Search

CN-122003505-A - Method for treating local and remote tumors

CN122003505ACN 122003505 ACN122003505 ACN 122003505ACN-122003505-A

Abstract

The present invention relates to a method of treating cancer by delivering Lipid Nanoparticles (LNPs) encapsulating immunostimulant mRNA and bispecific antibody mRNA into cancer cells or tumors. The method comprises the step of administering LNP encapsulating mRNA to a tumor lesion of a cancer subject by injection. The immunostimulant mRNA-LNP activates immune cells at and around the tumor site that act synergistically with bispecific antibody mRNA-LNP encoded antibodies and PBMCs to effectively target local and distant metastases. Preferred immunostimulants are granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12), and combinations thereof.

Inventors

  • WU LIJUN
  • Vita Gruboskaya

Assignees

  • 英创安博生物技术有限公司

Dates

Publication Date
20260508
Application Date
20240725
Priority Date
20230726

Claims (14)

  1. 1. A method of treating cancer comprising the steps of: Preparing a first Lipid Nanoparticle (LNP) that encapsulates a first class of mRNA encoding a bispecific antigen binding molecule comprising a first scFv and a CD3 ε scFv, wherein the first scFv targets EPCAM, her-2, mesothelin, EGFR, PLAP, CD147, 4-1BB, c-Met, CD19/CD37, PSMA, CD47, CD19, BCMA or Claudin 18.2; preparing a second class of LNPs, said second class of LNPs harboring a second mRNA encoding GM-CSF; Preparing a third class of LNPs, said third class of LNPs harboring a third mRNA encoding IL-12; administering the first, second, and third types of LNP of the entrapped mRNA to a tumor lesion of a subject having cancer.
  2. 2. A method of treating cancer comprising the steps of: Obtaining Lipid Nanoparticles (LNP) encapsulating (a) a first mRNA encoding a bispecific antigen binding molecule comprising a first ScFv and a CD3 epsilon ScFv, wherein the first ScFv targets EPCAM, her-2, mesothelin, EGFR, PLAP, CD147, 4-1BB, c-Met, CD19/CD37, PSMA, CD47, CD19, BCMA or Claudin 18.2, (b) a second mRNA encoding GM-CSF, and (c) a third mRNA encoding IL-12; administering the mRNA encapsulated Lipid Nanoparticle (LNP) to a tumor lesion of a subject having cancer.
  3. 3. The method of claim 1 or 2, wherein the cancer is a metastatic solid tumor.
  4. 4. The method of any one of claims 1-3, wherein the method treats a tumor at the site of administration and a tumor at the distal site by activating T cells and/or NK cells in the tumor microenvironment.
  5. 5. The method of any one of claims 1-3, wherein the bispecific antigen binding molecule comprises a monovalent humanized antibody EPCAM SCFV and a monovalent CD3 epsilon scFv, and the bispecific antigen binding molecule is fused to a human Fc fragment, wherein the Fc fragment is optionally mutated.
  6. 6. The method of any one of claims 1-3, wherein the bispecific antigen binding molecule comprises a monovalent humanized anti-Her-2 scFv and a monovalent CD3 epsilon scFv, and the bispecific antigen binding molecule is fused to a human Fc segment, wherein the Fc segment is optionally mutated.
  7. 7. The method of any one of claims 1-4, wherein the bispecific antigen binding molecule comprises a monovalent humanized anti-Mesothelin scFv and a monovalent CD3 epsilon scFv, and the bispecific antigen binding molecule is fused to a human Fc fragment, wherein the Fc fragment is optionally mutated.
  8. 8. The method of claim 1, wherein the first, second, and third types of LNPs are encapsulated for simultaneous administration to the subject.
  9. 9. The method of claim 1, wherein the first, second, and third types of LNPs are encapsulated for sequential administration to the subject.
  10. 10. The method of claim 1, wherein the first mRNA is transcribed from a DNA sequence comprising (a) a promoter coding sequence, (b) a 5 'UTR (untranslated region) coding sequence, (c) a sequence encoding the bispecific antigen binding molecule, (d) a 3' UTR coding sequence, and (e) a poly A tail sequence.
  11. 11. The method of claim 1, wherein the second mRNA is transcribed from a DNA sequence comprising (a) a promoter coding sequence, (b) a5 'UTR (untranslated region) coding sequence, (c) a sequence encoding GM-CSF, (d) a 3' UTR coding sequence, and (e) a poly A tail sequence.
  12. 12. The method of claim 1, wherein the third mRNA is transcribed from a DNA sequence comprising (a) a promoter coding sequence, (b) a 5 'UTR (untranslated region) coding sequence, (c) a sequence encoding IL-12, (d) a 3' UTR coding sequence, and (e) a poly A tail sequence.
  13. 13. The method of claim 1 or 2, wherein the cancer is colorectal cancer, ovarian cancer, pancreatic cancer, breast cancer, or lung cancer.
  14. 14. The method of claim 1, wherein the Lipid Nanoparticle (LNP) comprises 8- [ (2-hydroxyethyl) [ 6-oxo-6- (undecyloxy) hexyl ] amino ] octanoate-1-octyl nonyl ester (SM-102), distearoyl phosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

Description

Method for treating local and remote tumors Technical Field The present invention relates to the preparation of mRNA-LNP nanoparticles (mRNA-LNP) encoding a variety of immunostimulants, a tumor antigen targeting antibody and a T cell adaptor for targeting cancer cells or tumor cell delivery, aimed at activating immune cells (T cells, NK cells, dendritic cells, macrophages) and thus attacking local and distant tumor cells. The messenger ribonucleic acid-lipid nanoparticle encoding an immunostimulant delivered to a tumor site can effect expression of an dockerin (expressed on the cell surface) or a secreted protein and promote local and distant tumor regression. Background Immunotherapy is becoming a promising approach to tumor treatment. T cells (also known as T lymphocytes) act as the "defensive main" of the human immune system, and can continuously recognize foreign antigens and distinguish abnormal cells (cancer cells or infected cells) from normal cells. The technical means of immunotherapy mainly comprise monoclonal antibodies 1, immune checkpoint inhibitors 2, tumor vaccines 3, chimeric antigen receptor T cells (CAR-T) and chimeric antigen receptor natural killer cell (CAR-NK) immunotherapy 4. Tumor vaccines are capable of efficiently delivering tumor-specific antigens (TSAs) to Antigen Presenting Cells (APCs), thereby activating tumor-specific T cells. The vaccine can help organisms build long-acting anti-tumor immune memory, realize local tumor regression and remove remote metastasis. Failure of tumor vaccines can be attributed to insufficient tumor immunogenicity, which can lead to failure of the body to produce sufficient numbers of potent T cells, a key element in inducing a long-lasting anti-tumor immune response. One strategy to improve the efficacy of tumor derived vaccines is to deliver tumor antigens in combination with natural, synthetic, or genetically engineered immunostimulatory molecules. Drawings FIG. 1 shows a schematic of a DNA vector template (A) for in vitro transcription of a protein or bispecific antibody RNA (B). The 5'UTR, i.e.the 5' untranslated region, the 3'UTR, i.e.the 3' untranslated region, the poly A tail (poly A tail) for improved RNA stability. FIG. 2 shows the bispecific antibody structure of EPCAM single chain variable region fragment-human CD3 single chain variable region fragment-human Fc fragment (EPCAM SCFV-CD3 scFv-human Fc). The aforementioned human Fc fragment may comprise L234A, L a and P239G mutations to reduce the Fc fragment-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) effect. FIG. 3 shows the detection of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein expression by enzyme-linked immunosorbent assay (ELISA) after transfection of mRNA-LNP into human embryonic kidney 293 cells (293 cells). The supernatant of transfected cells was collected and protein detection in the supernatant was performed using GM-CSF enzyme-linked immunosorbent assay (ELISA) kit. FIG. 4 shows IL-12 secretion detected by ELISA after IL-12 mRNA-LNP transfection into 293 cells. FIG. 5 shows that NK cell expansion was enhanced upon transfection with IL-12 mRNA-LNP. IL-12-mRNA-LNP transfected NK cells secreting IL-12 in supernatant medium when they die, expand at significantly higher levels than cytokine-free NK cells in the medium. Figure 6 shows that gamma-interferon (IFN-gamma) secretion levels were significantly increased against OVCAR-5 cells transfected with immunostimulant mRNA-LNP in killing experiments performed with EpCAM-CD3 antibodies and Peripheral Blood Mononuclear Cells (PBMCs). FIGS. 7A-7B show the effect of EpCAM-CD3-mRNA-LNP on local and distant tumor sites. EpCAM-CD3, intratumorally injected to the left tumor, had an inhibitory effect on growth of only the left tumor (fig. 7A), while no inhibitory effect on the right tumor that did not receive RNA-LNP treatment (fig. 7B). FIGS. 8A-8B show that local administration of the four immunostimulants mixed with mRNA-LNP and EpCAM-CD3-hFc mRNA-LNP and T cells inhibited growth of tumors on the local administration side (left tumor) in mice (FIG. 8A), and that the preparation also inhibited growth of tumors on the opposite distal end (right tumor) that were not administered (FIG. 8B). * p <0.05 the growth inhibitory effect of EpCAM-CD3 hFc mRNA-LNP combined with either immunostimulant mixture 1 or mixture 2 treated experimental group was compared to the Enhanced Green Fluorescent Protein (EGFP) mRNA-LNP treated control group using Student's t assay. FIGS. 9A-9B show that immunostimulant mixture 1A (IL-12+GM-CSF) mRNA-LNP, in combination with EpCAM-CD3-hFc mRNA-LNP topical administration and PBMC intravenous treatment, inhibited tumor growth in the topical (left) side of mice (FIG. 9A), and also inhibited growth in the opposite distal tumor (right) side of the mice that was not administered (FIG. 9B). FIGS. 9C-9D show that the local administration of the immunostimulant mixture 1B (chemokine ligand