WO2022057941A1 - Cellule effectrice immunitaire exprimant une protéine de liaison à l'il-7r - Google Patents

Cellule effectrice immunitaire exprimant une protéine de liaison à l'il-7r 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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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

Une cellule effectrice immunitaire génétiquement modifiée, dans laquelle la cellule exprime une protéine de liaison à l'IL-7R ou à l'IL-7, et exprime également un récepteur qui reconnaît spécifiquement un antigène cible.
PCT/CN2021/119634 2020-09-21 2021-09-22 Cellule effectrice immunitaire exprimant une protéine de liaison à l'il-7r WO2022057941A1 (fr)

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