CN112204135A

CN112204135A - Immune cell expressing CD3 antibody receptor complex and use thereof

Info

Publication number: CN112204135A
Application number: CN202080003009.1A
Authority: CN
Inventors: 王文博; 郭佩佩; 冯爱华; 林彦妮
Original assignee: Cure Genetics Co ltd
Current assignee: Cure Genetics Co ltd
Priority date: 2019-07-06
Filing date: 2020-07-03
Publication date: 2021-01-08

Abstract

一种改造的免疫细胞，其不表达T细胞受体(TCR)，并包含CD3抗体受体复合物。还涉及包含所述改造的免疫细胞和双特异性抗体的药物组合物，以及所述药物组合物在制备药物中的用途。An engineered immune cell that does not express the T cell receptor (TCR) and contains the CD3 antibody receptor complex. It also relates to a pharmaceutical composition comprising the engineered immune cells and the bispecific antibody, and the use of the pharmaceutical composition in the preparation of a medicament.

Description

Immune cell expressing CD3 antibody receptor complex and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to an immune cell expressing a CD3 antibody receptor complex, wherein the CD3 antibody receptor complex can be independent of TCR expression.

Background

In recent years, immunotherapy approaches to chimeric antigen receptor T cell (CAR-T) therapy have become hot of research worldwide due to significant clinical efficacy in the field of cancer therapy. At present, the clinical preparation of CAR-T cells is generally from patients per se, and therefore, the CAR-T cells are autologous. However, the autologous CAR-T has many problems in clinical application, such as long preparation period and high preparation cost. When the immune function state of a patient is poor, CAR-T cells cannot be successfully prepared.

There is also a method for preparing universal CAR-T by specifically knocking out certain immune related genes by using CRISPR/Cas9 nuclears and other gene editing tools. However, the long-standing CAR-T cells may cause loss of normal B cells and immunoglobulins, and CAR-T targeting solid tumors presents safety problems such as target-free and tumor-free cytotoxicity.

Therefore, there is still a need to develop a more safe and controllable cell therapy.

Disclosure of Invention

The present application provides an engineered immune cell comprising a CD3 antibody receptor complex, and which does not express a T Cell Receptor (TCR). The CD3 antibody receptor complex described herein can be expressed in a TCR-independent format. And can be recognized by a common CD3 antibody. The engineered immune cells described herein are capable of being activated and secreting cytokines upon stimulation by the CD3 antibody. The engineered immune cells described herein are also capable of killing tumor cells in combination with anti-CD 3 anti-CD 19 bispecific antibodies.

In one aspect, the present application provides an engineered immune cell comprising a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein, wherein the first CD3 recombinant protein comprises: (1) a first extracellular domain comprising an extracellular domain derived from a CD3epsilon domain, (2) a first transmembrane domain, (3) a first intracellular domain; the second CD3 recombinant protein comprises: (1) a second extracellular domain comprising an extracellular domain derived from any one selected from the group consisting of a CD3gamma domain and a CD3delta domain, (2) a second transmembrane domain, (3) a second intracellular domain, and which does not express a T Cell Receptor (TCR).

In certain embodiments, the engineered immune cells comprise T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells.

In certain embodiments, the extracellular domain of the CD3epsilon domain comprises the amino acid sequence set forth in SEQ ID No. 1.

In certain embodiments, the second extracellular domain comprises an extracellular domain derived from a CD3gamma domain.

In certain embodiments, the extracellular domain of the CD3gamma domain comprises the amino acid sequence set forth in SEQ ID No. 2.

In certain embodiments, the second extracellular domain comprises an extracellular domain derived from the delta domain of CD 3.

In certain embodiments, the extracellular domain of the delta domain of CD3 comprises the amino acid sequence set forth in SEQ ID No. 4.

In certain embodiments, the first transmembrane domain and the second transmembrane domain are the same or different.

In certain embodiments, the transmembrane domain does not comprise a transmembrane domain derived from CD3

In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from any one of the proteins selected from the group consisting of: CD8 alpha, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3epsilon, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.

In certain embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO 7.

In certain embodiments, the first intracellular domain and the second intracellular domain are the same or different.

In certain embodiments, at least one of the first intracellular domain and the second intracellular domain comprises a co-stimulatory domain and/or a signaling domain.

In certain embodiments, the co-stimulatory domain comprised in the first intracellular domain and the co-stimulatory domain comprised in the second intracellular domain are the same or different.

In certain embodiments, the co-stimulatory domain comprises a co-stimulatory domain derived from any one or more proteins selected from the group consisting of: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ε RI γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, CD40 and MyD 88.

In certain embodiments, the co-stimulatory domain comprises an amino acid sequence as set forth in SEQ ID NO 8.

In certain embodiments, the signaling domain comprised in the first intracellular domain and the signaling domain comprised in the second intracellular domain are the same or different.

In certain embodiments, the signaling domain comprises at least one Immunoreceptor Tyrosine Activation Motif (ITAM).

In certain embodiments, the signaling domain comprises a signaling domain derived from any one or more proteins selected from the group consisting of: CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FceRI gamma, FceRI beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, Simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma Herpesvirus (HSKV), DAP10, and DAP-12.

In certain embodiments, the signaling domain comprises the amino acid sequence set forth as SEQ ID No. 9.

In certain embodiments, the extracellular domain and the transmembrane domain further comprise a hinge region therebetween.

In certain embodiments, the hinge region comprises a hinge region derived from any one or more proteins selected from the group consisting of: CD8 alpha, CD28, 4-1BB, CD4, CD27, CD7 and PD-1.

In certain embodiments, the hinge region comprises an amino acid sequence set forth in any one of SEQ ID NOs 6.

In certain embodiments, one of the first intracellular domain and the second intracellular domain comprises a peptide stretch of at least two amino acids.

In certain embodiments, the first CD3 recombinant protein includes an extracellular domain derived from the CD3epsilon domain, a transmembrane region derived from CD28, an intracellular domain derived from CD28, and an intracellular domain derived from CD3 zeta. In certain embodiments, the first CD3 recombinant protein comprises the amino acid sequence set forth in SEQ ID No. 10.

In certain embodiments, the second CD3 recombinant protein comprises an extracellular domain derived from CD3gamma domain, a transmembrane region derived from CD28, and an intracellular domain derived from CD3 gamma.

In certain embodiments, the second CD3 recombinant protein comprises the amino acid sequence set forth in SEQ ID No. 11.

In certain embodiments, the second CD3 recombinant protein comprises an extracellular domain derived from the CD3gamma domain, a transmembrane region derived from CD28, and the peptide stretch.

In certain embodiments, the second CD3 recombinant protein comprises the amino acid sequence set forth in SEQ ID No. 12.

In certain embodiments, the engineered immune cell further comprises a third CD3 recombinant protein, the third CD3 recombinant protein comprising: (1) a third extracellular domain comprising an extracellular domain derived from any one selected from the group consisting of a CD3gamma domain or a CD3delta domain, (2) a third transmembrane domain, and (3) a third intracellular domain.

In certain embodiments, the third extracellular domain is the same or different from the second extracellular domain.

In certain embodiments, the third transmembrane domain is the same as or different from the first transmembrane domain and/or the second transmembrane domain.

In certain embodiments, the third intracellular domain is the same as or different from the first intracellular domain and/or the second intracellular domain.

In certain embodiments, the engineered immune cell has down-regulated expression and/or activity of the Major Histocompatibility Complex (MHC).

In certain embodiments, the MHC complex comprises B2M.

In certain embodiments, the engineered immune cell comprises a Chimeric Antigen Receptor (CAR) and/or a chimeric autoantibody receptor (CAAR).

In another aspect, the present application provides a pharmaceutical composition comprising said engineered immune cell and a pharmaceutically acceptable adjuvant.

In certain embodiments, the pharmaceutical composition comprises an antibody.

In certain embodiments, the antibody is capable of recognizing and/or binding to the CD3 antibody receptor complex.

In certain embodiments, the antibody comprises a bispecific antibody.

In certain embodiments, the bispecific antibody is derived from the immune cell.

In certain embodiments, the bispecific antibody is capable of recognizing and/or binding to a receptor on the surface of a target cell.

In certain embodiments, the target cell is a tumor cell.

In certain embodiments, the receptor on the surface of the target cell is selected from one of the following: CD, VEGFR, CD44V, CD79, CD123, CD133, CD137, CD151, CD171, CD276, CLL, B7H, BCMA, VEGFR-2, EGFR, GPC, PMSA, CEACAM, c-Met, EGFRvIII, ErbB, HER-2, HER, ErbB/HER-4, EphA, IGF1, GD, O-acetyl GD, GHRHR, GHR, GHFlt, KDR, Flt, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR, TGTGTGEAR, IL6, gp130, Lewis, TNFR, PD-L, LRPCL, MUCA, HVLA, TGFBR, TFR, TFFBR, TFR, TW-LRP-C, TRPC, TLR, TRPC, Robol, Frizzled receptor (Frizzled), OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin18.2, folate receptor alpha, folate receptor beta, GPC2, CD70, BAFF-R, and TROP-2.

In another aspect, the present application provides nucleic acid molecules encoding the CD3 antibody receptor complex in the engineered immune cells.

In another aspect, the present application provides a vector comprising said nucleic acid molecule.

In certain embodiments, the vector is a viral vector.

In certain embodiments, the vector is a lentiviral vector.

In another aspect, the present application provides a cell comprising said nucleic acid molecule and/or said vector.

In another aspect, the present application provides the use of said modified immune cell and/or said pharmaceutical composition for the preparation of a medicament for the treatment of a tumor.

In certain embodiments, the tumor comprises a solid tumor and a non-solid tumor.

In certain embodiments, the tumor is selected from the group consisting of: lymphoma, leukemia, and multiple myeloma.

In another aspect, the present application provides a method of treating a tumor, the method comprising administering the engineered immune cell, the pharmaceutical composition, to a subject in need thereof.

In certain embodiments, the tumor is selected from the group consisting of: lymphoma, leukemia, and multiple myeloma. .

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIG. 1 shows a schematic representation of the killing of target cells by an engineered immune cell in combination with a bispecific antibody as described herein.

Figure 2 shows engineered immune cells expressing the CD3 antibody receptor complex described herein.

Figure 3 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody UCHT1 in human primary T cells of TCR KO.

Figure 4 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody HIT3a in human primary T cells of TCR KO.

FIG. 5 shows that the CD3 antibody receptor complex is not recognized by the CD3 antibody SP34-2 in the human primary T cells of TCR KO

Figure 6 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody OKT3 in human primary T cells of TCR KO.

FIG. 7 shows that the engineered immune cells described herein are activated by the CD3 antibody to express the cell activation signature CD 137.

Figure 8 shows the tumor cell activation mediated by anti-CD 3 anti-CD 19 bispecific antibodies of engineered immune cells described herein.

Figure 9 shows that engineered immune cells described herein mediate killing of tumor cells by anti-CD 3 anti-CD 19 bispecific antibodies.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "CD 3 antibody" generally refers to an antibody that specifically recognizes the CD3 subunit (e.g., CD3epsilon, CD3gamma, CD3delta, or complexes thereof), which may be a monoclonal antibody that recognizes only CD3, or a multi-target antibody that recognizes both CD3 and other targets.

In the present application, the term "CD 3 antibody receptor complex" generally refers to a receptor capable of being recognized by a CD3 antibody, which may comprise at least 2 (e.g., 3) CD3 subunits (e.g., CD3epsilon and CD3delta, or CD3epsilon and CD3 gamma). The CD3 antibody receptor complex consists of at least 2 (e.g., 3, 4, 5, 6, or more) CD3 recombinant proteins that may comprise the extracellular, transmembrane, and intracellular domains of a CD3 subunit (e.g., CD3epsilon, CD3gamma, and/or CD3 delta) for the CD3 recombinant protein. The conformation of CD3epsilon and CD3gamma or CD3delta extracellular domain after binding is close to the native conformation of CD3epsilon heterodimers in TCR complexes, including the epitopes recognized by most CD3 antibodies such as UCHT1, OKT3, and the like. The conformation formed by the extracellular domain of CD3epsilon alone does not include the binding epitope of most CD3 antibody clones.

In the present application, the term "first CD3 recombinant protein" generally refers to a recombinant protein comprising an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (e.g., CD3 epsilon). The CD3 antibody receptor complex can comprise 1 or more (e.g., 2, 3, 4, or more) first CD3 recombinant proteins.

In the present application, the term "first extracellular domain" generally refers to the portion of the extracellular domain of the first CD3 recombinant protein that may comprise the extracellular domain derived from the CD3epsilon domain.

In the present application, the term "second CD3 recombinant protein" generally refers to a recombinant protein comprising an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (e.g., CD3delta and or CD3 gamma). The CD3 antibody receptor complex can comprise 1 or more (e.g., 2, 3, 4, or more) second CD3 recombinant proteins.

In the present application, the term "second extracellular domain" generally refers to the portion of the extracellular domain of the second CD3 recombinant protein that may comprise the extracellular domain derived from CD3delta and or CD3gamma domains.

In the present application, the term "third CD3 recombinant protein" generally refers to a recombinant protein comprising an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (e.g., CD3delta and or CD3 epsilon). The CD3 antibody receptor complex can comprise 1 or more (e.g., 2, 3, 4, or more) third CD3 recombinant proteins.

In the present application, the term "third extracellular domain" generally refers to the portion of the extracellular domain of a third CD3 recombinant protein that may comprise the extracellular domain derived from CD3delta and or CD3epsilon domain. The third ectodomain may be derived from the same or different CD3 subunit (e.g., CD3delta and or CD3 gamma) as the second ectodomain.

In the present application, the term "antibody" generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen. For example, an antibody may comprise an immunoglobulin of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dabs), single chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., Fab', and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, as well as any antigen-binding antibody fragments and derivatives thereof described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further distinguished as hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with more conserved regions termed Framework Regions (FRs). Each VH and VL may be composed of three CDRs and four FR regions, which may be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

In the present application, the term "transmembrane domain" generally refers to a segment of a cell membrane-spanning sequence in a cell surface protein, which may comprise a hydrophobic alpha helix. The transmembrane domain may be linked to an intracellular signalling domain and serves to transmit signals in this application, and may be derived from any type I, type II or type III transmembrane protein. In the present application, the transmembrane domain may not comprise a transmembrane protein derived from CD3 (e.g., CD3epsilon, CD3gamma, and/or CD3 delta).

In the present application, the term "Immunoreceptor Tyrosine Activation Motif (ITAM)" generally refers to a conserved sequence of more than ten amino acids, which is often found in the intracellular region of transmembrane proteins of certain cells of the immune system. TAMs are important transduction signals for immune cells. Thus, they are often found in the intracellular regions of important cellular signaling molecules, such as the CD3 and zeta chains of the T cell receptor complex, the CD79 α and β chains of the B cell receptor complex, and certain Fc receptors. When these receptor molecules interact with their ligands, tyrosine residues on intracellular ITAMs are phosphorylated, and the phosphorylated ITAMs bind to the encapsulated free protein with SH2 domain, allowing immune cell signaling to be transmitted to downstream signaling molecules.

In the present application, the term "Chimeric Antigen Receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding an Antigen and at least one intracellular domain. A CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which can include an antigen (e.g., a tumor-specific antigen and/or a tumor-associated antigen) binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In the present application, the CAR may be combined with a T cell receptor activating intracellular domain based on the antigen (e.g., CD70) specificity of the antibody. Genetically modified CAR-expressing T cells can specifically recognize and eliminate malignant cells that express a target antigen. For a description of CAR and CAR-T cells see, e.g., Sadelain M, Brentjens R, Rivi' ere i, the basic principles of molecular antigen receiver design, cancer disease v.2013; 388-; turtle CJ, Hudecek M, Jensen MC, ridsell sr. engineered T cells for anti-cancer therapy. curr Opin immunol.2012; 24(5) 633-639; dotti G, Gottschalk S, Savoldo B, Brenner mk.design and depth of thermal using polymeric antigen receivers-expressing T cells. immunological rev.2014; (257) 107-126; and WO2013154760, WO 2016014789.

In the present application, the term "chimeric autoantibody receptor (CAAR)" generally refers to proteins comprising autoantigens capable of being recognized by autoantibodies, known in the english name of chimeric autoantibody receptors. CAAR can direct immune cells genetically modified to express CAAR to attack B cells expressing antibodies capable of recognizing the antigen.

In the present application, the term "bispecific antibody" generally refers to an antibody having binding sites for two different antigens within a single antibody molecule. For example, one of the antigens may be CD 3.

In the present application, the term "peptide fragment" generally refers to a polypeptide that is composed of at least two (e.g., four, five, six, or more each) amino acids. It can include any whole molecule having amino functionality and acid functionality and comprising naturally occurring amino acid polymers, including natural amino acids and artificial amino acids.

In the present application, the term "co-stimulatory domain" generally refers to an intracellular domain that can provide an immune co-stimulatory molecule, a cell surface molecule required for an effective response of lymphocytes to an antigen.

In the present application, the term "hinge region" generally refers to the connection region between an extracellular domain (e.g., CD3 extracellular domain) and a transmembrane region.

In the present application, the term "signaling domain" generally refers to a domain that is located inside a cell and is capable of transducing a signal. In the present application, the intracellular signaling domain may conduct a signal into a cell. In general, a signaling domain is any contiguous stretch of amino acids that is used to direct protein targeting. In some cases, the signaling domain may be derived from CD3 ζ. CD3 ζ can form a T cell receptor-CD 3 complex with T cell receptor subunits and CD3-gamma, -delta, and-epsilon. CD3 ζ contains three ITAM motifs, the ITAM sequences mediating intracellular signaling activation of the TCR. The zeta chain is a protein tyrosine kinase substrate activated by receptor, and can be quickly subjected to tyrosine phosphorylation after a TCR receptor is combined with a polypeptide MHC complex to participate in the transduction of lymphocyte activation signals. CD3 ζ therefore plays a key role in antigen recognition and TCR signaling.

In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such formulations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and/or additives that may be used in the formulations described herein include: for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, proteinaceous excipients (e.g., serum albumin, gelatin, casein), salt-forming counterions (e.g., sodium), and the like.

In the present application, the term "immune cell" generally refers to a cell involved in an immune response, e.g., promoting an immune effector response. Examples of immune cells include, but are not limited to, T cells, B cells, Natural Killer (NK) cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages. The term also includes engineered immune cells, such as immune cells that are genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.

In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, for transferring the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly for inserting a DNA or RNA into a cell, a vector mainly for replicating a DNA or RNA, and a vector mainly for expression of transcription and/or translation of a DNA or RNA. The vector also includes vectors having a plurality of the above-described functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell containing the vector. The vector may encompass additional features beyond the transgene insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. Vectors referred to as expression vectors (expression constructs) are used in particular for expressing transgenes in target cells and generally have control sequences. The vectors described herein may be expression vectors, and may include viral vectors (lentiviral and/or retroviral vectors), phage vectors, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) of P1 origin, and/or plasmids.

In the present application, the term "treatment" generally means: (i) preventing the occurrence of a disease, disorder, or condition in a patient who may be predisposed to the disease, disorder, and/or condition, but has not yet been diagnosed as having the disease; (ii) inhibiting, i.e., arresting the development of, the disease, disorder or condition; and (iii) ameliorating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition and/or symptoms associated with the disease, disorder, and/or condition.

In the present application, the term "Major Histocompatibility Complex (MHC)" generally refers to a series of proteins located on the surface of cells that help the immune system recognize foreign substances, and MHC mainly comprises MHC class I and MHC class II molecules. Class I MHC molecules can span the membrane of almost all cells of an organism, while class II molecules are usually present on immune cells. Wherein MHC class I molecules, also known as class I major histocompatibility complex, heterodimeric glycoproteins consisting of two peptide chains joined by a non-covalent bond; one of them is called heavy chain, the structure is polymorphic, and the other is light chain or called beta 2 microglobulin (B2M). Functionally, MHC class I molecules present polypeptides that are degraded intracellularly and are not self-proteins, thereby activating the immune system. Human class I MHC molecules are classified into classical HLA molecules (HLA-A, HLA-B, HLA-C) and non-classical HLA molecules (HLA-E, HLA-G, HLA-F). In the present application, the engineered immune cell may not express active MHC, and the "not expressing active MHC" may include loss of activity of the expressed MHC to activate the immune system and/or loss of MHC class I molecules on the cell surface (e.g., loss of HLA-a/B/C/E/F/G). In some cases, the cell surface can be depleted of MHC class I molecules by editing the B2M or the corresponding heavy chain gene.

In the present application, the term "B2M" refers generally to β -2 microglobulin, and refers generally to the light chain of MHC class I molecules, and is therefore an integral part of the Major Histocompatibility Complex (MHC). In the human genome, B2M is encoded by the B2m gene located on chromosome 15, while other MHC genes exist as gene clusters on chromosome 6. Human B2M protein has 119 amino acids (see UniProt database code P61769). In a mouse model lacking β -2 microglobulin, it can be shown that B2M is essential for MHC class I molecule presentation on the cell surface, stability of the polypeptide binding groove. MHC class I molecules are present on the surface of all nucleated cells in the human body, mismatch of MHC causes immune rejection, resulting in destruction of grafts, and removal of MHC class I molecules on the cell surface by knocking out BETA 2 μm gene can prevent the occurrence of mismatch.

In the present application, the term "CD 3" generally refers to a CD3 protein multi-subunit complex, which CD3 protein multi-subunit complex is composed of 6 different polypeptide chains (subunits). In mammals, the polypeptide chain of CD3 can comprise one CD3gamma (γ) chain, one CD3gamma (δ) chain, two CD3epsilon (ε) chains and two CD3zeta (ζ) chains.

In the present application, the term "CD 3" refers to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). The term encompasses "full-length" and unprocessed CD3 protein as well as any form of protein or one or more CD3 chains (polypeptides) (e.g., mature polypeptides) that results from processing in a cell. The term also encompasses naturally occurring variants and isoforms of CD3, such as splice variants or allelic variants.

Likewise, the terms "CD 3 epsilon", "CD 3 gamma", "CD 3 gamma" and/or "CD 3 zeta" in the present application refer to any native CD3 from any vertebrate source, including mammals, such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). "CD 3 epsilon", "CD 3 gamma", "CD 3 gamma" and/or "CD 3 zeta" encompass "full-length" and unprocessed CD3epsilon "," CD3gamma "," CD3gamma "and/or" CD3zeta "proteins, respectively, as well as any form of CD3 chain (polypeptide) (e.g., mature polypeptide) derived from processing in a cell. The term also encompasses naturally occurring variants and isoforms of the CD3 chain, such as splice variants or allelic variants. For example, the amino acid sequence of exemplary CD3delta can be found in UniProt database accession number P04234, the amino acid sequence of exemplary CD3epsilon can be found in UniProt database accession number P07766, and the amino acid sequence of exemplary CD3gamma can be found in UniProt database accession number P09693.

In the present application, the term "T Cell Receptor (TCR)" generally refers to a transmembrane protein complex involved in activation of T cells upon recognition of an antigen. TCRs are heterodimers formed by two different protein subunits. In humans, 95% of T cells express an alpha (α) chain and a beta (β) chain. The remaining 5% express gamma (. gamma.) and delta (. delta.) chains. Each chain of the TCR molecule may comprise two extramembranous domains: variable and constant regions. The variable region binds to the polypeptide/major histocompatibility complex, and the variable regions of both the alpha and beta chains contain three Complementarity Determining Regions (CDRs) that are responsible for recognition by the antigen/MHC complex. The constant region is adjacent to the cell membrane and is connected to the transmembrane region. Both extracellular constant domains of the TCR heterologous subunits comprise short binding sequences with cysteine residues within the sequence. Disulfide bonds are formed between cysteine residues to bind the two heterologous TCR subunits together. The TCR transmembrane domain contains positively charged amino acids responsible for binding to the CD3 molecule. The intracellular domain of the TCR is short and has no active domain.

TCRs recognize processed polypeptide fragments bound to MHC molecules, also referred to as MHC restriction, since recognition requires presentation of the MHC molecule. When the MHC molecules of the donor and the recipient are different, the TCR is able to recognize the difference in MHC and cause activation and expansion of T cells, possibly causing graft versus host disease (GvHD). Knocking out the TRAC gene can remove the expression of TCR alpha chain, thus removing TCR complex from the surface of T cells, thus preventing graft-versus-host disease caused by TCR recognition of alloantigen.

The CD3 multi-subunit complex and TCR can form a functional complex through non-covalent bonds, referred to as the TCR-CD3 complex. During formation of the TCR-CD3 complex, CD3epsilon molecules form heterodimers with CD3gamma and CD3delta, respectively, and CD3zeta forms homodimers with itself. A TCR-CD3 complex includes a CD epsilon: delta heterodimer, a CD epsilon: gamma heterodimer and a CD zeta: zeta homodimer and a TCR alpha: beta heterodimer. Thus, these four dimers are in a 1:1:1:1 relationship in a TCR-CD3 complex. The transmembrane region of the CD3 molecule is negatively charged by the inclusion of aspartate residues, allowing CD3 to bind to a pair of TCR subunits that are positively charged across the membrane. The CD3gamma, CD3delta, and CD3epsilon molecules are highly related cell membrane proteins of the immunoglobulin superfamily, all comprising a single extracellular immunoglobulin domain, whereas the extracellular region of CD3zeta is very short. The intracellular domains of CD3gamma, CD3delta, and CD3epsilon molecules all contain a single conserved domain Immunoreceptor Tyrosine Activation Motif (ITAM). The CD3zeta chain contains three ITAM motifs. The single TCR-CD3 complex contained 10 ITAM motifs in total, which determine the extent of TCR activation.

In the present application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may comprise: disulfide bond formation, glycosylation, lipidation (acetylation), acetylation, phosphorylation, or any other manipulation (e.g., binding to a labeling component). The term "amino acid" includes natural and/or unnatural or synthetic amino acids, including glycine as well as D and L optical isomers, as well as amino acid analogs and peptidomimetics.

In the present application, the terms "polynucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined according to ligation analysis, exons, introns, messenger RNA (mrna), transfer RNA, ribosomal RNA, short interfering RNA (sirna), short hairpin RNA (shrna), micro-RNA (mirna), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeled moiety.

In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functional variants, derivatives, analogs, homologs, and fragments thereof.

The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a naturally occurring sequence or is encoded by substantially the same nucleotide sequence and is capable of one or more of the activities of a naturally occurring sequence. In the context of this application, a variant of any given sequence refers to a sequence in which the particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one endogenous function. Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally occurring protein and/or polynucleotide, so long as the original functional activity is retained.

In the present application, the term "derivative" generally refers to a polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and/or addition of one (or more) amino acid residues from/to the sequence, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

In the present application, the term "analog" generally with respect to a polypeptide or polynucleotide includes any mimetic of a polypeptide or polynucleotide, i.e., a chemical compound that possesses at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics.

Typically, amino acid substitutions, such as at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence substantially retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.

The proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues which produce silent changes and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues, as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with similar hydrophilicity values without an electrically polar head group include asparagine, glutamine, serine, threonine, and tyrosine.

In the present application, the term "homologue" generally refers to an amino acid sequence or a nucleotide sequence having a certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" may be equivalent to sequence "identity". A homologous sequence can include an amino acid sequence that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence. Typically, homologues will comprise the same active site etc. as the subject amino acid sequence. Homology may be considered in terms of similarity (i.e., amino acid residues with similar chemical properties/functions), or may be expressed in terms of sequence identity. In the present application, a sequence having a percent identity of any one of SEQ ID NOs of the referenced amino acid sequence or nucleotide sequence refers to a sequence having said percent identity over the entire length of the referenced SEQ ID NOs.

To determine sequence identity, sequence alignments can be performed, which can be performed by various means known to those skilled in the art, e.g., using BLAST, BLAST-2, ALIGN, needlet, or megalign (dnastar) software, etc. One skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment over the full-length sequences being compared.

In this application, the term "and/or" should be understood to mean either one of the options or both of the options.

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

Detailed Description

The present application develops a regulatable cell therapy that takes full advantage of the advantages of bispecific antibody therapy and cell therapy as a combination therapy of bispecific antibody therapy and cell therapy. As used herein, a T cell is a universal T cell that expresses one of the CD3 antibody receptors and is subject to gene knockout. The CD3 antibody receptor is composed of two recombinant CD3 protein molecules. The T cells express a CD3 antibody receptor that can be recognized by bispecific antibodies, which differs from similar chimeric antibody receptor therapies in that the CD3 antibody receptor of the present application can be recognized by bispecific antibodies and expressed independently of the TCR complex, directly on the T cell surface.

The application provides the following technical scheme:

1. an engineered lymphocyte that is deficient in a TCR molecule, the lymphocyte expressing a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising at least two CD3 recombinant proteins; wherein the first CD3 recombinant protein comprises:

a. an extracellular domain comprising a CD3epsilon domain;

b. a transmembrane domain;

c. an intracellular domain; and

wherein the second CD3 recombinant protein comprises:

d. an extracellular domain comprising CD3delta and/or CD3gamma domains;

e. a transmembrane domain;

f. an intracellular domain;

wherein the intracellular domain consists of an intracellular co-stimulatory signaling domain and an intracellular signaling domain, or the intracellular domain is a peptide stretch of at least two amino acids; wherein at least one intracellular domain of the first CD3 recombinant protein and the second CD3 recombinant protein consists of an intracellular costimulatory signaling domain and an intracellular signaling domain.

2. The lymphocyte of claim 1, wherein the lymphocyte is a T cell, a B cell, an NK cell, or a macrophage.

3. The lymphocyte of claim 2, wherein the lymphocyte is a T cell.

4. The lymphocyte of any of the preceding claims, wherein the a. extracellular domain comprises the extracellular domain of CD3epsilon shown in SEQ ID No:1, or a variant thereof.

5. The lymphocyte of claim 4, wherein the a. extracellular domain is the extracellular domain of CD3epsilon shown in SEQ ID No. 1.

6. The lymphocyte of any of the preceding claims, wherein the d.ectodomain comprises the ectodomain of CD3gamma domain.

7. The lymphocyte of claim 6, wherein the d. extracellular domain comprises the extracellular domain of CD3gamma shown in SEQ ID No. 2, or a variant thereof.

8. The lymphocyte of claim 7, wherein the d. extracellular domain is the extracellular domain of CD3gamma shown in SEQ ID No. 2.

9. The lymphocyte of any of the preceding claims, wherein the d.extracellular domain comprises the extracellular domain of CD3delta domain, or a variant thereof.

10. The lymphocyte of claim 9, wherein the d. extracellular domain comprises the extracellular domain of CD3delta shown in SEQ ID No. 4, or a variant thereof.

11. The lymphocyte of claim 10, wherein the d. extracellular domain is the extracellular domain of CD3delta shown in SEQ ID No. 4.

12. The lymphocyte of any of the preceding claims, wherein transmembrane domains b and e comprise at least one of: a transmembrane domain of CD8 α, a transmembrane domain of CD28, a transmembrane domain of 4-1BB, a transmembrane domain of CD4, a transmembrane domain of CD27, a transmembrane domain of CD7, a transmembrane domain of PD-1, a transmembrane domain of TRAC, and a transmembrane domain of TRBC.

13. The lymphocyte of claim 12, wherein the transmembrane domain of CD28 comprises the transmembrane domain of CD28 shown in SEQ ID No. 7, or a variant thereof.

14. The lymphocyte of any of the preceding claims, wherein the intracellular co-stimulatory signaling domain comprises at least one of: co-stimulatory molecules consisting of co-stimulatory signaling regions in CD28, 4-1BB, CD40L, TIM1, CD226, DR3, SLAM, ICOS, OX40, NKG2D, 2B4, CD244, fcsry, BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT and DAP12, and combinations thereof.

15. The lymphocyte of claim 14, wherein the intracellular costimulatory signaling domain comprises the costimulatory signaling region of CD28, shown in SEQ ID No:8, or a variant thereof.

16. The lymphocyte of claim 14, wherein the intracellular costimulatory signaling domain comprises the costimulatory signaling region of 4-1BB, or a variant thereof.

17. The lymphocyte of any of the preceding claims, wherein the intracellular signaling domain comprises at least one of: CD3zeta active region, CD3delta active region, CD3gamma active region, FceRI active region, immunologlulin alpha (Iga) active region, Igbeta active region, bovine leukavirus gp30 active region, Ep-stein-Barr virus (EBV) LMP2A active region, simian immunodeficiencyvirus PBj14 Nef active region, HSKV active region, DAP-12 active region, a domain comprising at least one ITAM (tyrosine activation motif), and domains formed by combinations of the above domains.

18. The lymphocyte of claim 17, wherein the CD3zeta active region comprises the sequence shown in SEQ ID No. 9, or a variant thereof.

19. The lymphocyte of any preceding claim, wherein the recombinant CD3 protein further comprises a hinge region between the extracellular domain and the transmembrane region.

20. The lymphocyte of claim 19, wherein the hinge region comprises at least one of: the hinge region of CD8 alpha, CD28, 4-1BB, CD4, CD27, CD7 and PD-1.

21. The lymphocyte of any of the preceding claims, wherein the intracellular domain is a peptide stretch of at least two amino acids.

22. The lymphocyte of any of the preceding claims, wherein the first recombinant CD3 protein is selected from one of the following recombinant proteins: CD3epsilon ectodomain-CD 8 alpha hinge region-CD 8 alpha transmembrane region-4-1 BB costimulatory signaling region-CD 3zeta active region, CD3epsilon ectodomain-CD 8 alpha transmembrane region-4-1 BB costimulatory signaling region-CD 3zeta active region, CD3epsilon ectodomain-CD 28 hinge region-CD 28 transmembrane region-CD 28 costimulatory signaling region-CD 3zeta active region, and CD3epsilon ectodomain-CD 28 transmembrane region-CD 28 costimulatory signaling region-CD 3zeta active region.

23. The lymphocyte of claim 22, wherein the first recombinant protein of CD3 has the sequence shown in SEQ ID No. 10, or a variant thereof.

24. The lymphocyte of any of the preceding claims, wherein the second recombinant CD3 protein is selected from one of the following recombinant proteins: CD3 γ extracellular region-CD 8 α hinge region-CD 8 α transmembrane region-CD 3 γ costimulatory signaling region, CD3 γ extracellular region-CD 8 α transmembrane region-CD 3 γ costimulatory signaling region, CD3 γ extracellular region-CD 28 hinge region-CD 28 transmembrane region-CD 3 γ costimulatory signaling region, and CD3 γ extracellular region-CD 28 transmembrane region-CD 3 γ costimulatory signaling region, CD3 γ extracellular region-CD 28 hinge region-CD 28 transmembrane region-peptide segment, wherein the peptide segment can be at least 2, 4 or at least 6 amino acids.

25. The lymphocyte of claim 24, wherein the second recombinant protein of CD3 has the sequence shown in SEQ ID No. 11 and SEQ ID No. 12, or a variant thereof.

26. The lymphocyte of any of the preceding claims 1-25, wherein the second recombinant CD3 protein is selected from one of the following recombinant proteins: CD3 δ extracellular region-CD 8 α hinge region-CD 8 α transmembrane region-CD 3 δ costimulatory signaling region, CD3 δ extracellular region-CD 8 α transmembrane region-CD 3 δ costimulatory signaling region, CD3 δ extracellular region-CD 28 hinge region-CD 28 transmembrane region-CD 3 δ costimulatory signaling region, and CD3 δ extracellular region-CD 28 transmembrane region-CD 3 δ costimulatory signaling region.

27. The lymphocyte of any of the preceding claims, which is devoid of an MHC molecule.

28. The lymphocyte of any of the preceding claims, wherein the intracellular domain of the first recombinant CD3 protein consists of an intracellular costimulatory signaling domain and an intracellular signaling domain.

29. The lymphocyte of any of the preceding claims, wherein the intracellular domain of the second recombinant CD3 protein consists of an intracellular costimulatory signaling domain and an intracellular signaling domain.

30. The lymphocyte of any of the preceding claims, the CD3 antibody receptor complex comprising three recombinant CD3 proteins, the first recombinant CD3 protein comprising:

a. an extracellular domain comprising a CD3epsilon domain;

b. a transmembrane domain;

c. an intracellular domain; and

wherein the second CD3 recombinant protein comprises:

d. an extracellular domain comprising the delta domain of CD 3;

e. a transmembrane domain;

f. an intracellular domain; and

wherein the third CD3 recombinant protein comprises:

g. an extracellular domain comprising a CD3gamma domain;

h. a transmembrane domain;

i. an intracellular domain;

wherein the intracellular domain consists of an intracellular co-stimulatory signaling domain and an intracellular signaling domain, or the intracellular domain is a peptide stretch of at least two amino acids; wherein at least one intracellular domain of the three CD3 recombinant proteins consists of an intracellular costimulatory signaling domain and an intracellular signaling domain.

31. A pharmaceutical composition comprising a lymphocyte according to any of claims 1-30, and a bispecific antibody that binds to the CD3 antibody receptor complex on the surface of the lymphocyte and simultaneously binds to a receptor on the surface of a target cell.

32. The pharmaceutical composition of claim 31, wherein the receptor of the target cell is selected from one of the following: CD19, CD20, CD22, CD123, CD33, BCMA, IL13R alpha, PSMA, EGFR, HER2, Mesothelin, and Claudin 18.2.

33. The pharmaceutical composition of claim 31 or 32, wherein the bispecific antibody binds to a CD3 antibody receptor complex on the surface of lymphocytes and simultaneously binds to a CD19 receptor on the surface of a target cell.

34. The pharmaceutical composition of any of claims 31-33, wherein the target cell is a tumor cell.

35. The pharmaceutical composition of any of claims 31-34, wherein the lymphocyte is a T cell, a B cell, an NK cell, or a macrophage.

36. Use of the lymphocytes according to any one of claims 1-24 in the preparation of a medicament for the treatment of a tumor.

37. The use of claim 30, wherein the medicament further comprises a bispecific antibody that binds to a CD3 antibody receptor complex on the surface of lymphocytes and simultaneously binds to a receptor on the surface of a target cell.

38. A method of treating a disease, the method comprising administering to a subject:

c. an effective amount of the lymphocyte of any of claims 1-30, and

d. an effective amount of a bispecific antibody that binds to a CD3 antibody receptor complex on the surface of lymphocytes and simultaneously binds to a receptor on the surface of a target cell.

39. The method of claim 38, wherein the receptor of the target cell is selected from one of the following: CD19, CD20, CD22, CD123, CD33, BCMA, IL13R alpha, PSMA, EGFR, HER2, Mesothelin, and Claudin 18.2.

40. The method of claim 39, wherein the target cell is a tumor cell.

CD3 antibody receptor complex

The present application provides an engineered immune cell that can comprise a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein.

In the present application, the first CD3 recombinant protein may comprise (1) a first extracellular domain, (2) a first transmembrane domain, and (3) a first intracellular domain; the second CD3 recombinant protein may comprise (1) a second extracellular domain, (2) a second transmembrane domain, and (3) a second intracellular domain.

In certain instances, the first extracellular domain can comprise an extracellular domain derived from a CD3epsilon domain, e.g., the first extracellular domain can comprise an amino acid sequence as set forth in SEQ ID No. 1. For example, the first extracellular domain can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 1.

In certain instances, the second extracellular domain can comprise an extracellular domain derived from a CD3gamma domain, e.g., the second extracellular domain can comprise an amino acid sequence as set forth in SEQ ID No. 2. For example, the second extracellular domain can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 2.

In certain instances, the second extracellular domain can comprise an extracellular domain derived from the delta domain of CD3, e.g., the second extracellular domain can comprise an amino acid sequence as set forth in SEQ ID No. 4. For example, the second extracellular domain can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID No. 4.

In the present application, the CD3 recombinant protein (e.g., the first CD3 recombinant protein and/or the second CD3 recombinant protein) may comprise a transmembrane domain (e.g., the first transmembrane domain and/or the second transmembrane domain). The transmembrane domain (e.g., the first transmembrane domain and/or the second transmembrane domain) may be derived from any type I transmembrane protein, so long as it is not a transmembrane domain of CD 3. In the present application, exemplary of the transmembrane domains (e.g., the first transmembrane domain and/or the second transmembrane domain) may include, but are not limited to, transmembrane domains derived from the following histones: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, zeta chain of T cell receptor, CD3epsilon, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, SLAM, and any other transmembrane domain independent of TCR expression, or a mutant of the above transmembrane domains.

For example, the transmembrane domain (e.g., the first transmembrane domain and/or the second transmembrane domain) may be a transmembrane domain derived from human CD 28. For example, the first transmembrane domain may comprise the amino acid sequence shown as SEQ ID NO 7. For example, the first transmembrane domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 7.

In the present application, the CD3 recombinant protein may comprise an intracellular domain (e.g., a first intracellular domain and/or a second intracellular domain). In certain instances, the intracellular domain (e.g., the first intracellular domain and/or the second intracellular domain) can comprise an intracellular co-stimulatory domain and/or an intracellular signaling domain.

In the present application, the intracellular signaling domain may comprise a domain having at least one ITAM motif. Exemplary signaling domains can be derived from a signaling domain selected from the group consisting of, but not limited to, CD3zeta, CD3delta, CD3gamma, CD3epsilon, CD79a, CD79b, FceRI gamma, FceRI beta, fcyriia, bovine leukemia virus gp30 activation region, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, kaposi's sarcoma Herpesvirus (HSKV), DAP10, and DAP-12, and variants of the foregoing.

For example, the signaling domain may be a signaling domain from the CD3zeta endodomain. For example, the signaling domain from the CD3zeta endodomain may comprise the signaling domain shown in SEQ ID NO 9. For example, the signaling domain from the CD3zeta endodomain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 9.

The CD3 antibody receptor complex, when stimulated, can transmit activation signals into the cell. Sometimes, activation of the signaling domain is not sufficient to provide sufficient activation signal, and a co-stimulatory domain is also required to provide a stimulatory signal. The co-stimulatory domain may include, but is not limited to, the following groups: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ε RI γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, co-stimulatory signaling regions in CD40 and MyD88 and combinations thereof.

For example, the costimulatory domain can be a costimulatory domain from the intracellular domain of human CD 28. For example, the costimulatory domain of the endodomain of human CD28 can comprise the costimulatory domain shown in SEQ ID NO. 8. For example, the co-stimulatory domain of the intracellular domain of human CD28 can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 8.

For example, the endodomain (e.g., first endodomain and/or second endodomain) can comprise a costimulatory domain from the human CD28 endodomain described above and a signaling domain from the CD3zeta endodomain described above.

For another example, the intracellular domain (e.g., the first intracellular domain and/or the second intracellular domain) can comprise an intracellular domain from CD3 gamma. For example, the intracellular domain of CD3gamma may comprise the amino acid sequence shown in SEQ ID NO. 3. For example, the intracellular domain of CD3gamma can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 3.

In certain instances, the intracellular domain (e.g., the first intracellular domain and/or the second intracellular domain) can comprise a peptide stretch of at least two (e.g., at least four, at least six, or more) amino acids. In certain instances, the intracellular domain can be a peptide stretch of at least two (e.g., at least four, at least six, or more) amino acids. For example, the peptide fragment may comprise the amino acid sequence shown in SEQ ID NO 19.

In the CD3 antibody receptor complex of the present application, the other portions (e.g., transmembrane domain, intracellular domain) may be the same or different for the first CD3 recombinant protein and the second CD3 recombinant protein except for the extracellular region. For example, the first transmembrane domain of the first CD3 recombinant protein and the second transmembrane domain of the second CD3 recombinant protein may be the same or different, and the first intracellular domain of the first CD3 recombinant protein and the second intracellular domain of the second CD3 recombinant protein may be the same or different. For example, the signaling domain of the first intracellular domain and the signaling domain of the second intracellular domain may be the same or different. For example, the co-stimulatory domain of the first intracellular domain and the co-stimulatory domain of the second intracellular domain may be the same or different. As long as at least one (e.g., at least two, three, four, or more) of the first and second intracellular domains comprises a co-stimulatory domain and/or a signaling domain, or is capable of providing sufficient signaling for antibody stimulation to activate the engineered immune cell.

In some cases, the intracellular domain may be an intracellular domain from CD3gamma and/or CD3delta, e.g., the intracellular domain may comprise an intracellular domain as set forth in any one of SEQ ID NOs 3 and 5. Alternatively, the intracellular domain may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in any one of SEQ ID NOs 3 and 5.

In the CD3 antibody receptor complex of the present application, the first intracellular domain of the first CD3 recombinant protein may comprise a costimulatory domain and a signaling domain, and the first intracellular domain of the second CD3 recombinant protein may be a peptide stretch of at least two (e.g., at least four, at least six, or more) amino acids.

In the CD3 antibody receptor complex of the present application, the first intracellular domain of the second CD3 recombinant protein may comprise a costimulatory domain and a signaling domain, and the first intracellular domain of the second CD3 recombinant protein may be a peptide stretch of at least two (e.g., at least four, at least six, or more) amino acids.

In the present application, the CD3 recombinant protein (first CD3 recombinant protein and/or second CD3 recombinant protein) may further comprise a hinge region. The hinge region may be between the extracellular domain and the transmembrane domain. For example, the hinge region may comprise a hinge region derived from any one or more proteins selected from the group consisting of: CD8 alpha, CD28, 4-1BB, CD4, CD27, CD7 and PD-1.

For example, the hinge region may comprise an amino acid sequence as set forth in any one of SEQ ID NOs 6. Or the hinge region may comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in any one of SEQ ID NO. 6.

For example, in the present application, the first CD3 recombinant protein may be derived from the extracellular domain of the CD3epsilon domain, from the transmembrane region of CD28, from the intracellular domain of CD28 and from the intracellular domain of CD3 zeta. For example, the first CD3 recombinant protein may comprise the amino acid sequence shown in SEQ ID NO. 10. Alternatively, the first CD3 recombinant protein can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 10.

For example, the second CD3 recombinant protein may comprise an extracellular domain derived from CD3gamma domain, a transmembrane region derived from CD28, and an intracellular domain derived from CD3 gamma. For example, the second CD3 recombinant protein may comprise the amino acid sequence shown in SEQ ID NO. 11. Alternatively, the second CD3 recombinant protein can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 11.

For example, the second CD3 recombinant protein may comprise an extracellular domain derived from CD3gamma domain, a transmembrane region derived from CD28, and the peptidyl fragment. For example, the second CD3 recombinant protein may comprise the amino acid sequence shown in SEQ ID NO. 12. Alternatively, the second CD3 recombinant protein can comprise an amino acid sequence having at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence homology to the amino acid sequence set forth in SEQ ID NO. 12.

In the present application, the CD3 antibody receptor complex may further comprise one or more (e.g., two, three, four, or more) first recombinant proteins. In the present application, the CD3 antibody receptor complex may also comprise one or more (e.g., two, three, four, or more) second recombinant proteins. In certain instances, the CD3 antibody receptor complex may further comprise a third histone, which may comprise (1) a third ectodomain comprising an ectodomain derived from any one selected from the group consisting of a CD3gamma domain or a CD3delta domain, (2) a third transmembrane domain, and (3) a third intracellular domain. The third transmembrane domain may be within the scope of the transmembrane domain described above and may be the same or different from the first transmembrane domain and/or the second transmembrane domain. The third intracellular domain may be within the scope of the intracellular domains described above, and may be the same as or different from the first intracellular domain and/or the second intracellular domain. Provided that the endodomain of at least one (e.g., at least two, three, four, or more) CD3 recombinant protein in said CD3 antibody receptor complex comprises a costimulatory domain and/or a signaling domain, or alternatively, is capable of providing sufficient signaling for antibody stimulation to activate engineered immune cells.

Immune cell

In another aspect, the present application provides a modified immune cell. The immune cells may include T cells, B cells, Natural Killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells.

In some cases, the immune cells may include T lymphocytes. The T lymphocytes may include thymocytes, natural T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell may be a helper T cell (Th), such as a helper T cell 1(Th1) or a helper T cell 2(Th2) cell. The T lymphocyte may be CD4⁺Helper T cells (HTL; CD 4)⁺T cells), cytotoxic T cells (CTL; CD8⁺T cells), tumor infiltrating cytotoxic T cells (TIL; CD8⁺T cells), CD4⁺/CD8⁺T cell, CD4^-/CD8^-T cells or any other T lymphocyte subtype. In certain instances, the modified T cell is a human T cell. Prior to expansion and genetic modification of the cells of the present application, the cell source may be obtained from a subject, e.g., a patient, by various non-limiting methods. T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain instances, any number of T cell lines available and known to those skilled in the art may be used. In other cases, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In other cases, the cell is part of a mixed population of cells presenting different phenotypic characteristics.

In some cases, the immune cells may include B cells. In some cases, the B cells may include effector B cells (plasma cells), memory B cells. The B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, regulatory B cells. In some cases, the immune cells may include macrophages. The B cells may include type I macrophages (M1), type II macrophages (e.g., M2a, M2B, M2 c).

In certain instances, the immune cells can include NK cells. In certain instances, the NK cells may comprise CD56bright and CD56 dim. In certain instances, the NK cells can include NK1 and NK 2. In certain instances, the NK cells can include A-NK and NA-NK.

In some cases, the immune cells may comprise leukocytes. Leukocytes generally refer to nucleated blood cells that have active motility and can migrate from within a blood vessel to outside the blood vessel, or from extravascular tissue to within the blood vessel. In addition to the blood, leukocytes can also be present in the lymphatic system, spleen, tonsils and other tissues of the body. In the present application, the leukocytes may include granulocytes (e.g., neutrophils, eosinophils, basophils), agranulocytes (e.g., lymphocytes, monocytes, macrophages, phagocytes, mast cells).

In certain instances, the immune cells can include lymphocytes, which can include any mononuclear cells, non-phagocytic leukocytes found in blood, lymph, and lymphoid tissues, e.g., B lymphocytes, T lymphocytes, Natural Killer (NK) cells.

In certain instances, the immune cells can include peripheral blood mononuclear cells, which can include any cell having a mononuclear in peripheral blood. For example, in the present application, the peripheral blood mononuclear cells may include T cells, B cells, NK cells, lymphocytes, monocytes, and dendritic cells.

In some cases, the immune cells may include macrophages. Macrophages are a substance that phagocytose and digest cell debris, microorganisms, cancer cells, and all other substances that lack surface markers expressed on the surface of normal cells, a process called phagocytosis. Macrophages are present in almost all tissues and seek potential pathogens through amoebic movements. In addition to their important role in nonspecific innate immune responses, they may also help to initiate adaptive immunity by recruiting other immune cell types, such as lymphocytes.

An engineered immune cell as described herein that does not express a T Cell Receptor (TCR). The non-expression of a T Cell Receptor (TCR) may comprise down-regulation of expression and/or activity of a T Cell Receptor (TCR). The downregulation can include expressing no active TCR, expressing no endogenous TCR, expressing no exogenous TCR, comprising a TCR structure, comprising an inactivated TCR, and/or deleting a TCR.

In certain instances, expression and/or activity of a T cell receptor alpha constant region protein and/or a T cell receptor beta constant region protein in the immune cell may be down-regulated. In certain instances, the downregulating can include downregulating expression and/or activity of a nucleic acid molecule encoding the cellular receptor alpha constant region protein and/or the T cell receptor beta constant region protein; and/or, comprising downregulating expression and/or activity of said cellular receptor alpha constant region protein and/or T cell receptor beta constant region protein. In the engineered immune cells described herein, the expression of the CD3 antibody receptor complex may be independent of TCR expression.

Downregulating expression and/or activity of MHC complexes in the immune cell. In certain instances, the downregulating may include downregulating expression and/or activity of a nucleic acid molecule encoding the cell MHC complex; and/or, comprising down-regulating the expression and/or activity of said cell MHC complex protein.

In some cases, the downregulation can be by gene knockout (knock out), gene knock down (knock down), gene mutation, gene deletion, gene silencing or any combination thereof to downregulate the expression and/or activity of TCR and/or MHC complexes of the immune cell.

For example, down-regulation may be achieved by administering to the immune cells one or more agents selected from the group consisting of: antisense RNA, siRNA, shRNA, CRISPR/Cas systems, RNA editing systems such as RNA Adenosine Deaminase (ADAR), RNA-guided endonucleases, Zinc Finger Nucleases (ZFNs), Mega-TAL nucleases, transcription activator-like effector nucleases (TALENs), meganucleases (Meganuclasees), base editing, CRISPR interference, and, Zinc finger protein (Zinc finger) gene repressor and/or transcription activator-like effector (TALE) gene repressor-mediated transcriptional inhibition.

In certain instances, the downregulating can include administering to the immune effector cell a guide RNA that targets an exon portion of the nucleic acid molecule (e.g., a nucleic acid molecule encoding an MHC complex of the cell). Guide RNAs targeted to nucleic acid molecules encoding the B2M may use guide RNAs in the prior art, the entire content of WO2019/011118 being incorporated herein by reference.

The engineered immune cells described herein can further comprise a Chimeric Antigen Receptor (CAR) and/or a chimeric autoantibody receptor (CAAR).

Preparation method

The present application provides methods of making engineered lymphocytes, which can comprise the steps of: 1. obtaining peripheral blood T cells from a healthy donor; 2. activating T cells using magnetic beads loaded with CD3 and CD28 antibodies; after T cells are activated, transferring the chimeric antibody receptor gene into the T cells by using lentivirus; 4. removing the magnetic beads; 5. knocking out TRAC and B2M which are important genes for generating immune rejection by using a gene editing technology; 6. the culture was continued and the cells were harvested.

The TRAC and B2M genes in lymphocytes are inactivated by gene editing, so that the immunological rejection of the lymphocytes in allogeneic cell treatment can be effectively reduced, and two recombinant CD3 surface proteins are expressed in the lymphocytes simultaneously, wherein the two co-expressed CD3 surface proteins are CD3 antibody receptors, and the modified lymphocytes can be combined with bispecific antibodies.

The method of making the engineered lymphocytes comprises: (i) preparing lymphocytes with the surfaces lacking class I MHC molecules and TCR molecules; (ii) two recombinant CD3 surface proteins were expressed on the surface of lymphocytes.

In another aspect, the present application provides a vector useful for transferring an isolated nucleic acid molecule encoding the CD3 antibody receptor complex into a cell. In the present application, the vector may be selected from one or more of a plasmid, a retroviral vector and a lentiviral vector. The vector may also contain other genes, such as marker genes that allow for selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may contain expression control elements that allow for the proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements that regulate gene transcription or mRNA translation, among others. In certain embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but typically comprises 5 ' non-transcribed sequences and 5 ' and 3 ' non-translated sequences, such as TATA box, capping sequences, CAAT sequences, etc., which are involved in initiation of transcription and translation, respectively. For example, the 5' non-transcriptional expression control sequence may comprise a promoter region that may comprise a promoter sequence for a transcriptional control functional linkage nucleic acid. One or more of the nucleic acid molecules described herein can be operably linked to the expression control element.

Non-viral delivery methods of nucleic acids include lipofection, nuclear transfection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycationic or lipid nucleic acid conjugates, naked DNA, artificial virosomes, and agents that enhance DNA uptake. Nucleic acid delivery can be achieved using RNA or DNA virus based systems, for example, by using the property of viruses to target specific cells in the body, to efficiently load (payload) the virus into the nucleus. The viral vector may be administered directly to the patient (in vivo) or may be administered to the patient (ex vivo), for example, by treating the cells with the virus in vitro and then administering the treated cells to the patient. Conventional virus-based systems may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors and herpes simplex viral vectors for gene transfer. In some cases, retroviral, lentiviral and adeno-associated viral approaches can be used to integrate gene transfer into the host genome, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. The lentiviral vector may comprise a long terminal repeat 5 'LTR and a truncated 3' LTR, an RRE, a rev response element (cPPT), a Central Termination Sequence (CTS) and/or a post-translational regulatory element (WPRE). The molecule can be constructed into a lentiviral vector by digestion with BamHI and SalI.

In another aspect, the present application provides a pharmaceutical composition. The pharmaceutical composition can comprise an engineered immune cell as described herein, and a pharmaceutically acceptable carrier. In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration of the immune cells and/or cell populations of the present application. Any conventional media or agent is contemplated for use in the pharmaceutical compositions of the present application, except as incompatible with the immune cells and/or cell populations of the present application.

The pharmaceutical compositions described herein may include an antibody. The antibody may be present in the same or a different container as the engineered immune cell. The antibody may be administered before, during or after administration of the engineered immune cells. For example, the antibody can be a bispecific antibody. The bispecific antibody can bind two targets simultaneously, and the two targets can be on the same target protein or different target proteins. The T cell engager is a relatively specific bispecific antibody. The T cell engager may include two linked scfvs, one targeting CD3 on the surface of the T cell and the other targeting a receptor on the surface of a target cell (e.g., tumor cell), thereby mediating T cell killing of the tumor cell. Receptors on the surface of target cells may include, but are not limited to, CD, CLL, CD44V, CD79, CD123, CD133, CD137, CD151, CD171, CD276, CLL, B7H, BCMA, VEGFR-2, EGFR, GPC, PMSA, CEACAM, c-Met, EGFRvIII, ErbB/HER, ErbB-2, HER, ErbB/HER-4, EphA, IGF1, GD, O-acetyl GD, GHRGD, GHRr, Flt, KDr, Flt, FBR, CEA, CA125, CTLA-4, GITR, BTLA, TGTGFR, TGFBR, TGFR, FBLIGP, Lewis, TNFR, PD, NYLPR, MULRP-L, PSLRP-4, TFLRP, PSRC, TFRC, TFR, TFLRP-1, TW-1, LTRPR, TFR, TFLRP, TFPR, TFLRP-4, TFLRP, TFR, TFLRP-L, TFR, TFLRP-R, MUC16, TCR α, TCR β, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled receptor (Frizzled), OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, and TROP-2.

The rationale behind this combination therapy is that bispecific antibodies are responsible for targeting, directing T cells to target cells, while engineered immune cells are responsible for the potent killing of target cells. In primary T cells, the TCR was not knocked out, and bispecific antibodies could mediate killing of target cells by control T cells through naturally expressed TCR-CD3 complex. As shown in figure 1, in the universal CD3 antibody receptor T cells, the CD3 antibody receptor was expressed on the surface of engineered immune cells. Bispecific antibodies are capable of promoting tumor cell killing and T cell proliferation by activating both a co-activation signal and a CD3zeta activation signal through the CD3 antibody receptor. The advantage of the combination is that the cell therapy can be combined with bispecific antibodies in a multi-target combination, while being easy to regulate, making the therapy safer. In certain instances, the bispecific antibody can be derived from an engineered immune cell as described herein. For example, the bispecific antibody can comprise a bispecific antibody secreted by an engineered immune cell described herein itself.

The application can be applied to universal T cells, and can also be applied to other killer cells without TCR structures, such as NK cells, or phagocytes, such as macrophages and the like. Current recurrence after CAR-T cell use includes target protein negative recurrence and target protein positive recurrence. Target protein positive relapse is mainly caused by failure of CAR-T cells after reinfusion. The therapy can realize continuous application of bispecific antibody after cell regression, and suppress tumor recurrence by chimeric antibody receptor T cell and human body self T cell. The patient with poor later-stage physical state can be flexibly regulated and controlled, good treatment effect is achieved on the basis of ensuring safety, and the application range of the patient is enlarged.

Method and use

In another aspect, the present application also provides the use of the modified immune cell and/or the pharmaceutical composition in the preparation of a medicament for treating a tumor.

In another aspect, the present application also provides a method of treating a tumor, the method comprising administering the engineered immune cell, the pharmaceutical composition, to a subject in need thereof.

The subject may first be subjected to a certain chemotherapy pretreatment and then the engineered lymphocytes are infused intravenously to the subject, either simultaneously or sequentially with the bispecific antibody. In the case of cell infusion, cells are infused before the bispecific antibody is administered at different doses. The input dose of bispecific antibody is adjusted if the patient has side effects. The infusion of engineered lymphocytes and diabodies can be repeated until remission or severe side effects occur. The engineered lymphocytes of the present application, in combination with bispecific antibodies, are capable of killing cancer cells, such as hematological and solid tumor cancer cells.

In the present application, the tumor may include non-solid tumors, which may include, but are not limited to, leukemia, lymphoma, and/or multiple myeloma, as well as solid tumors, including, but not limited to, lung cancer, stomach cancer, esophageal cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck tumors, gliomas, soft tissue sarcomas, and the like.

For example, the tumor may comprise a lymphoma.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Examples

EXAMPLE 1 design of CD3 antibody receptor Complex molecules and construction of plasmids

(1) Design molecules

Gene sequence information (Table 1) was obtained from a search of the NCBI website database (https:// www.ncbi.nlm.nih.gov /) and the gene CG-UST-1(SEQ ID NO.13) encoding the CD3 antibody receptor complex was designed, the CG-UST-1 comprising two parts, being a tandem of two CD3 recombinant protein genes, the first CD3 recombinant protein comprising a CD3epsilon ectodomain, a CD28 transmembrane region, a CD28 endodomain and a CD3zeta endodomain (SEQ ID NO: 10). The second CD3 recombinant protein comprises a CD3gamma ectodomain, a CD28 transmembrane region, and a CD3gamma endodomain (SEQ ID NO: 11). The two CD3 recombinant proteins are connected through a connecting molecule T2A gene (SEQ ID NO. 16).

The CD3gamma intracellular domain of CG-UST-1 gene coding the second CD3 recombinant protein is replaced by a gene coding a short peptide segment to obtain CG-UST-2(SEQ ID NO.14) and code the second CD3 recombinant protein (SEQ ID NO: 12). Meanwhile, a nucleic acid molecule CG-UST-3(SEQ ID NO.15) of a first CD3 recombinant protein of CG-UST-1 is designed. For convenient detection, a Flag tag is added into the gene sequence.

(2) Construction of plasmids

The CG-UST-1 gene sequence was synthesized by Nanjing Kinsery and cloned into a pUC57 vector (Nanjing Kinsery). When synthesizing the gene, adding specific restriction enzyme cutting sites at two ends of the gene: BamH1 and Sal 1. The recombinant plasmid obtained by gene synthesis was digested simultaneously with restriction enzymes BamH1 (NEB; R3136S) and Sal1 (NEB; R3138S), and the gene fragments were separated by agarose gel electrophoresis and purified by gel recovery (QIAGEN; 28706). The concentration of the recovered gene fragment was measured. The synthetic gene sequence was ligated into the BamH1-Sal1 site of a lentiviral vector (Addgene; cat # 12252) using T4 DNA ligase (NEB; M0202S). The cloned lentiviral vector was named: pL-CG-UST-1. After cloning, sequencing verification is carried out on the slow virus vector plasmid, and the recombinant plasmid sequencing primer is as follows: Lenti-For (TCAAGCCTCAGACAGTGGTTC; SEQ ID NO:17) and Lenti-Rev (CCTCATAAAGAGACAGCAACCAGG; SEQ ID NO: 18). The same construction process is adopted for the construction of the CG-UST-2 and CG-UST-3 plasmids. The constructed lentiviral vector plasmids are respectively called: pL-CG-UST-2 and pL-CG-UST-3.

Example 2 preparation of lentiviruses

(1) Extraction of plasmids

The lentivirus vector plasmid constructed above was transformed into E.coli again. Picking the monoclonals from the transformed plate to a shaking tube of 3ml of liquid LB culture medium containing ampicillin, rotating the shaking tube at 220rpm, and shaking and culturing for 8 h; and (3) sucking 500 mu l of the activated bacterial liquid, inoculating the liquid into 250ml of liquid LB culture medium containing ampicillin, and carrying out shaking culture on a shaking table at 220rpm for 12-16 h. Plasmid extraction was performed using the Qiagen high speed Plasmid Maxi Kit (cat # 12662) following the protocol provided in the Kit. After plasmid extraction, plasmid concentration was checked using nanodrop (thermo Fisher scientific) and supercoiled plasmid content was checked by DNA agarose gel.

(2) Culturing 293T cells

After removing the frozen 293T cells (ATCC) from the liquid nitrogen, they were thawed by shaking in a 37 ℃ water bath. Transferring into 15ml centrifuge tube containing 10ml preheated DMEM complete culture medium, and gently blowing; centrifuging at 1000rpm for 3min, and sucking and removing supernatant; adding 10ml DMEM complete culture medium, gently blowing uniformly, inoculating into a 10cm dish, and culturing in a cell culture box containing 5% CO2 at 37 ℃; when the cell density reaches 80% -90%, removing the culture medium, and washing with 10ml PBS for 1 time; adding 3ml trypsin containing 0.25% EDTA, placing in incubator for 1-2min (during which time it is necessary to take out and observe whether the cells become round under microscope); after the cells were rounded, 1ml of DMEM complete medium was added to terminate the trypsinization, the cells were transferred to a 15ml centrifuge tube, centrifuged at 1000rpm for 3min, and the supernatant was discarded. Subculturing at a ratio of 1:3 or 1:5, inoculating into new 10cm dishes, or freezing according to experiment requirements.

(3) 293T cells were transfected and lentiviruses harvested

1) Day 1, 293T cells were seeded: according to about 15-16X 10⁶Cells were seeded in T175 flasks (35-40ml medium).

2) On day 2, plasmid transfection: the medium before transfection was changed to medium with 10% FBS but no double antibody. First, a plasmid complex is prepared: the following plasmids were added to 1.5ml of Opti-MEM (Thermo Fisher Scientific; 31985-. Viral vector plasmid: 18 μ g of psPAX2 plasmid (Addgene; cat # 12260): 9 μ g, pMD2.G plasmid (Addgene; cat # 12259): 18 μ g. Preparing a transfection reagent complex: adding 100 μ l Lipofectamine 2000 (invitrogen; 11668-; then adding the plasmid compound into the transfection reagent compound, uniformly mixing, and standing for 25 min; finally, the transfection complex was added to the cell culture medium and shaken gently.

3) On day 4, the virus was harvested: collecting cell supernatant, centrifuging at 2000rpm for 10 min; filtering the supernatant with 0.45um filter membrane, transferring the filtrate into a special centrifuge tube, and balancing; performing ultracentrifugation for 2-3h by using an ultracentrifuge at 20000 rpm; after decanting the supernatant, the lentiviruses were resuspended in serum-free medium, aliquoted and stored at-80 ℃. According to the process, lentiviruses containing CG-UST-1, CG-UST-2 and CG-UST-3 are respectively prepared.

Example 3 preparation of Universal T cells expressing the CD3 antibody receptor Complex

PMBCs of peripheral blood of healthy donors (purchased from Miaotong organisms) were isolated using a apheresis machine. PBMCs were diluted to 2X 106. T cells were activated using CD3/CD28 magnetic beads (Thermo Fisher Scientific) at a cell to magnetic bead ratio of 1:3, with IL-2 added (PeproTech; 200-02). On day 3 post-activation, concentrated lentivirus was added to T cell culture flasks to transfect T cells. At day 5 after T cell activation, universal T cells were constructed using CRISPR/Cas9 to knock out TCR and B2M in T cells. The gRNA sequences used and the operating procedure were carried out with reference to patent WO2019/011118, example 3.

Example 4 detection of the expression of CD3 antibody receptor in cells

The expression of the CD3 antibody receptor complex in cells is detected by fluorescent antibody staining and flow cytometry, and the basic steps are as follows: and (3) respectively centrifugally collecting a certain volume of cultured modified T cells, staining the cells by using a Flag antibody (BioLegend; 637309) and an APC-TCR antibody (BioLegend; 306718), incubating the cells in the absence of light for 30min, washing the cells once by using PBS, then resuspending the cells by using a proper volume of PBS, and finally detecting the expression condition of the CD3 antibody receptor complex in the T cells and the expression condition of the CD3 antibody receptor complex in TCR negative cells by using a flow cytometer.

The staining results of the Flag antibody and the APC-TCR antibody are shown in FIG. 2. In the untransfected control group, when the TCR was not knocked out, the TCR positive rate was 95.09%; after TCR knockout, TCR positive rate was 1.95%. Indicating that the knockout efficiency is high. In the TCR knockout group, the expression efficiency of the CD3 antibody receptor complex was: CG-UST-1: 27.21%, CG-UST-2: 25.41% and CG-UST-3: 68.88 percent. In the TCR knockout group, TCR negative T cells, the expression efficiency of the CD3 antibody receptor complex was: CG-UST-1: 30.2%, CG-UST-2: 25.0% and CG-UST-3: 65.6% (FIG. 2). In the knockout group, the expression efficiency was similar to that of the corresponding knockout-free group, and thus the CD3 antibody receptor complex was independently expressed in the human T cell in a TCR-independent manner.

T cells expressing CD3 antibody receptors were stained with the different CD3 antibodies UCHT1(BD Biosciences; 555335), HIT3a (BD Biosciences; 561804), SP34-2(BD Biosciences; 552127) and OKT3(BD Biosciences; 566686) and analyzed with flow cytometry. The results are shown in FIGS. 3-6, respectively. The results show that T cells expressing CG-UST-1 and CG-UST-2 can be recognized by the common CD3 antibody clone UCHT 1. As shown in FIG. 3, the TCR knockout group, 22.0% and 19.9% of CG-UST-1 and CG-UST-2 cells, respectively, were recognized by UCHT1 in TCR negative T cells. Using the same staining and analysis methods, it was found that T cells expressing CG-UST-1 and CG-UST-2 were recognized by the CD3 antibody HIT3a clone (see FIG. 4), but not by the clone SP34-2 (see FIG. 5). Further simultaneous staining of cells with SP34-2 and OKT3 antibodies revealed that these cells were recognized by OKT3 antibody (see fig. 6). However, T cells expressing CG-UST-3 alone, which comprises the ectodomain of CD3epsilon membrane, are not recognized by a common CD3 antibody clone (see fig. 3-6).

Thus, the CD3 antibody receptor can be expressed in TCR-independent form in TCR-knocked out primary T cells. T cells co-expressing the CD3 antibody receptor complex of CD3epsilon and the extracellular domain of CD3gamma can be recognized by conventional CD3 antibodies, including the CD3 antibodies UCHT1, HIT3a, and OKT 3.

Example 5 activation of Universal CD3 antibody receptor T cells by the CD3 antibody OKT3

(1) Coating the flat plate. OKT3 antibody was diluted to 0.25. mu.g/ml with PBS. The diluted antibody was added to a 96-well plate in 100. mu.l per well and incubated at 37 ℃ for 3 hours. After incubation was complete, the plates were washed with 1 × PBS and PBS was removed.

(2) T cells are activated. The T cell density of different groups was adjusted to 1X 10⁶And/ml. Cells were then plated in 100. mu.l wells and incubated at 37 ℃ for 24 hours.

(3) Activation of T cells is detected. After cell incubation was complete, a volume of cells was taken, stained with CD137 fluorescent antibody (BD Biosciences; 555956) and TCR antibody (Biolegend; 306718), and flow cytometry was used to detect expression of the activation-tagged CD137 protein in TCR knockout T cells. Among the T cells expressing the CD3 antibody receptors CG-UST-1 and CG-UST-2, the TCR-negative cut CD 137-positive cells were 7.01% and 4.05%, respectively, as shown in FIG. 7. Indicating that the coated CD3 antibody can activate universal CD3 antibody receptor T cells.

(4) Cytokine secretion was detected. Supernatants from the above cells were transferred to a new 96-well plate, and secretion of IFN-. gamma.cytokines from T cells was detected using an ELISA kit (Thermo Fisher Scientific; cat. No. 88-7316). Plate preparation and detection of supernatant cytokines were performed according to the protocol provided in the kit.

As a result, the OKT3 antibody stimulates the universal antibody receptor T cells expressing CG-UST-1 and CG-UST-2 to secrete 341.28 and 248.76pg/ml of IFN-gamma, and the secretion is shown in Table 1. Thus, the coated CD3 antibody can stimulate the secretion of the cytokine IFN-. gamma.by general T cells expressing the CD3 antibody receptor complexes CG-UST-1 and CG-UST-2.

TABLE 1 stimulation of cytokine IFN-. gamma.secretion (pg/ml) by general CD3 antibody receptor T cells by OKT3 antibody

	Control group	CG-UST-1	CG-UST-2	CG-UST-3
					Antibody-free antibodies	-64.32	-60.24	-61.68	-53.52
Adding antibodies	6.48	341.28	248.76	-6.6

Example 6 Universal CD3 antibody receptor Complex T cells in combination with anti-CD 3 anti-CD 19 bispecific antibody killing of tumor cells

anti-CD 3 anti-CD 19 bispecific antibody was purchased from Invivogen (cat # bimab-hcd19CD 3). The double antibody can simultaneously bind to CD3epsilon and CD19, and mediate T cell killing of target cells expressing CD 19. The clone number of the CD3 antibody used by the double antibody is L2K-07.

(1) And (4) co-culturing the cells.

First, cell co-culture was performed. The method comprises the following steps: the concentration of T cells in different groups was adjusted to 1X 10⁶T cells were then plated in 96-well plates in 100. mu.l per well. The seeded T cells were temporarily incubated at 37 ℃; the concentration of Raji cells (cell bank of Chinese academy of sciences) expressing CD19 was adjusted to 1X 10⁶Perml, then the Raji cells were seeded to 9 containing T cells in 100. mu.l wells6-hole plate. The final T cell to tumor cell ratio was 1: 1. An anti-CD 3 anti-CD 19 bispecific antibody was added to the cells. The final concentration of the double antibody was 50 ng/ml. After mixing well, the mixture was centrifuged at 500rpm for 3 minutes. Cells were incubated at 37 ℃ for 24 hours.

(2) CD137 expression assay.

A volume of cells was taken, stained with CD19 antibody (eBioscience, 11-0199-42), CD137 fluorescent antibody and TCR antibody, and detected using flow cytometry. The CD19 negative cells were T cells. By detecting the cell membrane activation label CD137, the Raji cells added with double antibodies can activate the universal T cells expressing the CD3 antibody receptors CG-UST-1 and CG-UST-2, and the figure is 8. In TCR-negative T cells, the CD137 positivity rates were 7.15% and 6.47%, respectively.

(3) And (5) detecting the secretion of the cell factor.

After completion of incubation, the supernatant was taken and subjected to ELISA assay as in example 5. The results show that the anti-CD 3 anti-CD 19 bispecific antibody can mediate the secretion of cytokine IFN- γ by universal CD3 antibody receptor T cells, see table 2. In the case of TCR knockdown, the dual-antibody combination tumor cells stimulate the secretion of 287.639 and 286.512pg/ml IFN-. gamma.by CG-UST-1 and CG-UST-2 expressing universal antibody receptor T cells, respectively.

TABLE 2 Dual-antibody mediated tumor cell stimulation general type CD3 chimeric antibody receptor T cell secretion cytokine IFN-gamma (pg/ml)

	Control group	CG-UST-1	CG-UST-2	CG-UST-3
					Without double antibody	-32.478	-18.807	-28.117	-6.018
Adding double antibody	50.773	287.639	286.512	195.127

(4) CD107a expression assay.

1) Taking 96-well plate, adding T cells and target cells 2X 10 respectively per well⁵After centrifugation, 100. mu.l of RPMI-1640 complete medium was used for resuspension, and anti-CD 3 and anti-CD 19 diabody were added to the cells. The final concentration of the double antibody was 50 ng/ml. CD107a-PE antibody (BD Biosciences; 555801) was added to each well at a ratio of 1:50 and incubated for one hour. Then adding BD GolgiStop diluted at a ratio of 1:30,000 (BD Biosciences; 554724), and incubating at 37 ℃ for 2.5 hours;

2) the samples were centrifuged to remove the medium, the cells were washed once with serum-free medium and centrifuged at 1600rpm for 6 minutes. The supernatant was discarded, the cells were resuspended, and the volume of the resuspension was 100. mu.l. Adding a proper amount of CD3 antibody UCHT1 into each tube, and incubating for 30 minutes at 4 ℃ in a dark place;

3) cells were washed 1 time with PBS per tube and centrifuged at 1600rpm for 5 minutes. Carefully sucking off the supernatant;

4) adding a proper amount of PBS to resuspend the cells, and detecting the expression level of CD107a by a flow cytometer.

The results showed that CG-UST-1 and CG-UST-2 expressing universal antibody receptor T cells could be recognized by UCHT1, while anti-CD 3 anti-CD 19 bispecific antibody could mediate the release of killer particles from universal antibody receptor T cells (see FIG. 9). 9.49% and 10.75% of the universal CD3 antibody receptor T cells expressing CG-UST-1 and CG-UST-2 expressed CD107a after double anti-and tumor cell stimulation, respectively. Therefore, the universal CD3 antibody receptor T cell can be used in combination with bispecific antibody to kill tumor cells.

Sequence listing

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<213> Homo sapiens

<400> 2

Gln Ser Ile Lys Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu

1 5 10 15

Asp Gly Ser Val Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr

20 25 30

Trp Phe Lys Asp Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys

35 40 45

Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln

50 55 60

Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg

65 70 75 80

Met Cys Gln Asn Cys Ile Glu Leu Asn Ala Ala Thr Ile Ser

85 90

<210> 3

<211> 45

<212> PRT

<213> Homo sapiens

<400> 3

Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys Gln Thr Leu

1 5 10 15

Leu Pro Asn Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp

20 25 30

Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn

35 40 45

<210> 4

<211> 84

<212> PRT

<213> Homo sapiens

<400> 4

Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys

1 5 10 15

Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser

20 25 30

Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly

35 40 45

Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr

50 55 60

Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp Pro

65 70 75 80

Ala Thr Val Ala

<210> 5

<211> 45

<212> PRT

<213> Homo sapiens

<400> 5

Gly His Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu

1 5 10 15

Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala

20 25 30

Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

35 40 45

<210> 6

<211> 42

<212> PRT

<213> Homo sapiens

<400> 6

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro

35 40

<210> 7

<211> 27

<212> PRT

<213> Homo sapiens

<400> 7

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 8

<211> 41

<212> PRT

<213> Homo sapiens

<400> 8

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 9

<211> 113

<212> PRT

<213> Homo sapiens

<400> 9

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 10

<211> 284

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> first CD3 recombinant protein (CD 3epsilon ectodomain + CD28 transmembrane region + CD28 endodomain + CD3zeta endodomain)

<400> 10

Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys Val

1 5 10 15

Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro Gly

20 25 30

Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp Glu

35 40 45

Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys Glu

50 55 60

Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly

65 70 75 80

Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg Val

85 90 95

Cys Glu Asn Cys Met Glu Met Asp Phe Trp Val Leu Val Val Val Gly

100 105 110

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

115 120 125

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

130 135 140

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

145 150 155 160

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe

165 170 175

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

180 185 190

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

195 200 205

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

210 215 220

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

225 230 235 240

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

245 250 255

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

260 265 270

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

275 280

<210> 11

<211> 166

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> second CD3 recombinant protein 1 (CD 3gamma ectodomain + CD28 transmembrane region + CD3gamma endodomain)

<400> 11

Gln Ser Ile Lys Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu

1 5 10 15

Asp Gly Ser Val Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr

20 25 30

Trp Phe Lys Asp Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys

35 40 45

Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln

50 55 60

Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg

65 70 75 80

Met Cys Gln Asn Cys Ile Glu Leu Asn Ala Ala Thr Ile Ser Phe Trp

85 90 95

Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val

100 105 110

Thr Val Ala Phe Ile Ile Phe Trp Val Gly Gln Asp Gly Val Arg Gln

115 120 125

Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr

130 135 140

Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly

145 150 155 160

Asn Gln Leu Arg Arg Asn

165

<210> 12

<211> 129

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> second CD3 recombinant protein 2 (CD 3gamma ectodomain + CD28 transmembrane region + short peptide)

<400> 12

Gln Ser Ile Lys Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu

1 5 10 15

Asp Gly Ser Val Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr

20 25 30

Trp Phe Lys Asp Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys

35 40 45

Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln

50 55 60

Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg

65 70 75 80

Met Cys Gln Asn Cys Ile Glu Leu Asn Ala Ala Thr Ile Ser Phe Trp

85 90 95

Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val

100 105 110

Thr Val Ala Phe Ile Ile Phe Trp Val Gly Gln Asp Gly Val Arg Gln

115 120 125

Ser

<210> 13

<211> 1590

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CG-UST-1

<400> 13

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggatggta atgaagaaat gggtggtatt acacagacac catataaagt ctccatctct 120

ggaaccacag taatattgac atgccctcag tatcctggat ctgaaatact atggcaacac 180

aatgataaaa acataggcgg tgatgaggat gataaaaaca taggcagtga tgaggatcac 240

ctgtcactga aggaattttc agaattggag caaagtggtt attatgtctg ctaccccaga 300

ggaagcaaac cagaagatgc gaacttttat ctctacctga gggcaagagt gtgtgagaac 360

tgcatggaga tggatttttg ggtgctggtg gtggttgggg gagtcctggc ttgctatagc 420

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 480

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 540

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 600

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 660

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 720

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 780

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 840

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 900

gccctgcccc ctcgcggcag cggagagggc agaggaagtc ttctaacatg cggtgacgtg 960

gaggagaatc ccggccctag gatgcttctc ctggtgacaa gccttctgct ctgtgagtta 1020

ccacacccag cattcctcct gatcccaggc ggcagcgact acaaagacga tgacgacaag 1080

ggtggctccc agtcaatcaa aggaaaccac ttggttaagg tgtatgacta tcaagaagat 1140

ggttcggtac ttctgacttg tgatgcagaa gccaaaaata tcacatggtt taaagatggg 1200

aagatgatcg gcttcctaac tgaagataaa aaaaaatgga atctgggaag taatgccaag 1260

gaccctcgag ggatgtatca gtgtaaagga tcacagaaca agtcaaaacc actccaagtg 1320

tattacagaa tgtgtcagaa ctgcattgaa ctaaatgcag ccaccatatc tttttgggtc 1380

cttgtcgttg tcgggggcgt cttggcgtgt tatagcctcc tcgtcaccgt agcattcatt 1440

atattctggg tgggacagga tggagttcgc cagtcgagag cttcagacaa gcagactctg 1500

ttgcccaatg accagctcta ccagcccctc aaggatcgag aagatgacca gtacagccac 1560

cttcaaggaa accagttgag gaggaattga 1590

<210> 14

<211> 1479

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CG-UST-2

<400> 14

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggatggta atgaagaaat gggtggtatt acacagacac catataaagt ctccatctct 120

ggaaccacag taatattgac atgccctcag tatcctggat ctgaaatact atggcaacac 180

aatgataaaa acataggcgg tgatgaggat gataaaaaca taggcagtga tgaggatcac 240

ctgtcactga aggaattttc agaattggag caaagtggtt attatgtctg ctaccccaga 300

ggaagcaaac cagaagatgc gaacttttat ctctacctga gggcaagagt gtgtgagaac 360

tgcatggaga tggatttttg ggtgctggtg gtggttgggg gagtcctggc ttgctatagc 420

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 480

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 540

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 600

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 660

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 720

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 780

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 840

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 900

gccctgcccc ctcgcggcag cggagagggc agaggaagtc ttctaacatg cggtgacgtg 960

gaggagaatc ccggccctag gatgcttctc ctggtgacaa gccttctgct ctgtgagtta 1020

ccacacccag cattcctcct gatcccaggc ggcagcgact acaaagacga tgacgacaag 1080

ggtggctccc agtcaatcaa aggaaaccac ttggttaagg tgtatgacta tcaagaagat 1140

ggttcggtac ttctgacttg tgatgcagaa gccaaaaata tcacatggtt taaagatggg 1200

aagatgatcg gcttcctaac tgaagataaa aaaaaatgga atctgggaag taatgccaag 1260

gaccctcgag ggatgtatca gtgtaaagga tcacagaaca agtcaaaacc actccaagtg 1320

tattacagaa tgtgtcagaa ctgcattgaa ctaaatgcag ccaccatatc tttttgggtc 1380

cttgtcgttg tcgggggcgt cttggcgtgt tatagcctcc tcgtcaccgt agcattcatt 1440

atattctggg tgggacagga tggagttcgc cagtcgtga 1479

<210> 15

<211> 960

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CG-UST-3

<400> 15

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgggcggca gcgactacaa agacgatgac gacaagggtg gctccgatgg taatgaagaa 120

atgggtggta ttacacagac accatataaa gtctccatct ctggaaccac agtaatattg 180

acatgccctc agtatcctgg atctgaaata ctatggcaac acaatgataa aaacataggc 240

ggtgatgagg atgataaaaa cataggcagt gatgaggatc acctgtcact gaaggaattt 300

tcagaattgg agcaaagtgg ttattatgtc tgctacccca gaggaagcaa accagaagat 360

gcgaactttt atctctacct gagggcaaga gtgtgtgaga actgcatgga gatggatttt 420

tgggtgctgg tggtggttgg gggagtcctg gcttgctata gcttgctagt aacagtggcc 480

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 540

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 600

gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 660

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 720

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 780

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 840

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 900

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctga 960

<210> 16

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> T2A

<400> 16

ggcagcggag agggcagagg aagtcttcta acatgcggtg acgtggagga gaatcccggc 60

cctagg 66

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Lenti-For

<400> 17

tcaagcctca gacagtggtt c 21

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Lenti-Rev

<400> 18

cctcataaag agacagcaac cagg 24

<210> 19

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> short peptide

<400> 19

Gly Gln Asp Gly Val Arg Gln Ser

1 5

Claims

1. An engineered immune cell comprising a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein, wherein the first CD3 recombinant protein comprises:

(1) a first extracellular domain comprising an extracellular domain derived from a CD3 epsilon domain,

(2) the first transmembrane domain,

(3) the first intracellular domain;

The second CD3 recombinant protein comprises:

(1) a second extracellular domain comprising an extracellular domain derived from any one selected from the group consisting of CD3 gamma domain and CD3 delta domain,

(2) the second transmembrane domain,

(3) a second intracellular domain; and,

It does not express the T cell receptor (TCR).

2. The engineered immune cell of claim 1, comprising T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes cells, leukocytes and/or peripheral blood mononuclear cells.

3. The engineered immune cell of any one of claims 1-2, wherein the extracellular domain of the CD3 epsilon domain comprises the amino acid sequence set forth in SEQ ID NO:1.

4. The engineered immune cell of any one of claims 1-3, wherein the second extracellular domain comprises an extracellular domain derived from a CD3 gamma domain.

5. The engineered immune cell of any one of claims 1-4, wherein the extracellular domain of the CD3 gamma domain comprises the amino acid sequence shown in SEQ ID NO:2.

6. The engineered immune cell of any one of claims 1-5, wherein the second ectodomain comprises an ectodomain derived from a CD3 delta domain.

7. The engineered immune cell of any one of claims 1-6, wherein the extracellular domain of the CD3 delta domain comprises the amino acid sequence set forth in SEQ ID NO:4.

8. The engineered immune cell of any one of claims 1-7, wherein the first transmembrane domain and the second transmembrane domain are the same or different.

9. The engineered immune cell of any one of claims 1-8, wherein the transmembrane domain does not comprise a CD3-derived transmembrane domain.

10. The engineered immune cell of any one of claims 1-9, wherein the transmembrane domain comprises a transmembrane domain derived from any one of the proteins selected from the group consisting of CD8α, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.

11. The engineered immune cell of any one of claims 1-10, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:7.

12. The engineered immune cell of any one of claims 1-11, wherein the first intracellular domain and the second intracellular domain are the same or different.

13. The engineered immune cell of any one of claims 1-12, wherein at least one of the first intracellular domain and the second intracellular domain comprises a costimulatory domain and/or or signal transduction domains.

14. The engineered immune cell of claim 13, wherein the costimulatory domain comprised in the first intracellular domain and the costimulatory domain comprised in the second intracellular domain are the same or different .

15. The engineered immune cell of any one of claims 13-14, wherein the costimulatory domain comprises a costimulatory domain derived from any one or more proteins selected from the group consisting of CD28, CD137 , CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L , TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands for CD83, CD40 and MyD88.

16. The engineered immune cell of any one of claims 13-15, wherein the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:8.

17. The engineered immune cell of any one of claims 13-16, wherein a signal transduction domain comprised in the first intracellular domain and a signal comprised in the second intracellular domain The transduction domains are the same or different.

18. The engineered immune cell of any one of claims 13-17, wherein the signal transduction domain comprises at least one immunoreceptor tyrosine activation motif (ITAM).

19. The engineered immune cell of any one of claims 13-18, wherein the signal transduction domain comprises a signal transduction domain derived from any one or more proteins selected from the group consisting of CD3zeta , CD3delta, CD3gamma, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.

20. The engineered immune cell of any one of claims 13-19, wherein the signal transduction domain comprises the amino acid sequence set forth in SEQ ID NO:9.

21. The engineered immune cell of any one of claims 1-20, wherein a hinge region is further included between the extracellular and transmembrane domains.

22. The engineered immune cell of claim 21, wherein the hinge region comprises a hinge region derived from any one or more proteins selected from the group consisting of CD8α, CD28, 4-1BB, CD4, CD27, CD7 and PD-1.

23. The engineered immune cell of any one of claims 21-22, wherein the hinge region comprises the amino acid sequence set forth in any one of SEQ ID NO:6.

24. The engineered immune cell of any one of claims 1-23, wherein one of the first intracellular domain and the second intracellular domain comprises a peptide stretch of at least two amino acids .

25. The engineered immune cell of any one of claims 1-24, wherein the first CD3 recombinant protein comprises an extracellular domain derived from a CD3 epsilon domain, a transmembrane region derived from CD28, a CD28 derived and the intracellular domain derived from CD3zeta.

26. The engineered immune cell of claim 25, wherein the first CD3 recombinant protein comprises the amino acid sequence shown in SEQ ID NO:10.

27. The engineered immune cell of any one of claims 1-26, wherein the second CD3 recombinant protein comprises an extracellular domain derived from a CD3 gamma domain, a transmembrane region derived from CD28, and a CD3 The intracellular domain of gamma.

28. The engineered immune cell of claim 27, wherein the second CD3 recombinant protein comprises the amino acid sequence shown in SEQ ID NO:11.

29. The engineered immune cell of any one of claims 1-26, wherein the second CD3 recombinant protein comprises an extracellular domain derived from a CD3 gamma domain, a transmembrane region derived from CD28, and the peptide part.

30. The engineered immune cell of claim 29, wherein the second CD3 recombinant protein comprises the amino acid sequence shown in SEQ ID NO:12.

31. The engineered immune cell of any one of claims 1-30, further comprising a third CD3 recombinant protein comprising:

(1) a third extracellular domain, at least a part of the third extracellular domain comprises an extracellular domain derived from any one selected from the CD3 gamma domain or the CD3delta domain,

(2) the third transmembrane domain, and

(3) The third intracellular domain.

32. The engineered immune cell of claim 31, wherein the third ectodomain is the same or different from the second ectodomain.

33. The engineered immune cell of any one of claims 31-32, wherein the third transmembrane domain is the same as or different from the first transmembrane domain and/or the second transmembrane domain.

34. The engineered immune cell of any one of claims 31-33, wherein the third intracellular domain is the same or different from the first intracellular domain and/or the second intracellular domain .

35. The engineered immune cell of any one of claims 1-34, wherein the expression and/or activity of the major histocompatibility complex (MHC) is down-regulated.

36. The engineered immune cell of claim 35, wherein the MHC complex comprises B2M.

37. The engineered immune cell of any one of claims 1-36, wherein the engineered immune cell comprises a chimeric antigen receptor (CAR) and/or a chimeric autoantibody receptor (CAAR).

38. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-37 and a pharmaceutically acceptable adjuvant.

39. The pharmaceutical composition of claim 38, comprising an antibody.

40. The pharmaceutical composition of any one of claims 38-39, wherein the antibody is capable of recognizing and/or binding to the CD3 antibody receptor complex.

41. The pharmaceutical composition of any one of claims 38-40, wherein the antibody comprises a bispecific antibody.

42. The pharmaceutical composition of any one of claims 38-41, wherein the bispecific antibody is derived from the engineered immune cell of any one of claims 1-37.

43. The pharmaceutical composition of any one of claims 38-42, wherein the bispecific antibody is capable of recognizing and/or binding CD3.

44. The pharmaceutical composition of any one of claims 38-43, wherein the bispecific antibody is capable of recognizing and/or binding to a receptor on the surface of a target cell.

45. The pharmaceutical composition of claim 44, wherein the target cells are tumor cells.

46. The pharmaceutical composition of any one of claims 44-45, wherein the receptor on the surface of the target cell is selected from one of the group consisting of CD19, CD2, CD3, CD4, CD5, CD7, CD8 , CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276 , CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O -Acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2 , PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα , TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin 18.2, folate receptor alpha, folate receptor beta, GPC2 , CD70, BAFF-R and TROP-2.

47. A nucleic acid molecule encoding a CD3 antibody receptor complex in the engineered immune cell of any one of claims 1-37.

48. A vector comprising the nucleic acid molecule of claim 47.

49. The vector of claim 48, which is a viral vector.

50. The vector of any one of claims 48-49, which is a lentiviral vector.

51. A cell comprising the nucleic acid molecule of claim 47 and/or the vector of any one of claims 48-50.

52. Use of the engineered immune cells of any one of claims 1-37 and/or the pharmaceutical composition of any one of claims 38-46 in the manufacture of a medicament for the treatment of tumors .

53. The use of claim 52, wherein the tumors comprise solid tumors and non-solid tumors.

54. The use of any one of claims 52-53, wherein the tumor is selected from the group consisting of lymphoma, leukemia and multiple myeloma.

55. A method of treating a tumor, the method comprising administering the engineered immune cell of any one of claims 1-37, the pharmaceutical combination of any one of claims 38-46 to a subject in need thing.

56. The method of claim 55, wherein the tumors comprise solid tumors and non-solid tumors.

57. The method of any one of claims 55-56, wherein the tumor is selected from the group consisting of lymphoma, leukemia, and multiple myeloma.