogeneic T cell transplant.

Other features, objects and advantages of the present disclosure will be apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, not limitation.

In one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, an intracellular signaling domain and an intracellular co-stimulatory domain, wherein the intracellular The costimulatory domain comprises a first costimulatory domain capable of binding and activating lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation.

A CAR may contain a co-stimulatory domain, for example, to increase signaling potency. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011) and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signaling through the TCR alone may not be sufficient to fully activate T cells, whereas secondary or co-stimulatory signals can increase activation. Thus, a CAR of the invention comprises one or more co-stimulatory domains that activate one or more immune cell effector functions, such as the innate immune cell effector functions described herein. A portion of such co-stimulatory domains may be used as long as the portion transduces an effector function signal.

The present inventors found that the cytoplasmic region of CD146 molecules in T cells can directly interact with LCK molecules, and can promote the activation of LCK by promoting the autophosphorylation of LCK. This direct interaction is at least partially dependent on the KKGK motif at the membrane-proximal end of the CD146 cytoplasmic region, especially amino acids 584-609 of the CD146 cytoplasmic region. Therefore, by integrating the CD146 cytoplasmic region or its membrane-proximal fragment (for example, the KKGK motif at the proximal membrane, especially the 584-609 amino acids of the CD146 cytoplasmic region) into the intracellular domain of the CAR molecule, the use of Its ability to bind and activate LCK recruits more activated LCK molecules to the intracellular domain of the CAR molecule, thereby increasing the activation level of CAR-T cells, thereby improving the tumor-killing ability of CAR-T cells.

Accordingly, in some embodiments of the CAR of the invention, the first co-stimulatory domain may comprise a sequence derived from the cytoplasmic region of the CD146 molecule.

In some embodiments, the first co-stimulatory domain may comprise a KKGK motif at the membrane-proximal end of the cytoplasmic region of CD146. In some embodiments, the first co-stimulatory domain may comprise the cytoplasmic region of CD146, or a membrane-proximal fragment thereof. In some embodiments, the first co-stimulatory domain may comprise a functional variant of the CD146 cytoplasmic region or its proximal membrane fragment, and the functional variant may comprise one or more amino acid insertions or deletions relative to the wild-type sequence , substitution or addition, so long as the variant retains the ability to bind and activate LCK to enhance immune cell activation. For example, the functional variant of the CD146 cytoplasmic region or its membrane-near end fragment may retain the KKGK motif and contain one or more amino acid insertions, deletions, or substitutions at other positions except the KKGK motif in the CD146 cytoplasmic region or added peptides.

The term "functional variant" refers to a polypeptide that has significant sequence identity to a parent polypeptide and that retains the biological activity of the parent polypeptide. Functional variants encompass, for example, a CD146 cytoplasmic region or its vicinity described herein that retains the ability to bind and activate LCK to a similar extent, to the same extent, or to a greater extent than the parent polypeptide. Variant of the membrane end fragment. The amino acid sequence of the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of the parent polypeptide. identity.

A functional variant may be a functional variant formed by insertion, deletion or substitution of one or more amino acids in a parent polypeptide. For example, a functional variant may comprise the amino acid sequence of a parent polypeptide with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, a conservative amino acid substitution can be an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly , Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid is replaced by another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain is replaced by a polar side chain Another amino acid (Asn, Cys, Gln, Ser, Thr, Tyr, etc.) etc.

The term "the membrane-proximal segment of the CD146 cytoplasmic region" refers to the cytoplasmic region fragment of CD146 near the transmembrane region, which means the fragment from the 584th amino acid to the 646th amino acid of the CD146 molecule.

For example, the membrane-proximal segment of the cytoplasmic region of CD146 may be 4-55 amino acids in length, preferably 10-45 amino acids in length, more preferably 15-35 amino acids in length, and even more preferably 15-26 amino acids in length. In some embodiments, the membrane-proximal segment of the cytoplasmic region of CD146 comprises amino acids 584-609 of the CD146 molecule.

In some embodiments, the first co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 7 or a functional variant thereof formed by insertion, deletion or substitution of one or more amino acids.

In some embodiments, the intracellular co-stimulatory domain can also comprise a second co-stimulatory domain. The second co-stimulatory domain may be selected from 4-1BB (CD137), OX40 (CD134), ICOS (CD278), 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumors Necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), MyD88, CD2, CD4, CD7, LIGHT, NKG2C, and the intracellular signaling region of B7-H3, or any combination thereof. In some embodiments, the second co-stimulatory domain is the intracellular signaling region of 41BB.

The amino acid sequences of the second co-stimulatory domains provided herein are known in the art. In certain instances, the second co-stimulatory domain can comprise the amino acid sequence set forth in SEQ ID NO:9.

In some embodiments, the first costimulatory domain is the cytoplasmic region of CD146, the second costimulatory domain is the intracellular signaling region of 41BB, and the C-terminus of the first costimulatory domain is connected to the N-terminal connection.

Intracellular signaling domains that can transduce signals upon antigen binding to immune cells are known, any of which can be included in the CARs of the present disclosure. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction upon binding of the TCR to an antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

In certain embodiments, suitable intracellular signaling domains include, but are not limited to, TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD28, CD29, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96, CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, DNAM1(CD226), GADS, IA4, ICAM-1, ICAM-1, CD79a, IL-2Rβ, IL-2Rγ, IL-7Rα, ICOS, Integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LTBR, NKp30, NKp44, NKp46, NKp80(KLRF1), PAG/Cbp, PD-1, PSGL1, SELPLG(CD162), signaling lymphocytes Sex activating molecule (SLAM protein) SLP-76, TNF receptor protein, TNFR2, TNFSF14, VLA1 or VLA-6, or fragments thereof or any combination thereof.

In some embodiments, an intracellular signaling domain of the invention may comprise a signaling domain selected from the group consisting of TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, ICOS (CD278), and CD66d. area or any combination thereof. In some embodiments, the intracellular signaling domain is preferably the signaling region of CD3ζ.

The amino acid sequences of the intracellular signaling domains provided herein are known in the art. In certain instances, the intracellular signaling domain can comprise the amino acid sequence set forth in SEQ ID NO: 11.

In some embodiments, the order of the first co-stimulatory domain, the second co-stimulatory domain and the intracellular signaling domain from the N-terminus to the C-terminus of the present invention is: first co-stimulatory domain-second co-stimulatory domain domain - intracellular signaling domain. They are optionally linked by linkers.

The type of transmembrane domain contained in the CAR of the present invention is not limited to any type. In some embodiments, a transmembrane domain is selected that is naturally associated with the antigen binding domain and/or the intracellular domain. In some cases, transmembrane domains comprise modifications (e.g., deletions, insertions, and/or substitutions) of one or more amino acids, e.g., to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex.

Transmembrane domains can be derived from natural sources or from synthetic sources. When the source is a natural source, the domains may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains can be derived from T cell receptor (TCR) alpha chain, TCR beta chain, TCR zeta chain, CD28, CD3ε, CD3ζ, CD3δ, CD3γ, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activated NK cell receptor , BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD276(B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f , CD69, CD84, CD96, CDS, CEACAM1, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fcγ receptors, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1 , CD79a, IL-2Rβ, IL-2Rγ, IL-7Rα, ICOS, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, bind to CD83 ligands, LIGHT, LTBR, Ly9 (CD229), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PD-1, PSGL1, SELPLG (CD162), SLAM ( SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, VLA1 or VLA- 6, or fragments or any combination thereof.

In some embodiments, the transmembrane domain may comprise an alpha chain selected from T cell receptor (TCR), beta chain of TCR, zeta chain of TCR, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD19 , CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137(41BB), CD152, CD154 and the transmembrane domain of PD1 or any combination thereof. Preferably, the transmembrane domain is that of CD8.

In some embodiments, the transmembrane domain may be synthetic (and may, for example, comprise predominantly hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked to the intracellular domain. In some embodiments, short oligopeptide or polypeptide linkers (eg, between 2 and 10 amino acids in length) can form a link between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.

The amino acid sequences of the transmembrane domains provided herein are known in the art. In some cases, the transmembrane domain can comprise the amino acid sequence set forth in SEQ ID NO:5.

The antigen-binding domain refers to the portion of the chimeric antigen receptor that recognizes an antigen. In some embodiments, an antigen binding domain of the invention can bind one or more tumor-associated antigens (TAAs). The TAA is preferably selected from 5T4, alpha-fetoprotein, BCMA, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, ROR1, GPC3, Tyrp-1, TACI, ALK, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, ERBB2, FLT3, melanoma-associated antigen, mesothelin, MUC-1, VEGFR2. More preferably, the antigen binding domain specifically binds CD19.

In some embodiments, the antigen binding domain may be selected from an antigen binding domain derived from an antibody against the antigen and an antigen binding domain derived from a natural ligand of the antigen. Preferably, the antigen binding domain derived from an antibody may be in the form of a scFv, Fab or domain antibody (dAb).

For example, the antigen binding domain may be in the form of a Fab fragment of a monoclonal antibody. A Fab CAR comprises two chains: one with an antibody light chain variable region (VL) and constant region (CL); and one with a heavy chain variable region (VH) and constant region (CH). One chain also contains a transmembrane domain and an intracellular domain. The association between CL and CH leads to the assembly of the receptor.

The two chains of a Fab CAR can have a general structure:

VH-CH-hinge region-transmembrane domain-intracellular domain; and VL-CL

or

VL-CL-hinge region-transmembrane domain-intracellular domain; and VH-CH.

For the Fab-type chimeric receptors described herein, the antigen binding domain consists of a VH from one polypeptide chain and a VL from the other polypeptide chain.

A polypeptide chain may comprise a linker between VH/VL and CH/CL. Linkers can be flexible and serve to spatially separate VH/VL from CH/CL.

Flexible linkers can consist of small non-polar residues such as glycine, threonine and serine. A linker may comprise one or more repeats of a glycine-serine linker, such as a ( _Gly4Ser ) _n linker, where n is the number of repeats. The or each linker may be less than 50, 40, 30, 20 or 10 amino acids in length.

The amino acid sequences of antigen binding domains targeting one or more TAAs described herein are known. For example, several anti-CD19 antibodies have been previously described in CAR format, such as fmc63, 4G7, SJ25C1, CAT19 (as described in WO2016/139487) and CD19ALAb (as described in WO2016/102965). These anti-CD19 antibodies and antigen-binding fragments thereof can be used in the CAR of the present invention. In some cases, the antigen binding domain can comprise the amino acid sequence set forth in SEQ ID NO: 1.

In some embodiments, the CAR of the present invention may also comprise a hinge region. The hinge region can be located between the antigen binding domain and the transmembrane domain.

The hinge region can be derived from natural sources or from synthetic sources. In some embodiments, the hinge region may be, be from, or be derived from CD2, CD3γ, CD3δ, CD3ε, CD3ζ, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d ( ITGAD), CD18(ITGB2), CD19(B4), CD27(TNFRSF7), CD28, CD28T, CD29(ITGB1), CD30(TNFRSF8), CD40(TNFRSF5), CD48(SLAMF2), CD49a(ITGA1), CD49d(ITGA4 ), CD49f(ITGA6), CD66a(CEACAM1), CD66b(CEACAM8), CD66c(CEACAM6), CD66d(CEACAM3), CD66e(CEACAM5), CD69(CLEC2), CD79A, CD79B, CD84(SLAMF5), CD96, CD100( SEMA4D), CD103(ITGAE), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD158A(KIR2DL1), CD158B1(KIR2DL2), CD158B2(KIR2DL3), CD158C(KIR3DP1), CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244(SLAMF4), CD258(LIGHT), CD268(BAFFR), CD270(TNFSF14), CD272(BTLA), CD276(B7-H3), CD279(PD-1), CD314(NKG2D), CD319(SLAMF7), CD335(NK-p46), CD336(NK-p44), CD337( NK-p30), CD352(SLAMF6), CD353(SLAMF8), CD355(CRTAM), CD357(TNFRSF18), ICOS, LFA-1(CD11a/CD18), NKG2C, DAP-10, ICAM-1, NKp80(KLRF1) , IL-2Rβ, IL-2Rγ, IL-7Rα, LFA-1, SLAMF9, LAT, GADS(GrpL), SLP-76(LCP2), PAG1/CBP, CD83 ligand, FcRγ, MHC class I molecule, MHC II Molecules, T NF receptor protein, immunoglobulin, cytokine receptor, integrin, NK cell activating receptor or Toll ligand receptor, or fragments thereof or any combination thereof.

In some embodiments, the hinge region may comprise a hinge or fragment thereof selected from the group consisting of CD8, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. Preferably, the hinge region may be a CD8 hinge.

The amino acid sequences of the hinge regions provided herein are known in the art. In some cases, the hinge region can comprise the amino acid sequence set forth in SEQ ID NO:3.

BD-hinge-TM-Costi-Singal;

in:

BD is a nucleotide sequence encoding an antigen-binding domain;

hinge is the nucleotide sequence encoding the hinge region;

TM is a nucleotide sequence encoding a transmembrane domain;

Costi is a nucleotide sequence encoding an intracellular co-stimulatory domain;

Signal is a nucleotide sequence encoding an intracellular signaling domain.

In some embodiments, the nucleic acid construct has the following structure:

BD-hinge-TM-Costi1-Costi2-Singal;

in:

BD is a nucleotide sequence encoding an antigen-binding domain;

hinge is the nucleotide sequence encoding the hinge region;

TM is a nucleotide sequence encoding a transmembrane domain;

Costi1 is a nucleotide sequence encoding the first co-stimulatory domain;

Costi2 is a nucleotide sequence encoding a second co-stimulatory domain;

Signal is a nucleotide sequence encoding an intracellular signaling domain.

In an embodiment of the nucleic acid construct of the invention, the antigen binding domain, hinge region, transmembrane domain, intracellular costimulatory domain, first costimulatory domain and second costimulatory domain are as defined above.

Preferably, the antigen binding domain specifically binds to CD19; the hinge region is the CD8 hinge region; the transmembrane domain is the CD8 transmembrane domain; the first co-stimulatory domain is the cytoplasmic region of the CD146 molecule; the second co-stimulatory domain is the 41BB intracellular signaling domain; and the intracellular signaling domain is the CD3ζ intracellular signaling domain.

In some embodiments, the nucleotide sequence encoding the antigen binding domain can be a nucleotide sequence as shown in SEQ ID NO: 2, or at least 80%, at least 85%, at least 90% identical to SEQ ID NO: 2 %, at least 95% or 99% sequence identity of nucleotide sequences.

In some embodiments, the nucleotide sequence encoding the hinge region can be a nucleotide sequence as shown in SEQ ID NO: 4, or have at least 80%, at least 85%, at least 90%, at least 90%, Nucleotide sequences with at least 95% or 99% sequence identity.

In some embodiments, the nucleotide sequence encoding the transmembrane domain can be a nucleotide sequence as shown in SEQ ID NO:6, or have at least 80%, at least 85%, at least 90% of SEQ ID NO:6 %, at least 95% or 99% sequence identity of nucleotide sequences.

In some embodiments, the nucleotide sequence encoding the first co-stimulatory domain can be a nucleotide sequence as shown in SEQ ID NO: 8, or at least 80%, at least 85%, Nucleotide sequences having at least 90%, at least 95%, or 99% sequence identity.

In some embodiments, the nucleotide sequence encoding the second co-stimulatory domain can be a nucleotide sequence as shown in SEQ ID NO: 10, or at least 80%, at least 85%, Nucleotide sequences having at least 90%, at least 95%, or 99% sequence identity.

In some embodiments, the nucleotide sequence encoding the intracellular signaling domain can be a nucleotide sequence as shown in SEQ ID NO: 12, or have at least 80%, at least 85%, Nucleotide sequences having at least 90%, at least 95%, or 99% sequence identity.

Non-limiting examples of vectors include, but are not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia viral vectors, polyoma viral vectors, and adeno-associated vectors (AAV)), bacteriophage, phage Bacteroids, cosmids and artificial chromosomes (including BAC and YAC). The vector itself is usually a sequence of nucleotides, usually a DNA sequence containing the insert (transgene) and a larger sequence that acts as the "backbone" of the vector.

Engineered vectors typically contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). A vector may additionally comprise a promoter, genetic marker, reporter gene, targeting sequence and/or protein purification tag. As is known to those skilled in the art, a large number of suitable vectors are known to those skilled in the art, and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is hereby incorporated by reference in its entirety.

In some embodiments, the vector is preferably selected from lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors and artificial chromosomes.

In some embodiments, the cells are selected from lymphocytes (eg, T cells, NK cells) and monocytes (eg, PBMCs). In some embodiments, the cells are T cells. The T cell can be any T cell, such as a cultured T cell, such as a primary T cell or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, T cells can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus or other tissues or fluids. T cells can also be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are T cells isolated from humans. T cells can be of any type and at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (eg, cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (eg, central memory T cells and effector memory T cells), naive T cells, and the like. In some embodiments, the cells are immune cells, preferably T cells, NK cells or macrophages. In certain embodiments, the cells are stem cells, preferably pluripotent stem cells, induced pluripotent stem cells (iPSC), mesenchymal stem cells, hematopoietic stem cells or lymphoid progenitor cells.

In yet another aspect, the present invention provides a method of preparing the cells of the present invention, comprising the step of transducing or transfecting the cells with the vector of the present invention. In some embodiments, the method may further comprise the step of expanding and/or activating cells before or after said transducing or transfecting.

Compositions according to the invention are in particular compositions suitable for administration to humans. However, it generally also encompasses compositions suitable for administration to non-human animals. The composition and its components (i.e. the active agent and optionally the carrier or excipient) are preferably pharmaceutically acceptable, i.e. capable of eliciting the desired therapeutic effect in the recipient without causing any undesired Local or systemic action. A pharmaceutically acceptable composition of the invention may, for example, be sterile. In particular, the term "pharmaceutically acceptable" may mean approval by a regulatory agency or other recognized pharmacopoeia for use in animals, more particularly in humans.

Examples of excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners Agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity regulators. The selection of suitable excipients for the preparation of the compositions of the invention is known to those skilled in the art. Exemplary carriers for use in compositions of the invention include saline, buffered saline, dextrose and water. In general, the selection of suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired formulation of the composition.

Preferred examples of the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, Gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin; and peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as IL-2, chemokines such as IL-8, Platelet factor 4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; viral/bacterial protein domains and viral/bacterial peptides.

Methods for treating disease involve the therapeutic use of the cells of the invention. Herein, cells may be administered to a subject already suffering from a disease or condition to alleviate, reduce or ameliorate at least one symptom associated with the disease and/or to slow, reduce or block the progression of the disease.

The effective amount of cells administered can be determined by one skilled in the art using known techniques. A suitable dosage provides a sufficient amount of the active agent of the invention, and preferably is therapeutically effective, ie sufficient to elicit eg a therapeutic or prophylactic response in a subject or animal within a reasonable time frame. For example, the dose of cells of the invention should be within a period of about 2 hours or more, such as 12 hours to 24 hours or more (e.g., 6 months, 12 months, 24 months, etc.) from the time of administration Sufficient to bind a cancer antigen or to detect, treat or prevent cancer. In certain embodiments, the period of time can be even longer. Route, time and frequency of administration, time and frequency of administration of formulation, age, body weight, general health, sex, diet, disease state, as known in the art, for therapeutic purposes (e.g., alleviation of acute episodes of disease) Adjustments based on severity, drug combination, response sensitivity, and tolerance/response to therapy may be necessary.

Numerous assays for determining administered dosages are known in the art. For the purposes of the present invention, an assay can be used to determine the starting dose to be administered to a mammal, the assay comprising administering a given dose of T cells expressing a CAR of the invention to mammals in a group of mammals (each determined by After administration of different doses of T cells), the extent to which target cell lysis or IFN-γ secretion by such T cells is compared is compared. The extent to which target cell lysis or IFN-γ secretion is achieved following administration of a certain dose can be determined by methods known in the art. Dosages of the cells of the invention are also determined by the presence, nature and extent of any adverse side effects that may accompany the administration of the cells of the invention. Typically, the attending physician determines the dosage of the cells of the invention to be administered to each individual patient, taking into account factors such as age, body weight, general health, diet, sex, active agent to be administered, route of administration, and severity of the condition being treated degree. In some embodiments of the methods of treatment of the present disclosure, the number of cells administered per infusion can vary, for example, from about 1×10 ⁶ to about 1×10 ¹² cells or more. In certain embodiments, less than 1 x ¹⁰⁶ cells may be administered.

It will be appreciated that treatment may require a single administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of an agent of the invention. For example, some compositions may be administered every 3 to 4 days, weekly, or once every two weeks, or once in a month, depending on the formulation, half-life, and clearance rate of the particular composition.

The cells of the invention are suitable for a variety of routes of administration. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

A subject refers to any subject in need of treatment, especially a mammalian subject. Generally, mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like. However, it is readily understood that the cells and pharmaceutical compositions provided herein are specifically envisioned for use in the treatment of human subjects.

In embodiments of the methods of the invention, said cells are autologous or allogeneic to said subject. In some embodiments, the cells are CAR-T cells or CAR-NK cells.

A sample containing, for example, T cells can be isolated from the subject or from other sources, for example, from the patient's own peripheral blood (first party), or in a hematopoietic stem cell transplant from donor peripheral blood (second party). environment, or isolate cells from peripheral blood (third party) from an unrelated donor.

In some embodiments, the method can also include administering a second therapeutic agent. Preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs. Preferred examples of the second therapeutic agent are described above.

In certain embodiments, the cancer may be acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia ("BALL") ), blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Hairy Cell Leukemia, Hodgkin's Disease, Malignant Lymphoproliferative Conditions, MALT Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Significance Monoclonal gammopathy of unspecified (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma (NHL), plasma cell proliferative disorders (including asymptomatic myeloma ( smoldering multiple myeloma or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, plasmacytoma (including plasma cell dyscrasia; solitary myeloma; solitary plasmacytoma ; extramedullary plasmacytoma; and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase syndrome; Takatsuki disease; and PEP syndrome), primary mediastinal large B-cell lymphoma (PMBC), Small cell or large cell follicular lymphoma, splenic marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transforming filter Follicular lymphoma or Waldschl's macroglobulinemia, mantle cell lymphoma (MCL), transformed follicular lymphoma (TFL), primary mediastinal B-cell lymphoma (PMBCL), multiple myeloma, hair cell Lymphoma or leukemia.

In certain embodiments, the cancer may be alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or rectoanal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, Cancer of the gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cavity, vagina, vulva, chronic lymphocytic leukemia, chronic myeloid, colon, esophagus, cervix, gastrointestinal carcinoid tumors , glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, Oropharyngeal, ovarian, penile, pancreatic, peritoneal, omental, and mesenteric, pharynx, prostate, rectum, kidney, skin, small intestine, soft tissue, stomach, testis, thyroid cancer of the uterus, ureter, or bladder.

In another aspect, the present invention provides a method for enhancing the activation of immune cells, which comprises introducing the CAR of the present invention into the immune cells. In some embodiments, the immune cells are T cells or NK cells. In some embodiments, the method includes the step of transducing or transfecting immune cells with a vector of the invention.

In some embodiments of the uses of the present invention, the cancer may be selected from lymphoma, multiple myeloma, leukemia and solid tumors. Non-limiting examples of cancer are as described above.

The present invention is further illustrated by the following specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples, by according to routine conditions in this field, for example, people such as Sambrook and Russeii, molecular cloning: laboratory handbook (third edition) (2001), described in CSHL publishing house conditions, or as recommended by the manufacturer. Experimental materials and reagents used in the following examples are commercially available unless otherwise stated.

Example 1. Interaction assay between CD146 and LCK

Studies have shown that CD146 is related to the activation of T cells during the immune response (Pickl WF, et al. MUC18/MCAM (CD146), an activation antigen of human T lymphocytes. Journal of immunology (Baltimore, Md: 1950). 1997; 158 (5):2107-15; Elshal MF, et al. CD146(Mel-CAM), an adhesion marker of endothelial cells, is a novel marker of lymphocyte subset activation in normal peripheral blood. Blood. 2005; 106(8): 2923-4; Brucklacher-Waldert V, et al. Phenotypical and functional characterization of T helper 17 cells in multiple sclerosis. Brain. 2009; 132(Pt 12):3329-41). To determine the mechanism by which CD146 regulates TCR signaling, immunoprecipitation (IP) was first used to detect whether CD146 interacts with TCR signaling-related molecules (such as CD3, CD4, Zap70, LAT, PLCγ, Fyn, and LCK). The results showed that CD146 did not interact with CD3, CD4, Zap70, LAT, PLCγ, and Fyn, but interacted with LCK (data not shown).

In order to determine whether the interaction between CD146 and LCK is direct or indirect, two recombinants of CD146 cytoplasmic region were constructed, in which GST protein was linked at the C-terminal or N-terminal of CD146 cytoplasmic region (respectively CD146 cytoplasmic region-linker-GST or GST-linker-CD146 cytoplasmic region), the structure of which is shown in Figure 1A. These two recombinants were used together with recombinant LCK-His protein (Abacan, Cat. No. ab82185) for Pull-down experiments. The recombinant CD146 protein was attached to the GST beads, and after washing three times, the recombinant LCK-His protein was added and incubated at 4°C for 1 hour. Then wash three times with PBS/lysis buffer (9:1), dissolve the sample in 1X SDS reducing sample buffer, and then perform western blot analysis with anti-GST and anti-LCK antibodies, the results are shown in Figure 1B. The results showed that both recombinants directly interacted with LCK, which indicated a direct interaction between CD146 and LCK.

To determine which residues of CD146 are involved in its interaction with LCK, three truncated parts of CD146 were constructed, namely C19 (627 amino acids, deletion of amino acids 628-646), C37 (609 amino acids, deletion of 610 -646 amino acids) and C63 (583 amino acids, deletion of 584-646 amino acids) (Figure 2A). Plasmids encoding these CD146 truncations were transfected into LCK stably transfected 293T cells. Cells were lysed with cell lysate, cell lysates were pre-cleared with protein A/G Sepharose beads, and supernatants were immunoprecipitated overnight at 4°C with anti-CD146 antibody AA1. Protein A/G Sepharose beads were added and samples were incubated for an additional 2 hours. After washing 3 times with PBS/lysis buffer (9:1), samples were dissolved in 1x SDS reducing sample buffer and subjected to (10%) SDS-PAGE. Western blot analysis was performed using antibodies against CD146 and LCK, and the results are shown in Figures 2B-2C. The results showed that only C63 weakened the interaction between CD146 and LCK, but C19 and C37 did not weaken the interaction, which indicated that the interaction site between CD146 and LCK was located at amino acids 584-609 of the cytoplasmic domain.

Previous studies have shown that the conserved positively charged amino acid cluster KKGK motif in the membrane-proximal region of the CD146 cytoplasmic tail acts as a binding site for ERM proteins (Luo Y, Zheng C, Zhang J, Lu D, Zhuang J, Xing S , et al. Recognition of CD146 as an ERM-binding protein offers novel mechanisms for melanoma cell migration. Oncogene. 2012; 31(3):306-21). Therefore, it was envisioned that this KKGK motif might also be involved in the interaction with LCK. To test this conjecture, a mutant KKGK-AAGA was constructed (Fig. 2A). A plasmid encoding this mutant was transfected into 293T cells that had been stably transfected with a plasmid encoding LCK, and immunoprecipitation experiments were performed as described above. The results showed that the KKGK mutant significantly weakened the interaction with LCK, suggesting that KKGK is indeed required for LCK interaction. At the same time, another mutant RRS-AAS with a positively charged amino acid cluster RRS motif (which is similar to the motif RRR interacting with LCK in CD4 or CD44) was constructed, and similar experiments were carried out. The results showed that the mutation The body did not lead to weakened interaction with LCK (Fig. 2B-C). Therefore, the interaction of CD146 with LCK is mainly dependent on the KKGK motif.

These results indicate that in T cells, the intracellular domain (cytoplasmic region) of CD146 molecules can directly interact with LCK molecules, and can promote the activation of LCK by promoting the autophosphorylation of LCK. This direct interaction depends on the KKGK motif at the proximal end of the CD146 cytoplasmic region, especially amino acids 584-609 of the CD146 cytoplasmic region. Therefore, it is envisaged to apply this discovery to CAR molecules—by modifying the structure of the intracellular segment of CAR molecules, the CD146 cytoplasmic region or its membrane-proximal fragments (for example, the KKGK motif at the proximal membrane end, especially the CD146 cytoplasmic region 584-609 amino acids of the cytoplasmic region) into the intracellular domain of the CAR molecule, and use its ability to bind and activate LCK to recruit more activated forms of LCK molecules to the intracellular domain of the CAR molecule, thereby It can better activate the CD3ζ domain of the intracellular domain of the CAR molecule to increase the activation level of CAR-T cells, thereby improving the tumor-killing ability of CAR-T cells.

Example 2. Construction of CAR-encoding vectors

The second-generation CAR was engineered so that its intracellular domain (eg, at the membrane-proximal end) contained the cytoplasmic region of CD146. As an example, a vector encoding an anti-CD19 CAR was constructed using the Lenti-EF1a-CD19(FMC63)-2nd-CAR(4-1BB)-EGFRt vector (Aikond Biomedical Technology (Suzhou) Co., Ltd.). The vector is designed to contain anti-CD19 scFv (FMC63), CD8 hinge region, CD8 transmembrane domain, CD146 cytoplasmic region and 41BB-CD3ζ intracellular signal region. The CAR molecule is connected to the EGFRt fragment through the P2A linker to realize the simultaneous transcription and translation of the inserted gene fragment and the EGFRt marker molecule, so that subsequent experiments can detect the lentiviral transfection efficiency through the EGFRt marker molecule. Vectors were subjected to lentiviral packaging for subsequent transduction of T cells according to the manufacturer's (and Metabiota's) instructions.

The amino acid sequence and coding sequence of each component of the CAR molecule are shown below.

Amino acid sequence (SEQ ID NO: 1) of the anti-CD19 antigen-binding domain (FMC63):

Coding sequence (SEQ ID NO: 2) of anti-CD19 antigen-binding domain (FMC63):

Amino acid sequence (SEQ ID NO:3) of CD8 hinge region:

Coding sequence of CD8 hinge region (SEQ ID NO:4):

Amino acid sequence (SEQ ID NO:5) of CD8 transmembrane domain:

Coding sequence (SEQ ID NO:6) of CD8 transmembrane domain:

Amino acid sequence of CD146 cytoplasmic region (SEQ ID NO: 7):

The coding sequence of CD146 cytoplasmic region (SEQ ID NO:8):

Amino acid sequence (SEQ ID NO: 9) of the 41BB intracellular signal region:

Coding sequence (SEQ ID NO: 10) of 41BB intracellular signal region:

Amino acid sequence (SEQ ID NO: 11) of CD3ζ intracellular signal region:

Coding sequence (SEQ ID NO: 12) of CD3ζ intracellular signal region:

Example 3. Construction of CAR-T cells

T cells that can stably express the CAR molecule of the present invention are constructed by the following procedure:

(1) Petri dish coating: Coat the cell culture plate overnight at 4°C in advance with anti-human CD3 antibody (1 μg/ml), anti-human CD28 antibody (1 μg/ml) and recombinant human fibrin fragment (5 μg/ml);

(2) T cell acquisition: Peripheral blood mononuclear cells (PBMC) from healthy individuals were provided by the Department of Hematology, Peking University People's Hospital, and the use of this sample complied with the ethical regulations of the Institutional Review Board. The mononuclear cells were isolated by using the human peripheral blood lymphocyte isolation medium (CB6100) of Jinclon brand, and then the T cells were sorted out by MojoSort ^TM Human CD3 T Cell Isolation Kit (Cat. No. 480021).

(3) T cell activation culture: T cell proliferation medium (recipe: RPMI 1640 medium + 10% heat-inactivated fetal bovine serum + sodium pyruvate + non-essential amino acids + penicillin/streptomycin + 1000IU/ml human IL2) Resuspend T cells to a concentration of 1×10 ⁶ cells/ml. 500 μl per well was inoculated into a 48-well cell culture plate coated with anti-human CD3 antibody, anti-human CD28 antibody and recombinant human fibrin fragment at 4°C overnight, and activated in a 37°C, 5% CO ₂ incubator. After 24-36 hours of activation, the T cells are attached to the bottom of the cell culture plate under the action of antibodies and fibronectin. The cell volume becomes larger, and the shape gradually changes from round to polarized. At this time, the cells are about to start to proliferate rapidly. in lentiviral infection.

(4) Lentivirus infection: add the concentrated lentivirus of Example 1 into T cell culture wells (MOI=10-30), then centrifuge the T cell culture plate at 32°C and 1700rpm for 100 minutes, and then Put it back into the carbon dioxide incubator to continue culturing. After 36-48 hours of centrifugal infection, replace the T cell proliferation medium for T cells (the formula is RPMI 1640 medium + 10% heat-inactivated fetal bovine serum + sodium pyruvate + non-essential amino acids + penicillin/streptomycin + 1000IU /ml human IL2), and transferred to a new culture plate without anti-CD3 antibody, anti-CD28 antibody and fibronectin at a density of 2×10 ⁶ cells/ml for expansion culture. Supplement T cells with a fresh equal volume of medium every other day to maintain the cell density at 1×10 ⁶ to 3×10 ⁶ cells/ml. After 4-5 days of centrifugal infection, the gene transduced by lentivirus was stably expressed in T cells (the results are shown in Figure 3), which can be used for subsequent sorting for in vitro experiments or direct animal experiments.

Example 4. Determination of CAR-T cell activation

Untransfected T cells, T cells transfected with 41BB-CD3ζ second-generation CAR molecules, T cells transfected with CD146 cytoplasmic region (CD146cyt)-41BB-CD3ζ CAR molecules were compared with Raji cells highly expressing CD19 molecules. (Human Burkitt lymphoma cell line) were co-cultured for 24 hours at an effector cell-target cell ratio (E:T ratio) of 1:1. Then, by flow cytometry, the expression of CD69 molecules (a marker of T cell activation) on the surface of the three T cells was detected by using FITC-anti-human CD69 antibody, and the results are shown in FIG. 4A . Further, the CD19 antigen was coated on a 48-well plate according to the specified concentration (1 μg/ml, 2 μg/ml, 4 μg/ml), and then the above-mentioned T cells transfected with 41BB-CD3ζ second-generation CAR molecule, transfected with CD146cyt T cells with -41BB-CD3ζCAR molecules were added into the well plate, and the CD69 ⁺ cell ratios of the two T cells were detected by flow cytometry after 5 hours, and the results are shown in Figure 4B.

The results showed that after co-culture with target cells, the activation efficiency of T cells transfected with CD146cyt-41BB-CD3ζ CAR molecules was significantly increased from 41.4% compared with T cells transfected with 41BB-CD3ζ second-generation CAR molecules. increased to 50% (Fig. 4A). After co-culture with CD19 antigen, the proportion of CD69 ⁺ T cells transfected with CD146cyt-41BB-CD3ζ CAR molecule increased by more than 50% compared with the proportion of CD69 ⁺ T cells transfected with 41BB-CD3ζ second-generation CAR molecule (Fig. 4B ). These results indicated that the introduction of CD146 cytoplasmic region significantly promoted the activation of CAR-T cells.

Example 5. Cytokine secretion of CAR-T cells

T cells transfected with 41BB-CD3ζ second-generation CAR molecules and T cells transfected with CD146cyt-41BB-CD3ζ CAR molecules were co-cultured with Raji cells highly expressing CD19 molecules at an E:T ratio of 1:1 for 48 h. Then, the production of TNF-α, IL-2 and IFN-γ in T cells was detected by flow cytometry, and the results are shown in FIG. 5 .

The results showed that compared with T cells transfected with 41BB-CD3ζ second-generation CAR molecules, the cytokine secretion of T cells transfected with CD146cyt-41BB-CD3ζ CAR molecules was significantly increased, and the secretion of TNF-α increased from 20% to 31.3%; IL-2 secretion increased from 19.7% to 34.2%; IFN-γ secretion increased from 38.8% to 42%. This indicates that the introduction of CD146 cytoplasmic region significantly promotes the release of cytokines in CAR-T cells.

Example 6. In vitro tumor killing activity of CAR-T cells

Untransfected T cells, T cells transfected with 41BB-CD3ζ second-generation CAR molecules, T cells transfected with CD146cyt-41BB-CD3ζ CAR molecules were compared with Raji cells highly expressing CD19 molecules at a ratio of 1:1 and 2 After 36 hours of co-cultivation at an E:T ratio of 1, the supernatant and cells were collected by centrifugation.

Lactate dehydrogenase in the culture supernatant was detected by using a non-isotope-labeled cell death kit (CytoTox96 Non-radioactive Cytotoxicity Assay) (the detection principle is that after cell death, intracellular lactate dehydrogenase (LDH ) is released into the medium, so the level of LDH in the medium is detected by enzymatic discoloration reaction, and the number of cell death can be converted). The target cell maximum release group deducted the volume to correct the control group, and after the experimental group and the self-release group deducted the medium blank control group, the killing rate was calculated according to the following formula:

Killing rate (%)=(experimental group release-spontaneous release of effector cells-spontaneous release of target cells/maximum release of target cells-spontaneous release of target cells)×100%.

The results showed that compared with T cells transfected with 41BB-CD3ζ second-generation CAR molecules, the tumor killing efficiency of T cells transfected with CD146cyt-41BB-CD3ζ CAR molecules was significantly increased, indicating that the introduction of CD146 cytoplasmic region increased CAR - T cell killing activity of tumors (Fig. 6). This indicates that the introduction of the CD146 cytoplasmic region into the intracellular domain of existing CAR molecules can further improve the tumor killing activity of CAR-T cells, which can be used in the treatment of various cancers.

Example 7. In vivo killing activity of CAR-T cells against hematological tumors

In this example, female NSG immunodeficient mice were used as a model. NSG mice lack T cells, B cells, NK cells, and the complement system, and their macrophages and dendritic cells are also defective. NSG mice have severe immunodeficiency and are widely used in preclinical research on T cell therapy because they do not have rejection reactions to transplanted tumors and T cells. In this example, 6-week-old female NSG mice were used, and the difference in body weight of each batch of experiments was controlled within 2 g. Mice were housed in individually ventilated cages within specific pathogen-free (SPF) clean-grade barriers, provided with a normal diet and drinking water with an acidic pH to prevent pathogen contamination.

In order to construct a xenograft hematological tumor model, Raji-luc cells with high expression of CD19 were selected as tumor-bearing cells, which express the luciferase gene, so they can be monitored in real time by means of fluorescein chemiluminescence and in vivo imaging in mice Changes in the development of blood tumors. In order to fully prove the effect of CD146cyt-41BB-CD3ζ CAR-T cells on tumor treatment, experiments were carried out at the effect-to-target ratio of 1:1 and 1:2, respectively. The specific experimental procedures are as follows.

Day 0: Raji-luc cells were injected into NSG mice by tail vein at an inoculation dose of 1.5*10 ⁶ (effect-to-target ratio 1:1) or 1*10 ⁶ (effect-target ratio 1:2) cells/mouse ;

Day 4: The positive rate of CAR-T cells in each group was detected by flow cytometry (detection of the percentage of EGFR ⁺ T cells in the total T cells);

Day 5: The tumor-bearing mice were numbered and randomly divided into 3 groups, with three mice in each group. The three groups of mice were: human T cell group (injection of untransfected human primary T cells), 41BB CAR-T group (injection of FMC63-4-1BB-2 ^nd -CAR T cells), CD146cyt-41BB-CAR -T group (injection of FMC63-CD8TM-CD146cyt-4-1BB-CD3-CAR T cells);

41BB CAR-T group and CD146cyt-41BB-CAR-T group were inoculated with 1.5*10 ⁶ (effect-target ratio 1:1) or 5*10 ⁵ (effect-target ratio 1:2) CAR-T cells/only Dosage CAR-T cells were injected into the tail vein (the total number of T cells injected = 1.5*10 ⁶ or 5*10 ⁵ /CAR-T cell positive rate); the human T cell group was injected into the tail vein with a total T cell number similar to that of the other two groups Human primary T cells served as controls.

On the 8th day, 12th day, 16th day, 20th day, and 24th day, the growth of Raji-luc cells in each mouse was detected by the small animal in vivo imager lumina3 to evaluate CAR - In vivo tumor killing efficiency of T cells, the results are shown in FIG. 7 .

The results showed that when the effect-to-target ratio was 1:1, both the CD146cyt-41BB CAR-T group and the 41BB CAR-T group could inhibit the growth of hematological tumors, with similar effects; while when the effect-to-target ratio was 1:2, CD146cyt- The 41BB CAR-T group had a more significant tumor inhibitory effect than the 41BB CAR-T group.

Example 8. In vivo killing activity of CAR-T cells against solid tumors

In order to construct a solid tumor model, NSG mice were used in this example to subcutaneously inoculate the aforementioned Raji-luc cells with tumors, and each mouse was inoculated with 3* ¹⁰⁶ cells. On the 7th day after inoculation, the tumor-bearing mice were numbered and randomized. Divide into 3 groups with two mice in each group. The three groups of mice were: human T cell group (injection of untransfected human primary T cells), 41BB CAR-T group (injection of FMC63-4-1BB-2 ^nd -CAR T cells), CD146cyt-41BB-CAR -T group (injection of FMC63-CD8TM-CD146cyt-4-1BB-CD3-CAR T cells). Each mouse in each group was injected with 4*10 ⁶ corresponding T cells, and the images were detected on the 13th day and the 18th day after inoculation respectively, and the results are shown in FIG. 8 .

The results showed that compared with the human T cell group, the tumor fluorescence in the 41BB CAR-T group was significantly reduced, indicating that the 41BB second-generation CAR-T cells had a certain ability to kill solid tumors; compared with the 41BB CAR-T group, The tumor fluorescence in the CD146cyt-41BB-CAR-T group was also significantly reduced, indicating that CD146cyt-41BB-CAR T cells were superior to 41BB second-generation CAR T cells in killing solid tumors.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any incorporated reference and this specification, this specification shall prevail. Furthermore, any particular embodiment of the invention which falls within the scope of the prior art may be expressly excluded from any one or more claims. Since said embodiments are considered known to those skilled in the art, they may be excluded even if said exclusions are not explicitly listed in the present application. Any particular embodiment of the invention may be excluded from any claim for any reason, whether related to the existence of prior art or not.

While the invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition, method, method step to the objective, spirit and scope of the invention. All such modifications are intended to be within the scope of the claims.

Claims

A chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, an intracellular signaling domain and an intracellular co-stimulatory domain, wherein the intracellular co-stimulatory domain comprises a domain capable of binding and First co-stimulatory domain that activates lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation.
The CAR according to claim 1, wherein said first co-stimulatory domain comprises a sequence derived from the cytoplasmic region of the CD146 molecule.
The CAR according to claim 1 or 2, wherein the first co-stimulatory domain comprises a KKGK motif at the proximal end of the CD146 cytoplasmic region.
The CAR according to any one of claims 1-3, wherein the first co-stimulatory domain comprises a CD146 cytoplasmic region, or a membrane proximal fragment thereof.
The CAR according to claim 4, wherein the length of the near-membrane fragment of the CD146 cytoplasmic region is 4-55 amino acids, preferably 10-45 amino acids, more preferably 15-35 amino acids, still more preferably 15-26 amino acid.
The CAR according to claim 4 or 5, wherein the membrane proximal fragment of the CD146 cytoplasmic region comprises amino acids 584-609 of the CD146 molecule.
The CAR according to any one of claims 1-6, wherein the intracellular co-stimulatory domain further comprises a second co-stimulatory domain selected from the group consisting of 4-1BB (CD137), OX40 ( CD134), ICOS (CD278), 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA- 1), the intracellular signaling regions of MyD88, CD2, CD4, CD7, LIGHT, NKG2C and B7-H3, or any combination thereof.
The CAR according to claim 7, wherein the second co-stimulatory domain is the intracellular signaling region of 41BB.
The CAR according to claim 7 or 8, wherein the C-terminus of the first costimulatory domain is linked to the N-terminus of the second costimulatory domain, optionally via a linker.
The CAR according to claim 7, wherein the first costimulatory domain is CD146 cytoplasmic region, the second costimulatory domain is the intracellular signaling region of 41BB, and the C-terminus of the first costimulatory domain Linked to the N-terminus of the second co-stimulatory domain.
The CAR according to any one of claims 1-10, wherein the intracellular signaling domain comprises a domain selected from the group consisting of TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, ICOS (CD278 ) and the signaling region of CD66d, or any combination thereof.
The CAR according to claim 11, wherein the intracellular signaling domain comprises the signaling region of CD3ζ.
The CAR according to any one of claims 7-12, wherein the arrangement order of the first co-stimulatory domain, the second co-stimulatory domain and the intracellular signaling domain from the N-terminus to the C-terminus is:

First Costimulatory Domain-Second Costimulatory Domain-Intracellular Signaling Domain.
The CAR according to any one of claims 1-13, wherein the transmembrane domain comprises an alpha chain selected from T cell receptor (TCR), a beta chain of TCR, a zeta chain of TCR, CD3ε, CD3ζ, CD4, The transmembrane domain of CD5, CD8α, CD9, CD16, CD19, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (41BB), CD152, CD154 and PD1 or any combination thereof.
The CAR according to any one of claims 1-14, wherein the antigen binding domain binds one or more tumor associated antigens (TAAs).
The CAR according to claim 15, wherein the TAA is selected from 5T4, alpha-fetoprotein, BCMA, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123 , CD138, c-Met, CSPG4, ROR1, GPC3, Tyrp-1, TACI, ALK, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, ERBB2, FLT3, melanoma-associated antigen, mesothelin , MUC-1, VEGFR2, preferably, the antigen-binding domain specifically binds to CD19.
The CAR according to any one of claims 1-16, wherein the antigen binding domain is selected from an antigen binding domain derived from an antibody against the antigen and an antigen binding domain derived from a natural ligand of the antigen .
The CAR according to claim 17, wherein the antigen-binding domain derived from an antibody is in the form of a scFv, Fab or domain antibody (dAb).
The CAR according to any one of claims 1-18, wherein the CAR further comprises a hinge region, preferably the hinge region comprises a hinge selected from CD8, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE and IgM or its fragment.
A nucleic acid sequence encoding a CAR according to any one of claims 1-19.
A nucleic acid construct comprising a nucleic acid sequence encoding the CAR according to any one of claims 1-19.
The nucleic acid construct according to claim 21, which has the following structure:

BD-hinge-TM-Costi-Singal;

in:

BD is the nucleotide sequence encoding the antigen binding domain;

hinge is the nucleotide sequence encoding the hinge region;

TM is a nucleotide sequence encoding a transmembrane domain;

Costi is a nucleotide sequence encoding an intracellular co-stimulatory domain;

Signal is a nucleotide sequence encoding an intracellular signaling domain.
The nucleic acid construct according to claim 22, which has the following structure:

BD-hinge-TM-Costi1-Costi2-Singal;

in:

BD is the nucleotide sequence encoding the antigen binding domain;

hinge is the nucleotide sequence encoding the hinge region;

TM is a nucleotide sequence encoding a transmembrane domain;

Costi1 is a nucleotide sequence encoding the first co-stimulatory domain;

Costi2 is a nucleotide sequence encoding a second co-stimulatory domain;

Signal is a nucleotide sequence encoding an intracellular signaling domain.
A vector comprising a nucleic acid sequence according to claim 20 or a nucleic acid construct according to any one of claims 21-23.
The vector according to claim 24, wherein said vector is selected from the group consisting of lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors and artificial chromosomes.
A cell expressing a CAR according to any one of claims 1-19.
Cells according to claim 26, wherein said cells are immune cells, preferably T cells, NK cells or macrophages.
The cell according to claim 26, wherein said cell is a stem cell, preferably a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a mesenchymal stem cell, a hematopoietic stem cell or a lymphoid progenitor cell.
A method of producing a cell according to any one of claims 26-28, comprising the step of transducing or transfecting the cell with the vector of claim 24 or 25.
The method according to claim 29, further comprising the step of expanding and/or activating cells before or after said transduction or transfection.
A composition comprising the CAR according to any one of claims 1-19, the nucleic acid sequence according to claim 20, the nucleic acid construct according to any one of claims 21-23, the CAR according to claim 24 or 25 A carrier, or a cell according to any one of claims 26-28, and a pharmaceutically acceptable carrier or excipient.
The composition according to claim 31, wherein said composition further comprises a second therapeutic agent, preferably said second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.
A method of treating cancer in a subject comprising administering to said subject an effective amount of a cell according to any one of claims 26-28.
The method according to claim 33, wherein said cells are autologous or allogeneic to said subject.
The method according to claim 34, comprising the steps of:

(i) isolating a sample containing cells from said subject;

(ii) transducing or transfecting said cells with the vector of claim 24 or 25; and

(iii) administering the cells obtained in step (ii) to a subject.
The method according to any one of claims 33-35, wherein said method further comprises administering a second therapeutic agent, preferably said second therapeutic agent is selected from the group consisting of antibodies, chemotherapeutic agents and small molecule drugs.
The method according to any one of claims 33-36, wherein the cancer is selected from lymphoma, multiple myeloma, leukemia and solid tumors.