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CN-122012398-A - Enhanced SNTC cell targeting CD33 and CD38 antigens, preparation method and application thereof

CN122012398ACN 122012398 ACN122012398 ACN 122012398ACN-122012398-A

Abstract

The invention discloses an enhanced SNTC cell targeting CD33 and CD38 antigens, a preparation method and application thereof, and in particular provides a pluripotent stem cell, which comprises (1) down-regulating the expression of CD33 and CD38, (2) over-expressing a chimeric antigen receptor targeting CD33 and CD38, and/or over-expressing the chimeric antigen receptor targeting CD33 and the chimeric antigen receptor targeting CD38, and (3) over-expressing at least one of FB22 and IL-15 RF. According to the embodiment of the invention, the pluripotent stem cells can be directionally differentiated into the artificially modified innate immune cells which can avoid self-injury and kill, accurately identify and remove tumor cells and enhance survival and migration capacity, and compared with NK cells in the prior art, the pluripotent stem cells are low in transformation cost, and the acquired innate immune cells after differentiation can be used for effectively treating tumors and have higher value for clinical treatment.

Inventors

  • TANG XIAOYAN
  • SUN JINGXIN
  • GAO JUNSHUANG
  • XU PENGFEI

Assignees

  • 北京京科信拓生物技术有限公司

Dates

Publication Date
20260512
Application Date
20251231

Claims (16)

  1. 1. A pluripotent stem cell, which comprises a cell line, characterized by comprising the following steps: (1) Down-regulation of CD33 and CD38 expression; (2) Over-expressing a CD 33-and CD 38-targeting chimeric antigen receptor and/or over-expressing a CD 33-targeting chimeric antigen receptor and a CD 38-targeting chimeric antigen receptor, and (3) At least one of FB22 and IL-15RF is overexpressed.
  2. 2. The pluripotent stem cell of claim 1, wherein the pluripotent stem cell is selected from at least one of a human embryonic stem cell, a human induced pluripotent stem cell, and a chemically induced reprogrammed pluripotent stem cell; Optionally, the chimeric antigen receptor targeting CD33 and CD38 comprises a single chain antibody targeting CD33 and a single chain antibody targeting CD 38; optionally, the CD 33-targeting chimeric antigen receptor comprises a CD 33-targeting single chain antibody; Optionally, the CD 38-targeting chimeric antigen receptor comprises a CD 38-targeting single chain antibody; optionally, the CD 33-targeting single chain antibody comprises a first heavy chain variable region and a first light chain variable region; Optionally, the first heavy chain variable region and the first light chain variable region comprise a heavy chain variable region and a light chain variable region selected from Gemtuzumab ozogamicin, lintuzumab (SGN-33), or M195.34 antibodies; Optionally, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is linked to the N-terminus of the first heavy chain variable region; optionally, the first heavy chain variable region has an amino acid sequence as set forth in SEQ ID No. 12 or an amino acid sequence at least 80% identical thereto, and the first light chain variable region has an amino acid sequence as set forth in SEQ ID No. 11 or an amino acid sequence at least 80% identical thereto; Optionally, the C-terminus of the first heavy chain variable region is linked to the N-terminus of a linker peptide 4, the C-terminus of the linker peptide 4 is linked to the N-terminus of the first light chain variable region, or the C-terminus of the first light chain variable region is linked to the N-terminus of the linker peptide 4, the C-terminus of the linker peptide 4 is linked to the N-terminus of the first heavy chain variable region; Optionally, the CD 33-targeting single chain antibody has an amino acid sequence as shown in SEQ ID No.1 or an amino acid sequence having at least 80% identity thereto; optionally, the CD 38-targeting single chain antibody comprises a second heavy chain variable region and a second light chain variable region; Optionally, the second heavy chain variable region and the second light chain variable region comprise a heavy chain variable region and a light chain variable region selected from Daratumumab, isatuximab or CM313 antibodies; Optionally, the C-terminus of the second heavy chain variable region is linked to the N-terminus of the second light chain variable region, or the C-terminus of the second light chain variable region is linked to the N-terminus of the second heavy chain variable region; Optionally, the second heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO. 14 or an amino acid sequence at least 80% identical thereto, and the second light chain variable region has an amino acid sequence as set forth in SEQ ID NO. 13 or an amino acid sequence at least 80% identical thereto; Optionally, the C-terminus of the second heavy chain variable region is linked to the N-terminus of a linker peptide 5, the C-terminus of the linker peptide 5 is linked to the N-terminus of the second light chain variable region, or the C-terminus of the second light chain variable region is linked to the N-terminus of the linker peptide 5, the C-terminus of the linker peptide 5 is linked to the N-terminus of the second heavy chain variable region; Optionally, the CD 38-targeting single chain antibody has an amino acid sequence as shown in SEQ ID No. 2 or an amino acid sequence having at least 80% identity thereto.
  3. 3. The pluripotent stem cell of claim 1, wherein the CD33 and CD38 targeting chimeric antigen receptor has the C-terminus of the CD33 targeting single chain antibody linked to the N-terminus of the CD38 targeting single chain antibody; Optionally, the C-terminus of the CD 33-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, the C-terminus of the linker peptide 3 being linked to the N-terminus of the CD 33-targeting single-chain antibody; Optionally, the C-terminus of the CD 38-targeting single-chain antibody is linked to the N-terminus of the CD 33-targeting single-chain antibody; optionally, the C-terminus of the CD 38-targeting single-chain antibody is linked to the N-terminus of the linker peptide 3, the C-terminus of the linker peptide 3 being linked to the N-terminus of the CD 38-targeting single-chain antibody; optionally, the C-terminus of the first light chain variable region is linked to the N-terminus of the CD 38-targeting single chain antibody, and the C-terminus of the CD 38-targeting single chain antibody is linked to the N-terminus of the first heavy chain variable region; Optionally, the C-terminus of the first light chain variable region is linked to the N-terminus of a linker peptide 1, the C-terminus of the linker peptide 1 is linked to the N-terminus of the second heavy chain variable region, the C-terminus of the second heavy chain variable region is linked to the N-terminus of the linker peptide 2, the C-terminus of the linker peptide 2 is linked to the N-terminus of the second light chain variable region, the C-terminus of the second light chain variable region is linked to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the first heavy chain variable region; optionally, the C-terminus of the second heavy chain variable region is linked to the N-terminus of the CD 33-targeting single chain antibody, and the C-terminus of the CD 33-targeting single chain antibody is linked to the N-terminus of the second light chain variable region; Optionally, the C-terminus of the second heavy chain variable region is linked to the N-terminus of the connecting peptide 1, the C-terminus of the connecting peptide 1 is linked to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is linked to the N-terminus of the connecting peptide 2, the C-terminus of the connecting peptide 2 is linked to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is linked to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is linked to the N-terminus of the second light chain variable region; Optionally, the linker peptide 1, linker peptide 2, linker peptide 3, linker peptide 4, linker peptide 5 is a flexible linker peptide or a rigid linker peptide; Optionally, the amino acid sequences of the connecting peptide 1, the connecting peptide 3, the connecting peptide 4 and the connecting peptide 5 are (GGGGS) n, wherein n is a positive integer not less than 1, preferably n is 1-6; Preferably, the amino acid sequence of the connecting peptide 1 is shown as SEQ ID NO. 15; preferably, the amino acid sequence of the connecting peptide 2 is shown as SEQ ID NO. 16; preferably, the amino acid sequence of the connecting peptide 3 is shown as SEQ ID NO. 17; preferably, the amino acid sequence of the connecting peptide 4 is shown as SEQ ID NO. 22; preferably, the amino acid sequence of the connecting peptide 5 is shown as SEQ ID NO. 35.
  4. 4. A pluripotent stem cell according to any of claims 1 to 3, wherein the CD 33-and CD 38-targeting chimeric antigen receptor and/or the CD 33-targeting chimeric antigen receptor and/or the CD 38-targeting chimeric antigen receptor further comprises at least one of a signal peptide 1, a hinge region, a transmembrane region and an intracellular region; optionally, the C-terminus of the signal peptide 1 is linked to the CD 33-and CD 38-targeting chimeric antigen receptor and/or the CD 33-targeting chimeric antigen receptor and/or the CD 38-targeting chimeric antigen receptor at the N-terminus; Optionally, the N-terminus of the hinge region is linked to the C-terminus of the CD 33-and CD 38-targeting chimeric antigen receptor and/or the CD 33-targeting chimeric antigen receptor and/or the CD 38-targeting chimeric antigen receptor; optionally, the C-terminal of the hinge region is connected to the N-terminal of the transmembrane region; optionally, the C-terminus of the transmembrane region is linked to the N-terminus of the intracellular region; optionally, the signal peptide 1 has an amino acid sequence as shown in SEQ ID NO. 21 or an amino acid sequence having at least 80% identity thereto; optionally, the hinge region comprises a CD8 hinge region; Optionally, the hinge region has an amino acid sequence as shown in SEQ ID NO. 18 or an amino acid sequence having at least 80% identity thereto; optionally, the transmembrane region comprises a CD8 transmembrane region; Optionally, the transmembrane region has an amino acid sequence as shown in SEQ ID NO. 19 or an amino acid sequence having at least 80% identity thereto; Optionally, the intracellular region comprises cd3ζ; Optionally, the intracellular region has the amino acid sequence shown in SEQ ID NO. 20 or an amino acid sequence having at least 80% identity thereto; optionally, the CD 33-targeting chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID No. 3 or an amino acid sequence having at least 80% identity thereto; Optionally, the CD 38-targeting chimeric antigen receptor has an amino acid sequence as set forth in SEQ ID No.4 or an amino acid sequence having at least 80% identity thereto; optionally, the chimeric antigen receptor targeting CD33 and CD38 has an amino acid sequence as shown in any one of SEQ ID NO 24-27 or an amino acid sequence having at least 80% identity thereto; Optionally, the FB22 has an amino acid sequence as shown in SEQ ID No. 5 or an amino acid sequence at least 80% identical thereto; Optionally, the IL-15RF includes signal peptide 2, IL-15, connecting peptide 6 and IL-15R alpha; Optionally, the signal peptide 2 has the amino acid sequence shown as SEQ ID NO. 28 or an amino acid sequence having at least 80% identity thereto; Optionally, the IL-15 has an amino acid sequence as set forth in SEQ ID NO. 29 or an amino acid sequence having at least 80% identity thereto; Optionally, the linker peptide 6 has the amino acid sequence shown as SEQ ID NO. 23 or an amino acid sequence having at least 80% identity thereto; Optionally, the IL-15Rα has an amino acid sequence as set forth in SEQ ID NO. 30 or an amino acid sequence having at least 80% identity thereto; Optionally, the IL-15RF has an amino acid sequence as set forth in SEQ ID NO. 6 or an amino acid sequence having at least 80% identity thereto.
  5. 5. The pluripotent stem cell of claim 1, wherein the pluripotent stem cell carries: (1) Nucleotide sequences shown in SEQ ID NO. 7 and 8, or a nucleotide sequence shown in any one of SEQ ID NO. 31-34; (2) At least one of the nucleotide sequences shown in SEQ ID NO 9 and 10.
  6. 6. A method of preparing the pluripotent stem cell of any one of claims 1 to 5, comprising: Down-regulating the expression of CD33 and CD38 in the pluripotent stem cells to be treated; overexpressing a CD 33-and CD 38-targeting chimeric antigen receptor in the pluripotent stem cells to be treated and/or overexpressing a CD 33-targeting chimeric antigen receptor and a CD 38-targeting chimeric antigen receptor; And overexpressing at least one of FB22 and IL-15RF, so as to obtain the pluripotent stem cells.
  7. 7. The method of claim 6, wherein the down-regulating the expression of CD33 and CD38 in the pluripotent stem cells to be treated is achieved by at least one of gene silencing, gene editing, small molecule inhibitors, antibody drugs; Optionally, the overexpression of the CD 33-and CD 38-targeting chimeric antigen receptor, and/or the overexpression of the CD 33-targeting chimeric antigen receptor and the CD 38-targeting chimeric antigen receptor is achieved by introducing a nucleic acid molecule encoding the CD 33-and CD 38-targeting chimeric antigen receptor, and/or the overexpression of the CD 33-targeting chimeric antigen receptor and the CD 38-targeting chimeric antigen receptor into the pluripotent stem cell to be treated; optionally, the over-expressing at least one of FB22 and IL-15RF is achieved by introducing a nucleic acid molecule encoding at least one of FB22 and IL-15RF into the pluripotent stem cell to be treated.
  8. 8. The gene editing according to claim 7 is selected from at least one of CRISPR-Cas9, CRISPR-Cas12a, CRISPR-Cas13, base editor and PRIME EDITING, said introducing being by means of at least one of electrotransfection, transfection and infection.
  9. 9. A method of preparing an artificially modified innate immune cell comprising: The pluripotent stem cells according to any one of claims 1 to 5 or the pluripotent stem cells prepared by the method according to any one of claims 6 to 8 are subjected to directional differentiation culture so as to obtain artificially modified innate immune cells.
  10. 10. The method of claim 9, wherein the directed differentiation culture is performed by at least one of monolayer induction, embryoid body induction, and organoid induction.
  11. 11. An artificially modified innate immune cell comprising: (1) Down-regulation of CD33 and CD38 expression; (2) Over-expressing a CD 33-and CD 38-targeting chimeric antigen receptor and/or over-expressing a CD 33-targeting chimeric antigen receptor and a CD 38-targeting chimeric antigen receptor, and (3) At least one of FB22 and IL-15RF is overexpressed.
  12. 12. An artificially modified innate immune cell, characterized in that it is produced by the method of claim 9 or 10.
  13. 13. A pharmaceutical composition, in the form of a capsule, characterized by comprising the following steps: the pluripotent stem cell according to any one of claims 1 to 5, or the artificially modified innate immune cell according to claim 11 or 12.
  14. 14. A method of enhancing killing, proliferation, activation and migration of artificially modified innate immune cells comprising: a method of preparing pluripotent stem cells by the method of any one of claims 6 to 8, and And performing directional differentiation culture on the pluripotent stem cells.
  15. 15. The method of claim 14, wherein the directed differentiation culture is performed by at least one of monolayer induction, embryoid body induction, and organoid induction.
  16. 16. Use of the pluripotent stem cell of any one of claims 1 to 5, the artificially modified innate immune cell of claim 11 or 12 or the pharmaceutical composition of claim 13 in the manufacture of a medicament for the treatment and/or prevention of tumors.

Description

Enhanced SNTC cell targeting CD33 and CD38 antigens, preparation method and application thereof Technical Field The invention relates to the field of biotechnology, in particular to an enhanced SNTC cell targeting CD33 and CD38 antigens, a preparation method and application thereof. Background The CAR structure of CAR-NK cells is typically composed of three parts, namely an extracellular antigen binding region (typically scFv), a transmembrane domain, and an intracellular activation domain, similar to the CAR structure used by CAR-T cells. Currently, most CAR-T therapies widely studied and used are autologous CAR-T cells. The research discovers that the CAR-T cells prepared from the single-target CAR can have the phenomenon of tumor cell antigen escape in tumor treatment, namely the antigen targeted by the CAR is combined with the antigen to have reduced expression or even no expression in the tumor cells, so that the CAR-T cells can not recognize the tumor cells, and the treatment effect of the CAR-T cells is limited. Therefore, a double-target CAR is designed aiming at the phenomenon, namely a CAR designed aiming at two antigens of the same tumor cell, and the CAR-T cell prepared by the double-target CAR can effectively avoid the antigen escaping phenomenon, so that the tumor cell is well cleared. In addition, the CAR-NK cells applied to adoptive cell therapy have the advantages of no GvHD (graft versus host disease), higher safety, uniform quality, high aging, small side effect and the like. In the treatment of myeloid leukemia, many CD33 CAR-NK cells are currently being studied. CD33 is a myeloid cell differentiation antigen with a molecular weight of 67KD and is predominantly distributed in myeloid blood cells, especially in the early stages of differentiation. The intracellular region contains an Immune Tyrosine Inhibitory Motif (ITIM), so it may have the function of regulating cell growth and differentiation by recruiting signaling molecules. CD33 is expressed in most acute myeloid leukemia patients, but not on the surface of hematopoietic stem cells, nor on mature granulocytes and other tissues, and thus CD33 is a good target for myeloid leukemia treatment. However, since NK cells partially express CD33, the phenomenon of self-killing and reduction of amplification efficiency occurs in the preparation of CD33 CAR-NK (PMID: 35058934). This is also a current challenge to be addressed in the preparation of CD33 CAR-NK using natural NK cells. CD38 is a transmembrane glycoprotein involved in signal transduction and cell adhesion, and is highly expressed on the cell surface of hematological malignancies, multiple Myeloma (MM), acute Myelogenous Leukemia (AML), burkitt's Lymphoma (BL), and T-cell acute lymphoblastic leukemia (T-ALL). CD38 CAR-T cells are mostly used for research in the treatment of multiple myeloma. However, since mature myeloid cells and their precursor cells also express CD38, anti-CD 38 CARs can have potential myelosuppressive effects. NK cells also highly express CD38, so that self-phase killing also occurs between the prepared CD38 CAR-NK cells. And single-target treatment can cause tumor antigen escape. Therefore, in order to solve the above problems, there is still a need for further development of CAR cells which can effectively treat tumors without the phenomenon of self-killing and decrease in amplification efficiency. Disclosure of Invention The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the present invention provides a pluripotent stem cell. The present invention has been completed based on the following findings by the inventors: Currently, chimeric Antigen Receptors (CARs) designed for the CD33 target are effective in killing CD33 expressing myeloid leukemia cells after loading into either T cells or NK cells. However, during the construction of CD33 CAR-NK, since NK cells themselves also partially express CD33 antigen, a "self-phase killing" phenomenon between CAR-NK cells occurs and its ability to expand in vitro is significantly impaired. In addition, NK cells commonly express CD38 molecules at high levels. This property not only causes self-killing between CD38 CAR-NK cells, but also presents challenges in clinical combination, in that when patients receive anti-CD 38 monoclonal antibody therapy, the drug may accidentally injure infused CD38 CAR-NK cells, thereby impairing its actual tumor clearance. The direct knockout of CD33 and CD38 genes in natural NK cells is inefficient, costly, and prior art multi-focused single modifications (such as knockout of CD33, CD38 only or expression of CAR only) lack synergistic design. To overcome the above problems, the inventors performed gene manipulation at the stem cell stage, knocked out CD33, CD38 and over-expressed functional molecules FB22 and IL-15RF, improved efficiency and uniformity of knocked out genes, remarkably reduced cost of in