WO2022057941A1 - Immune effector cell expressing il-7r binding protein - Google Patents

Immune effector cell expressing il-7r binding protein Download PDF

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WO2022057941A1
WO2022057941A1 PCT/CN2021/119634 CN2021119634W WO2022057941A1 WO 2022057941 A1 WO2022057941 A1 WO 2022057941A1 CN 2021119634 W CN2021119634 W CN 2021119634W WO 2022057941 A1 WO2022057941 A1 WO 2022057941A1
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cancer
cell
antigen
cells
receptor
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PCT/CN2021/119634
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French (fr)
Chinese (zh)
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蒋华
孙艳莎
李宗海
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克莱格医学有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of cell therapy, and relates to immune effector cells expressing IL-7R binding protein.
  • CAR-T cells can specifically kill tumors in a MHC-unrestricted manner, and show good application prospects in tumor immunotherapy, but they still have many limitations, such as poor efficacy on solid tumors, Drug candidates that exhibit excellent effects in vitro often fail to exhibit corresponding effects in vivo.
  • Immunotherapy drugs and regimens involve the various stages in which the body's immune system recognizes and attacks cancer cells.
  • Existing immuno-oncology drugs include the following types: antibodies targeting cancer cells, adoptive cell therapy, oncolytic viruses, dendritic cell-related therapy, tumor vaccines at the DNA and protein levels, immune-activating cytokines, and other immune modulators compound.
  • T cell-based genetic engineering includes CAR-T and TCR-T.
  • the former requires the construction of a chimeric antigen receptor, usually by linking a single-chain antibody to the intracellular segment of CD3 ⁇ through a hinge region and a transmembrane segment, and then transducing the virus to induce T cells, which bind to the antigen on the surface of the tumor cell through the single-chain antibody.
  • the intracellular signal of CD3 ⁇ is activated, which in turn causes the killing effect of the transduced T cells on tumor cells.
  • TCR tumor antigen peptide/MHC complexes
  • the purpose of the present invention is to provide an immune effector cell that co-expresses IL-7 and TFP.
  • a first aspect of the present invention provides a genetically engineered immune effector cell, which expresses IL-7R binding protein or IL-7; and also expresses a receptor that specifically recognizes a target antigen.
  • the receptor that specifically recognizes the target antigen is a TCR fusion protein (TFP).
  • TCP TCR fusion protein
  • the IL-7R-binding protein is an exogenous IL-7R-binding protein, and the exogenous IL-7R-binding protein can specifically bind to IL-7R and enhance the activity of IL-7R; preferably, The IL-7R binding protein is IL-7, and further preferably, the amino acid sequence of the IL-7R is shown in SEQ ID NO: 1;.
  • the IL-7 is native IL-7, or a truncated fragment of native IL-7 having the same function as native IL-7, or a mutant of native IL-7;
  • the nucleic acid sequence of IL-7 has at least 90% identity with the sequence shown in SEQ ID NO: 2 or 3, or is a truncated fragment of the nucleic acid sequence shown in SEQ ID NO: 2 or 3; or
  • the amino acid sequence of the IL-7 has at least 80% identity with the sequence shown in SEQ ID NO: 13 or 19, or a truncated fragment of the amino acid sequence shown in SEQ ID NO: 13 or 19.
  • the IL7 is constitutively expressed or inducible; preferably, the expression of the IL-7 is induced by an inducible promoter; preferably, the promoter is the NFAT6 promoter; preferably, The NFAT6 promoter is reverse regulated.
  • the TFP includes:
  • TCR subunits or TCR units
  • TCR subunit and the antigen recognition unit are operably linked.
  • the TCR subunit comprises:
  • a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domains of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , or a combination thereof;
  • the TCR subunit comprises: a CD3 ⁇ TCR subunit, a CD3 ⁇ TCR subunit, a CD3 ⁇ TCR subunit, a CD3 ⁇ TCR subunit, a CD3 ⁇ TCR subunit, or a combination thereof.
  • the target antigen is selected from: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); ); interleukin 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1 ; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; Specific embryonic antigen-4 (SSEA-4); cell surface associated mucin
  • the target antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; wherein, the viral antigens are selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen or influenza virus antigen.
  • the antigen recognition unit includes an antibody or a fragment thereof, preferably, the antibody or fragment thereof is a Fab, Fab', F(ab')2, Fv fragment, scFv, sdFv, composed of VH and CH1.
  • Fd fragments composed of domains, linear antibodies, single-domain antibodies, or camelid VHH domains, more preferably, the antibody is an scFv.
  • the antigen recognition unit recognizes GPC3 or recognizes Claudin18.2,
  • the light chains LCDR1, LCDR2 and LCDR3 recognizing the amino acid sequence of the antigen recognition unit of GPC3 are independently selected from or have 70-100% sequence identity with the light chains LCDR1, LCDR2 and LCDR3 shown in the table below, and/or
  • the heavy chains HCDR1, HCDR2 and HCDR3 recognizing the amino acid sequence of the antigen recognition unit of GPC3 are independently selected from or have 70-100% sequence identity with the heavy chains HCDR1, HCDR2 and HCDR3 shown in the following table;
  • the light chains LCDR1, LCDR2 and LCDR3 that recognize the amino acid sequence of the antigen recognition unit of claudin18.2 are independently selected from or have 70-100% sequence identity to the light chains LCDR1, LCDR2 and LCDR3 shown in the table below, and/or recognize
  • the heavy chain HCDR1, HCDR2 and HCDR3 of the amino acid sequence of the antigen recognition unit of claudin18.2 are independently selected from or have 70-100% sequence identity with the heavy chains HCDR1, HCDR2 and HCDR3 shown in the following table:
  • the light chain variable region that recognizes the amino acid sequence of the antigen recognition unit of GPC3 is independently selected from the light chain variable region shown in the following table or has 70-100% sequence identity with it, and/or recognizes
  • the heavy chain variable region of the amino acid sequence of the antigen recognition unit of GPC3 is independently selected from the heavy chain variable region shown in the following table or has 70-100% sequence identity therewith;
  • the light chain variable region that recognizes the amino acid sequence of the antigen recognition unit of claudin18.2 is independently selected from the light chain variable region shown in the following table or has 70-100% sequence identity with it, and/or recognizes claudin18
  • the heavy chain variable region of the amino acid sequence of the antigen recognition unit of .2 is independently selected from the heavy chain variable region shown in the following table or has 70-100% sequence identity therewith;
  • amino acid sequence of the antigen recognition unit that recognizes GPC3 is selected from the sequences shown in the following table or has 70-100% sequence identity with it;
  • amino acid sequence of the antigen recognition unit that recognizes claudin18.2 is selected from the sequence shown in the following table or has 70-100% sequence identity with it;
  • SEQ ID NO. 136 SEQ ID NO. 145 SEQ ID NO. 154 SEQ ID NO. 163 SEQ ID NO. 172 SEQ ID NO. 181 SEQ ID NO.100
  • the coding sequence of the linker has the nucleic acid sequence shown in SEQ ID NO: 8 or 11; or the amino acid sequence shown in SEQ ID NO: 12.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain included in the TFP are from the same TCR subunit.
  • the TFP and IL7 are expressed by the same nucleic acid molecule, or expressed by different nucleic acid molecules;
  • the TFP and IL7 are expressed from the same nucleic acid molecule, and the expression cassette of IL7 and TFP, as well as between the expression cassettes, are directly connected or connected by tandem fragments selected from F2A, PA2, T2A, and/or or E2A.
  • the TFP includes a protein containing an immunoreceptor tyrosine activation motif (ITAM);
  • ITAM-containing protein includes a protein or a part thereof selected from the group consisting of: CD3 ⁇ TCR subunit, CD3 ⁇ TCR Subunit, CD3 ⁇ TCR subunit, CD3 ⁇ TCR subunit, Fc ⁇ receptor 1 chain, Fc ⁇ receptor 2 chain, Fc ⁇ receptor 1 chain, Fc ⁇ receptor 2a chain, Fc ⁇ receptor 2b1 chain, Fc ⁇ receptor 2b2 chain, Fc ⁇ receptor 3a chain, Fc ⁇ receptor 3b chain, Fc ⁇ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d or functional fragments thereof;
  • the TFP molecule further comprises a leader sequence, preferably, the leader sequence comprises the nucleic acid sequence shown in SEQ ID NO:7.
  • the TFP includes the nucleic acid sequence shown in SEQ ID NO: 9, 10 or 16; or the TFP includes the amino acid sequence shown in SEQ ID NO: 14 or 15.
  • the second aspect of the present invention provides a nucleic acid molecule encoding the IL-7R binding protein or IL-7 according to the first aspect of the present invention; the nucleic acid molecule also encodes any one of claims 1-17 the receptor that specifically recognizes the target antigen;
  • the nucleic acid consists of DNA and/or RNA
  • the nucleic acid is mRNA
  • the nucleic acid comprises a nucleotide analog.
  • the third aspect of the present invention provides a vector, comprising the nucleic acid molecule described in the second aspect of the present invention,
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, Rous Sarcoma virus (RSV) vector or retroviral vector.
  • RSV Rous Sarcoma virus
  • the fourth aspect of the present invention provides a cell comprising the vector of the third aspect of the present invention or the nucleic acid molecule of the first aspect of the present invention integrated into its genome, preferably the cell is a human T cell, preferably , for allogeneic T cells.
  • the fifth aspect of the present invention provides a method for preparing cells, the method comprising transducing T cells with the vector described in the third aspect of the present invention or the nucleic acid molecule described in the second aspect of the present invention.
  • a sixth aspect of the present invention provides a method of producing an RNA engineered cell population, the method comprising introducing into a cell an in vitro transcribed RNA or a synthetic RNA, wherein the RNA comprises the nucleic acid of the second aspect of the present invention.
  • the seventh aspect of the present invention provides a method for providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of the cells of the first and fourth aspects of the present invention, the second aspect of the present invention
  • the nucleic acid molecule of the aspect, the vector of the third aspect of the present invention preferably, the mammal is a human.
  • the eighth aspect of the present invention provides a method for treating a mammal suffering from a disease related to the expression of GPC3 or claudin18.2, the method comprising administering to the mammal an effective amount of the first and fourth aspects of the present invention the cell, the nucleic acid molecule described in the second aspect of the present invention, and the vector described in the third aspect of the present invention;
  • the disease related to the expression of GPC3 or claudin18.2 is selected from colon cancer, rectal cancer, renal cell cancer, liver cancer, lung cancer, small bowel cancer, esophagus cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer , skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, endocrine system cancer, thyroid cancer Carcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, tumor angiogenesis, spinal tumor, brain stem glial tumor, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma,
  • the cells of the first and fourth aspects of the present invention are administered in combination with an agent that increases the efficacy of the cells of the first and fourth aspects of the present invention
  • the cells of the first and fourth aspects of the present invention are administered in combination with an agent that ameliorates one or more side effects associated with the administration of the cells of the first and fourth aspects of the present invention;
  • the cells described in the first and fourth aspects of the present invention are administered in combination with the agent for treating the disease associated with GPC3 or claudin18.2.
  • the cells described in the first and fourth aspects of the present invention, the nucleic acid molecules described in the second aspect of the present invention, and the vectors described in the third aspect of the present invention are used as medicines.
  • the cells provided by the present invention can improve the tumor cell killing ability of CAR T cells.
  • Figure 1 shows the schematic diagrams of pMSCV-GPC3-mCD3 ⁇ , pMSCV-GPC3-mCD3 ⁇ -F2A-mIL7 plasmids;
  • Figure 2 shows the in vitro killing results of GPC3-CD3 ⁇ -F2A-IL7 and GPC3-CD3 ⁇ ;
  • Figures 3A-3F are cytokine secretion results.
  • Figures 4A and 4B are the results of tumor volume and mouse body weight changes in GPC3-CD3 ⁇ -F2A-IL7 and GPC3-CD3 ⁇ in vivo killing experiments, respectively.
  • the application provides a technical solution for enhancing the treatment of solid tumors by cells expressing TCR fusion protein (also known as TFP protein).
  • TCR fusion protein also known as TFP protein
  • “about” may mean plus or minus 1% or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% or greater than 30 %.
  • T cell (antigen) receptor also known as TCR subunit, or TCR unit
  • TCR TCR subunit
  • TCR unit is a characteristic marker on the surface of all T cells that binds non-covalently to CD3 to form TCR- CD3 complex.
  • the TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCR is a heterodimer composed of two different peptide chains, consisting of ⁇ and ⁇ peptide chains.
  • the cytoplasmic region is characterized by a short cytoplasmic region.
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (V ⁇ , V ⁇ ) has three hypervariable regions, CDR1, CDR2, and CDR3.
  • CDR3 has the largest variation, which directly determines the antigen of TCR. binding specificity.
  • TCR recognizes the MHC-antigen peptide complex
  • CDR1 and CDR2 recognize and bind to the side wall of the antigen-binding groove of the MHC molecule, while CDR3 directly binds to the antigen peptide.
  • TCRs are divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, ⁇ and ⁇ , and TCR2 is composed of two chains, ⁇ and ⁇ .
  • the "affinity” and combat effectiveness of these TCRs to the corresponding TAAs, ie high-affinity TCRs, can be improved by means of partial genetic modification.
  • the "genetically modified TCR” technology is also called “Affinity-Enhanced TCR” technology (Affinity-Enhanced TCR).
  • the gene modified T cell receptor (Gene Modified TCR) replaces the constant region domains of its beta chain and alpha chain with the constant region domains of the antibody heavy and light chains, which belong to the same immunoglobulin superfamily as the TCR molecule.
  • a chimeric TCR molecule was developed.
  • TCR fusion protein also known as TFP protein
  • TFP protein includes recombinant proteins derived from various proteins of TCR, generally capable of: i) binding to surface antigens on target cells; ii) binding to intact TCR when localized to T cells
  • TFP T cells or TFP-T cells are T cells that have been transduced (eg, according to the methods disclosed herein) and express TFP.
  • the T cells are CD4+ T cells, CD8+ T cells, or CD4+/CD8+ T cells.
  • the TFP T cells are NK cells.
  • the TFP T cells are ⁇ T cells.
  • the "TCR fusion protein (also called TFP protein)" of the present invention comprises an extracellular antigen binding domain (also called an antigen recognition unit), a TCR transmembrane domain and an intracellular domain, wherein the antigen binding domain is a continuous polypeptide Chains, including, for example, single-domain antibody fragments (sdAbs), a portion of single-chain antibodies (scFvs) derived from murine, humanized or human antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al, 1988, Proc. Natl. Acad. Sci.
  • sdAbs single-domain antibody fragments
  • scFvs single-chain antibodies
  • the antigen binding domain of the TFP composition of the invention comprises an antibody or antibody fragment.
  • the TFP includes an antibody or antibody fragment comprising an scFv or sdAb.
  • the present invention includes recombinant DNA constructs encoding TFP, wherein the TFP comprises an antibody fragment that specifically binds to GPC3, wherein the sequence of the antibody fragment is contiguous and in reading frame with the nucleic acid sequence encoding the TCR unit or portion thereof.
  • the TFPs provided herein are capable of associating with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR units in order to form functional TCR complexes.
  • the TFP of the invention comprises a target antigen-specific binding element, also referred to as an antigen recognition unit.
  • the choice of the antigen recognition unit depends on the type and number of target antigens on the surface of the target cells.
  • the antigen recognition unit can be selected to recognize the target antigen as a cell surface marker on the target cell that is associated with a particular disease state.
  • examples of cell surface markers that can be target antigens for the antigen recognition units in the TFPs of the present invention include markers associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancerous diseases (eg, malignant diseases).
  • the extracellular domains of the TFPs of the invention can be derived from natural sources or from recombinant sources.
  • the domain may be derived from any protein, especially a membrane-bound protein or a transmembrane protein.
  • the extracellular domain is capable of associating with the transmembrane domain.
  • the extracellular domain in the present invention may comprise at least the following extracellular domains: for example the alpha, beta or delta chains of T cell receptors, or CD3 ⁇ , CD3 ⁇ or CD3 ⁇ , or in alternative embodiments CD28, CD45, CD4 , CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and/or CD154.
  • the transmembrane domains of the TFPs of the present invention may be derived from natural sources or from recombinant sources. In the case of natural sources, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the TFP binds to the target antigen.
  • the transmembrane domain in the present invention may comprise at least the following transmembrane domains: for example the alpha, beta or delta chains of T cell receptors, or CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and/or CD154.
  • the transmembrane domain can be linked to the extracellular region of TFP (eg, the antigen binding domain of TFP) via a hinge (eg, from a human protein).
  • a hinge eg, from a human protein.
  • the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge or a CD8a hinge.
  • Linkers of the Invention can form a link between the transmembrane domain and the cytoplasmic region of TFP.
  • Glycine-serine doublets provide particularly suitable linkers.
  • the cytoplasmic domain of the TFP may include an intracellular signaling domain; the TCR ⁇ and TCR ⁇ subunits typically lack the signaling domain.
  • Intracellular signaling domains are generally responsible for activating at least one normal effector function of immune cells into which TFP has been introduced.
  • effector function refers to a specialized function of a cell.
  • the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.
  • the TFP-T cells can secrete large amounts of IFN- ⁇ , Granzyme-B, IL2, TNF- ⁇ and GM-CSF; recognize the same antigens Compared with cell-constructed CAR T cells, TFP-T cells expressing TFP protein maintained the same significant cytotoxicity, but the secretion of cytokines was significantly reduced, effectively reducing the possibility of cytokine storm.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs cells to perform specialized functions. While the entire intracellular signaling domain can generally be employed, in many cases it is not necessary to use the entire chain. In the case of the use of truncated portions of intracellular signaling domains, such truncated portions can be used in place of the full chain, so long as they transduce effector function signals. Thus, the term “intracellular signaling domain” includes any truncated portion of an intracellular signaling domain sufficient to signal effector function.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Examples of ITAM-containing primary intracellular signaling domains in the present invention include the intracellular signaling domains of CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the TFP of the invention comprises an intracellular signaling domain, eg, the primary signaling domain of CD3 ⁇ .
  • the primary signaling domain comprises a modified ITAM domain, eg, a mutant ITAM domain with altered (eg, increased or decreased) activity compared to the native ITAM domain.
  • the primary signaling domain includes a primary intracellular signaling domain containing a modified ITAM, eg, a primary intracellular signaling domain containing an optimized and/or truncated ITAM.
  • the primary signaling domain comprises one, two, three, four or more ITAM motifs.
  • the intracellular signaling domain of a TFP may comprise the CD3delta signaling domain alone, or it may be combined with any other desired intracellular signaling domain useful in the context of the TFP of the invention.
  • the intracellular signaling domain of the TFP may comprise a CD3 ⁇ chain moiety and a costimulatory signaling domain.
  • a costimulatory signaling domain refers to an intracellular domain comprising a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient lymphocyte response to an antigen.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, DAP10, DAP12, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83, etc.
  • intracellular signaling sequences within the cytoplasmic portion of the TFP of the invention may be linked to each other in random or specified order.
  • short oligopeptide or polypeptide linkers eg, 2 to 10 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can be intracellular Links are formed between signaling sequences.
  • the TFP-expressing cells described herein may further express another factor, such as a cytokine, transcription factor, chemokine, and/or a combination thereof, to increase T cell proliferation, cell survival, anti-apoptosis Anti-tumor activity can be enhanced by the effects of apoptosis and tumor infiltration.
  • the TFP-expressing T cells that also express the cytokine IL-7, and the TFP-T cells co-incubated with tumor cells expressing the TFP-targeted antigen the TFP-T cells can significantly increase the response to the TFP-targeting antigen.
  • the in vitro killing toxicity of tumor cells can significantly inhibit the in vivo subcutaneous transplanted tumors formed by the tumor cells; the in vitro cytokine secretion test shows that the TFP-T cells can secrete a large amount of IFN- ⁇ , Granzyme-B, IL2, TNF- ⁇ and GM-CSF.
  • TFP-T cells expressing IL-7 maintained the same significant cytotoxicity, but the secretion of cytokines was significantly reduced, effectively reducing the cytokine storm. possible.
  • IL-7 Interleukin7, interleukin 7 or IL7
  • IL-7 has one of the following characteristics: (i) is the amino acid sequence of a naturally occurring mammalian IL-7 or a fragment thereof, eg, SEQ ID NO: 19 (human) or The amino acid sequence set forth in SEQ ID NO: 13 (murine) or a fragment thereof; (ii) substantially the same as the amino acid set forth in SEQ ID NO: 19 (human) or SEQ ID NO: 13 (murine) or a fragment thereof, for example, at least 85%, 90%, 95% %, 96%, 97%, 98%, 99% homology of amino acid sequences; (iii) from naturally occurring mammalian IL-7 nucleotide sequences or fragments thereof (eg SEQ ID NO: 2 (human) or fragments thereof) The encoded amino acid sequence, or the amino acid sequence encoded by SEQ ID NO: 3 (mouse) or a fragment thereof; (iv) by the nucleotide sequence
  • IL7 can interact (eg, bind) to an IL-7R (preferably from a mammal, such as a murine or human IL-7R), preferably from a mammal, such as Murine or human. IL7 can also exert anti-tumor effects through non-IL-7R pathways.
  • IL-7R preferably from a mammal, such as a murine or human IL-7R
  • IL7 can also exert anti-tumor effects through non-IL-7R pathways.
  • Exogenous IL-7R binding protein refers to all proteins that can specifically bind to IL-7R and enhance the activity of IL-7R.
  • “Enhancing IL-7R activity” is understood to mean that an IL-7R binding protein is capable of enhancing any one or more activities of naturally occurring IL-7R, including but not limited to stimulation of NK cell proliferation, cytotoxicity or maturation; stimulation of Proliferation or differentiation of B cells and T cells; stimulation of antibody production and affinity maturation in B cells; stimulation of cytotoxicity of CD8+ T cells; stimulation of interferon gamma production in T cells and NK cells; inhibition of dendritic cells (DC) Activation and maturation; inhibits the release of inflammatory mediators from mast cells; enhances phagocytosis of macrophages; inhibits the production or survival of TReg cells (regulatory T cells); and stimulates proliferation of myeloid progenitor cells.
  • DC dendritic cells
  • the invention also provides nucleic acid molecules encoding one or more of the co-expressing IL7 and TFP constructs described herein.
  • TFP and IL7 are expressed from the same nucleic acid molecule, or from different nucleic acid molecules.
  • TFP and IL7 are expressed by the same nucleic acid molecule, and the expression cassette of IL7 and TFP, as well as between the expression cassettes are directly connected or connected by tandem fragments selected from F2A, PA2, T2A , and/or E2A.
  • the present invention also provides a vector into which the DNA of the present invention is inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • the present invention is not limited to the use of constitutive promoters, but also contemplates inducible promoters.
  • an inducible promoter provides a molecular switch that enables expression of a polynucleotide sequence to which it is operably linked when expression is desired, or turns off expression when expression is not desired.
  • inducible promoters include, but are not limited to, NFAT6 promoters, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters.
  • T cells A source of T cells is obtained from the subject prior to expansion and genetic modification. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the invention, any number of T cell lines available in the art can be used. In certain aspects of the invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as the FicollTM separation technique.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps.
  • T cells of the invention can be expanded by surface contact with an agent that stimulates signals associated with the CD3/TCR complex and ligands that stimulate co-stimulatory molecules on the surface of the T cells.
  • T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by a protein kinase C activator (eg, bryostatin) and calcium ionophore contacts.
  • immune effector cells refers to cells that perform effector functions during an immune response. For example, it includes immune cells that secrete cytokines and/or chemokines, kill microorganisms, secrete antibodies, and recognize or eliminate tumor cells.
  • immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, or stem cell-derived immune effector cells.
  • the term "immune effector function" includes any function mediated by components of the immune system that can result in inhibition of tumor growth and/or inhibition of tumorigenesis, including inhibition of tumor spread and metastasis.
  • the immune effector function kills tumor cells.
  • the immune effector function in the present invention is antibody-mediated, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis ( ADCP), induction of apoptosis in cells bearing tumor-associated antigens (eg, by binding of antibodies to surface antigens), inhibition of CD40L-mediated signaling (eg, by antibodies interacting with CD40 receptor or CD40 ligand (CD40L) binding), and/or inhibit the proliferation of cells bearing tumor-associated antigens, preferably ADCC and/or CDC.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • antibodies capable of mediating one or more immune effector functions are preferably capable of lysis and/or ADCC-mediated lysis) to mediate killing of tumor cells.
  • Antibodies can also act simply by binding to tumor-associated antigens on the surface of cancer cells.
  • antibodies can block the function of tumor-associated antigens or induce apoptosis by binding to tumor-associated antigens on the surface of tumor cells.
  • APC antigen presenting cell
  • T cells can recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • anti-tumor effect refers to a biological effect that can be manifested in various ways, including, but not limited to, for example, decreased tumor volume, decreased tumor cell number, decreased number of metastases, increased life expectancy, decreased tumor cell proliferation, tumor cell survival rate, or improvement in various physical symptoms associated with cancerous conditions.
  • Anti-tumor effect can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent tumorigenesis in the first place.
  • autologous refers to any material derived from an individual to be later reintroduced into that same individual.
  • allogeneic refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some aspects, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • genetically engineered cell refers to a cell that has been engineered by means of genetic engineering.
  • terapéuticaally effective amount refers to a compound effective to achieve a specified biological result as described herein , formulation, substance or composition in an amount such as, but not limited to, an amount or dose sufficient to promote a T cell response.
  • an "immunologically effective amount”, “anti-tumor effective amount”, “tumor inhibitory effective amount” or “therapeutically effective amount” is indicated, the precise amount of immune effector cells, therapeutic agents of the invention to be administered can be determined by a physician Determined taking into account the individual's age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).
  • An effective amount of immune effector cells refers to, but is not limited to, increasing, enhancing or prolonging the anti-tumor activity of immune effector cells; increasing the number of anti-tumor immune effector cells or activated immune effector cells; promoting IFN- ⁇ secretion; tumor regression and tumor shrinkage , the number of tumor necrotic immune effector cells.
  • the co-receptor for CD3 (cluster of differentiation 3) T cells is a protein complex that consists of four distinct chains. In mammals, this complex contains one CD3 ⁇ chain, CD3 ⁇ chain, and two CD3 ⁇ chains.
  • the CD3 molecule is connected to the T cell receptor (TCR) through a salt bridge to form a TCR-CD3 complex, which is involved in the signal transduction of T cells and is mainly used to label thymocytes, T lymphocytes and T cell lymphomas.
  • the CD3 cytoplasmic segment contains an immunoreceptor tyrosine-based activation motif (ITAM), and TCR recognizes and binds to antigenic peptides presented by MHC (major histo-compatibility complex) molecules, resulting in the activation of CD3 ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • MHC major histo-compatibility complex
  • CD3 ITAM immunoreceptor tyrosine-based activation motif
  • conserveed sequence tyrosine residues are phosphorylated by the tyrosine protein kinase p56lck in T cells, which can then recruit other SH2 (Scr homology 2) domain-containing tyrosine protein kinases (eg, ZAP-70).
  • SH2 Str homology 2 domain-containing tyrosine protein kinases
  • an exogenous receptor that can bind to a target antigen and that can trigger activation of CD3 signaling comprises at least one CD3 binding site and at least one additional response to bacterial substances, viral proteins, autoimmune markers, or proteins present on specific cells.
  • Antigens eg, cell surface proteins of B cells, T cells, natural killer (NK) cells, myeloid cells, phagocytes, or tumor cells
  • NK natural killer
  • Such exogenous receptors are capable of cross-linking two cells and can be used to direct T cells to specific target antigens and trigger T cell cytotoxic activity against target cells. Examples of such target antigens can be tumor cells or infectious agents such as viral or bacterial pathogens.
  • stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (eg, a TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, via TCR/CD3 Signal transduction by the CD3 complex.
  • a stimulatory domain or stimulatory molecule eg, a TCR/CD3 complex
  • Stimulation can mediate changes in the expression of certain molecules and/or reorganization of cytoskeletal structures, among others.
  • stimulation molecule or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides a primary cytoplasmic signal that modulates primary activation of the TCR complex in a stimulatory manner directed against at least some aspects of the T cell signaling pathway conduction sequence.
  • primary signaling is initiated by, for example, binding of the TCR/CD3 complex to peptide-loaded MHC molecules, and it results in the mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like.
  • Primary cytoplasmic signaling sequences (also referred to as “primary signaling domains”) that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or "ITAMs.”
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS” ”) and those of CD66d.
  • Intracellular signaling domain refers to the intracellular portion of a molecule. Intracellular signaling domains generate signals that promote the immune effector function of TFP-containing cells, such as TFP-expressing T cells. Examples of immune effector functions, eg, in TFP-expressing T cells, include cytolytic activity and T helper cell activity, including secretion of cytokines.
  • the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include intracellular signaling domains derived from molecules responsible for primary stimulation or antigen-dependent stimulation.
  • the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include intracellular signaling domains derived from molecules responsible for costimulatory signaling or antigen-independent stimulation.
  • the primary intracellular signaling domain may comprise an ITAM ("immunoreceptor tyrosine-based activation motif").
  • ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.
  • costimulatory molecule refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors, and DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18 ) and 4-1BB (CD137).
  • the costimulatory intracellular signaling domain can be the intracellular portion of the costimulatory molecule.
  • Costimulatory molecules can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors.
  • TNF receptor proteins CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, Lymphocyte Function Associated Antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C , SLAMF7, NKp80, CD160, B7-H3 and ligands that specifically bind to CD83, etc.
  • the intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment thereof.
  • the term "4-1BB” refers to a member of the TNFR superfamily having the amino acid sequence provided under GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc.;"
  • the 4-1BB costimulatory domain is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • encoding refers to the inherent property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA or mRNA to serve as a template in biological processes for the synthesis of other polymers and macromolecules with a defined Nucleotide sequences (eg, rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties.
  • a gene, cDNA or RNA encodes a protein if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system.
  • nucleotide sequences encoding amino acid sequences include all nucleotide sequences that are in degenerate form of each other and that encode the same amino acid sequence.
  • a "nucleotide sequence” encoding a protein or RNA may also include introns, ie, a nucleotide sequence encoding a protein may, in some forms, contain one or more introns.
  • expression refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.
  • transfer vector refers to a composition comprising an isolated nucleic acid and a substance that can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes autonomously replicating plasmids or viruses.
  • the term should also be construed to further encompass non-plasmid and non-viral compounds that facilitate transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide sequence to be expressed.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all expression vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses) incorporating recombinant polynucleotides virus and adeno-associated virus).
  • homologous refers to a subunit sequence between two polymer molecules, eg, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules identity.
  • a subunit position in two molecules is occupied by the same monomeric subunit; for example, if the position of each of two DNA molecules is occupied by an adenine, they are homologous or identical at that position.
  • Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are Homologous, then the two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) are matched or homologous, the two sequences are 90% homologous .
  • percent identity refers to the two or more sequences that are identical.
  • percent identity refers to the two or more sequences that are identical.
  • sequence comparison algorithms e.g., 60% identity over a specified region, or, if not specified, the entire sequence, optionally 70%, 71%, 72%, 73%, 74% %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity), then the two sequences are "substantially identical".
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably 100 to 500 or 1000 or more nucleotides in length ( or 20, 50, 200 or more amino acids).
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be specified.
  • the sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Sequence alignment methods for comparison are well known in the art.
  • the invention contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, scFv) that produces a functionally equivalent molecule.
  • an anti-GPC3 or Claudin18.2 binding domain such as the VH or VL of a scFv, contained in a TFP can be modified to retain at least about 70% of the starting VH or VL framework region of the anti-GPC3 or Claudin18.2 binding domain, such as the scFv , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the present invention contemplates modification of the entire TFP construct, eg, modification of one or more amino acid sequences of individual domains of the TFP construct, in order to generate functionally equivalent molecules.
  • the TFP construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the starting TFP construct , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity.
  • sequence of the anti-GPC3 antibody or fragment thereof used in the present invention is SEQ ID NO: 4, 20-99.
  • sequence of the anti-Claudin18.2 antibody or fragment thereof used in the present invention is SEQ ID NO: 100-189.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or peptide that occurs naturally in a living animal is not “isolated”, but the same nucleic acid or peptide that is partially or completely separated from the material with which it occurs in nature is “isolated.”
  • An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as a host cell.
  • operably linked refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which results in the expression of the latter.
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is arranged in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the sequence.
  • Operably linked DNA sequences can be contiguous to each other and, for example, where it is desired to join two protein coding regions, the DNA sequences are in the same reading frame.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form.
  • the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements, as well as explicitly indicated sequences.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues.
  • peptide refers to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds.
  • the term refers to both short chains, also commonly known in the art as eg peptides, oligopeptides, and oligomers, and long chains, commonly known in the art as proteins, of which many exist Types of.
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.
  • promoter/regulatory sequence refers to a nucleic acid sequence required for the expression of a gene product to which the promoter/regulatory sequence is operably linked.
  • constitutive refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in production of the gene product in a cell under most or all physiological conditions of the cell.
  • inducible promoter means that, when operably linked to a polynucleotide encoding or specifying a gene product, it results in production of a gene in a cell substantially only when an inducer corresponding to the promoter is present in the cell The nucleotide sequence of the product.
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen.
  • Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources.
  • Antibodies can be tetramers of immunoglobulin molecules.
  • antibody fragment refers to at least a portion of an antibody that retains the ability to specifically interact (eg, through binding, steric hindrance, stabilization/destabilization, steric distribution) with an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, Linear antibodies, single domain antibodies such as sdAbs (VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments (eg bivalent fragments comprising two Fab fragments linked by disulfide bonds at the hinge region) and An isolated CDR or other epitope binding fragment of an antibody.
  • Antigen-binding fragments can also be incorporated into single-domain antibodies, maximal antibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, " Nature Biotechnology (23): 1126-1136, 2005).
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N-terminus and C-terminus of the polypeptide), the scFv may include a VL-linker-VH or VH-linker-VL can be included.
  • antibody heavy chain refers to the larger of two polypeptide chains that are present in an antibody molecule in its naturally occurring configuration and that generally determine the class to which the antibody belongs.
  • antibody light chain refers to the smaller of two polypeptide chains present in an antibody molecule in its naturally occurring configuration. Kappa (k) and lambda (l) light chains refer to the two major antibody light chain isotypes.
  • recombinant antibody refers to antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed by phage or yeast expression systems.
  • the term should also be interpreted to refer to an antibody that has been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or the amino acid sequence of the specified antibody, wherein the DNA or amino acid sequence has been using recombinant DNA or is available in the art and well-known amino acid sequence techniques.
  • antibodies or antibody fragments of the invention can be further modified such that they vary in amino acid sequence (eg, relative to wild type), but not in the desired activity.
  • additional nucleotide substitutions can be made in the protein, resulting in amino acid substitutions at "non-essential" amino acid residues.
  • a non-essential amino acid residue in a molecule can be substituted with another amino acid residue from the same side chain family.
  • the amino acid sequence may be substituted with a sequence that is structurally similar but differs in sequence and/or composition from side chain family members, eg, conservative substitutions may be made in which amino acid residues are replaced by amino acid residues with similar side chains base substituted.
  • Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • antigen refers to a molecule that elicits an immune response.
  • the immune response may involve antibody production or activation of specific immunocompetent cells or both.
  • any macromolecule including virtually any protein or peptide, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA that includes a nucleotide sequence or a portion of a nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen".
  • the antigen need not be encoded solely by the full-length nucleotide sequence of the gene.
  • the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response.
  • the antigen need not be encoded by a "gene”.
  • antigens can be produced synthetically, or can be derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.
  • the term "antigen recognition unit” refers to immunoglobulin molecules and immunologically active portions of immunological molecules, ie, containing an antigen binding site that specifically binds ("immunoreactive") an antigen molecule.
  • immunoglobulin molecules derived from various species including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibodies (eg, IgG) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • the immunoglobulin representation includes several types of molecules, such as IgD, IgG, IgA, IgM and IgE.
  • immunoglobulin molecule includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen-binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen recognition units". Also included in the term “antigen recognition unit” is any polypeptide chain-containing molecular structure having a specific shape that conforms to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the relationship between the molecular structure and the epitope. Complex.
  • antigen recognition units examples include Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, bivalent fragments (F(ab) comprising two Fab fragments linked by a disulfide bond on the hinge region 2 fragments); Fd fragments composed of VH and CH1 domains, Fv fragments composed of the VL and VH domains of the one-armed antibody; dAb fragments composed of VH domains (Ward et al., Nature, 341:544- 546, 1989); and an isolated complementarity determining region (CDR) or any fusion protein comprising such an antigen recognition unit.
  • CDR complementarity determining region
  • An antigen-recognition unit "specifically binds" or is “immunoreactive” with an antigen if it binds the antigen with greater affinity or avidity as compared to other reference antigens, including polypeptides or other substances ".
  • Tumor antigen refers to an antigen common to specific hyperproliferative diseases.
  • the hyperproliferative disorder antigens of the invention are derived from cancer or tumors.
  • the tumor antigens of the present invention include, but are not limited to: Thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease Ser
  • the pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
  • tumor heterogeneity means that during the growth process of a tumor, after multiple divisions and proliferations, its daughter cells exhibit molecular biological or genetic changes, which make the tumor's growth rate, invasive ability, and drug sensitivity. gender, prognosis, etc. It is one of the characteristics of malignant tumors.
  • cancer refers to a broad class of disorders characterized by hyperproliferative cell growth in vitro (eg, transformed cells) or in vivo.
  • Conditions that can be treated or prevented by the methods of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like.
  • cancer examples include, but are not limited to: blood cancer, colon cancer, rectal cancer, renal cell cancer, liver cancer, non-small cell cancer of the lung, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, Head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureter cancer, renal pelvis Carcinoma, Central Nervous System (CNS) Tumor, Primary CNS Lymphoma, Tumor Angiogenesis, Spinal Tumor, Brains
  • transfected or “transformed” or “transduced” refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cells include primary subject cells and their progeny.
  • the term "specifically binds” refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample .
  • a binding partner eg, tumor antigen
  • Refractory refers to a disease, eg, cancer, that does not respond to treatment.
  • the refractory cancer may be resistant to treatment prior to or at initiation of treatment.
  • refractory cancers may become resistant during treatment.
  • Refractory cancers are also called resistant cancers.
  • refractory cancers include, but are not limited to, radiotherapy-insensitive, relapsed after radiotherapy, chemotherapy-insensitive, relapsed after chemotherapy, insensitive to CAR-T therapy, or relapsed after treatment. Refractory or relapsed malignancies can be treated with the treatment regimens described herein.
  • relapsed refers to a return to a disease (eg, cancer) or signs and symptoms of a disease such as cancer over a period of improvement, eg, after prior treatment with therapy, eg, cancer therapy.
  • a disease eg, cancer
  • signs and symptoms of a disease such as cancer over a period of improvement, eg, after prior treatment with therapy, eg, cancer therapy.
  • a “patient” is a subject having or at risk of having a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
  • enhancing refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein.
  • an enhanced response can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the responsiveness %, 75%, 80%, 85%, 90%, 95% or 98% or more increase.
  • enhancing can also refer to increasing the number of subjects that respond to treatment, eg, immune effector cell therapy.
  • an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentages are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
  • treatment is judged by clinical outcome, and may also be increased, enhanced or prolonged by the anti-tumor activity of T cells; an increase in the number of anti-tumor T cells or activated T cells compared to the number before treatment, promotes IFN- ⁇ , Granzyme-B, IL2, TNF- ⁇ , GM-CSF secretion, or a combination thereof.
  • the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor expansion, recurrence or spread, or a combination thereof.
  • the therapeutic effect is predicted by the presence of T cells, the presence of genetic markers indicative of T cell inflammation, promotion of IFN-gamma secretion, or a combination thereof.
  • Cells as disclosed herein can be administered to an individual by various routes, including, for example, orally or parenterally, such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral, nasal Intravasally, intradermally, or passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
  • routes including, for example, orally or parenterally, such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral, nasal Intravasally, intradermally, or passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
  • the total amount of agent to be administered in practicing the methods of the invention may be administered to the subject as a single dose as a bolus injection or by infusion over a relatively short period of time, or may be administered using a graded treatment regimen, wherein over an extended period of time Administer multiple doses.
  • Those of skill in the art will know that the amount of the composition to treat a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, the technician will adjust the specific dose as needed. In general, initially, Phase I and Phase II clinical trials are used to determine the formulation of the composition as well as the route and frequency of administration.
  • Ranges Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be regarded as an inexorable limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, the description of a range such as from 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as within that range. individual values such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6.
  • a range such as 95-99% identity includes ranges having 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the width of the range.
  • GPC3 or "Glypican 3" as used herein is a member of the Glypican family, gene accession numbers: NM_016697.3, NP_057906.2, which play an important role in regulating cell growth and differentiation.
  • the abnormal expression of GPC3 is closely related to the occurrence and development of various tumors, such as abnormal expression in liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, etc.
  • TFP co-expressing IL7 targets GPC3-positive tumors.
  • the tumor includes, but is not limited to, liver cancer, gastric cancer, lung cancer, esophageal cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, Adrenal cancer, schwannoma, malignant fibrous histiocytoma, esophageal cancer.
  • the GPC3-positive expressing tumor or GPC-positive tumor described herein is liver cancer, gastric cancer, lung cancer, esophageal cancer.
  • the present invention also provides T cells modified with a TCR fusion protein that co-expresses IL7, which is transduced with a nucleic acid encoding the IL7 and TCR fusion protein or transduced with the above-mentioned recombinant plasmid comprising the nucleic acid, or a plasmid comprising the plasmid Virus.
  • Conventional nucleic acid transduction methods in the art including non-viral and viral transduction methods, can be used in the present invention.
  • Non-viral-based transduction methods include electroporation and transposon methods.
  • the method of transduction of immune effector cells to achieve genetic modification of IL7 and TCR fusion proteins is a virus-based transduction method such as retrovirus or lentivirus.
  • the method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro cultured immune effector cells to reach clinical level.
  • the transduced nucleic acid is expressed on its surface through transcription and translation.
  • Modified immune effector cells have a highly specific tumor cell killing effect (also known as cytotoxicity) and can effectively survive in tumor tissue. Therefore, the nucleic acid encoding the chimeric antigen receptor of the present invention, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic immune effector cells transduced with the nucleic acid, plasmid or virus can be effectively used for tumor immunotherapy.
  • the T cells modified by the TCR fusion protein of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents.
  • the composition may also comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • some substances which may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose Sodium, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycols; alginic acid; emulsifiers such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions and phosphate buffers, etc
  • composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by a physician at a dose beneficial to the patient according to factors such as the type, age, weight and general disease state of the patient, and the mode of administration.
  • the mode of administration can be by injection or other therapeutic methods.
  • the cells of the present invention may be used in combination with chemotherapeutic drugs when used in tumor therapy.
  • TCR fusion proteins of the present invention can be selected from the following sequential linkages:
  • Antigen recognition unit-TCR ⁇ Antigen recognition unit-TCR ⁇
  • the CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ and TCR ⁇ are the full lengths including the extracellular region, the transmembrane region and the intracellular region.
  • the present invention also includes nucleic acids encoding said TCRs.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
  • the immune effector cells co-expressing IL7 and TCR fusion proteins targeting specific antigens of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents.
  • the composition may also comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the immune effector cells provided herein have more excellent killing effect on solid tumor cells and in vitro expansion performance. It can effectively increase the proliferation, survival and function of the immune effector cells in the tumor; reduce the expression of inhibitory immune checkpoints, thereby alleviating the exhaustion of T cells.
  • Exemplary antigen receptors of the present invention and methods for engineering and introducing the receptors into cells, refer to, for example, Chinese Patent Application Publication Nos. CN106397593A, CN106467573A, CN104140974A, CN 108884459 A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN 109908176 A, CN109880803A, CN 110055275 A, CN110123837A, CN 110438082 A, CN 110468105 A; international Patent application Publication No.
  • WO2017186121A1 WO2018006882A1, WO2015172339A8, WO2018 / 018958A1, WO2014180306 A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, WO2018045811A1, WO2018108106A1, WO2018 / 219299, WO2018 / 210279, WO2019 / 024933, WO2019 / 114751, WO2019 / 114762, Those disclosed in WO2019/141270, WO2019/149279, WO2019/170147A1, WO 2019/210863, WO2019/219029.
  • the scFv used in this example is an antibody targeting GPC3, the amino acid sequence is shown in SEQ ID NO: 4, and the nucleic acid sequence is shown in SEQ ID NO: 6.
  • the anti-GPC3 single-chain antibody GPC3 and CD3 ⁇ were connected by inserting a short linker to form an anti-GPC3 retroviral plasmid.
  • mCD8 signal peptide SEQ ID NO: 190
  • anti-GPC3 antibody SEQ ID NO: 6
  • linker 1 SEQ ID NO: 8
  • mCD3 ⁇ SEQ ID NO: 9
  • the gene sequences of these three fragments were joined together by bridging PCR to form the fragment GPC3-mCD3 ⁇ (SEQ ID NO: 10).
  • the vector was digested with the restriction enzymes EcoRI&HindIII to obtain the linearized vector pMSCV-EcoRI&HindIII, and the homologous recombinase was used to circularize the digested vector and the fragment to form the plasmid pMSCV-GPC3-mCD3 ⁇ .
  • GPC3-mCD3 ⁇ sequentially links F2A (SEQ ID NO:5) and mIL7 (SEQ ID NO:3).
  • the gene sequences F2A and mIL7 were linked together by bridging PCR to form F2A-mIL7.
  • Inoculate 293T cells in a petri dish using conventional techniques in the art, respectively use plasmids pMSCV-GPC3-mCD3 ⁇ , pMSCV-GPC3-mCD3 ⁇ -F2A-mIL7 to transfect 293T cells, 48h after transfection, collect virus supernatant , the retroviruses GPC3-CD3 ⁇ , GPC3-CD3 ⁇ -F2A-IL7 were obtained.
  • Example 2 In vitro killing toxicity detection and in vitro cytokine secretion detection
  • CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. For specific methods, refer to the instructions of CytoTox 96 non-radioactive cytotoxicity detection kit.
  • the epitope human-derived mouse GPC3 was transferred into mouse hepatoma cells Hepa 1-6 (Cell Collection Center, Chinese Academy of Sciences (Shanghai) and mouse breast cancer cell E0771, respectively, to construct Hepa 1-6 overexpressing GPC3 protein. 6 GPC3 and E0771 GPC3 cells.
  • the number of target cells was (Hepa 1-6:10000/well; Hepa 1-6 GPC3:10000/well, E0771 GPC3:10000/well), and the effector-target ratio was 1:1, 1:3, and co-cultured with effector cells for 18h Detection (1640+10% FBS, 200ul system), the effector cells were GPC3-CD3 ⁇ T cells and GPC3-CD3 ⁇ -F2A-IL7T cells expressing TCR fusion protein.
  • ELISA was used to detect the cytokine secretion after co-incubation of T cells expressing TCR fusion protein with liver cancer Hepa 1-6 GPC3 or E0771-GPC3 cells, and co-incubated for 24 hours according to the effect-target ratio of 1:1, and the cytokine expression in the cell culture supernatant was detected.
  • IL7, IFN- ⁇ , Granzyme-B, IL2, TNF- ⁇ and GM-CSF secretion are shown in Figure 3A-F, respectively, in which Granzyme-B represents T cell degranulation, GM-CSF is T cell activation released cytokines.
  • Figure 3A shows that the expression of IL7 was basically undetectable in the supernatant of GPC3-CD3 ⁇ T cells; IL7 secretion was significantly increased.
  • Figures 3B-F show that the cytokines IFN- ⁇ , Granzyme-B, IL2, TNF- ⁇ and GM-CSF secreted by T cells expressing TCR fusion protein after co-incubation with GPC3-expressing hepatoma cells were significantly increased relative to UTD.
  • Hepa 1-6 GPC3 were inoculated subcutaneously in the right axilla of female C57BL/6 mice (Shanghai Sipple-Bike Laboratory Animal Co., Ltd.), and divided into 3 groups, 6 mice in each group, inoculation diary D0.
  • the 3 groups were: UTD group, GPC3-CD3 ⁇ T cell group, GPC3-CD3 ⁇ -F2A-IL7 T cell group.
  • T cells expressing TCR fusion protein were injected into the tail vein, and the injection dose was 1.5 ⁇ 10 6 cells/only.
  • the blank control group was injected with 1.5 ⁇ 10 6 cells/only.
  • T cells are used in the above embodiments, other immune cells can also be selected, such as NK cells, NK-T cells, and specific subtypes of immune cells can also be selected, such as ⁇ / ⁇ T cells.

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Abstract

A genetically engineered immune effector cell, wherein the cell expresses an IL-7R binding protein or IL-7, and also expresses a receptor that specifically recognizes a target antigen.

Description

表达IL-7R结合蛋白的免疫效应细胞Immune effector cells expressing IL-7R binding protein 技术领域technical field
本发明属于细胞治疗领域，涉及表达IL-7R结合蛋白的免疫效应细胞。The invention belongs to the field of cell therapy, and relates to immune effector cells expressing IL-7R binding protein.
背景技术Background technique
CAR-T细胞能够以MHC非限制性的方式进行肿瘤特异性地杀伤，在肿瘤免疫治疗中显示出较好的应用前景，但其仍有较多局限性，如对实体瘤的疗效不佳、在体外表现出优良效果的候选药物，在体内往往无法表现出相应的效果。CAR-T cells can specifically kill tumors in a MHC-unrestricted manner, and show good application prospects in tumor immunotherapy, but they still have many limitations, such as poor efficacy on solid tumors, Drug candidates that exhibit excellent effects in vitro often fail to exhibit corresponding effects in vivo.
当前免疫治疗已经成为肿瘤临床治疗中不可或缺的环节。免疫治疗的药物和方案涉及到机体免疫***识别和攻击癌细胞的各个阶段。已有的肿瘤免疫药物包括以下多个类型：靶向癌细胞的抗体、过继细胞治疗、溶瘤病毒、树突状细胞相关治疗、DNA和蛋白水平的肿瘤疫苗、免疫激活细胞因子以及其他免疫调节化合物。At present, immunotherapy has become an indispensable link in the clinical treatment of tumors. Immunotherapy drugs and regimens involve the various stages in which the body's immune system recognizes and attacks cancer cells. Existing immuno-oncology drugs include the following types: antibodies targeting cancer cells, adoptive cell therapy, oncolytic viruses, dendritic cell-related therapy, tumor vaccines at the DNA and protein levels, immune-activating cytokines, and other immune modulators compound.
基于T细胞的基因工程改造包括CAR-T和TCR-T。前者需要构建嵌合抗原受体，通常是将单链抗体通过铰链区和跨膜段与CD3δ的胞内段相连接然后通过病毒转导致T细胞，通过单链抗体和肿瘤细胞表面的抗原相结合从而激活CD3δ的胞内信号，继而引起转导的T细胞对肿瘤细胞杀伤作用。后者通常是需要转导一个能够特异性识别肿瘤抗原肽/MHC复合物的TCR于T细胞，然后利用这些TCR与T细胞内在的CD3亚基形成新的TCR复合物，从而使T细胞能特异性的靶向肿瘤细胞，激活整个TCR复合物的信号通路，达到杀伤肿瘤细胞的目的。T cell-based genetic engineering includes CAR-T and TCR-T. The former requires the construction of a chimeric antigen receptor, usually by linking a single-chain antibody to the intracellular segment of CD3δ through a hinge region and a transmembrane segment, and then transducing the virus to induce T cells, which bind to the antigen on the surface of the tumor cell through the single-chain antibody. Thus, the intracellular signal of CD3δ is activated, which in turn causes the killing effect of the transduced T cells on tumor cells. The latter usually requires the transduction of a TCR that can specifically recognize tumor antigen peptide/MHC complexes into T cells, and then use these TCRs to form new TCR complexes with the CD3 subunit inherent in T cells, so that T cells can be specific. It can specifically target tumor cells, activate the signaling pathway of the entire TCR complex, and achieve the purpose of killing tumor cells.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种共表达IL-7和TFP的免疫效应细胞。The purpose of the present invention is to provide an immune effector cell that co-expresses IL-7 and TFP.
本发明第一方面提供了一种基因工程化的免疫效应细胞，所述细胞表达IL-7R结合蛋白或IL-7；还表达特异性识别靶抗原的受体。A first aspect of the present invention provides a genetically engineered immune effector cell, which expresses IL-7R binding protein or IL-7; and also expresses a receptor that specifically recognizes a target antigen.
在一优选例中，所述特异性识别靶抗原的受体为TCR融合蛋白(TFP)。In a preferred embodiment, the receptor that specifically recognizes the target antigen is a TCR fusion protein (TFP).
在一优选例中，所述IL-7R结合蛋白为外源性IL-7R结合蛋白，所述外源性IL-7R结合蛋白能特异性结合IL-7R且增强IL-7R活性；优选地，所述的IL-7R结合蛋白为IL-7，进一步优选地所述IL-7R的氨基酸序列如SEQ ID NO:1所示；。In a preferred embodiment, the IL-7R-binding protein is an exogenous IL-7R-binding protein, and the exogenous IL-7R-binding protein can specifically bind to IL-7R and enhance the activity of IL-7R; preferably, The IL-7R binding protein is IL-7, and further preferably, the amino acid sequence of the IL-7R is shown in SEQ ID NO: 1;.
在一优选例中，所述IL-7为天然IL-7、或与天然IL-7具有相同功能的天然IL-7的截短片段、或天然IL-7的突变体；In a preferred embodiment, the IL-7 is native IL-7, or a truncated fragment of native IL-7 having the same function as native IL-7, or a mutant of native IL-7;
优选地，所述IL-7的核酸序列与SEQ ID NO:2或3所示的序列具有至少90％同一性、或是SEQ ID NO:2或3所示的核酸序列的截短片段；或所述IL-7的氨基酸序列与SEQ ID NO:13或19所示的序列具有至少80％同一性、或是SEQ ID NO:13或19所示的氨基酸序列的截短片段。Preferably, the nucleic acid sequence of IL-7 has at least 90% identity with the sequence shown in SEQ ID NO: 2 or 3, or is a truncated fragment of the nucleic acid sequence shown in SEQ ID NO: 2 or 3; or The amino acid sequence of the IL-7 has at least 80% identity with the sequence shown in SEQ ID NO: 13 or 19, or a truncated fragment of the amino acid sequence shown in SEQ ID NO: 13 or 19.
在一优选例中，所述IL7为组成性表达或诱导性表达；优选地，为诱导型启动子诱导所述IL-7的表达；优选地，所述启动子是NFAT6启动子；优选地，所述NFAT6启动子为反向调控。In a preferred embodiment, the IL7 is constitutively expressed or inducible; preferably, the expression of the IL-7 is induced by an inducible promoter; preferably, the promoter is the NFAT6 promoter; preferably, The NFAT6 promoter is reverse regulated.
在一优选例中，所述TFP包含：In a preferred embodiment, the TFP includes:
(a)TCR亚基(或称TCR单元)；和(a) TCR subunits (or TCR units); and
(b)识别靶抗原的抗原识别单元；(b) an antigen recognition unit that recognizes the target antigen;
其中所述TCR亚基和所述抗原识别单元可操作地连接。wherein the TCR subunit and the antigen recognition unit are operably linked.
在一优选例中，所述TCR亚基包含：In a preferred embodiment, the TCR subunit comprises:
(i)TCR细胞外结构域的至少一部分，和(i) at least a portion of the TCR extracellular domain, and
(ii)包含来自CD3ε、CD3γ、CD3δ、TCRα、TCRβ的细胞内信号传导结构域的刺激性结构域的TCR细胞内结构域或其组合；(ii) a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ, TCRα, TCRβ, or a combination thereof;
或所述TCR亚基包含：CD3αTCR亚基、CD3βTCR亚基、CD3εTCR亚基、CD3γTCR亚基、CD3δTCR亚基或其组合。Or the TCR subunit comprises: a CD3α TCR subunit, a CD3β TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, or a combination thereof.
在一优选例中，所述靶抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；***干细胞抗原(PSCA)；***特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记25 1(TEM1/CD248)；肿 瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoHglycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受5体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；***蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变10体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)；In a preferred embodiment, the target antigen is selected from: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); ); interleukin 11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1 ; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; Specific embryonic antigen-4 (SSEA-4); cell surface associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion carbonic anhydrase IX (CAIX); LMP2; ephrin A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; Tumor Vascular Endothelial Marker 251 (TEM1/CD248); Tumor Vascular Endothelial Marker 7 Related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 Integrin; B Cell Maturation Antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligands ; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; Reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta specificity 1 (PLAC1); Hexose moiety of globoHglycoceramide (GloboH); Mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor 5 body β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis Antigen-1 (MAD-CT-1); Melanoma Cancer Testis Antigen-2 (MAD-CT-2); Fos-Associated Antigen 1; p53 Mutation 10; Human Telomerase Reverse Transcriptase (hTERT); site breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box Protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytomatosis virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C ( RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin Binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase-anchored protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocytes Related immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type agglutination Protein domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); containing EGF-like module mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Carbohydrate-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLL1);
或，所述靶抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；其中，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原或流感病毒抗原。Or, the target antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; wherein, the viral antigens are selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen or influenza virus antigen.
在一优选例中，所述抗原识别单元包括抗体或其片段，优选的，所述抗体或其片段为Fab， Fab'、F(ab')2、Fv片段、scFv、sdFv、由VH和CH1结构域组成的Fd片段、线性抗体、单域抗体、或camelid VHH结构域，更优选的，所述抗体为scFv。In a preferred embodiment, the antigen recognition unit includes an antibody or a fragment thereof, preferably, the antibody or fragment thereof is a Fab, Fab', F(ab')2, Fv fragment, scFv, sdFv, composed of VH and CH1. Fd fragments composed of domains, linear antibodies, single-domain antibodies, or camelid VHH domains, more preferably, the antibody is an scFv.
在一优选例中，所述抗原识别单元识别GPC3或识别Claudin18.2，In a preferred embodiment, the antigen recognition unit recognizes GPC3 or recognizes Claudin18.2,
优选地，识别GPC3的抗原识别单元的氨基酸序列的轻链LCDR1、LCDR2和LCDR3独立选自下表所示轻链LCDR1、LCDR2和LCDR3或与之具有70-100％的序列同一性，和/或Preferably, the light chains LCDR1, LCDR2 and LCDR3 recognizing the amino acid sequence of the antigen recognition unit of GPC3 are independently selected from or have 70-100% sequence identity with the light chains LCDR1, LCDR2 and LCDR3 shown in the table below, and/or
识别GPC3的抗原识别单元的氨基酸序列的重链HCDR1、HCDR2和HCDR3独立选自下表所示重链HCDR1、HCDR2和HCDR3或与之具有70-100％的序列同一性；The heavy chains HCDR1, HCDR2 and HCDR3 recognizing the amino acid sequence of the antigen recognition unit of GPC3 are independently selected from or have 70-100% sequence identity with the heavy chains HCDR1, HCDR2 and HCDR3 shown in the following table;
LCDR1LCDR1 LCDR2LCDR2 LCDR3LCDR3 HCDR1HCDR1 HCDR2HCDR2 HCDR3HCDR3
SEQ ID No.32SEQ ID No.32 SEQ ID No.33SEQ ID No. 33 SEQ ID No.34SEQ ID No.34 SEQ ID No.29SEQ ID No. 29 SEQ ID No.30SEQ ID No.30 SEQ ID No.31SEQ ID No.31
SEQ ID No.41SEQ ID No.41 SEQ ID No.42SEQ ID No.42 SEQ ID No.43SEQ ID No.43 SEQ ID No.38SEQ ID No. 38 SEQ ID No.39SEQ ID No. 39 SEQ ID No.40SEQ ID No.40
SEQ ID No.50SEQ ID No.50 SEQ ID No.51SEQ ID No. 51 SEQ ID No.52SEQ ID No. 52 SEQ ID No.47SEQ ID No.47 SEQ ID No.48SEQ ID No.48 SEQ ID No.49SEQ ID No.49
SEQ ID No.59SEQ ID No. 59 SEQ ID No.60SEQ ID No.60 SEQ ID No.61SEQ ID No.61 SEQ ID No.56SEQ ID No. 56 SEQ ID No.57SEQ ID No. 57 SEQ ID No.58SEQ ID No. 58
SEQ ID No.68SEQ ID No.68 SEQ ID No.69SEQ ID No.69 SEQ ID No.70SEQ ID No.70 SEQ ID No.65SEQ ID No.65 SEQ ID No.66SEQ ID No.66 SEQ ID No.67SEQ ID No. 67
SEQ ID No.77SEQ ID No. 77 SEQ ID No.78SEQ ID No. 78 SEQ ID No.79SEQ ID No. 79 SEQ ID No.74SEQ ID No. 74 SEQ ID No.75SEQ ID No. 75 SEQ ID No.76SEQ ID No. 76
SEQ ID No.86SEQ ID No. 86 SEQ ID No.87SEQ ID No. 87 SEQ ID No.88SEQ ID No. 88 SEQ ID No.83SEQ ID No. 83 SEQ ID No.84SEQ ID No. 84 SEQ ID No.85SEQ ID No. 85
SEQ ID No.23SEQ ID No. 23 SEQ ID No.24SEQ ID No. 24 SEQ ID No.25SEQ ID No. 25 SEQ ID No.20SEQ ID No.20 SEQ ID No.21SEQ ID No. 21 SEQ ID No.22SEQ ID No. 22
SEQ ID No.95SEQ ID No. 95 SEQ ID No.96SEQ ID No. 96 SEQ ID No.97SEQ ID No. 97 SEQ ID No.92SEQ ID No. 92 SEQ ID No.93SEQ ID No. 93 SEQ ID No.94SEQ ID No. 94
；或者;or
识别claudin18.2的抗原识别单元的氨基酸序列的轻链LCDR1、LCDR2和LCDR3独立选自下表所示轻链LCDR1、LCDR2和LCDR3或与之具有70-100％的序列同一性，和/或识别claudin18.2的抗原识别单元的氨基酸序列的重链HCDR1、HCDR2和HCDR3独立选自下表所示重链HCDR1、HCDR2和HCDR3或与之具有70-100％的序列同一性：The light chains LCDR1, LCDR2 and LCDR3 that recognize the amino acid sequence of the antigen recognition unit of claudin18.2 are independently selected from or have 70-100% sequence identity to the light chains LCDR1, LCDR2 and LCDR3 shown in the table below, and/or recognize The heavy chain HCDR1, HCDR2 and HCDR3 of the amino acid sequence of the antigen recognition unit of claudin18.2 are independently selected from or have 70-100% sequence identity with the heavy chains HCDR1, HCDR2 and HCDR3 shown in the following table:
Figure PCTCN2021119634-appb-000001
Figure PCTCN2021119634-appb-000001
在一优选例中，识别GPC3的抗原识别单元的氨基酸序列的轻链可变区独立选自下表所示轻链可变区或与之具有70-100％的序列同一性，和/或识别GPC3的抗原识别单元的氨基酸序列的重链可变区独立选自下表所示重链可变区或与之具有70-100％的序列同一性；In a preferred embodiment, the light chain variable region that recognizes the amino acid sequence of the antigen recognition unit of GPC3 is independently selected from the light chain variable region shown in the following table or has 70-100% sequence identity with it, and/or recognizes The heavy chain variable region of the amino acid sequence of the antigen recognition unit of GPC3 is independently selected from the heavy chain variable region shown in the following table or has 70-100% sequence identity therewith;
VHVH VLVL
SEQ ID No.35SEQ ID No.35 SEQ ID No.36SEQ ID No.36
SEQ ID No.44SEQ ID No.44 SEQ ID No.45SEQ ID No.45
SEQ ID No.53SEQ ID No. 53 SEQ ID No.54SEQ ID No. 54
SEQ ID No.62SEQ ID No.62 SEQ ID No.63SEQ ID No.63
SEQ ID No.71SEQ ID No. 71 SEQ ID No.72SEQ ID No. 72
SEQ ID No.80SEQ ID No.80 SEQ ID No.81SEQ ID No. 81
SEQ ID No.89SEQ ID No. 89 SEQ ID No.90SEQ ID No. 90
SEQ ID No.26SEQ ID No. 26 SEQ ID No.27SEQ ID No. 27
SEQ ID No.98SEQ ID No. 98 SEQ ID No.99SEQ ID No. 99
；或者，识别claudin18.2的抗原识别单元的氨基酸序列的轻链可变区独立选自下表所示轻链可变区或与之具有70-100％的序列同一性，和/或识别claudin18.2的抗原识别单元的氨基酸序列的重链可变区独立选自下表所示重链可变区或与之具有70-100％的序列同一性；Or, the light chain variable region that recognizes the amino acid sequence of the antigen recognition unit of claudin18.2 is independently selected from the light chain variable region shown in the following table or has 70-100% sequence identity with it, and/or recognizes claudin18 The heavy chain variable region of the amino acid sequence of the antigen recognition unit of .2 is independently selected from the heavy chain variable region shown in the following table or has 70-100% sequence identity therewith;
VHVH VLVL
SEQ ID No.116SEQ ID No. 116 SEQ ID No.117SEQ ID No. 117
SEQ ID No.125SEQ ID No. 125 SEQ ID No.126SEQ ID No. 126
SEQ ID No.134SEQ ID No. 134 SEQ ID No.135SEQ ID No. 135
SEQ ID No.143SEQ ID No. 143 SEQ ID No.144SEQ ID No. 144
SEQ ID No.152SEQ ID No. 152 SEQ ID No.153SEQ ID No. 153
SEQ ID No.161SEQ ID No. 161 SEQ ID No.162SEQ ID No. 162
SEQ ID No.170SEQ ID No. 170 SEQ ID No.171SEQ ID No. 171
SEQ ID No.179SEQ ID No. 179 SEQ ID No.180SEQ ID No. 180
SEQ ID No.188SEQ ID No. 188 SEQ ID No.189SEQ ID No. 189
SEQ ID No.107SEQ ID No. 107 SEQ ID No.108SEQ ID No. 108
在一优选例中，识别GPC3的抗原识别单元的氨基酸序列选自下表所示序列或与之具有70-100％的序列同一性；In a preferred embodiment, the amino acid sequence of the antigen recognition unit that recognizes GPC3 is selected from the sequences shown in the following table or has 70-100% sequence identity with it;
SEQ ID NO.28SEQ ID NO.28
SEQ ID NO.37SEQ ID NO.37
SEQ ID NO.46SEQ ID NO.46
SEQ ID NO.55SEQ ID NO.55
SEQ ID NO.64SEQ ID NO.64
SEQ ID NO.73SEQ ID NO.73
SEQ ID NO.82SEQ ID NO.82
SEQ ID NO.4SEQ ID NO.4
SEQ ID NO.91SEQ ID NO.91
；或者，;or,
识别claudin18.2的抗原识别单元的氨基酸序列选自下表所示序列或与之具有70-100％的序列同一性；The amino acid sequence of the antigen recognition unit that recognizes claudin18.2 is selected from the sequence shown in the following table or has 70-100% sequence identity with it;
SEQ ID NO.109SEQ ID NO.109
SEQ ID NO.118SEQ ID NO. 118
SEQ ID NO.127SEQ ID NO. 127
SEQ ID NO.136SEQ ID NO. 136
SEQ ID NO.145SEQ ID NO. 145
SEQ ID NO.154SEQ ID NO. 154
SEQ ID NO.163SEQ ID NO. 163
SEQ ID NO.172SEQ ID NO. 172
SEQ ID NO.181SEQ ID NO. 181
SEQ ID NO.100SEQ ID NO.100
在一优选例中，所述抗原识别单元通过连接子序列连接至所述TCR细胞外结构域的至少一部分；优选所述连接子序列包括(G 4S) n，其中n＝1至4； In a preferred embodiment, the antigen recognition unit is connected to at least a part of the extracellular domain of the TCR through a linker sequence; preferably, the linker sequence includes (G 4 S) n , wherein n=1 to 4;
进一步优选所述连接子的编码序列具有SEQ ID NO：8或11所示的核酸序列；或具有SEQ ID NO：12所示的氨基酸序列。Further preferably, the coding sequence of the linker has the nucleic acid sequence shown in SEQ ID NO: 8 or 11; or the amino acid sequence shown in SEQ ID NO: 12.
在一优选例中，所述TCR细胞外结构域的至少一部分、所述TFP包括的TCR跨膜结构域和TCR细胞内结构域中,至少两个结构域是来自同一TCR亚基。In a preferred embodiment, at least two of the TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain included in the TFP are from the same TCR subunit.
在一优选例中，所述TFP与IL7由同一核酸分子表达，或由不同核酸分子表达；In a preferred embodiment, the TFP and IL7 are expressed by the same nucleic acid molecule, or expressed by different nucleic acid molecules;
优选所述TFP与IL7由同一核酸分子表达，所述IL7的表达盒与TFP之间、以及表达盒之间直接连接或由串联片段连接，所述串联片段选自F2A、PA2、T2A、和/或E2A。Preferably, the TFP and IL7 are expressed from the same nucleic acid molecule, and the expression cassette of IL7 and TFP, as well as between the expression cassettes, are directly connected or connected by tandem fragments selected from F2A, PA2, T2A, and/or or E2A.
在一优选例中，所述TFP包括含有免疫受体酪氨酸活化基序(ITAM)的蛋白；所述含有ITAM的蛋白包括选自由以下组成的组的蛋白质或其部分：CD3δTCR亚基、CD3εTCR亚基、CD3γTCR亚基、CD3δTCR亚基、Fcε受体1链、Fcε受体2链、Fcγ受体1链、Fcγ受体2a链、Fcγ受体2b1链、Fcγ受体2b2链、Fcγ受体3a链、Fcγ受体3b链、Fcβ受体1链、TYROBP(DAP12)、CD5、CD16a、CD16b、CD22、CD23、CD32、CD64、CD79a、CD79b、CD89、CD278、CD66d或其功能片段；In a preferred embodiment, the TFP includes a protein containing an immunoreceptor tyrosine activation motif (ITAM); the ITAM-containing protein includes a protein or a part thereof selected from the group consisting of: CD3δTCR subunit, CD3εTCR Subunit, CD3γTCR subunit, CD3δTCR subunit, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d or functional fragments thereof;
进一步优选，所述TFP分子还包含前导序列，优选地，所述前导序列包括如SEQ ID NO：7所示的核酸序列。Further preferably, the TFP molecule further comprises a leader sequence, preferably, the leader sequence comprises the nucleic acid sequence shown in SEQ ID NO:7.
在一优选例中，所述TFP包括SEQ ID NO：9、10或16所示的核酸序列；或TFP包括SEQ ID NO：14或15所示的氨基酸序列。In a preferred embodiment, the TFP includes the nucleic acid sequence shown in SEQ ID NO: 9, 10 or 16; or the TFP includes the amino acid sequence shown in SEQ ID NO: 14 or 15.
本发明第二方面提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的IL-7R结合蛋白或IL-7；所述核酸分子还编码权利要求1-17中任一项所述的特异性识别靶抗原的受体；The second aspect of the present invention provides a nucleic acid molecule encoding the IL-7R binding protein or IL-7 according to the first aspect of the present invention; the nucleic acid molecule also encodes any one of claims 1-17 the receptor that specifically recognizes the target antigen;
优选，所述核酸由DNA和/或RNA组成；Preferably, the nucleic acid consists of DNA and/or RNA;
进一步优选，所述核酸是mRNA；Further preferably, the nucleic acid is mRNA;
进一步优选，所述核酸包含核苷酸类似物。Further preferably, the nucleic acid comprises a nucleotide analog.
本发明第三方面提供了一种载体，包含本发明第二方面所述的核酸分子，The third aspect of the present invention provides a vector, comprising the nucleic acid molecule described in the second aspect of the present invention,
优选所述载体选自由以下组成的组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、劳氏肉瘤病毒(RSV)载体或逆转录病毒载体。Preferably the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, Rous Sarcoma virus (RSV) vector or retroviral vector.
本发明第四方面提供了一种细胞，其包含本发明第三方面所述的载体或其基因组上整合有本发明第而方面所述的核酸分子，优选所述细胞是人T细胞，优选地，为同种异体T细胞。The fourth aspect of the present invention provides a cell comprising the vector of the third aspect of the present invention or the nucleic acid molecule of the first aspect of the present invention integrated into its genome, preferably the cell is a human T cell, preferably , for allogeneic T cells.
本发明第五方面提供了一种制备细胞的方法，所述方法包括用本发明第三方面所述的载体或本发明第二方面所述的核酸分子转导T细胞。The fifth aspect of the present invention provides a method for preparing cells, the method comprising transducing T cells with the vector described in the third aspect of the present invention or the nucleic acid molecule described in the second aspect of the present invention.
本发明第六方面提供了一种产生RNA工程改造的细胞群的方法，所述方法包括将体外转录的RNA或合成RNA引入细胞中，其中所述RNA包括本发明第二方面所述的核酸。A sixth aspect of the present invention provides a method of producing an RNA engineered cell population, the method comprising introducing into a cell an in vitro transcribed RNA or a synthetic RNA, wherein the RNA comprises the nucleic acid of the second aspect of the present invention.
本发明第七方面提供了一种在哺乳动物体内提供抗肿瘤免疫性的方法，所述方法包括向所述哺乳动物施用有效量的本发明第一、第四方面所述细胞、本发明第二方面所述的核酸分子、本发明第三方面所述的载体；优选所述哺乳动物是人。The seventh aspect of the present invention provides a method for providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of the cells of the first and fourth aspects of the present invention, the second aspect of the present invention The nucleic acid molecule of the aspect, the vector of the third aspect of the present invention; preferably, the mammal is a human.
本发明第八方面提供了一种治疗患有与GPC3或者claudin18.2表达有关的疾病的哺乳动物的方法，所述方法包括向所述哺乳动物施用有效量的本发明第一、第四方面所述细胞、本发明第二方面所述的核酸分子、本发明第三方面所述的载体；The eighth aspect of the present invention provides a method for treating a mammal suffering from a disease related to the expression of GPC3 or claudin18.2, the method comprising administering to the mammal an effective amount of the first and fourth aspects of the present invention the cell, the nucleic acid molecule described in the second aspect of the present invention, and the vector described in the third aspect of the present invention;
优选所述与GPC3或者claudin18.2表达有关的疾病选自结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，***，***癌，内分泌***癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，***癌，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经***(CNS)瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌、与GPC3或者claudin18.2表达有关的非癌症相关适应症；优选的，选自肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、胰腺癌、食道癌；Preferably, the disease related to the expression of GPC3 or claudin18.2 is selected from colon cancer, rectal cancer, renal cell cancer, liver cancer, lung cancer, small bowel cancer, esophagus cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer , skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, endocrine system cancer, thyroid cancer Carcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, tumor angiogenesis, spinal tumor, brain stem glial tumor, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, non-cancer-related indications related to GPC3 or claudin18.2 expression; preferably, selected from liver cancer, lung cancer, breast cancer, ovarian cancer, Kidney cancer, thyroid cancer, stomach cancer, colorectal cancer, pancreatic cancer, esophageal cancer;
优选本发明第一、第四方面所述细胞和增加本发明第一、第四方面所述细胞功效的药剂组合施用；Preferably, the cells of the first and fourth aspects of the present invention are administered in combination with an agent that increases the efficacy of the cells of the first and fourth aspects of the present invention;
优选本发明第一、第四方面所述细胞与改善与施用本发明第一、第四方面所述细胞相关的一种或多种副作用的药剂联合施用；Preferably, the cells of the first and fourth aspects of the present invention are administered in combination with an agent that ameliorates one or more side effects associated with the administration of the cells of the first and fourth aspects of the present invention;
优选本发明第一、第四方面所述细胞和治疗所述与GPC3或者claudin18.2相关的疾病的药剂组合施用。Preferably, the cells described in the first and fourth aspects of the present invention are administered in combination with the agent for treating the disease associated with GPC3 or claudin18.2.
本发明第一、第四方面所述细胞、本发明第二方面所述的核酸分子、本发明第三方面所述的载体，用作药物。The cells described in the first and fourth aspects of the present invention, the nucleic acid molecules described in the second aspect of the present invention, and the vectors described in the third aspect of the present invention are used as medicines.
本发明所提供的细胞，能够提高CAR T细胞的肿瘤细胞杀伤能力。The cells provided by the present invention can improve the tumor cell killing ability of CAR T cells.
附图说明Description of drawings
图1显示了pMSCV-GPC3-mCD3ε、pMSCV-GPC3-mCD3ε-F2A-mIL7质粒简图；Figure 1 shows the schematic diagrams of pMSCV-GPC3-mCD3ε, pMSCV-GPC3-mCD3ε-F2A-mIL7 plasmids;
图2为GPC3-CD3ε-F2A-IL7和GPC3-CD3ε的体外杀伤结果；Figure 2 shows the in vitro killing results of GPC3-CD3ε-F2A-IL7 and GPC3-CD3ε;
图3A-3F为细胞因子分泌结果。Figures 3A-3F are cytokine secretion results.
图4A、4B分别为GPC3-CD3ε-F2A-IL7和GPC3-CD3ε体内杀伤实验的肿瘤体积、小鼠体重变化结果。Figures 4A and 4B are the results of tumor volume and mouse body weight changes in GPC3-CD3ε-F2A-IL7 and GPC3-CD3ε in vivo killing experiments, respectively.
具体实施方式detailed description
本申请提供的增强表达TCR融合蛋白(也称为TFP蛋白)的细胞对实体瘤治疗治疗的技术方案。The application provides a technical solution for enhancing the treatment of solid tumors by cells expressing TCR fusion protein (also known as TFP protein).
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的那些的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, where suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, unless otherwise specified, the materials, methods, and examples are illustrative only and not intended to be limiting.
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，WileyandsonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief， Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. These techniques are fully explained in the literature. See, eg, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wileyandson Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJGaited., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & S. J. Higginseds. 1984); Transcription And Translation (BD Hames & S. J. Higginseds. 1984 ); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, "Gene Expression Technology "(D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHiller and MP Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Exper imental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
为便于更好地理解本发明，对相关术语定义如下：For a better understanding of the present invention, relevant terms are defined as follows:
如本文所用，“约”可表示取决于具体情况并且由本领域技术人员已知或可知的，加或减小于1％或1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、25％、30％或大于30％。As used herein, "about" may mean plus or minus 1% or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% or greater than 30 %.
术语“T细胞(抗原)受体(T cell receptor,TCR)，也称为TCR亚基，或TCR单元”为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。TCR负责识别与主要组织相容性复合体分子结合的抗原。TCR是由两条不同肽链构成的异二聚体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等几部分；其特点是胞质区很短。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时，CDR1，CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。这些天然(或者通过其他手段制造的)的“抗癌”T细胞的识别能力常常比较弱，因此不能形成对癌细胞的有利攻击。在这种情况下，可以通过部分基因修改的方法来提高这些TCR对相应TAA的“亲和力”和战斗力，即高亲和力TCR。“基因修饰的TCR”技术也因此被称为“亲和力增强的TCR”技术(Affinity-Enhanced TCR)。基因修饰的T细胞受体(Gene Modified TCR)利用与TCR分子同属于免疫球蛋白超家族的抗体重链和轻链的恒定区结构域分别替换其β链和a链的恒定区结构域，形成了一种嵌合型的TCR分子(chim-TCR)。The term "T cell (antigen) receptor (TCR), also known as TCR subunit, or TCR unit" is a characteristic marker on the surface of all T cells that binds non-covalently to CD3 to form TCR- CD3 complex. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is a heterodimer composed of two different peptide chains, consisting of α and β peptide chains. The cytoplasmic region is characterized by a short cytoplasmic region. The TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (Vα, Vβ) has three hypervariable regions, CDR1, CDR2, and CDR3. Among them, CDR3 has the largest variation, which directly determines the antigen of TCR. binding specificity. When TCR recognizes the MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the side wall of the antigen-binding groove of the MHC molecule, while CDR3 directly binds to the antigen peptide. TCRs are divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, γ and δ, and TCR2 is composed of two chains, α and β. These natural (or by other means) "anti-cancer" T cells are often poorly recognized and thus unable to mount a favorable attack on cancer cells. In this case, the "affinity" and combat effectiveness of these TCRs to the corresponding TAAs, ie high-affinity TCRs, can be improved by means of partial genetic modification. The "genetically modified TCR" technology is also called "Affinity-Enhanced TCR" technology (Affinity-Enhanced TCR). The gene modified T cell receptor (Gene Modified TCR) replaces the constant region domains of its beta chain and alpha chain with the constant region domains of the antibody heavy and light chains, which belong to the same immunoglobulin superfamily as the TCR molecule. A chimeric TCR molecule (chim-TCR) was developed.
术语“TCR融合蛋白(也称为TFP蛋白)”包括来源于TCR的各种蛋白的重组蛋白，一般能够：i)结合至靶细胞上的表面抗原；ii)当定位于T细胞时与完整TCR复合物的其它多肽组分相互作用。“TFP T细胞或TFP-T细胞”是已被转导(例如，根据本文公开的方法)和表达TFP的T细胞。在一些实施方案中，该T细胞是CD4+T细胞、CD8+T细胞或CD4+/CD8+T细胞。在一些实施方案中，该TFP T细胞是NK细胞。在一些实施方案中，该TFP T细胞是γδT细胞。 本发明的“TCR融合蛋白(也称为TFP蛋白)”包含胞外的抗原结合结构域(也称为抗原识别单元)、TCR跨膜域及胞内域，其中抗原结合结构域是作为连续多肽链，包括例如单结构域抗体片段(sdAb)，来源于鼠类、人源化或人抗体的单链抗体(scFv)的一部分表达(Harlow等人,1999,Using Antibodies:ALaboratoryManual,Cold Spring Harbor Laboratory Press,N.Y.；Harlow等人,1989,Antibodies:A Laboratory Manual,Cold Spring Harbor,N.Y.；Houston等人,1988,Proc.Natl.Acad.Sci.USA 85:5879-5883；Bird等人,1988,Science 242:423-426)。在一个方面，本发明的TFP组合物的抗原结合结构域包括抗体或抗体片段。在另一方面，TFP包括含scFv或sdAb的抗体或抗体片段。The term "TCR fusion protein (also known as TFP protein)" includes recombinant proteins derived from various proteins of TCR, generally capable of: i) binding to surface antigens on target cells; ii) binding to intact TCR when localized to T cells The other polypeptide components of the complex interact. "TFP T cells or TFP-T cells" are T cells that have been transduced (eg, according to the methods disclosed herein) and express TFP. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or CD4+/CD8+ T cells. In some embodiments, the TFP T cells are NK cells. In some embodiments, the TFP T cells are γδ T cells. The "TCR fusion protein (also called TFP protein)" of the present invention comprises an extracellular antigen binding domain (also called an antigen recognition unit), a TCR transmembrane domain and an intracellular domain, wherein the antigen binding domain is a continuous polypeptide Chains, including, for example, single-domain antibody fragments (sdAbs), a portion of single-chain antibodies (scFvs) derived from murine, humanized or human antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al, 1988, Science 242:423-426). In one aspect, the antigen binding domain of the TFP composition of the invention comprises an antibody or antibody fragment. In another aspect, the TFP includes an antibody or antibody fragment comprising an scFv or sdAb.
本发明包括编码TFP的重组DNA构建体，其中该TFP包含与GPC3特异性结合的抗体片段，其中该抗体片段的序列与编码TCR单位或其部分的核酸序列邻接并在同一阅读框中。本文提供的TFP能够与一个或多个内源(或替代地，一个或多个外源，或者内源与外源的组合)TCR单位缔合，以便形成功能性TCR复合物。The present invention includes recombinant DNA constructs encoding TFP, wherein the TFP comprises an antibody fragment that specifically binds to GPC3, wherein the sequence of the antibody fragment is contiguous and in reading frame with the nucleic acid sequence encoding the TCR unit or portion thereof. The TFPs provided herein are capable of associating with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR units in order to form functional TCR complexes.
在一个方面，本发明的TFP包含靶抗原特异性结合元件，也被称为抗原识别单元。所述抗原识别单元的选择取决于靶细胞表面的靶抗原的类型和数目。例如，可选择抗原识别单元以识别作为靶细胞上与特定疾病状态相关的细胞表面标志物的靶抗原。因此，可作为本发明TFP中抗原识别单元的靶抗原的细胞表面标志物的实例包括与病毒、细菌和寄生物感染、自身免疫病和癌性疾病(例如，恶性疾病)相关的标志物。本发明所述抗原识别单元通过连接子序列连接至所述组成TFP的T细胞受体的细胞外结构域；优选所述连接子序列包括(G 4S) n，其中n＝1至4；进一步优选所述连接子的编码序列具有SEQ ID NO：8或11所示的核酸序列；或具有SEQ ID NO：12所示的氨基酸序列。 In one aspect, the TFP of the invention comprises a target antigen-specific binding element, also referred to as an antigen recognition unit. The choice of the antigen recognition unit depends on the type and number of target antigens on the surface of the target cells. For example, the antigen recognition unit can be selected to recognize the target antigen as a cell surface marker on the target cell that is associated with a particular disease state. Thus, examples of cell surface markers that can be target antigens for the antigen recognition units in the TFPs of the present invention include markers associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancerous diseases (eg, malignant diseases). The antigen recognition unit of the present invention is linked to the extracellular domain of the T cell receptor constituting TFP through a linker sequence; preferably, the linker sequence includes (G 4 S) n , wherein n=1 to 4; further Preferably, the coding sequence of the linker has the nucleic acid sequence shown in SEQ ID NO: 8 or 11; or the amino acid sequence shown in SEQ ID NO: 12.
本发明的TFP的细胞外结构域可衍生自天然来源或来自重组来源。在天然来源的情况下，该结构域可衍生自任何蛋白质，特别是膜结合蛋白质或跨膜蛋白质。在一个方面，细胞外结构域能够与跨膜结构域缔合。在本发明中的细胞外结构域可包括至少以下细胞外区域：例如T细胞受体的α、β或δ链，或者CD3ε、CD3γ或CD3δ，或者在替代实施方案中，包括CD28、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和/或CD154。The extracellular domains of the TFPs of the invention can be derived from natural sources or from recombinant sources. In the case of natural sources, the domain may be derived from any protein, especially a membrane-bound protein or a transmembrane protein. In one aspect, the extracellular domain is capable of associating with the transmembrane domain. The extracellular domain in the present invention may comprise at least the following extracellular domains: for example the alpha, beta or delta chains of T cell receptors, or CD3ε, CD3γ or CD3δ, or in alternative embodiments CD28, CD45, CD4 , CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and/or CD154.
本发明的TFP的跨膜结构域可衍生自天然来源或来自重组来源。在天然来源的情况下，该结构域可衍生自任何膜结合蛋白质或跨膜蛋白质。在一个方面，每当TFP与靶抗原结合时，跨膜结构域能够向细胞内结构域发信号。在本发明中跨膜结构域可包括至少以下跨膜区域：例如T细胞受体的α、β或δ链，或者CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、 CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和/或CD154。在一些情况下，跨膜结构域可经由铰链(例如来自人类蛋白质的铰链)与TFP的细胞外区域(例如TFP的抗原结合结构域)连接。例如，在一个实施方案中，该铰链可以是人免疫球蛋白(Ig)铰链，例如IgG4铰链或CD8a铰链。The transmembrane domains of the TFPs of the present invention may be derived from natural sources or from recombinant sources. In the case of natural sources, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the TFP binds to the target antigen. The transmembrane domain in the present invention may comprise at least the following transmembrane domains: for example the alpha, beta or delta chains of T cell receptors, or CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and/or CD154. In some cases, the transmembrane domain can be linked to the extracellular region of TFP (eg, the antigen binding domain of TFP) via a hinge (eg, from a human protein). For example, in one embodiment, the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge or a CD8a hinge.
本发明的连接体任选地，长度为2至10个氨基酸的短寡肽或多肽连接体可形成TFP的跨膜结构域与细胞质区域之间的连接。甘氨酸-丝氨酸双联体提供了特别合适的连接体。Linkers of the Invention Optionally, short oligopeptide or polypeptide linkers of 2 to 10 amino acids in length can form a link between the transmembrane domain and the cytoplasmic region of TFP. Glycine-serine doublets provide particularly suitable linkers.
如果TFP含有CD3γ、δ或ε多肽，则TFP的细胞质结构域可包括细胞内信号传导结构域；TCRα和TCRβ亚单位通常缺乏信号传导结构域。细胞内信号传导结构域通常负责激活其中已引入TFP的免疫细胞的至少一种正常效应物功能。术语“效应物功能”是指细胞的特化功能。例如，T细胞的效应物功能可以是细胞溶解活性或辅助活性，包括细胞因子的分泌。在一个实施例中，与过表达TFP靶向抗原的肿瘤细胞共孵育，所述TFP-T细胞能分泌大量IFN-γ，Granzyme-B，IL2，TNF-α及GM-CSF；与同样抗原识别单元构建的CAR T细胞相比，表达TFP蛋白的TFP-T细胞保持了同样显著的细胞毒性，但是细胞因子分泌量明显降低，有效的降低了细胞因子风暴的可能。If the TFP contains a CD3 gamma, delta, or epsilon polypeptide, the cytoplasmic domain of the TFP may include an intracellular signaling domain; the TCRα and TCRβ subunits typically lack the signaling domain. Intracellular signaling domains are generally responsible for activating at least one normal effector function of immune cells into which TFP has been introduced. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. In one example, co-incubated with tumor cells overexpressing TFP-targeted antigens, the TFP-T cells can secrete large amounts of IFN-γ, Granzyme-B, IL2, TNF-α and GM-CSF; recognize the same antigens Compared with cell-constructed CAR T cells, TFP-T cells expressing TFP protein maintained the same significant cytotoxicity, but the secretion of cytokines was significantly reduced, effectively reducing the possibility of cytokine storm.
因此，术语“细胞内信号传导结构域”是指蛋白质的部分，其转导效应物功能信号并引导细胞执行特化功能。虽然通常可采用整个细胞内信号传导结构域，但在许多情况下不必使用整条链。就使用细胞内信号传导结构域的截短部分来说，可使用这样的截短部分代替完整链，只要其转导效应物功能信号即可。因此，术语“细胞内信号传导结构域”包括足以转导效应物功能信号的细胞内信号传导结构域的任何截短部分。Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs cells to perform specialized functions. While the entire intracellular signaling domain can generally be employed, in many cases it is not necessary to use the entire chain. In the case of the use of truncated portions of intracellular signaling domains, such truncated portions can be used in place of the full chain, so long as they transduce effector function signals. Thus, the term "intracellular signaling domain" includes any truncated portion of an intracellular signaling domain sufficient to signal effector function.
以刺激方式起作用的初级细胞内信号传导结构域可含有信号传导基序，其被称为基于免疫受体酪氨酸的活化基序(ITAM)。在本发明中含有ITAM的初级细胞内信号传导结构域的实例包括CD3δ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的细胞内信号传导结构域。在一个实施方案中，本发明的TFP包含细胞内信号传导结构域，例如CD3ε的初级信号传导结构域。在一个实施方案中，初级信号传导结构域包含修饰的ITAM结构域，例如与天然ITAM结构域相比具有改变的(例如，升高或降低的)活性的突变ITAM结构域。在一个实施方案中，初级信号传导结构域包括含有修饰的ITAM的初级细胞内信号传导结构域，例如，含有优化的和/或截短的ITAM的初级细胞内信号传导结构域。在一个实施方案中，初级信号传导结构域包含一个、两个、三个、四个或更多个ITAM基序。Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM-containing primary intracellular signaling domains in the present invention include the intracellular signaling domains of CD3δ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the TFP of the invention comprises an intracellular signaling domain, eg, the primary signaling domain of CD3ε. In one embodiment, the primary signaling domain comprises a modified ITAM domain, eg, a mutant ITAM domain with altered (eg, increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain includes a primary intracellular signaling domain containing a modified ITAM, eg, a primary intracellular signaling domain containing an optimized and/or truncated ITAM. In one embodiment, the primary signaling domain comprises one, two, three, four or more ITAM motifs.
TFP的细胞内信号传导结构域可包含单独的CD3δ信号传导结构域，或者其可与在本发明 TFP的背景下有用的任何其他所需的细胞内信号传导结构域组合。在一个是实施例中，TFP的细胞内信号传导结构域可包含CD3ε链部分和共刺激信号传导结构域。共刺激信号传导结构域是指包含共刺激分子的细胞内结构域。共刺激分子是除了抗原受体或其配体之外的细胞表面分子，其为淋巴细胞对抗原的有效应答所需的。此类分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、DAP10、DAP12、CD30、CD40、PD1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3以及与CD83特异性结合的配体等。The intracellular signaling domain of a TFP may comprise the CD3delta signaling domain alone, or it may be combined with any other desired intracellular signaling domain useful in the context of the TFP of the invention. In one embodiment, the intracellular signaling domain of the TFP may comprise a CD3ε chain moiety and a costimulatory signaling domain. A costimulatory signaling domain refers to an intracellular domain comprising a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, DAP10, DAP12, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and ligands that specifically bind to CD83, etc.
本发明TFP的细胞质部分内的细胞内信号传导序列可以以随机或指定的顺序彼此连接。任选地，短的寡肽或多肽连接体，例如，长度为2至10个氨基酸(例如，2、3、4、5、6、7、8、9或10个氨基酸)，可在细胞内信号传导序列之间形成连接。The intracellular signaling sequences within the cytoplasmic portion of the TFP of the invention may be linked to each other in random or specified order. Optionally, short oligopeptide or polypeptide linkers, eg, 2 to 10 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), can be intracellular Links are formed between signaling sequences.
在另一个方面，本文所述的表达TFP的细胞可进一步表达另一种因子，例如细胞因子、转录因子、趋化因子、和/或其组合，来增加T细胞的增殖、细胞存活、抗凋亡作用、肿瘤浸润等作用来提高抗肿瘤活性。在一个实施例中，表达TFP的T细胞还表达细胞因子IL-7，所述TFP-T细胞与表达有TFP靶向抗原的肿瘤细胞共孵育，所述TFP-T细胞能明显增加对所述肿瘤细胞的体外杀伤毒性，能显著抑制所述肿瘤细胞形成的体内皮下移植瘤；对体外细胞因子分泌检测发现所述TFP-T细胞能分泌大量IFN-γ、Granzyme-B、IL2，TNF-α及GM-CSF。与表达IL-7且同样抗原识别单元构建的CAR T细胞相比，表达IL-7的TFP-T细胞保持了同样显著的细胞毒性，但是细胞因子分泌量明显降低，有效的降低了细胞因子风暴的可能。In another aspect, the TFP-expressing cells described herein may further express another factor, such as a cytokine, transcription factor, chemokine, and/or a combination thereof, to increase T cell proliferation, cell survival, anti-apoptosis Anti-tumor activity can be enhanced by the effects of apoptosis and tumor infiltration. In one example, the TFP-expressing T cells that also express the cytokine IL-7, and the TFP-T cells co-incubated with tumor cells expressing the TFP-targeted antigen, the TFP-T cells can significantly increase the response to the TFP-targeting antigen. The in vitro killing toxicity of tumor cells can significantly inhibit the in vivo subcutaneous transplanted tumors formed by the tumor cells; the in vitro cytokine secretion test shows that the TFP-T cells can secrete a large amount of IFN-γ, Granzyme-B, IL2, TNF-α and GM-CSF. Compared with CAR T cells expressing IL-7 and constructed with the same antigen recognition unit, TFP-T cells expressing IL-7 maintained the same significant cytotoxicity, but the secretion of cytokines was significantly reduced, effectively reducing the cytokine storm. possible.
本术语“IL-7(Interleukin7，白细胞介素7或IL7)”具有以下特征之一：(i)为天然存在的哺乳动物IL-7的氨基酸序列或其片段，例如SEQIDNO：19(人)或SEQIDNO：13(鼠)所示的氨基酸序列或其片段；(ii)基本上与SEQIDNO：19(人)或SEQIDNO：13(鼠)所示氨基酸或其片段具有例如至少85％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(iii)由天然存在的哺乳动物IL-7核苷酸序列或其片段(例如SEQIDNO：2(人)或其片段所编码的氨基酸序列、或SEQIDNO：3(鼠)或其片段所编码的氨基酸序列；(iv)由与SEQ ID NO:2(人)或SEQIDNO：3(鼠)所示核苷酸序列或其片段具有例如至少85％、90％、95％、96％、97％、98％、99％同源性的核苷酸序列所编码的氨基酸序列；(v)由与天然存在的IL-7核苷酸序列或其片段简并的核苷酸序列(例如SEQ ID NO：2(人)或其片段、或SEQIDNO：3(鼠)或其片段)所编码的氨基酸序列；或(vi)在严格性条件与前述核苷酸序列之一杂交的核苷酸序列。IL7可以与IL-7R(优选来自哺乳动物，例如鼠或人IL-7R)相互作用(例如结合)，优选来自哺乳动物，例如鼠或人。IL7也可以通过非IL-7R通路起抗肿瘤作用。The term "IL-7 (Interleukin7, interleukin 7 or IL7)" has one of the following characteristics: (i) is the amino acid sequence of a naturally occurring mammalian IL-7 or a fragment thereof, eg, SEQ ID NO: 19 (human) or The amino acid sequence set forth in SEQ ID NO: 13 (murine) or a fragment thereof; (ii) substantially the same as the amino acid set forth in SEQ ID NO: 19 (human) or SEQ ID NO: 13 (murine) or a fragment thereof, for example, at least 85%, 90%, 95% %, 96%, 97%, 98%, 99% homology of amino acid sequences; (iii) from naturally occurring mammalian IL-7 nucleotide sequences or fragments thereof (eg SEQ ID NO: 2 (human) or fragments thereof) The encoded amino acid sequence, or the amino acid sequence encoded by SEQ ID NO: 3 (mouse) or a fragment thereof; (iv) by the nucleotide sequence shown in SEQ ID NO: 2 (human) or SEQ ID NO: 3 (mouse) or its Fragments have, for example, an amino acid sequence encoded by a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous to a nucleotide sequence; The amino acid sequence encoded by a nucleotide sequence or a fragment thereof degenerate nucleotide sequence (e.g., SEQ ID NO: 2 (human) or a fragment thereof, or SEQ ID NO: 3 (murine) or a fragment thereof); or (vi) A nucleotide sequence that hybridizes to one of the aforementioned nucleotide sequences. IL7 can interact (eg, bind) to an IL-7R (preferably from a mammal, such as a murine or human IL-7R), preferably from a mammal, such as Murine or human. IL7 can also exert anti-tumor effects through non-IL-7R pathways.
“外源性IL-7R结合蛋白”是指能特异性结合IL-7R且增强IL-7R活性的所有蛋白质。 “增强IL-7R活性”应理解为意指IL-7R结合蛋白能够增强天然存在的IL-7R的任何一种或多种活性，包括但不限于刺激NK细胞的增殖、细胞毒性或成熟；刺激B细胞和T细胞的增殖或分化；刺激B细胞中的抗体产生和亲和力成熟；刺激CD8+T细胞的细胞毒性；刺激T细胞和NK细胞中干扰素γ产生；抑制树突状细胞(DC)活化和成熟；抑制炎性介质从肥大细胞释放；增强巨噬细胞的吞噬作用；抑制TReg细胞(调节性T细胞)的产生或存活；和刺激骨髓祖细胞的增殖。"Exogenous IL-7R binding protein" refers to all proteins that can specifically bind to IL-7R and enhance the activity of IL-7R. "Enhancing IL-7R activity" is understood to mean that an IL-7R binding protein is capable of enhancing any one or more activities of naturally occurring IL-7R, including but not limited to stimulation of NK cell proliferation, cytotoxicity or maturation; stimulation of Proliferation or differentiation of B cells and T cells; stimulation of antibody production and affinity maturation in B cells; stimulation of cytotoxicity of CD8+ T cells; stimulation of interferon gamma production in T cells and NK cells; inhibition of dendritic cells (DC) Activation and maturation; inhibits the release of inflammatory mediators from mast cells; enhances phagocytosis of macrophages; inhibits the production or survival of TReg cells (regulatory T cells); and stimulates proliferation of myeloid progenitor cells.
本发明还提供了编码本文所述的一种或多种共表达IL7和TFP构建体的核酸分子。TFP与IL7由同一核酸分子表达，或由不同核酸分子表达。在具体实施例中，TFP与IL7由同一核酸分子表达，所述IL7的表达盒与TFP之间、以及表达盒之间直接连接或由串联片段连接，所述串联片段选自F2A、PA2、T2A、和/或E2A。The invention also provides nucleic acid molecules encoding one or more of the co-expressing IL7 and TFP constructs described herein. TFP and IL7 are expressed from the same nucleic acid molecule, or from different nucleic acid molecules. In a specific embodiment, TFP and IL7 are expressed by the same nucleic acid molecule, and the expression cassette of IL7 and TFP, as well as between the expression cassettes are directly connected or connected by tandem fragments selected from F2A, PA2, T2A , and/or E2A.
本发明还提供了本发明DNA所***的载体。衍生自逆转录病毒如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因的长期、稳定的整合及其在子细胞中的繁殖。例如，核酸可克隆到包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒等载体中。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。本发明不限于使用组成型启动子，也考虑诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在需要表达时启动其可操作地连接的多核苷酸序列的表达，或者当不需要表达时关闭该表达。诱导型启动子的实例包括但不限于NFAT6启动子、金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素调节的启动子。The present invention also provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. The present invention is not limited to the use of constitutive promoters, but also contemplates inducible promoters. The use of an inducible promoter provides a molecular switch that enables expression of a polynucleotide sequence to which it is operably linked when expression is desired, or turns off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, NFAT6 promoters, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters.
在扩充和遗传修饰之前，从受试者获得T细胞来源。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可从许多来源获得，包括外周血单个核细胞、骨髓、***组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。在本发明的某些方面，可使用本领域可获得的任何数目的T细胞系。在本发明的某些方面，可使用本领域技术人员已知的任意数量的技术如FicollTM分离技术从收集自受试者的血液单位中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体的循环血液的细胞。单采血液成分术产物通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个方面，可洗涤通过单采血液成分术收集的细胞以去除血浆部分并将细胞置于适当的缓冲液或培养基中以供后续处理步骤。A source of T cells is obtained from the subject prior to expansion and genetic modification. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the invention, any number of T cell lines available in the art can be used. In certain aspects of the invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as the Ficoll™ separation technique. In a preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps.
通常，本发明的T细胞可通过与附着有刺激CD3/TCR复合物相关信号的药剂和刺激T细胞表面上的共刺激分子的配体的表面接触而扩充。特别地，可如本文所述刺激T细胞群体，诸如通过与抗CD3抗体或其抗原结合片段或固定在表面上的抗CD2抗体接触，或通过与蛋白 激酶C活化剂(例如，苔藓抑制素)以及钙离子载体接触。Generally, T cells of the invention can be expanded by surface contact with an agent that stimulates signals associated with the CD3/TCR complex and ligands that stimulate co-stimulatory molecules on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by a protein kinase C activator (eg, bryostatin) and calcium ionophore contacts.
术语“免疫效应细胞”是指在免疫反应期间发挥效应功能的细胞。例如，包括分泌细胞因子和/或趋化因子、杀伤微生物、分泌抗体、识别或清除肿瘤细胞的免疫细胞。在一些实施例中，免疫效应细胞包括T细胞(细胞毒性T细胞、辅助T细胞、肿瘤浸润性T细胞)、B细胞、NK细胞、NKT细胞、肥大细胞、巨噬细胞、树突细胞、CIK细胞、或干细胞衍生的免疫效应细胞。The term "immune effector cells" refers to cells that perform effector functions during an immune response. For example, it includes immune cells that secrete cytokines and/or chemokines, kill microorganisms, secrete antibodies, and recognize or eliminate tumor cells. In some embodiments, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, or stem cell-derived immune effector cells.
术语“免疫效应功能”包括免疫***的组成所介导的任何功能，可导致抑制肿瘤生长和/或抑制肿瘤发生，包括抑制肿瘤的扩散和转移。优选地，免疫效应功能杀伤肿瘤细胞。优选地，本发明中的免疫效应功能是抗体介导，包括补体依赖的细胞毒性作用(CDC)、抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞介导的吞噬作用(ADCP)、在携带肿瘤相关抗原的细胞中诱导凋亡(例如，通过抗体与表面抗原的结合)、抑制CD40L介导的信号转导(例如通过抗体与CD40受体或CD40配体(CD40L)相结合)，和/或抑制携带肿瘤相关抗原的细胞的增殖，优选ADCC和/或CDC。因此，能够介导一种或更多种免疫效应功能的抗体优选地能够通过诱导CDC介导的裂解、ADCC介导的裂解、凋亡、同型黏着和/或吞噬作用(优选通过诱导CDC介导的裂解和/或ADCC介导的裂解)来介导对肿瘤细胞的杀伤。抗体还可简单地通过与癌细胞表面上的肿瘤相关抗原结合而发挥作用。例如，抗体可通过与肿瘤细胞表面上的肿瘤相关抗原结合来阻断肿瘤相关抗原的功能或诱导凋亡。The term "immune effector function" includes any function mediated by components of the immune system that can result in inhibition of tumor growth and/or inhibition of tumorigenesis, including inhibition of tumor spread and metastasis. Preferably, the immune effector function kills tumor cells. Preferably, the immune effector function in the present invention is antibody-mediated, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis ( ADCP), induction of apoptosis in cells bearing tumor-associated antigens (eg, by binding of antibodies to surface antigens), inhibition of CD40L-mediated signaling (eg, by antibodies interacting with CD40 receptor or CD40 ligand (CD40L) binding), and/or inhibit the proliferation of cells bearing tumor-associated antigens, preferably ADCC and/or CDC. Thus, antibodies capable of mediating one or more immune effector functions are preferably capable of lysis and/or ADCC-mediated lysis) to mediate killing of tumor cells. Antibodies can also act simply by binding to tumor-associated antigens on the surface of cancer cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis by binding to tumor-associated antigens on the surface of tumor cells.
术语“抗原呈递细胞”或“APC”是指在表面上展示外来抗原与主要组织相容性复合物(MHC)的复合物的免疫***细胞，如辅助细胞(例如B细胞、树突状细胞等)。T细胞可以使用其T细胞受体(TCR)识别这些复合物。APC加工抗原并将其呈递至T细胞。The term "antigen presenting cell" or "APC" refers to a cell of the immune system, such as a helper cell (eg, B cells, dendritic cells, etc.) ). T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.
术语“抗肿瘤作用”是指可以通过各种方式表现的生物作用，包括但不限于，例如肿瘤体积减小、肿瘤细胞数量减少、转移数量减少、期望寿命增加、肿瘤细胞增殖减少、肿瘤细胞存活率降低，或与癌性病况相关的各种生理症状改善。“抗肿瘤作用”还可以由本发明的肽、多核苷酸、细胞及抗体在首先防止肿瘤发生方面的能力来表现。The term "anti-tumor effect" refers to a biological effect that can be manifested in various ways, including, but not limited to, for example, decreased tumor volume, decreased tumor cell number, decreased number of metastases, increased life expectancy, decreased tumor cell proliferation, tumor cell survival rate, or improvement in various physical symptoms associated with cancerous conditions. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent tumorigenesis in the first place.
术语“自体”是指来源于个体的任何材料稍后将被再引入该相同个体中。The term "autologous" refers to any material derived from an individual to be later reintroduced into that same individual.
术语“同种异体”是指任何材料来源于与引入该材料的个体相同物种的不同动物或不同患者。当在一个或多个基因座处的基因不同时，认为两个或更多个体彼此为同种异体的。在一些方面，来自同一物种的各个体的同种异体材料在基因上的不同可能足以发生抗原相互作用。The term "allogeneic" refers to any material derived from a different animal or different patient of the same species as the individual into which the material is introduced. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are different. In some aspects, allogeneic material from individuals of the same species may be genetically different enough for antigenic interactions to occur.
术语“异种”是指移植物来源于不同物种的动物。The term "xenogeneic" refers to a graft derived from an animal of a different species.
术语“基因工程化的细胞”，是指通过基因工程的手段改造的细胞。The term "genetically engineered cell" refers to a cell that has been engineered by means of genetic engineering.
术语“治疗有效量”、“治疗有效的”、“有效量”或“以有效的量”在本文中可互换地使用，并且是指如本文中所述有效地实现特定生物学结果的化合物、制剂、物质或组合物的量，例如但不限于足以促进T细胞应答的量或剂量。当指示“免疫学上有效量”、“抗肿瘤有效量”、“抑制肿瘤有效量”或“治疗有效量”时，将要被施用的本发明的免疫效应细胞、治疗剂的精确数量可以由医师在考虑个体在年龄、体重、肿瘤大小、感染或转移的程度以及患者(受试者)的状况的情况下确定。有效量的免疫效应细胞是指但不限于能使免疫效应细胞抗肿瘤活性增加、增强或延长；抗肿瘤免疫效应细胞或活化免疫效应细胞数目的增加；促进IFN-γ分泌；肿瘤消退、肿瘤缩小、肿瘤坏死的免疫效应细胞的数量。The terms "therapeutically effective amount", "therapeutically effective", "effective amount" or "in an effective amount" are used interchangeably herein and refer to a compound effective to achieve a specified biological result as described herein , formulation, substance or composition in an amount such as, but not limited to, an amount or dose sufficient to promote a T cell response. When an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount" or "therapeutically effective amount" is indicated, the precise amount of immune effector cells, therapeutic agents of the invention to be administered can be determined by a physician Determined taking into account the individual's age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). An effective amount of immune effector cells refers to, but is not limited to, increasing, enhancing or prolonging the anti-tumor activity of immune effector cells; increasing the number of anti-tumor immune effector cells or activated immune effector cells; promoting IFN-γ secretion; tumor regression and tumor shrinkage , the number of tumor necrotic immune effector cells.
CD3(分化簇3)T细胞的共受体是一种蛋白质复合物，它由四个不同的链组成。在哺乳动物中，该复合物含有一个CD3γ链，CD3δ链，和2个CD3ε链。CD3分子通过盐桥与T细胞抗原受体(T cell receptor，TCR)相连形成TCR-CD3复合物，参与T细胞的信号转导，主要用于标记胸腺细胞、T淋巴细胞及T细胞淋巴瘤。CD3胞质段含免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif,ITAM)，TCR识别并结合由MHC(major histo-compatibility complex)分子提呈的抗原肽，导致CD3的ITAM的保守序列的酪氨酸残基被T细胞内的酪氨酸蛋白激酶p56lck磷酸化，然后可募集其他含有SH2(Scr homology 2)结构域的酪氨酸蛋白激酶(如ZAP-70)。ITAM的磷酸化和与ZAP-70的结合是T细胞活化信号传导过程早期阶段的重要生化反应之一。因此，CD3分子的功能是转导TCR识别抗原所产生的活化信号。在本申请中，可结合靶抗原且可引发CD3信号活化的外源性受体包含至少一个CD3结合位点和至少一个另外的对细菌物质、病毒蛋白、自身免疫标记物或特定细胞上存在的抗原(例如B细胞、T细胞、自然杀伤(NK)细胞、骨髓细胞、吞噬细胞或肿瘤细胞的细胞表面蛋白)是特异性的抗原结合位点。这样的外源性受体能够交联两种细胞并且可以用于将T细胞导向特定的靶抗原，并触发T细胞对靶细胞的细胞毒活性。这种靶抗原的实例可以是肿瘤细胞或感染因子，例如病毒病原体或细菌病原体。The co-receptor for CD3 (cluster of differentiation 3) T cells is a protein complex that consists of four distinct chains. In mammals, this complex contains one CD3γ chain, CD3δ chain, and two CD3ε chains. The CD3 molecule is connected to the T cell receptor (TCR) through a salt bridge to form a TCR-CD3 complex, which is involved in the signal transduction of T cells and is mainly used to label thymocytes, T lymphocytes and T cell lymphomas. The CD3 cytoplasmic segment contains an immunoreceptor tyrosine-based activation motif (ITAM), and TCR recognizes and binds to antigenic peptides presented by MHC (major histo-compatibility complex) molecules, resulting in the activation of CD3 ITAM. Conserved sequence tyrosine residues are phosphorylated by the tyrosine protein kinase p56lck in T cells, which can then recruit other SH2 (Scr homology 2) domain-containing tyrosine protein kinases (eg, ZAP-70). Phosphorylation of ITAM and binding to ZAP-70 is one of the important biochemical reactions in the early stage of T cell activation signaling process. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by TCR recognition of the antigen. In the present application, an exogenous receptor that can bind to a target antigen and that can trigger activation of CD3 signaling comprises at least one CD3 binding site and at least one additional response to bacterial substances, viral proteins, autoimmune markers, or proteins present on specific cells. Antigens (eg, cell surface proteins of B cells, T cells, natural killer (NK) cells, myeloid cells, phagocytes, or tumor cells) are specific antigen binding sites. Such exogenous receptors are capable of cross-linking two cells and can be used to direct T cells to specific target antigens and trigger T cell cytotoxic activity against target cells. Examples of such target antigens can be tumor cells or infectious agents such as viral or bacterial pathogens.
术语“刺激”是指通过刺激结构域或刺激分子(例如，TCR/CD3复合物)与其同源配体结合而诱导的初级应答，从而介导信号转导事件，诸如但不限于，经由TCR/CD3复合物的信号转导。刺激可介导某些分子的表达改变和/或细胞骨架结构的重组等。The term "stimulation" refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (eg, a TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, via TCR/CD3 Signal transduction by the CD3 complex. Stimulation can mediate changes in the expression of certain molecules and/or reorganization of cytoskeletal structures, among others.
术语“刺激分子”或“刺激结构域”是指由T细胞表达的分子或其部分，其提供以针对T细胞信号传导途径的至少一些方面的刺激方式调节TCR复合物的初级活化的初级细胞质信号传导序列。在一个方面，初级信号通过例如TCR/CD3复合物与装载有肽的MHC分子的结合而起 始，并且其导致包括但不限于增殖、活化、分化等T细胞应答的介导。以刺激方式起作用的初级细胞质信号传导序列(也称为“初级信号传导结构域”)可含有信号传导基序，其被称为基于免疫受体酪氨酸的活化基序或“ITAM”。在本发明中特别有用的含有ITAM的初级细胞质信号传导序列的实例包括但不限于衍生自TCRδ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(也称为“ICOS”)和CD66d的那些序列。The term "stimulatory molecule" or "stimulatory domain" refers to a molecule or portion thereof expressed by a T cell that provides a primary cytoplasmic signal that modulates primary activation of the TCR complex in a stimulatory manner directed against at least some aspects of the T cell signaling pathway conduction sequence. In one aspect, primary signaling is initiated by, for example, binding of the TCR/CD3 complex to peptide-loaded MHC molecules, and it results in the mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or "ITAMs." Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from TCRδ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS" ”) and those of CD66d.
术语“细胞内信号传导结构域”是指分子的细胞内部分。细胞内信号传导结构域产生促进含TFP细胞例如表达TFP的T细胞的免疫效应物功能的信号。例如在表达TFP的T细胞中的免疫效应物功能的实例包括细胞溶解活性和T辅助细胞活性，包括细胞因子的分泌。在一个实施方案中，细胞内信号传导结构域可包含初级细胞内信号传导结构域。示例性的初级细胞内信号传导结构域包括衍生自负责初级刺激或抗原依赖性刺激的分子的细胞内信号传导结构域。在一个实施方案中，细胞内信号传导结构域可包含共刺激细胞内结构域。示例性的共刺激细胞内信号传导结构域包括衍生自负责共刺激信号或抗原非依赖性刺激的分子的细胞内信号传导结构域。The term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains generate signals that promote the immune effector function of TFP-containing cells, such as TFP-expressing T cells. Examples of immune effector functions, eg, in TFP-expressing T cells, include cytolytic activity and T helper cell activity, including secretion of cytokines. In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include intracellular signaling domains derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include intracellular signaling domains derived from molecules responsible for costimulatory signaling or antigen-independent stimulation.
初级细胞内信号传导结构域可包含ITAM(“基于免疫受体酪氨酸的活化基序”)。含有ITAM的初级细胞质信号传导序列的实例包括但不限于衍生自CD3δ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d DAP10和DAP12的那些序列。术语“共刺激分子”是指T细胞上的同源结合配偶体，其与共刺激配体特异性结合，从而介导T细胞的共刺激反应，诸如但不限于增殖。共刺激分子是有效免疫应答所需的抗原受体或其配体之外的细胞表面分子。共刺激分子包括但不限于MHC 1类分子、BTLA和Toll配体受体，以及DAP10、DAP12、CD30、LIGHT、OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。共刺激细胞内信号传导结构域可以是共刺激分子的细胞内部分。共刺激分子可在以下蛋白质家族中表示：TNF受体蛋白质、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导性淋巴细胞活化分子(SLAM蛋白)和激活性NK细胞受体。此类分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、GITR、CD30、CD40、ICOS、BAFFR、HVEM、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160、B7-H3以及与CD83特异性结合的配体等。细胞内信号传导结构域可包含其衍生的分子的整个细胞内部分或整个天然细胞内信号传导结构域，或其功能片段。术语“4-1BB”是指TNFR超家族的成员，其具有以GenBank登录号AAA62478.2提供的氨基酸序列，或来自非人物种如小鼠、啮齿动物、猴、猿等的等同残基；“4-1BB共刺激结构域”被定义为GenBank登录号AAA62478.2的氨基酸残基214-255，或来自非人物种如小鼠、啮齿动物、猴、猿等的等同残 基。The primary intracellular signaling domain may comprise an ITAM ("immunoreceptor tyrosine-based activation motif"). Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3δ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12. The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors, and DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18 ) and 4-1BB (CD137). The costimulatory intracellular signaling domain can be the intracellular portion of the costimulatory molecule. Costimulatory molecules can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, Lymphocyte Function Associated Antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C , SLAMF7, NKp80, CD160, B7-H3 and ligands that specifically bind to CD83, etc. The intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment thereof. The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided under GenBank Accession No. AAA62478.2, or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc.;" The 4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
术语“编码”是指多核苷酸如基因、cDNA或mRNA中的特定核苷酸序列用作在生物过程中合成其他聚合物和大分子的模板的固有性质，该聚合物和大分子具有确定的核苷酸序列(例如，rRNA、tRNA和mRNA)或确定的氨基酸序列以及由此产生的生物学性质。因此，如果对应于基因的mRNA的转录和翻译在细胞或其他生物***中产生蛋白质，则该基因、cDNA或RNA编码蛋白质。编码链及其核苷酸序列均与mRNA序列相同并且通常在序列表中提供，而用作基因或cDNA转录的模板的非编码链可以称为编码蛋白质或者该基因或cDNA的其他产物。除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括彼此为简并形式且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质或RNA的“核苷酸序列”也可包括内含子，即编码蛋白质的核苷酸序列在一些形式中可含有一个或多个内含子。The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA or mRNA to serve as a template in biological processes for the synthesis of other polymers and macromolecules with a defined Nucleotide sequences (eg, rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Thus, a gene, cDNA or RNA encodes a protein if the transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand and its nucleotide sequence are identical to the mRNA sequence and are generally provided in sequence listings, while the non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of that gene or cDNA. Unless stated otherwise, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are in degenerate form of each other and that encode the same amino acid sequence. A "nucleotide sequence" encoding a protein or RNA may also include introns, ie, a nucleotide sequence encoding a protein may, in some forms, contain one or more introns.
术语“表达”是指由启动子驱动的特定核苷酸序列的转录和/或翻译。The term "expression" refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.
术语“转移载体”是指包含分离的核酸并且可用于将所分离的核酸递送至细胞内部的物质的组合物。许多载体是本领域已知的，包括但不限于线性多核苷酸、与离子或两亲化合物缔合的多核苷酸、质粒和病毒。因此，术语“转移载体”包括自主复制的质粒或病毒。该术语还应解释为进一步包含促进将核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒转移载体的实例包括但不限于腺病毒载体、腺伴随病毒载体、逆转录病毒载体、慢病毒载体等。The term "transfer vector" refers to a composition comprising an isolated nucleic acid and a substance that can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be construed to further encompass non-plasmid and non-viral compounds that facilitate transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.
术语“表达载体”是指包含重组多核苷酸的载体，该重组多核苷酸包含与待表达的核苷酸序列可操作地连接的表达控制序列。表达载体包含足以用于表达的顺式作用元件；用于表达的其他元件可由宿主细胞或在体外表达***中提供。表达载体包括本领域已知的所有表达载体，包括粘粒、质粒(例如，裸露的或包含在脂质体中)和掺有重组多核苷酸的病毒(例如，慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses) incorporating recombinant polynucleotides virus and adeno-associated virus).
术语“同源”或“同一性”是指两个聚合物分子之间，例如，两个核酸分子如两个DNA分子或两个RNA分子之间，或两个多肽分子之间的亚单位序列同一性。当两个分子中的亚单位位置被相同的单体亚单位占据时；例如，如果两个DNA分子中的每一个的位置被腺嘌呤占据，则它们在该位置是同源的或相同的。两个序列之间的同源性是匹配或同源位置的数目的直接函数；例如，如果两个序列中的一半位置(例如，长度为10个亚单位的聚合物中的5个位置)是同源的，则这两个序列是50％同源的；如果90％的位置(例如，10个中的9个)是匹配的或同源的，则这两个序列是90％同源的。The term "homologous" or "identity" refers to a subunit sequence between two polymer molecules, eg, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules identity. When a subunit position in two molecules is occupied by the same monomeric subunit; for example, if the position of each of two DNA molecules is occupied by an adenine, they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a polymer of 10 subunits in length) are Homologous, then the two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) are matched or homologous, the two sequences are 90% homologous .
在两个或更多个核酸或多肽序列的上下文中，同一性百分比是指相同的两个或更多个序列。 当在比较窗口或指定区域上针对最大对应性进行比较和比对时，如使用以下序列比较算法中的一种或者通过手动比对和目视检查所测量的，如果两个序列具有指定百分比的相同的氨基酸残基或核苷酸(例如，在指定区域上，或者在未指定的情况下在整个序列上60％的同一性，任选地70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性)，则这两个序列“基本上相同”。任选地，同一性存在于长度为至少约50个核苷酸(或10个氨基酸)的区域上，或更优选地存在于长度为100至500个或1000个或更多个核苷酸(或者20、50、200个或更多个氨基酸)的区域上。对于序列比较，通常一个序列充当参考序列，测试序列与之进行比较。当使用序列比较算法时，将测试序列和参考序列输入计算机，在需要的情况下指定子序列坐标，并指定序列算法程序参数。可使用默认程序参数，或得可指定替代参数。随后，序列比较算法基于程序参数计算测试序列相对于参考序列的序列同一性百分比。用于比较的序列比对方法是本领域公知的。在一个方面，本发明考虑到产生功能上等同的分子的起始抗体或片段(例如，scFv)氨基酸序列的修饰。例如，可修饰TFP中包含的抗GPC3或Claudin18.2结合结构域例如scFv的VH或VL，以保留抗GPC3或Claudin18.2结合结构域例如scFv的起始VH或VL构架区的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。本发明考虑到整个TFP构建体的修饰，例如，TFP构建体的各个结构域的一个或多个氨基酸序列的修饰，以便产生功能上等同的分子。可修饰TFP构建体以保留起始TFP构建体的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。In the context of two or more nucleic acid or polypeptide sequences, percent identity refers to the two or more sequences that are identical. When compared and aligned for maximum correspondence over a comparison window or specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, if two sequences have a specified percentage of Identical amino acid residues or nucleotides (eg, 60% identity over a specified region, or, if not specified, the entire sequence, optionally 70%, 71%, 72%, 73%, 74% %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity), then the two sequences are "substantially identical". Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably 100 to 500 or 1000 or more nucleotides in length ( or 20, 50, 200 or more amino acids). For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Sequence alignment methods for comparison are well known in the art. In one aspect, the invention contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, scFv) that produces a functionally equivalent molecule. For example, an anti-GPC3 or Claudin18.2 binding domain, such as the VH or VL of a scFv, contained in a TFP can be modified to retain at least about 70% of the starting VH or VL framework region of the anti-GPC3 or Claudin18.2 binding domain, such as the scFv , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The present invention contemplates modification of the entire TFP construct, eg, modification of one or more amino acid sequences of individual domains of the TFP construct, in order to generate functionally equivalent molecules. The TFP construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% of the starting TFP construct , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identity.
在一个实施例中，本发明所使用的抗GPC3的抗体或其片段的序列是SEQ ID NO:4,20-99。In one embodiment, the sequence of the anti-GPC3 antibody or fragment thereof used in the present invention is SEQ ID NO: 4, 20-99.
在一个实施例中，本发明所使用的抗Claudin18.2的抗体或其片段的序列是SEQ ID NO:100-189。In one embodiment, the sequence of the anti-Claudin18.2 antibody or fragment thereof used in the present invention is SEQ ID NO: 100-189.
术语“分离的”意指从天然状态改变或移出的。例如，天然存在于活动物中的核酸或肽不是“分离的”，但与其天然状态下共同存在的物质部分或完全分离的相同核酸或肽是“分离的”。分离的核酸或蛋白质可以以基本上纯化的形式存在，或者可存在于非天然环境如宿主细胞中。The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the material with which it occurs in nature is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment such as a host cell.
术语“可操作地连接”或“转录控制”是指调节序列与异源核酸序列之间的功能性连接，其导致后者的表达。例如，当第一核酸序列与第二核酸序列以功能性关系布置时，第一核酸序列与第二核酸序列可操作地连接。例如，如果启动子影响编码序列的转录或表达，则该启动子与该 编码序列可操作地连接。可操作地连接的DNA序列可彼此邻接，并且例如，在需要连接两个蛋白质编码区的情况下，该DNA序列在相同的阅读框中。The term "operably linked" or "transcriptional control" refers to the functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is arranged in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the sequence. Operably linked DNA sequences can be contiguous to each other and, for example, where it is desired to join two protein coding regions, the DNA sequences are in the same reading frame.
术语“核酸”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物。除非明确限定，否则该术语包括含有天然核苷酸的已知类似物的核酸，其具有与参考核酸相似的结合特性，并以与天然存在的核苷酸相似的方式代谢。除非另有说明，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子置换)、等位基因、直向同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子置换可通过产生其中一个或多个选定的(或所有)密码子的第三位置被混合碱基和/或脱氧肌苷残基置换的序列来实现。The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless specifically defined, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complements, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues.
术语“肽”、“多肽”和“蛋白质”可互换使用，并且是指由通过肽键共价连接的氨基酸残基组成的化合物。蛋白质或肽必须含有至少两个氨基酸，并且对可构成蛋白质或肽序列的氨基酸的最大数目没有限制。多肽包括包含通过肽键彼此连接的两个或更多个氨基酸的任何肽或蛋白质。如本文所用，该术语是指短链和长链两者，短链在本领域中通常也称为例如肽、寡肽和寡聚体，长链在本领域中通常称为蛋白质，其存在很多类型。“多肽”包括例如生物活性片段、基本上同源的多肽、寡肽、同源二聚体、异源二聚体、多肽的变体、修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然肽、重组肽或其组合。The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly known in the art as eg peptides, oligopeptides, and oligomers, and long chains, commonly known in the art as proteins, of which many exist Types of. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.
术语“启动子/调节序列”是指表达与启动子/调节序列可操作地连接的基因产物所需的核酸序列。术语“组成型”启动子是指当与编码或指定基因产物的多核苷酸可操作地连接时，在细胞的大多数或所有生理条件下导致在细胞中产生基因产物的核苷酸序列。术语“诱导型”启动子是指当与编码或指定基因产物的多核苷酸可操作地连接时，基本上仅当对应于该启动子的诱导物存在于细胞中时才导致在细胞中产生基因产物的核苷酸序列。The term "promoter/regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product to which the promoter/regulatory sequence is operably linked. The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, results in production of the gene product in a cell under most or all physiological conditions of the cell. The term "inducible" promoter means that, when operably linked to a polynucleotide encoding or specifying a gene product, it results in production of a gene in a cell substantially only when an inducer corresponding to the promoter is present in the cell The nucleotide sequence of the product.
术语“抗体”是指源于特异性地结合抗原的免疫球蛋白分子的蛋白质或多肽序列。抗体可以为多克隆的或单克隆的、多链或单链、或完整的免疫球蛋白，并且可以来源于天然来源或重组来源。抗体可以为免疫球蛋白分子的四聚体。The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
术语“抗体片段”是指保留与抗原的表位特异性地相互作用(例如，通过结合、空间位阻、稳定化/去稳定化、空间分布)的能力的抗体的至少一部分。抗体片段的实例包括，但不限于Fab，Fab'、F(ab') 2、Fv片段、scFv抗体片段、二硫键-连接的Fvs(sdFv)、由VH和CH1结构域组成的Fd片段、线性抗体、单域抗体比如sdAb(VL或VH)、camelid VHH结构域、由抗体片段(例如包括在铰链区通过二硫键连接的两个Fab片段的二价片段)形成的多特异性抗体和抗体的分离的CDR或其它表位结合片段。抗原结合片段也可以被掺入单域抗体、最大抗体、微小抗体、纳米抗体、胞内抗体、双抗体、三抗体、四抗体、v-NAR和双-scFv(参见，例如Hollinger 和Hudson，《Nature Biotechnology》(23):1126-1136，2005)。 The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (eg, through binding, steric hindrance, stabilization/destabilization, steric distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, Linear antibodies, single domain antibodies such as sdAbs (VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments (eg bivalent fragments comprising two Fab fragments linked by disulfide bonds at the hinge region) and An isolated CDR or other epitope binding fragment of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maximal antibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, " Nature Biotechnology (23): 1126-1136, 2005).
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous ( For example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, a scFv may have the VL and VH variable regions described in any order (eg, with respect to the N-terminus and C-terminus of the polypeptide), the scFv may include a VL-linker-VH or VH-linker-VL can be included.
术语“抗体重链”是指以其天然存在的构型存在于抗体分子中且通常决定抗体所属类型的两种多肽链中较大者。The term "antibody heavy chain" refers to the larger of two polypeptide chains that are present in an antibody molecule in its naturally occurring configuration and that generally determine the class to which the antibody belongs.
术语“抗体轻链”是指以其天然存在构型存在于抗体分子中的两种多肽链的较小者。κ(k)和λ(l)轻链是指两种主要的抗体轻链的同种型。The term "antibody light chain" refers to the smaller of two polypeptide chains present in an antibody molecule in its naturally occurring configuration. Kappa (k) and lambda (l) light chains refer to the two major antibody light chain isotypes.
术语“重组抗体”是指使用重组DNA技术产生的抗体，比如例如由噬菌体或酵母菌表达***表达的抗体。该术语也应当解释为指已经通过合成编码抗体的DNA分子(且其中DNA分子表达抗体蛋白质)或指定抗体的氨基酸序列产生的抗体，其中所述DNA或氨基酸序列已经使用重组DNA或本领域可获得且熟知的氨基酸序列技术获得。The term "recombinant antibody" refers to antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed by phage or yeast expression systems. The term should also be interpreted to refer to an antibody that has been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or the amino acid sequence of the specified antibody, wherein the DNA or amino acid sequence has been using recombinant DNA or is available in the art and well-known amino acid sequence techniques.
本领域普通技术人员将会理解，可进一步修饰本发明的抗体或抗体片段，使得它们在氨基酸序列上(例如，相对于野生型)有所变化，但在所需活性上没有变化。例如，可对蛋白质进行另外的核苷酸置换，导致“非必需”氨基酸残基处的氨基酸置换。例如，分子中的非必需氨基酸残基可被来自相同侧链家族的另一个氨基酸残基取代。在另一个实施方案中，氨基酸序列可被结构相似但在顺序和/或组成上与侧链家族成员不同的序列取代，例如，可进行保守置换，其中氨基酸残基被具有相似侧链的氨基酸残基所取代。One of ordinary skill in the art will appreciate that the antibodies or antibody fragments of the invention can be further modified such that they vary in amino acid sequence (eg, relative to wild type), but not in the desired activity. For example, additional nucleotide substitutions can be made in the protein, resulting in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule can be substituted with another amino acid residue from the same side chain family. In another embodiment, the amino acid sequence may be substituted with a sequence that is structurally similar but differs in sequence and/or composition from side chain family members, eg, conservative substitutions may be made in which amino acid residues are replaced by amino acid residues with similar side chains base substituted.
本领域已经定义了具有相似侧链的氨基酸残基家族，包括碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine , valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
术语“抗原”或“Ag”是指引起免疫应答的分子。该免疫应答可以涉及抗体产生或有特异性免疫能力的细胞的活化或两者。本领域技术人员应当理解包括实际上所有蛋白质或肽的任何大分子都可以充当抗原。此外，抗原可以来源于重组或基因组DNA。当在本文中使用该术语时，本领域技术人员应当理解包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列的 任何DNA，编码“抗原”。此外，本领域技术人员应当理解抗原无需仅通过基因的全长核苷酸序列编码。本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列以不同组合排列以编码引发期望免疫应答的多肽。而且，本领域技术人员应当理解抗原也可以无需由“基因”编码。显而易见的是，抗原可以合成产生，或者可以来源于生物样品，或者可以是除了多肽之外的大分子。这样的生物样品包括，但不限于组织样品、肿瘤样品、具有其它生物组分的细胞或液体。The term "antigen" or "Ag" refers to a molecule that elicits an immune response. The immune response may involve antibody production or activation of specific immunocompetent cells or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. When the term is used herein, one of skill in the art will understand that any DNA that includes a nucleotide sequence or a portion of a nucleotide sequence encoding a protein that elicits an immune response, encodes an "antigen". Furthermore, those skilled in the art will understand that the antigen need not be encoded solely by the full-length nucleotide sequence of the gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit a desired immune response. Furthermore, those skilled in the art will appreciate that the antigen need not be encoded by a "gene". Obviously, antigens can be produced synthetically, or can be derived from biological samples, or can be macromolecules other than polypeptides. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.
本文所用的术语“抗原识别单元”(也称为抗原结合结构域)是指免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗原识别单元”中还包括各种物种来源的免疫球蛋白分子，包括无脊椎动物和脊椎动物。结构上，最简单的天然存在的抗体(例如IgG)包含四条多肽链，通过二硫键相互连接的两条重(H)链和两条轻链(L)。免疫球蛋白代表包括几种类型的分子，如IgD、IgG、IgA、IgM和IgE。术语“免疫球蛋白分子”包括例如杂交抗体或其改变的抗体及其片段。已经显示抗体的抗原结合功能可以通过天然存在的抗体的片段进行。这些片段统称为“抗原识别单元”。术语“抗原识别单元”中还包括具有与表位吻合并识别表位的特定形状的任何含多肽链的分子结构，其中一个或多个非共价结合相互作用稳定分子结构和表位之间的复合物。所述抗原识别单元的实例包括Fab片段，由VL、VH、CL和CH1结构域组成的单价片段，包含通过铰链区上的二硫健连接的两个Fab片段的双价片段(F(ab)2片段)；由VH和CH1结构域组成的Fd片段，由抗体的单臂的VL和VH结构域组成的Fv片段；由VH结构域组成的dAb片段(Ward等人，Nature,341:544-546,1989)；和分离的互补决定区(CDR)或包含这样的抗原识别单元的任何融合蛋白。As used herein, the term "antigen recognition unit" (also referred to as an antigen binding domain) refers to immunoglobulin molecules and immunologically active portions of immunological molecules, ie, containing an antigen binding site that specifically binds ("immunoreactive") an antigen molecule. Also included within the term "antigen recognition unit" are immunoglobulin molecules derived from various species, including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibodies (eg, IgG) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The immunoglobulin representation includes several types of molecules, such as IgD, IgG, IgA, IgM and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen-binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen recognition units". Also included in the term "antigen recognition unit" is any polypeptide chain-containing molecular structure having a specific shape that conforms to and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the relationship between the molecular structure and the epitope. Complex. Examples of such antigen recognition units include Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, bivalent fragments (F(ab) comprising two Fab fragments linked by a disulfide bond on the hinge region 2 fragments); Fd fragments composed of VH and CH1 domains, Fv fragments composed of the VL and VH domains of the one-armed antibody; dAb fragments composed of VH domains (Ward et al., Nature, 341:544- 546, 1989); and an isolated complementarity determining region (CDR) or any fusion protein comprising such an antigen recognition unit.
如果抗原识别单元以与其它参考抗原(包括多肽或其他物质)结合相比更大亲和力或亲合力结合抗原，则所述抗原识别单元与抗原“特异性结合”或与抗原是“免疫反应性的”。An antigen-recognition unit "specifically binds" or is "immunoreactive" with an antigen if it binds the antigen with greater affinity or avidity as compared to other reference antigens, including polypeptides or other substances ".
“肿瘤抗原”指的是特定过度增生性疾病共同的抗原。在某些方面，本发明的过度增生性病症抗原源自癌症或肿瘤。本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；***干细胞抗原(PSCA)；***特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表 皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoHglycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；***蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。"Tumor antigen" refers to an antigen common to specific hyperproliferative diseases. In certain aspects, the hyperproliferative disorder antigens of the invention are derived from cancer or tumors. The tumor antigens of the present invention include, but are not limited to: Thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); 11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosinase; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2); Lewis (Y) Antigen; CD24; Platelet-Derived Growth Factor Receptor β (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule ( sLe); ganglioside GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7 related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 Integrin; B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta specificity 1 (PLAC1); hexose moiety of globoHglycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP) Wilms tumor protein (WT1); ETS translocation variant 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2) ; Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase ( hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V ( NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase-anchored protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF ); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phospholipids Acyl-inositol-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1).
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。The pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
术语“肿瘤异质性”，是指肿瘤在生长过程中，经过多次***增殖，其子细胞呈现出分子生物学或基因方面的改变，从而使肿瘤的生长速度、侵袭能力、对药物的敏感性、预后等各方面产生差异。它是恶性肿瘤的特征之一。The term "tumor heterogeneity" means that during the growth process of a tumor, after multiple divisions and proliferations, its daughter cells exhibit molecular biological or genetic changes, which make the tumor's growth rate, invasive ability, and drug sensitivity. gender, prognosis, etc. It is one of the characteristics of malignant tumors.
术语“癌症”或“肿瘤”指特征在于在体外(例如经转化的细胞)或体内的过度增殖性细胞生长的广泛病症类别。可以通过本发明的方法治疗或预防的病况包括例如各种赘生物，包括良性或恶性肿瘤，各种增生等等。癌症的具体例子包括但不限于：血液癌症，结肠癌，直肠癌，肾细胞癌，肝癌，肺的非小细胞癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，***，***癌，***癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌***癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，***癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经***(CNS)瘤，原发性CNS淋巴瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶。The terms "cancer" or "tumor" refer to a broad class of disorders characterized by hyperproliferative cell growth in vitro (eg, transformed cells) or in vivo. Conditions that can be treated or prevented by the methods of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like. Specific examples of cancer include, but are not limited to: blood cancer, colon cancer, rectal cancer, renal cell cancer, liver cancer, non-small cell cancer of the lung, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, Head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureter cancer, renal pelvis Carcinoma, Central Nervous System (CNS) Tumor, Primary CNS Lymphoma, Tumor Angiogenesis, Spinal Tumor, Brainstem Glioma, Pituitary Adenoma, Kaposi's Sarcoma, Epidermoid Carcinoma, Squamous Cell Carcinoma, T Cellular lymphomas, environmentally induced cancers, combinations of such cancers and metastatic lesions of such cancers.
术语“转染的”或“转化的”或“转导的”是指外源性核酸通过其转移或引入到宿主细胞中的过程。“转染的”或“转化的”或“转导的”细胞是已经用外源性核酸转染、转化或转导的细胞。所述细胞包括原发性受试者细胞及其后代。The term "transfected" or "transformed" or "transduced" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cells include primary subject cells and their progeny.
术语“特异性地结合”是指识别并且结合存在于样品中的结合配偶体(例如肿瘤抗原)蛋白质的抗体或配体，但是该抗体或配体基本上不会识别或结合样品中的其它分子。The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample .
这里使用的“难治”指的是一种疾病，例如，癌症，其不应答治疗。在实施方案中，难治性癌症可以是对治疗开始前或开始时的治疗有抗性。在其他实施方案中，难治性癌症可以成为治疗期间抗性的。难治性癌症也称为抗性癌症。在本发明中，难治性癌症包括但不限于放疗不敏感、放疗后复发、化疗不敏感、化疗后复发、对CAR-T治疗不敏感或治疗后复发的癌症。难治性或复发性恶性肿瘤可以使用本文中描述的治疗方案。"Refractory" as used herein refers to a disease, eg, cancer, that does not respond to treatment. In embodiments, the refractory cancer may be resistant to treatment prior to or at initiation of treatment. In other embodiments, refractory cancers may become resistant during treatment. Refractory cancers are also called resistant cancers. In the present invention, refractory cancers include, but are not limited to, radiotherapy-insensitive, relapsed after radiotherapy, chemotherapy-insensitive, relapsed after chemotherapy, insensitive to CAR-T therapy, or relapsed after treatment. Refractory or relapsed malignancies can be treated with the treatment regimens described herein.
如本文所用“复发的”是指在一段改进期，例如，在疗法，例如癌症疗法的先前治疗后，返回疾病(例如癌症)或疾病如癌症的体征和症状。As used herein, "relapsed" refers to a return to a disease (eg, cancer) or signs and symptoms of a disease such as cancer over a period of improvement, eg, after prior treatment with therapy, eg, cancer therapy.
术语“个体”和“受试者”在本文中具有同等含义，可以是人和来自其他种属的动物。“患者”是患有疾病、病症或病况或者有患疾病、病症或病况的风险或者在其他方面需要本文提供的组合物和方法的受试者。The terms "individual" and "subject" have equivalent meanings herein and can be humans and animals from other species. A "patient" is a subject having or at risk of having a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强的应答可以包含应答性中5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、 65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫效应细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％更多。The term "enhancing" refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein. For example, an enhanced response can comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the responsiveness %, 75%, 80%, 85%, 90%, 95% or 98% or more increase. As used herein, "enhancing" can also refer to increasing the number of subjects that respond to treatment, eg, immune effector cell therapy. For example, an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentages are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
在一个方面，治疗由临床结果判断，也可以通过T细胞的抗肿瘤活性增加、增强或延长；与治疗前的数目相比较，抗肿瘤T细胞或活化T细胞数目的增加，促进IFN-γ、Granzyme-B、IL2、TNF-α、GM-CSF分泌,或其组合决定。在另一个方面，临床结果是肿瘤消退；肿瘤缩小；肿瘤坏死；通过免疫***的抗肿瘤应答；肿瘤扩大，复发或扩散或其组合。在一个另外方面，治疗效应通过T细胞的存在、指示T细胞炎症的基因标记的存在，促进IFN-γ分泌,或其组合预测。In one aspect, treatment is judged by clinical outcome, and may also be increased, enhanced or prolonged by the anti-tumor activity of T cells; an increase in the number of anti-tumor T cells or activated T cells compared to the number before treatment, promotes IFN-γ, Granzyme-B, IL2, TNF-α, GM-CSF secretion, or a combination thereof. In another aspect, the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor expansion, recurrence or spread, or a combination thereof. In an additional aspect, the therapeutic effect is predicted by the presence of T cells, the presence of genetic markers indicative of T cell inflammation, promotion of IFN-gamma secretion, or a combination thereof.
如本文公开的细胞可以通过各种途径施用于个体，包括例如经口或肠胃外，例如静脉内、肌内、皮下、眶内、囊内、腹膜内、直肠内、脑池内、瘤内、鼻内(intravasally)、皮内或者分别使用例如皮肤贴片或透皮离子电渗疗法通过皮肤的被动或促进吸收。Cells as disclosed herein can be administered to an individual by various routes, including, for example, orally or parenterally, such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral, nasal Intravasally, intradermally, or passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
在实践本发明的方法中待施用的试剂总量可以作为单一剂量以推注或通过在相对短时间段的输注，施用于受试者，或可以使用分级治疗方案进行施用，其中在延长时间段施用多个剂量。本领域技术人员将知道治疗受试者中的病理状况的组合物的量取决于许多因素，包括受试者的年龄和一般健康、以及施用途径和待施用的治疗次数。考虑到这些因素，技术人员将根据需要调整具体剂量。一般而言，最初，使用I期和II期临床试验测定组合物的配制以及施用途径和频率。The total amount of agent to be administered in practicing the methods of the invention may be administered to the subject as a single dose as a bolus injection or by infusion over a relatively short period of time, or may be administered using a graded treatment regimen, wherein over an extended period of time Administer multiple doses. Those of skill in the art will know that the amount of the composition to treat a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, the technician will adjust the specific dose as needed. In general, initially, Phase I and Phase II clinical trials are used to determine the formulation of the composition as well as the route and frequency of administration.
范围：在整个公开中，本发明的各个方面都可以以范围形式存在。应当理解，范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本发明的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。例如，范围的描述比如从1至6就应当被认为具体地公开了子范围比如1至3、1至4、1至5、2至4、2至6、3至6等，以及该范围内的单独数值，例如1、2、2.7、3、4、5、5.3和6。作为另一个实例，范围比如95-99％的同一性包括具有95％、96％、97％、98％或99％同一性的范围，并且包括子范围比如96～99％、96～98％、96～97％、97～99％、97～98％和98～99％的同一性。不考虑范围的宽度，这均适用。Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be regarded as an inexorable limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, the description of a range such as from 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as within that range. individual values such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95-99% identity includes ranges having 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the width of the range.
根据本公开内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等价的方法 和组分在本发明的范围内。事实上，本发明的各种修饰加上本文显示且描述的那些，根据前述描述对于本领域技术人员将变得明显。Based on the present disclosure, those of skill in the art should appreciate that many changes or changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The present invention is not limited in scope to the specific embodiments described herein, which are intended only as illustrations of aspects of the invention, and functionally equivalent methods and components are within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description.
本文所用的“GPC3”或“磷脂酰肌醇蛋白聚糖3”是Glypican家族的一员，基因登记号：NM_016697.3，NP_057906.2，其在调控细胞生长和分化方面起重要作用。GPC3异常表达与多种肿瘤的发生、发展关系密切，如在肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、等中呈现异常表达。"GPC3" or "Glypican 3" as used herein is a member of the Glypican family, gene accession numbers: NM_016697.3, NP_057906.2, which play an important role in regulating cell growth and differentiation. The abnormal expression of GPC3 is closely related to the occurrence and development of various tumors, such as abnormal expression in liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, etc.
在本发明中，共表达IL7的TFP靶向GPC3阳性表达的肿瘤。在具体的实施方式中，所述肿瘤包括但不限于肝癌、胃癌、肺癌、食道癌、头颈癌、膀胱癌、卵巢癌、***、肾癌、胰腺癌、***、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤、食道癌。本领域技术人员知晓，有些肿瘤细胞，例如肝癌细胞对很多药物不敏感，因此，即便在体外有效的药物，有时候在体内的效果也不佳，甚至没有效果。因此，在优选的实施方式中，本文所述的GPC3阳性表达的肿瘤或GPC阳性肿瘤是肝癌、胃癌、肺癌、食道癌。In the present invention, TFP co-expressing IL7 targets GPC3-positive tumors. In specific embodiments, the tumor includes, but is not limited to, liver cancer, gastric cancer, lung cancer, esophageal cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, Adrenal cancer, schwannoma, malignant fibrous histiocytoma, esophageal cancer. Those skilled in the art know that some tumor cells, such as liver cancer cells, are not sensitive to many drugs, so even drugs that are effective in vitro sometimes have poor or even no effect in vivo. Therefore, in a preferred embodiment, the GPC3-positive expressing tumor or GPC-positive tumor described herein is liver cancer, gastric cancer, lung cancer, esophageal cancer.
本发明还提供了共表达IL7的TCR融合蛋白修饰的T细胞，其被转导有编码所述IL7和TCR融合蛋白的核酸或被转导有上述包含该核酸的重组质粒，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。基于非病毒的转导方法包括电穿孔法和转座子法。在本发明的一个实施方案中，实现IL7和TCR融合蛋白基因修饰的免疫效应细胞的转导方法是基于病毒如逆转录病毒或慢病毒的转导方法。该方法具有转导效率高，外源基因能够稳定表达，且可以缩短体外培养免疫效应细胞到达临床级数量的时间等优点。在该转基因免疫效应细胞表面，转导的核酸通过转录、翻译表达在其表面。The present invention also provides T cells modified with a TCR fusion protein that co-expresses IL7, which is transduced with a nucleic acid encoding the IL7 and TCR fusion protein or transduced with the above-mentioned recombinant plasmid comprising the nucleic acid, or a plasmid comprising the plasmid Virus. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral-based transduction methods include electroporation and transposon methods. In one embodiment of the present invention, the method of transduction of immune effector cells to achieve genetic modification of IL7 and TCR fusion proteins is a virus-based transduction method such as retrovirus or lentivirus. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro cultured immune effector cells to reach clinical level. On the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface through transcription and translation.
修饰的免疫效应细胞具有高度特异性的肿瘤细胞杀伤效果(亦称细胞毒性)，且能够在肿瘤组织中有效存活。因此本发明的编码嵌合抗原受体的核酸，包含该核酸的质粒，包含该质粒的病毒和转导有上述核酸，质粒或病毒的转基因免疫效应细胞可以有效地用于肿瘤的免疫治疗。Modified immune effector cells have a highly specific tumor cell killing effect (also known as cytotoxicity) and can effectively survive in tumor tissue. Therefore, the nucleic acid encoding the chimeric antigen receptor of the present invention, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic immune effector cells transduced with the nucleic acid, plasmid or virus can be effectively used for tumor immunotherapy.
本发明的TCR融合蛋白修饰的T细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述免疫细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。The T cells modified by the TCR fusion protein of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents. In addition to comprising an effective amount of the immune cells, the composition may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、 山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如
Figure PCTCN2021119634-appb-000002
润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液和磷酸盐缓冲液等。 Specific examples of some substances which may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose Sodium, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycols; alginic acid; emulsifiers such as
Figure PCTCN2021119634-appb-000002
Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions and phosphate buffers, etc.
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式可以采用注射或其它治疗方式。The composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by a physician at a dose beneficial to the patient according to factors such as the type, age, weight and general disease state of the patient, and the mode of administration. The mode of administration can be by injection or other therapeutic methods.
在一些实施方案中，本发明的细胞在用于肿瘤治疗时可以与化疗药物联合应用。In some embodiments, the cells of the present invention may be used in combination with chemotherapeutic drugs when used in tumor therapy.
本发明的TCR融合蛋白可以选自按如下方式顺序连接：The TCR fusion proteins of the present invention can be selected from the following sequential linkages:
抗原识别单元-CD3ε，Antigen recognition unit - CD3ε,
抗原识别单元-CD3γ，antigen recognition unit - CD3γ,
抗原识别单元-CD3δ，antigen recognition unit - CD3δ,
抗原识别单元-TCRα,Antigen recognition unit-TCRα,
抗原识别单元-TCRβ；及其组合。所述CD3ε、CD3γ、CD3δ、TCRα、TCRβ为包含胞外区、跨膜区、胞内区的全长。本发明也包括编码所述TCR的核酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。Antigen recognition unit-TCRβ; and combinations thereof. The CD3ε, CD3γ, CD3δ, TCRα and TCRβ are the full lengths including the extracellular region, the transmembrane region and the intracellular region. The present invention also includes nucleic acids encoding said TCRs. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
本发明的共表达IL7和靶向特异抗原的TCR融合蛋白的免疫效应细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述免疫细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。The immune effector cells co-expressing IL7 and TCR fusion proteins targeting specific antigens of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents. In addition to comprising an effective amount of the immune cells, the composition may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
本发明的优点：Advantages of the present invention:
本文所提供的免疫效应细胞对实体瘤细胞具备更优异的杀伤效果，及体外扩增性能。可有效地增加所述免疫效应细胞在肿瘤内的增殖、存活及功能；降低抑制性免疫检查点的表达，从而缓解T细胞的耗竭。The immune effector cells provided herein have more excellent killing effect on solid tumor cells and in vitro expansion performance. It can effectively increase the proliferation, survival and function of the immune effector cells in the tumor; reduce the expression of inhibitory immune checkpoints, thereby alleviating the exhaustion of T cells.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.
本发明的示例性的抗原受体，以及用于工程改造和将受体导入细胞中的方法，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、 CN106397593A、CN106467573A、CN104140974A、CN 108884459 A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN 109908176 A、CN109880803A、CN 110055275 A、CN110123837A、CN 110438082 A、CN 110468105 A；国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306 A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的那些。Exemplary antigen receptors of the present invention, and methods for engineering and introducing the receptors into cells, refer to, for example, Chinese Patent Application Publication Nos. CN106397593A, CN106467573A, CN104140974A, CN 108884459 A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN 109908176 A, CN109880803A, CN 110055275 A, CN110123837A, CN 110438082 A, CN 110468105 A; international Patent application Publication No. WO2017186121A1 , WO2018006882A1, WO2015172339A8, WO2018 / 018958A1, WO2014180306 A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, WO2018045811A1, WO2018108106A1, WO2018 / 219299, WO2018 / 210279, WO2019 / 024933, WO2019 / 114751, WO2019 / 114762, Those disclosed in WO2019/141270, WO2019/149279, WO2019/170147A1, WO 2019/210863, WO2019/219029.
实施例1.靶向GPC3的表达TCR融合蛋白的T细胞的构建Example 1. Construction of T cells expressing TCR fusion proteins targeting GPC3
采用本领域常规分子生物学方法，本实施例采用的scFv为靶向GPC3的抗体，氨基酸序列如SEQ ID NO：4所示，核酸序列如SEQ ID NO:6所示。Using conventional molecular biology methods in the art, the scFv used in this example is an antibody targeting GPC3, the amino acid sequence is shown in SEQ ID NO: 4, and the nucleic acid sequence is shown in SEQ ID NO: 6.
1.质粒构建1. Plasmid Construction
以pMSCV(购自addgene)为载体，通过***短连接子连接的抗GPC3的单链抗体GPC3与CD3ε形成抗GPC3的逆转录病毒质粒。Using pMSCV (purchased from addgene) as a vector, the anti-GPC3 single-chain antibody GPC3 and CD3ε were connected by inserting a short linker to form an anti-GPC3 retroviral plasmid.
顺序连接mCD8信号肽(SEQ ID NO:190)，抗GPC3的抗体(SEQ ID NO:6)，连接子1(SEQ ID NO：8)，mCD3ε(SEQ ID NO：9)。通过搭桥PCR将这三个片段的基因序列连接在一起形成片段GPC3-mCD3ε(SEQ ID NO：10)。使用限制性内切酶EcoRI&HindIII双酶切载体，获得线性化载体pMSCV-EcoRI&HindIII，使同源重组酶进行酶切载体与片段的环化，形成质粒pMSCV-GPC3-mCD3ε。Sequentially linked mCD8 signal peptide (SEQ ID NO: 190), anti-GPC3 antibody (SEQ ID NO: 6), linker 1 (SEQ ID NO: 8), mCD3ε (SEQ ID NO: 9). The gene sequences of these three fragments were joined together by bridging PCR to form the fragment GPC3-mCD3ε (SEQ ID NO: 10). The vector was digested with the restriction enzymes EcoRI&HindIII to obtain the linearized vector pMSCV-EcoRI&HindIII, and the homologous recombinase was used to circularize the digested vector and the fragment to form the plasmid pMSCV-GPC3-mCD3ε.
GPC3-mCD3ε顺序连接F2A(SEQ ID NO：5)和mIL7(SEQ ID NO：3)。通过搭桥PCR将基因序列F2A和mIL7连接在一起形成F2A-mIL7。使用限制性内切酶EcoRI&HindIII双酶切载体，获得线性化载体pMSCV-EcoRI&HindIII，使同源重组酶进行酶切载体与片段GPC3-mCD3ε、F2A-mIL7的环化，形成质粒pMSCV-GPC3m-CD3ε-F2A-mIL7(图1)。GPC3-mCD3ε sequentially links F2A (SEQ ID NO:5) and mIL7 (SEQ ID NO:3). The gene sequences F2A and mIL7 were linked together by bridging PCR to form F2A-mIL7. Use the restriction endonucleases EcoRI&HindIII to cut the vector to obtain the linearized vector pMSCV-EcoRI&HindIII, and use the homologous recombinase to carry out the circularization of the digestion vector and the fragments GPC3-mCD3ε and F2A-mIL7 to form the plasmid pMSCV-GPC3m-CD3ε- F2A-mIL7 (Figure 1).
2、表达TCR融合蛋白的T细胞的构建2. Construction of T cells expressing TCR fusion protein
1)接种293T细胞于培养皿中，采用本领域常规的技术，分别采用质粒pMSCV-GPC3-mCD3ε、pMSCV-GPC3-mCD3ε-F2A-mIL7转染293T细胞，转染48h后，收集 病毒上清液，得到逆转录病毒GPC3-CD3ε、GPC3-CD3ε-F2A-IL7。1) Inoculate 293T cells in a petri dish, using conventional techniques in the art, respectively use plasmids pMSCV-GPC3-mCD3ε, pMSCV-GPC3-mCD3ε-F2A-mIL7 to transfect 293T cells, 48h after transfection, collect virus supernatant , the retroviruses GPC3-CD3ε, GPC3-CD3ε-F2A-IL7 were obtained.
2)将上述逆转录病毒分别感染鼠T细胞，得到GPC3-CD3ε细胞、GPC3-CD3ε-F2A-IL7细胞，经流式检测，分别筛选出GPC3-CD3ε细胞、GPC3-CD3ε-F2A-IL7细胞阳性细胞。2) Infect mouse T cells with the above-mentioned retroviruses to obtain GPC3-CD3ε cells and GPC3-CD3ε-F2A-IL7 cells, and through flow detection, respectively screen out GPC3-CD3ε cells and GPC3-CD3ε-F2A-IL7 cells as positive cell.
实施例2.体外杀伤毒性检测及体外细胞因子分泌检测Example 2. In vitro killing toxicity detection and in vitro cytokine secretion detection
采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。具体方法参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。利用常规分子生物学技术分别将表位人源的鼠源GPC3转入鼠肝癌细胞Hepa 1-6(中国科学院细胞典藏中心(上海)以及鼠乳腺癌细胞E0771构建成过表达GPC3蛋白的Hepa 1-6 GPC3和E0771 GPC3细胞。CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. For specific methods, refer to the instructions of CytoTox 96 non-radioactive cytotoxicity detection kit. Using conventional molecular biology techniques, the epitope human-derived mouse GPC3 was transferred into mouse hepatoma cells Hepa 1-6 (Cell Collection Center, Chinese Academy of Sciences (Shanghai) and mouse breast cancer cell E0771, respectively, to construct Hepa 1-6 overexpressing GPC3 protein. 6 GPC3 and E0771 GPC3 cells.
靶细胞数为(Hepa 1-6:10000/孔；Hepa 1-6 GPC3:10000/孔，E0771 GPC3:10000/孔)，按效靶比1:1、1:3，与效应细胞共培养18h检测(1640+10％FBS，200ul体系)，效应细胞分别为表达TCR融合蛋白的GPC3-CD3ε T细胞和GPC3-CD3ε-F2A-IL7T细胞。The number of target cells was (Hepa 1-6:10000/well; Hepa 1-6 GPC3:10000/well, E0771 GPC3:10000/well), and the effector-target ratio was 1:1, 1:3, and co-cultured with effector cells for 18h Detection (1640+10% FBS, 200ul system), the effector cells were GPC3-CD3ε T cells and GPC3-CD3ε-F2A-IL7T cells expressing TCR fusion protein.
实验结果如图2所示，对于不表达GPC3的Hepa 1-6细胞，上述表达TCR融合蛋白的T细胞在不同的效靶比情况下均无细胞毒性杀伤作用。对于表达靶抗原GPC3的Hepa 1-6 GPC3细胞和E0771-GPC3细胞在效靶比为1:1或1:3时，GPC3-CD3ε T细胞和GPC3-CD3ε-F2A-IL7 T细胞均能实现更好的杀伤，表现出非常显著的特异性细胞毒性作用。The experimental results are shown in Figure 2. For Hepa 1-6 cells that do not express GPC3, the above-mentioned T cells expressing TCR fusion protein have no cytotoxic killing effect under different effector-target ratios. For Hepa 1-6 GPC3 cells and E0771-GPC3 cells expressing the target antigen GPC3, when the effector-target ratio was 1:1 or 1:3, both GPC3-CD3ε T cells and GPC3-CD3ε-F2A-IL7 T cells could achieve more Good killing, showing very significant specific cytotoxicity.
ELISA检测表达TCR融合蛋白的T细胞与肝癌Hepa 1-6 GPC3或E0771-GPC3细胞共孵育后的细胞因子分泌情况，按照效靶比1:1共孵育24h,检测细胞培养上清中细胞因子表达，IL7,IFN-γ，Granzyme-B，IL2，TNF-α及GM-CSF的分泌情况分别如图3A-F所示，其中Granzyme-B代表T细胞脱颗粒，GM-CSF是T细胞活化后释放的细胞因子。图3A提示，GPC3-CD3ε T细胞上清液中基本检测不到IL7的表达；不与靶细胞孵育的GPC3-CD3ε-F2A-IL7 T细胞上清液中检测到IL7，而与靶细胞共孵育时IL7分泌量显著增加。图3B-F显示，相对UTD，与表达GPC3的肝癌细胞共孵育后的表达TCR融合蛋白的T细胞分泌的细胞因子IFN-γ，Granzyme-B，IL2，TNF-α及GM-CSF明显增加。ELISA was used to detect the cytokine secretion after co-incubation of T cells expressing TCR fusion protein with liver cancer Hepa 1-6 GPC3 or E0771-GPC3 cells, and co-incubated for 24 hours according to the effect-target ratio of 1:1, and the cytokine expression in the cell culture supernatant was detected. , IL7, IFN-γ, Granzyme-B, IL2, TNF-α and GM-CSF secretion are shown in Figure 3A-F, respectively, in which Granzyme-B represents T cell degranulation, GM-CSF is T cell activation released cytokines. Figure 3A shows that the expression of IL7 was basically undetectable in the supernatant of GPC3-CD3ε T cells; IL7 secretion was significantly increased. Figures 3B-F show that the cytokines IFN-γ, Granzyme-B, IL2, TNF-α and GM-CSF secreted by T cells expressing TCR fusion protein after co-incubation with GPC3-expressing hepatoma cells were significantly increased relative to UTD.
实施例3.皮下移植瘤的抗肿瘤治疗实验Example 3. Anti-tumor treatment experiment of subcutaneously transplanted tumor
一、Hepa 1-6 GPC3肝癌细胞的C57BL/6小鼠皮下移植瘤1. Subcutaneous transplantation of Hepa 1-6 GPC3 hepatoma cells in C57BL/6 mice
分别接种1×10 7的肝癌细胞Hepa 1-6 GPC3于雌性C57BL/6小鼠(上海西普尔－必凯实验动物有限公司)右侧腋部皮下，分3组，每组6只，接种日记为D0。3组分别为：UTD组、GPC3-CD3ε T细胞组、GPC3-CD3ε-F2A-IL7 T细胞组。 1×10 7 hepatocellular carcinoma cells Hepa 1-6 GPC3 were inoculated subcutaneously in the right axilla of female C57BL/6 mice (Shanghai Sipple-Bike Laboratory Animal Co., Ltd.), and divided into 3 groups, 6 mice in each group, inoculation diary D0. The 3 groups were: UTD group, GPC3-CD3ε T cell group, GPC3-CD3ε-F2A-IL7 T cell group.
皮下接种瘤组织后D7天，肿瘤平均体积约355-373mm 3。尾静脉注射表达TCR融合蛋白的T细胞，注射剂量：1.5×10 6细胞/只。空白对照组注射1.5×10 6细胞/只。 On D7 days after subcutaneous inoculation of tumor tissue, the average tumor volume was about 355-373 mm 3 . T cells expressing TCR fusion protein were injected into the tail vein, and the injection dose was 1.5×10 6 cells/only. The blank control group was injected with 1.5×10 6 cells/only.
给予表达TCR融合蛋白的T细胞后，每隔3-4天测量Hepa 1-6 GPC3移植瘤体积的大小，记录每组小鼠瘤体积的变化，瘤体积计算公式为：(长×宽 2)/2。结果如图4A所示，在D27天，与UTD组相比，GPC3-CD3ε T细胞和GPC3-CD3ε-F2A-IL7 T细胞的抑瘤率分别为40.6％和94.1％；相对GPC3-CD3ε T细胞组，GPC3-CD3ε-F2A-IL7 T细胞显示出更加明显的杀伤效果(P<0.001)。小鼠体重与对照组相比没有明显变化(图4B)。 After administration of T cells expressing TCR fusion protein, the size of Hepa 1-6 GPC3 transplanted tumor volume was measured every 3-4 days, and the change of tumor volume in each group of mice was recorded. The tumor volume calculation formula is: (length × width 2 ) /2. The results are shown in Figure 4A, on D27, the tumor inhibition rates of GPC3-CD3ε T cells and GPC3-CD3ε-F2A-IL7 T cells were 40.6% and 94.1%, respectively, compared with the UTD group; relative to GPC3-CD3ε T cells Group, GPC3-CD3ε-F2A-IL7 T cells showed more obvious killing effect (P<0.001). There was no significant change in the body weight of the mice compared with the control group (Fig. 4B).
作为示例性的，上述实施例虽然采用的是T细胞，但还可以选择其他免疫细胞，如NK细胞、NK-T细胞，还可以具体选择免疫细胞的特定亚型，如γ/δT细胞等。As an example, although T cells are used in the above embodiments, other immune cells can also be selected, such as NK cells, NK-T cells, and specific subtypes of immune cells can also be selected, such as γ/δ T cells.
本发明所用的序列总结于下表：The sequences used in the present invention are summarized in the following table:
Figure PCTCN2021119634-appb-000003
Figure PCTCN2021119634-appb-000003
Figure PCTCN2021119634-appb-000004
Figure PCTCN2021119634-appb-000004
Figure PCTCN2021119634-appb-000005
Figure PCTCN2021119634-appb-000005
Figure PCTCN2021119634-appb-000006
Figure PCTCN2021119634-appb-000006
Figure PCTCN2021119634-appb-000007
Figure PCTCN2021119634-appb-000007
Figure PCTCN2021119634-appb-000008
Figure PCTCN2021119634-appb-000008
Figure PCTCN2021119634-appb-000009
Figure PCTCN2021119634-appb-000009
Figure PCTCN2021119634-appb-000010
Figure PCTCN2021119634-appb-000010
Figure PCTCN2021119634-appb-000011
Figure PCTCN2021119634-appb-000011
Figure PCTCN2021119634-appb-000012
Figure PCTCN2021119634-appb-000012
本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (25)

  1. 一种基因工程化的免疫效应细胞，其特征在于，所述细胞表达IL-7R结合蛋白或IL-7；还表达特异性识别靶抗原的受体。A genetically engineered immune effector cell, characterized in that the cell expresses IL-7R binding protein or IL-7; and also expresses a receptor that specifically recognizes a target antigen.
  2. 如权利要求1所述的细胞，其特征在于，所述特异性识别靶抗原的受体为TCR融合蛋白(TFP)。The cell of claim 1, wherein the receptor that specifically recognizes the target antigen is a TCR fusion protein (TFP).
  3. 如权利要求1所述的细胞，其特征在于，所述IL-7R结合蛋白为外源性IL-7R结合蛋白，所述外源性IL-7R结合蛋白能特异性结合IL-7R且增强IL-7R活性；优选地，所述的IL-7R结合蛋白为IL-7，进一步优选地所述IL-7R的氨基酸序列如SEQ ID NO:1所示；。The cell of claim 1, wherein the IL-7R binding protein is an exogenous IL-7R binding protein, and the exogenous IL-7R binding protein can specifically bind to IL-7R and enhance IL-7R -7R activity; preferably, the IL-7R binding protein is IL-7, and further preferably, the amino acid sequence of the IL-7R is shown in SEQ ID NO: 1;.
  4. 如权利要求1所述的细胞，其特征在于，所述IL-7为天然IL-7、或与天然IL-7具有相同功能的天然IL-7的截短片段、或天然IL-7的突变体；The cell of claim 1, wherein the IL-7 is native IL-7, or a truncated fragment of native IL-7 having the same function as native IL-7, or a mutation of native IL-7 body;
    优选地，所述IL-7的核酸序列与SEQ ID NO:2或3所示的序列具有至少90％同一性、或是SEQ ID NO:2或3所示的核酸序列的截短片段；或所述IL-7的氨基酸序列与SEQ ID NO:13或19所示的序列具有至少80％同一性、或是SEQ ID NO:13或19所示的氨基酸序列的截短片段。Preferably, the nucleic acid sequence of IL-7 has at least 90% identity with the sequence shown in SEQ ID NO: 2 or 3, or is a truncated fragment of the nucleic acid sequence shown in SEQ ID NO: 2 or 3; or The amino acid sequence of the IL-7 has at least 80% identity with the sequence shown in SEQ ID NO: 13 or 19, or a truncated fragment of the amino acid sequence shown in SEQ ID NO: 13 or 19.
  5. 如权利要求4所述的细胞，其特征在于，所述IL7为组成性表达或诱导性表达；优选地，为诱导型启动子诱导所述IL-7的表达；优选地，所述启动子是NFAT6启动子；优选地，所述NFAT6启动子为反向调控。The cell of claim 4, wherein the IL7 is constitutively expressed or inducible; preferably, the expression of the IL-7 is induced by an inducible promoter; preferably, the promoter is NFAT6 promoter; preferably, the NFAT6 promoter is reverse regulated.
  6. 如权利要求2所述的细胞,其特征在于，所述TFP包含：The cell of claim 2, wherein the TFP comprises:
    (a)TCR亚基(或称TCR单元)；和(a) TCR subunits (or TCR units); and
    (b)识别靶抗原的抗原识别单元；(b) an antigen recognition unit that recognizes the target antigen;
    其中所述TCR亚基和所述抗原识别单元可操作地连接。wherein the TCR subunit and the antigen recognition unit are operably linked.
  7. 如权利要求6所述的细胞，其特征在于，所述TCR亚基包含：The cell of claim 6, wherein the TCR subunit comprises:
    (i)TCR细胞外结构域的至少一部分，和(i) at least a portion of the TCR extracellular domain, and
    (ii)包含来自CD3ε、CD3γ、CD3δ、TCRα、TCRβ的细胞内信号传导结构域的刺激性结构域的TCR细胞内结构域或其组合；(ii) a TCR intracellular domain comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ, TCRα, TCRβ, or a combination thereof;
    或所述TCR亚基包含：CD3αTCR亚基、CD3βTCR亚基、CD3εTCR亚基、CD3γTCR亚基、CD3δTCR亚基或其组合。Or the TCR subunit comprises: a CD3α TCR subunit, a CD3β TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, or a combination thereof.
  8. 如权利要求1所述的细胞，其特征在于，所述靶抗原选自：促甲状腺激素受体(TSHR)； CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD22；CD30；CD70；CD123；CD138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；***干细胞抗原(PSCA)；***特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记251(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoHglycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受5体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；***蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变10体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞 状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)；The cell of claim 1, wherein the target antigen is selected from: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); -13Rα); Interleukin 11 Receptor Alpha (IL-11Rα); Prostate Stem Cell Antigen (PSCA); Prostate Specific Membrane Antigen (PSMA); Carcinoembryonic Antigen (CEA); NY-ESO-1; HIV-1 Gag; MART -1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta) ; Stage-specific embryonic antigen-4 (SSEA-4); cell surface associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural Cell Adhesion Molecule (NCAM); Carbonic Anhydrase IX (CAIX); LMP2; Ephrin Type A Receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis Adhesion Molecule (sLe); Ganglioside Lipid GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); ortho-acetyl GD2 ganglioside (OAcGD2); folic acid receptor body; tumor endothelial marker 251 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation Antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D body; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; Open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PL AC1); hexose moiety of globoHglycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); ; pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms cytoplasmin (WT1); ETS translocation variant 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanin Tumor Cancer Testis Antigen-1 (MAD-CT-1); Melanoma Cancer Testis Antigen-2 (MAD-CT-2); Fos-Associated Antigen 1; p53 Mutation 10; Human Telomerase Reverse Transcriptase (hTERT) ; Sarcoma translocation breakpoint; Melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17) ; paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family Member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); Squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5 ); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase-anchored protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); containing EGF-like module mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glycan-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin λ-like polypeptide 1 (IGLL1);
    或，所述靶抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；其中，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原或流感病毒抗原。Or, the target antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; wherein, the viral antigens are selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen or influenza virus antigen.
  9. 如权利要求6所述的细胞，其特征在于，所述抗原识别单元包括抗体或其片段，优选的，所述抗体或其片段为Fab，Fab'、F(ab')2、Fv片段、scFv、sdFv、由VH和CH1结构域组成的Fd片段、线性抗体、单域抗体、或camelid VHH结构域，更优选的，所述抗体为scFv。The cell of claim 6, wherein the antigen recognition unit comprises an antibody or a fragment thereof, preferably, the antibody or a fragment thereof is Fab, Fab', F(ab')2, Fv fragment, scFv , sdFv, Fd fragment consisting of VH and CH1 domains, linear antibody, single domain antibody, or camelid VHH domain, more preferably, the antibody is an scFv.
  10. 如权利要求6所述的细胞，其特征在于，所述抗原识别单元识别GPC3或识别Claudin18.2，The cell of claim 6, wherein the antigen recognition unit recognizes GPC3 or recognizes Claudin18.2,
    优选地，识别GPC3的抗原识别单元的氨基酸序列的轻链LCDR1、LCDR2和LCDR3独立选自下表所示轻链LCDR1、LCDR2和LCDR3或与之具有70-100％的序列同一性，和/或Preferably, the light chains LCDR1, LCDR2 and LCDR3 recognizing the amino acid sequence of the antigen recognition unit of GPC3 are independently selected from or have 70-100% sequence identity with the light chains LCDR1, LCDR2 and LCDR3 shown in the table below, and/or
    识别GPC3的抗原识别单元的氨基酸序列的重链HCDR1、HCDR2和HCDR3独立选自下表所示重链HCDR1、HCDR2和HCDR3或与之具有70-100％的序列同一性；The heavy chains HCDR1, HCDR2 and HCDR3 recognizing the amino acid sequence of the antigen recognition unit of GPC3 are independently selected from or have 70-100% sequence identity with the heavy chains HCDR1, HCDR2 and HCDR3 shown in the following table;
    Figure PCTCN2021119634-appb-100001
    Figure PCTCN2021119634-appb-100001
    Figure PCTCN2021119634-appb-100002
    Figure PCTCN2021119634-appb-100002
    ；或者;or
    识别claudin18.2的抗原识别单元的氨基酸序列的轻链LCDR1、LCDR2和LCDR3独立选自下表所示轻链LCDR1、LCDR2和LCDR3或与之具有70-100％的序列同一性，和/或识别claudin18.2的抗原识别单元的氨基酸序列的重链HCDR1、HCDR2和HCDR3独立选自下表所示重链HCDR1、HCDR2和HCDR3或与之具有70-100％的序列同一性：The light chains LCDR1, LCDR2 and LCDR3 that recognize the amino acid sequence of the antigen recognition unit of claudin18.2 are independently selected from or have 70-100% sequence identity to the light chains LCDR1, LCDR2 and LCDR3 shown in the table below, and/or recognize The heavy chain HCDR1, HCDR2 and HCDR3 of the amino acid sequence of the antigen recognition unit of claudin18.2 are independently selected from or have 70-100% sequence identity with the heavy chains HCDR1, HCDR2 and HCDR3 shown in the following table:
    Figure PCTCN2021119634-appb-100003
    Figure PCTCN2021119634-appb-100003
  11. 如权利要求6所述的细胞，其特征在于，识别GPC3的抗原识别单元的氨基酸序列的轻链可变区独立选自下表所示轻链可变区或与之具有70-100％的序列同一性，和/或识别GPC3 的抗原识别单元的氨基酸序列的重链可变区独立选自下表所示重链可变区或与之具有70-100％的序列同一性；The cell according to claim 6, wherein the light chain variable region recognizing the amino acid sequence of the antigen recognition unit of GPC3 is independently selected from the light chain variable region shown in the following table or a sequence having 70-100% thereof The identity, and/or the heavy chain variable region that recognizes the amino acid sequence of the antigen recognition unit of GPC3 is independently selected from or has 70-100% sequence identity to the heavy chain variable region shown in the following table;
    Figure PCTCN2021119634-appb-100004
    Figure PCTCN2021119634-appb-100004
    ；或者，识别claudin18.2的抗原识别单元的氨基酸序列的轻链可变区独立选自下表所示轻链可变区或与之具有70-100％的序列同一性，和/或识别claudin18.2的抗原识别单元的氨基 酸序列的重链可变区独立选自下表所示重链可变区或与之具有70-100％的序列同一性；Or, the light chain variable region of the amino acid sequence of the antigen recognition unit that recognizes claudin18.2 is independently selected from the light chain variable region shown in the following table or has 70-100% sequence identity with it, and/or recognizes claudin18 The heavy chain variable region of the amino acid sequence of the antigen recognition unit of .2 is independently selected from the heavy chain variable region shown in the following table or has 70-100% sequence identity therewith;
    Figure PCTCN2021119634-appb-100005
    Figure PCTCN2021119634-appb-100005
  12. 如权利要求6所述的细胞，其特征在于，识别GPC3的抗原识别单元的氨基酸序列选 自下表所示序列或与之具有70-100％的序列同一性；The cell of claim 6, wherein the amino acid sequence of the antigen recognition unit recognizing GPC3 is selected from the sequence shown in the following table or has 70-100% sequence identity with it;
    Figure PCTCN2021119634-appb-100006
    Figure PCTCN2021119634-appb-100006
    ；或者，;or,
    识别claudin18.2的抗原识别单元的氨基酸序列选自下表所示序列或与之具有70-100％的序列同一性；The amino acid sequence of the antigen recognition unit that recognizes claudin18.2 is selected from the sequence shown in the following table or has 70-100% sequence identity with it;
    Figure PCTCN2021119634-appb-100007
    Figure PCTCN2021119634-appb-100007
    Figure PCTCN2021119634-appb-100008
    Figure PCTCN2021119634-appb-100008
  13. 如权利要求7所述的细胞，其特征在于，所述抗原识别单元通过连接子序列连接至所述TCR细胞外结构域的至少一部分；优选所述连接子序列包括(G 4S) n，其中n＝1至4； The cell of claim 7, wherein the antigen recognition unit is linked to at least a portion of the TCR extracellular domain via a linker sequence; preferably the linker sequence comprises (G 4 S) n , wherein n=1 to 4;
    进一步优选所述连接子的编码序列具有SEQ ID NO：8或11所示的核酸序列；或具有SEQ  ID NO：12所示的氨基酸序列。Further preferably, the coding sequence of the linker has the nucleic acid sequence shown in SEQ ID NO: 8 or 11; or the amino acid sequence shown in SEQ ID NO: 12.
  14. 如权利要求6-13任一所述的细胞，其特征在于，所述TCR细胞外结构域的至少一部分、所述TFP包括的TCR跨膜结构域和TCR细胞内结构域中,至少两个结构域是来自同一TCR亚基。The cell according to any one of claims 6-13, wherein at least a part of the TCR extracellular domain, the TCR transmembrane domain and the TCR intracellular domain included in the TFP, at least two structures The domains are from the same TCR subunit.
  15. 如权利要求2所述的细胞，其特征在于，所述TFP与IL7由同一核酸分子表达，或由不同核酸分子表达；The cell of claim 2, wherein the TFP and IL7 are expressed by the same nucleic acid molecule, or are expressed by different nucleic acid molecules;
    优选所述TFP与IL7由同一核酸分子表达，所述IL7的表达盒与TFP之间、以及表达盒之间直接连接或由串联片段连接，所述串联片段选自F2A、PA2、T2A、和/或E2A。Preferably, the TFP and IL7 are expressed from the same nucleic acid molecule, and the expression cassette of IL7 and TFP, as well as between the expression cassettes, are directly connected or connected by tandem fragments selected from F2A, PA2, T2A, and/or or E2A.
  16. 如权利要求7-15任一所述的细胞，其特征在于，所述TFP包括含有免疫受体酪氨酸活化基序(ITAM)的蛋白；所述含有ITAM的蛋白包括选自由以下组成的组的蛋白质或其部分：CD3δTCR亚基、CD3εTCR亚基、CD3γTCR亚基、CD3δTCR亚基、Fcε受体1链、Fcε受体2链、Fcγ受体1链、Fcγ受体2a链、Fcγ受体2b1链、Fcγ受体2b2链、Fcγ受体3a链、Fcγ受体3b链、Fcβ受体1链、TYROBP(DAP12)、CD5、CD16a、CD16b、CD22、CD23、CD32、CD64、CD79a、CD79b、CD89、CD278、CD66d或其功能片段；The cell of any one of claims 7-15, wherein the TFP comprises a protein containing an immunoreceptor tyrosine activation motif (ITAM); the ITAM-containing protein comprises a protein selected from the group consisting of The protein or part thereof: CD3δTCR subunit, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ receptor 2a chain, Fcγ receptor 2b1 Chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89 , CD278, CD66d or their functional fragments;
    进一步优选，所述TFP分子还包含前导序列，优选地，所述前导序列包括如SEQ ID NO：7所示的核酸序列。Further preferably, the TFP molecule further comprises a leader sequence, preferably, the leader sequence comprises the nucleic acid sequence shown in SEQ ID NO:7.
  17. 如权利要求16所述的细胞，其特征在于，所述TFP包括SEQ ID NO：9、10或16所示的核酸序列；或TFP包括SEQ ID NO：14或15所示的氨基酸序列。The cell of claim 16, wherein the TFP comprises the nucleic acid sequence shown in SEQ ID NO: 9, 10 or 16; or the TFP comprises the amino acid sequence shown in SEQ ID NO: 14 or 15.
  18. 一种核酸分子，所述核酸分子编码权利要求1-17中任一项所述的IL-7R结合蛋白或IL-7；所述核酸分子还编码权利要求1-17中任一项所述的特异性识别靶抗原的受体；A nucleic acid molecule encoding the IL-7R binding protein or IL-7 of any one of claims 1-17; the nucleic acid molecule also encoding the IL-7R binding protein of any one of claims 1-17 Receptors that specifically recognize the target antigen;
    优选，所述核酸由DNA和/或RNA组成；Preferably, the nucleic acid consists of DNA and/or RNA;
    进一步优选，所述核酸是mRNA；Further preferably, the nucleic acid is mRNA;
    进一步优选，所述核酸包含核苷酸类似物。Further preferably, the nucleic acid comprises a nucleotide analog.
  19. 一种载体，包含权利要求18所述的核酸分子，A carrier, comprising the nucleic acid molecule of claim 18,
    优选所述载体选自由以下组成的组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、劳氏肉瘤病毒(RSV)载体或逆转录病毒载体。Preferably the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, Rous Sarcoma virus (RSV) vector or retroviral vector.
  20. 一种细胞，其包含权利要求19所述的载体或其基因组上整合有权利要求18所述的核酸分子，优选所述细胞是人T细胞，优选地，为同种异体T细胞。A cell comprising the vector of claim 19 or the nucleic acid molecule of claim 18 integrated into its genome, preferably the cell is a human T cell, preferably an allogeneic T cell.
  21. 一种制备细胞的方法，所述方法包括用权利要求19所述的载体或权利要求18所述的核酸 分子转导T细胞。A method of preparing cells comprising transducing T cells with the vector of claim 19 or the nucleic acid molecule of claim 18.
  22. 一种产生RNA工程改造的细胞群的方法，所述方法包括将体外转录的RNA或合成RNA引入细胞中，其中所述RNA包括权利要求18所述的核酸。A method of producing an RNA-engineered cell population, the method comprising introducing into a cell an in vitro transcribed RNA or a synthetic RNA, wherein the RNA comprises the nucleic acid of claim 18.
  23. 一种在哺乳动物体内提供抗肿瘤免疫性的方法，所述方法包括向所述哺乳动物施用有效量的权利要求1-17、20中任一项所述细胞、权利要求18所述的核酸分子、权利要求19所述的载体；优选所述哺乳动物是人。A method for providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of the cells of any one of claims 1-17 and 20, the nucleic acid molecule of claim 18 . The vector of claim 19; preferably the mammal is a human.
  24. 一种治疗患有与GPC3或者claudin18.2表达有关的疾病的哺乳动物的方法，所述方法包括向所述哺乳动物施用有效量的权利要求1-17、20中任一项所述细胞、权利要求18所述的核酸分子、权利要求20所述的载体；A method for treating a mammal suffering from a disease related to GPC3 or claudin18.2 expression, the method comprising administering to the mammal an effective amount of the cells, claim The nucleic acid molecule of claim 18, the vector of claim 20;
    优选所述与GPC3或者claudin18.2表达有关的疾病选自结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，***，***癌，内分泌***癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，***癌，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经***(CNS)瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌、与GPC3或者claudin18.2表达有关的非癌症相关适应症；优选的，选自肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、胰腺癌、食道癌；Preferably, the disease related to the expression of GPC3 or claudin18.2 is selected from colon cancer, rectal cancer, renal cell cancer, liver cancer, lung cancer, small bowel cancer, esophagus cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer , skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, endocrine system cancer, thyroid cancer Carcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, tumor angiogenesis, spinal tumor, brain stem glial tumor, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, non-cancer-related indications related to GPC3 or claudin18.2 expression; preferably, selected from liver cancer, lung cancer, breast cancer, ovarian cancer, Kidney cancer, thyroid cancer, stomach cancer, colorectal cancer, pancreatic cancer, esophageal cancer;
    优选权利要求1-17、20中任一项所述细胞和增加权利要求1-17、20中任一项所述细胞功效的药剂组合施用；Preferably the cells of any one of claims 1-17, 20 are administered in combination with an agent that increases the efficacy of the cells of any one of claims 1-17, 20;
    优选权利要求1-17、20中任一项所述细胞与改善与施用权利要求1-17、20中任一项所述细胞相关的一种或多种副作用的药剂联合施用；Preferably the cells of any one of claims 1-17, 20 are administered in combination with an agent that ameliorates one or more side effects associated with administration of the cells of any one of claims 1-17, 20;
    优选权利要求1-17、20中任一项所述细胞和治疗所述与GPC3或者claudin18.2相关的疾病的药剂组合施用。Preferably, the cells of any one of claims 1-17 and 20 are administered in combination with an agent for treating the disease associated with GPC3 or claudin18.2.
  25. 权利要求1-17、20中任一项所述细胞、权利要求18所述的核酸分子、权利要求19所述的载体，用作药物。The cell of any one of claims 1-17 and 20, the nucleic acid molecule of claim 18, and the vector of claim 19, for use as a medicament.
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