WO2022089443A1 - 修饰的免疫细胞及其用途 - Google Patents

修饰的免疫细胞及其用途 Download PDF

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WO2022089443A1
WO2022089443A1 PCT/CN2021/126480 CN2021126480W WO2022089443A1 WO 2022089443 A1 WO2022089443 A1 WO 2022089443A1 CN 2021126480 W CN2021126480 W CN 2021126480W WO 2022089443 A1 WO2022089443 A1 WO 2022089443A1
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cells
cell
immune cell
modified immune
tumor
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PCT/CN2021/126480
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French (fr)
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谷为岳
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北京卡替医疗技术有限公司
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Priority to EP21885170.7A priority Critical patent/EP4257676A1/en
Priority to JP2023525626A priority patent/JP2023548106A/ja
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Definitions

  • the invention belongs to the technical field of biomedicine, in particular to the field of immune cell therapy.
  • T cells are one of the most important specific immune cells in the human body. In recent years, they have played an important role in the field of high-profile cell therapy. For example, cell therapy can be performed by modifying and infusing the patient's autologous T cells. However, given the heterogeneity of the tumor, the individuality of the patient's MHC, and the stochastic nature of the T cell receptor (TCR) production process (VDJ rearrangement), the T cell populations that specifically recognize harmful cells vary from patient to patient, in other words, the A T cell population comprising a highly individualized cohort of TCRs. How to quickly obtain T cell populations with the ability to specifically recognize harmful cells (such as cancer cells or viral host cells) from patients, and modify them on this basis, and then apply them to cell therapy more efficiently and safely, is a technical problem. .
  • PD-1 positive T cells are a group of cells in a suppressed state, and such cells are likely to be able to recognize target cells and have undergone PD-1 interaction with target cells.
  • L1-interacting T cells L1-interacting T cells.
  • PD-1 + T cells can be viewed as a collection of T cells that have attacked harmful cells. Because the T cells in this collection have come into contact with harmful cells, these T cells can be considered to have the ability to target harmful cells.
  • the processed PD-1 + T cells in peripheral blood are expected to be used to treat various diseases, especially tumors and infectious diseases.
  • PD-1 + T cells have better recognition characteristics of harmful cells, but the disadvantage is that the high expression of PD-1 makes these cells in a state of exhaustion, their function is inhibited, and it is difficult to kill them.
  • the present invention proposes several modification methods to overcome the suppressive state of PD-1 + T cells, thus completing the present invention.
  • the present application relates to a modified immune cell which is a PD-1 positive T (PD-1 + T) cell derived from peripheral blood.
  • the PD-1 + T cells are derived from peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the PD-1 + T cells are cells obtained by sorting PBMCs, eg, by PD-1 and CD3 as markers.
  • the modification is one that reduces or eliminates the suppressive state of PD-1 + T cells.
  • the modification can be carried out by genetic modification and/or protein modification of the immune cells.
  • the modification is a genetic modification of a PD1 + T cell such that the PD-1 expression of the PD1 + T cell is absent or reduced.
  • the genetic modification can be performed in vivo or in vitro. In one embodiment, the genetic modification is performed in vivo by gene editing or gene therapy. In another embodiment, the genetic modification is performed in vitro, eg, during the preparation of the PD-1 + T cells.
  • the genetic modification may be a knockout or knockdown of endogenous PD-1 of the PD-1 + T cell.
  • the means of knocking out the endogenous PD-1 gene include but are not limited to CRISPR/Cas method, TALEN, etc.
  • the modification is a modification of PD-1 on the surface of PD-1 + T cells such that the binding capacity of PD-1 on the surface of PD-1 + T cells to its ligand is relative to unmodified decreased before.
  • the PD-1 antibody competitively binds to PD-1 on the surface of the PD-1 + T cells, thereby reducing the interaction between PD-1 and PD-1 + T cells. The binding capacity of the ligand, thereby completing the modification.
  • the modification is performed in vivo by co-administration with a PD-1 antibody.
  • the modification is performed in vitro, such as by adding PD-1 antibody during the production of PD-1 + T cells.
  • the PD-1 antibody is preferably a PD-1 monoclonal antibody or a single-chain antibody.
  • the modified PD-1 + T cell has an enhancer receptor (ER) comprising: an extracellular domain (ECD) and an intracellular domain (ICD), wherein
  • the ICD comprises a costimulatory molecule that elicits an immune cell activation signal
  • the ECD comprises a moiety that specifically binds to a target cell of the immune cell.
  • the moiety that specifically binds to a target cell of an immune cell is selected from receptors, ligands and antibodies of membrane proteins of the target cell, or a portion or fragment thereof having a binding function to the target cell.
  • the ECD comprises a partial sequence of PD1 or an anti-PD-L1 antibody, preferably an anti-PD-L1 scFv, and/or the ICD is derived from CD28.
  • the modified immune cells are targeted to target cells in the subject.
  • the target cells are tumor cells, particularly cancer cells.
  • the target cell is a host cell infected with a virus, such as a hepatitis virus, preferably a hepatitis B virus, a hepatitis C virus, a hepatitis D virus, or a human papilloma virus (HPV).
  • a virus such as a hepatitis virus, preferably a hepatitis B virus, a hepatitis C virus, a hepatitis D virus, or a human papilloma virus (HPV).
  • the target cells of the modified immune cells are selected from one or more of the group consisting of tumor cells, cancer cells, virus-infected cells.
  • the modified PD-1 + T cell expresses a chimeric antigen receptor (CAR) that specifically recognizes another species different from that recognized by the immune cell's native TCR antigen.
  • CAR chimeric antigen receptor
  • the further antigen is CD19.
  • the modified PD-1 + T cells comprise other modifications, such as suicide switches.
  • the application provides a cell population comprising the modified immune cells of the first aspect.
  • the present application provides a method for preparing modified immune cells, the method comprising: (a) sorting PD-1 + T cells from peripheral blood; and (b) dividing the step (a) into Selected PD-1 + T cells are treated with one or more of the following: i. Knock out or knock down PD-1 expression; ii. Mix with PD-1 antibody; iii. Make it express enhancer receptor (ER) iv. Make it express a chimeric antigen receptor (CAR); v. Make it express modifications that control cell death, such as a suicide switch.
  • the treatment of step (b) includes at least causing expression of an enhancer receptor (ER).
  • the processing of the step (b) includes making it express a chimeric antigen receptor (CAR).
  • the treatment of step (b) comprises knocking down or knocking down the expression of PD-1 and/or mixing with PD-1 antibody.
  • the processing of step (b) includes causing it to express a suicide switch.
  • the treatment can be performed in vivo or in vitro.
  • the treatment can be performed before, after, or concurrently with the reinfusion of the modified immune cells to the subject.
  • the sorting of step (a) of the method comprises PD-1 + cell sorting and CD3 + cell sorting in any order from peripheral blood mononuclear cells.
  • the treatment comprises at least i. knocking out or knocking down the expression of PD-1.
  • the application relates to modified immune cells prepared by the method of the third aspect.
  • the present application relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the modified immune cells of the first aspect or the fourth aspect, or the cell population of the second aspect.
  • the application relates to the use of the immune cells of the first aspect or the fourth aspect or the cell population of the second aspect in therapy, or in the manufacture of a medicament.
  • the treatment or medicament is for (1) treating a tumor, and/or (2) treating or preventing a disease or symptom associated with a viral infection, or preventing the recurrence of a disease or symptom associated with a viral infection.
  • the viral infection is a chronic viral infection.
  • the virus is a hepatitis virus, preferably a hepatitis B virus.
  • the virus is human papillomavirus.
  • the immune cells of the present invention can be used to remove residual viral components after treatment in a subject and prevent the recurrence of viral infection.
  • FIG. 1 is a photograph showing the tumor-bearing condition of PDX mice in Example 1.
  • FIG. 1 is a photograph showing the tumor-bearing condition of PDX mice in Example 1.
  • FIG. 2 is a photograph showing tumor-bearing mice dissected to isolate tumor tissue in Example 1.
  • FIG. 2 is a photograph showing tumor-bearing mice dissected to isolate tumor tissue in Example 1.
  • Figures 3A-B show the results of an ELISPOT assay to detect IFN ⁇ secretion using PD-1 + T cells or PD-1 ⁇ T cells from patient peripheral blood co-cultured with patient tumor tissue, showing two T cells Differences in tumor recognition and responsiveness;
  • A ELISPOT staining photos and ELISPOT statistical results after 24 hours of co-culture;
  • B ELISPOT staining photos and ELISPOT statistical results after 48 hours of co-culture.
  • Figures 4A-E show the results of in vitro experiments examining the effect of PD-1 knockdown on PD-1 + T cell function.
  • A The sorting efficiency of PD-1 was detected by flow cytometry;
  • B the expression of TCR was detected by flow cytometry, and cells transfected with virus not expressing TCR were used as control (PD-1 + NOTD);
  • PD-1 knockout effect was detected by flow cytometry in PD-1 + T cells expressing and not expressing TCR, both cells were used as control without treatment and cells subjected to empty electric shock;
  • DE ELISA experiment was used to detect the relationship between T cells and J82 - Secretion of IFN ⁇ (D) and IL-2 (E) by T cells after co-culture with NY tumor cells.
  • Figures 5A-D show the results of in vitro experiments examining the effect of enhancing receptors on PD-1 + T cell function.
  • A The sorting efficiency of PD-1 was detected by flow cytometry;
  • BC ELISA assay detected the IFN ⁇ (B) and IL- 2 (C) secretion;
  • D ELISA assay to detect IFN ⁇ secretion in 96-well plates coated with OKT3 and/or PD-L1 protein by T cells expressing and not expressing the enhancer receptor.
  • Figures 6A-F show the results of the in vivo experiments in Example 4 in mice inoculated with J82-NY-ESO1 tumor cells, demonstrating the effect of enhanced receptors on the tumor suppressor function of PD-1 + T cells.
  • A PBS control;
  • B PD-1 + NOTD;
  • C PD-1 - TCR-T cells;
  • D PD-1 + TCR-T cells;
  • E PD-1 + -V1E-TCR - T cells;
  • Integral plot of group means means.
  • Figures 7A-C show the results of the in vivo experiments in Example 4 in mice inoculated with tumor tissue from colon cancer patients, demonstrating the effect of enhancing receptors on the tumor suppressor function of PD-1 + T cells.
  • A PBS control;
  • B PD-1-T cells;
  • C PD-1 + T cells;
  • D PD-1 + -V2E-T cells.
  • FIG. 8 shows liver function and HBV-related indicators of patients (A-B) and control subjects (C) in Example 6.
  • Figure 9 shows the biological process of HBV in host hepatocytes.
  • FIG. 10 shows cytotoxic T lymphocyte (CTL) challenge and target cell immune escape.
  • CTL cytotoxic T lymphocyte
  • Figure 11 shows that ScTIL clears HBV residual components (excluding active virus) in hepatocytes.
  • Figure 12 is a plasmid map of the lentiviral vector construct pLenti-TCR-NY ESO1 used to introduce the TCR recognizing NY-ESO-1.
  • Figure 13 is a plasmid map of the lentiviral vector construct pLenti-V1E-T2A-TCR used to introduce the enhancer receptor V1E.
  • Figure 14 is a plasmid map of the lentiviral vector construct pLenti-V2E-T2A-TCR used to introduce the enhancer receptor V2E.
  • Figure 15 is a schematic diagram of the structure of the CD19 CAR used as an amplification factor.
  • Example 16 is the imaging examination results of patients 1-4 in Example 5 before and after treatment.
  • the term “about” means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or greater than 1 standard deviation, according to the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of the given value. Alternatively, particularly for biological systems or processes, the term may represent an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a numerical value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean within an acceptable error range for the particular value.
  • Genomic DNA refers herein to a nucleic acid (eg, DNA, eg, genomic DNA and cDNA) and its corresponding nucleotide sequence encoding an RNA transcript.
  • Genomic DNA herein includes intervening non-coding regions as well as regulatory regions, and may include 5' and 3' ends.
  • the term includes transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns.
  • the transcribed region will contain an "open reading frame" encoding the polypeptide.
  • a “gene” comprises only the coding sequences necessary to encode a polypeptide, such as an "open reading frame” or “coding region.” In some cases, the genes do not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene” or a native gene in its natural location in the genome of an organism. A gene may refer to a "foreign gene” or a non-native gene.
  • rRNA ribosomal RNA genes
  • tRNA transfer RNA
  • a non-native gene can refer to a gene not normally found in the host organism but introduced into the host organism by gene transfer.
  • a non-native gene can also refer to a gene that is not in its natural location in the genome of an organism.
  • a non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that contains mutations, insertions and/or deletions (eg, non-native sequences).
  • nucleotide generally refers to a base-sugar-phosphate combination. Nucleotides can include analogs or derivatives of nucleotides as well as synthetic nucleotides. Nucleotides can be labeled for detection by known techniques. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels.
  • polynucleotide refers to a polymeric form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof, which Can be in single, double or multi-strand form.
  • the polynucleotide can be exogenous or endogenous to the cell.
  • a polynucleotide can be a gene or a fragment thereof.
  • Polynucleotides can be DNA or RNA.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • a polynucleotide may contain one or more analogs (eg, altered backbones, sugars or nucleobases).
  • expression refers to one or more processes by which polynucleotides are transcribed from a DNA template (eg, into mRNA or other RNA transcripts) and/or the transcribed mRNA is then translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.
  • Up-regulation or “down-regulation” of expression generally refers to an increase or decrease in the level of expression of a polynucleotide (eg, RNA, eg, mRNA) and/or polypeptide sequence relative to its level of expression in the wild-type state.
  • a polynucleotide eg, RNA, eg, mRNA
  • polypeptide sequence relative to its level of expression in the wild-type state.
  • modulate in reference to expression or activity refers to altering the level of expression or activity. Regulation can occur at the transcriptional and/or translational level.
  • peptide refers to a polymer of at least two amino acid residues linked by peptide bonds.
  • peptide refers to a polymer of at least two amino acid residues linked by peptide bonds.
  • peptide refers to a polymer of at least two amino acid residues linked by peptide bonds.
  • the term does not imply a specific length of polymer, nor does it imply that the polymer is natural, modified or synthetic. In some cases, polymers can be interrupted by non-amino acids.
  • the term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and/or tertiary structure (eg, domains).
  • amino acid polymers that have been modified, eg, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation, eg, conjugation to labeling components.
  • amino acid as used herein generally refers to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs.
  • amino acid includes D-amino acids and L-amino acids.
  • fusion can refer to proteins and/or nucleic acids comprising one or more non-native sequences (eg, portions). Fusions may comprise one or more identical or different non-native sequences. Fusions can be chimeras. Fusions may comprise nucleic acid affinity tags, barcodes or peptide affinity tags. Fusions can provide a signal that localizes the polypeptide to a subcellular site, e.g., a nuclear localization signal (NLS) for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, and Plasma reticulum (ER) retention signal, etc. Fusions can provide non-native sequences (eg, affinity tags) that can be used for tracking or purification. Fusions can contain small molecules such as biotin or dyes such as Alexa fluorine dyes, Cyanine3 dyes, Cyanine5 dyes.
  • an antigen refers to a molecule or fragment thereof capable of being bound by a selective binding agent.
  • an antigen can be a ligand that can be bound by a selective binding agent such as a receptor.
  • an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immunological protein (eg, an antibody).
  • Antigen can also refer to a molecule or fragment thereof that can be used in an animal to generate antibodies capable of binding the antigen.
  • antibody refers to a protein-binding molecule with immunoglobulin-like functions.
  • the term antibody includes antibodies (eg, monoclonal and polyclonal antibodies) and derivatives, variants and fragments thereof.
  • Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgGl, IgG2, etc.).
  • Ig immunoglobulins
  • Derivatives, variants or fragments thereof may refer to functional derivatives, variants or fragments which retain the binding specificity (eg whole and/or part) of the corresponding antibody.
  • Antigen-binding fragments include Fab, Fab', F(ab') 2 , variable fragments (Fv), single chain variable fragments (scFv), minibodies, diabodies and single domain antibodies ("sdAb” or “nanobodies” ” or “camel antibody”).
  • the term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR is the basis of T cell-mediated antigen recognition and is a key link in the immune system. TCRs are highly diverse, and the relationship between this diversity and disease is a research hotspot in the field of immunology.
  • the collection of TCRs that T cells contain under a certain condition or at a certain time point can be called a TCR repertoire or TCR profile, which may vary greatly with the onset and progression of disease .
  • subject preferably a mammal, such as a human.
  • Mammals include, but are not limited to, rodents, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • treatment refers to a method for obtaining beneficial or desired results, including but not limited to therapeutic benefit and/or prophylactic benefit.
  • treatment can include administration of a modified immune cell of the invention or a population of cells comprising the immune cell.
  • Therapeutic benefit refers to any treatment-related improvement in one or more diseases or symptoms being treated.
  • the modified immune cells of the present invention can be administered to subjects at risk of developing a particular disease or symptom, or to a subject with one or more physiological signs of a disease, even though the disease or symptom may not already be present show.
  • an effective amount refers to an amount of a composition, such as an amount of a composition comprising a modified immune cell of the present invention, such as lymphocytes (eg, T lymphocytes and/or NK cells), at This amount is sufficient to produce the desired activity when administered to a subject in need thereof.
  • a modified immune cell of the present invention such as lymphocytes (eg, T lymphocytes and/or NK cells)
  • the immune cells of the present invention are derived from peripheral blood, such as peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subsets, including but not limited to T cells, natural killer cells, monocytes, monocytes Cell precursor cells, hematopoietic stem cells or non-pluripotent stem cells.
  • PBMC peripheral blood mononuclear cells
  • PBL peripheral blood lymphocytes
  • other blood cell subsets including but not limited to T cells, natural killer cells, monocytes, monocytes Cell precursor cells, hematopoietic stem cells or non-pluripotent stem cells.
  • an immune cell of the invention can be any immune cell, including any T cell, such as tumor infiltrating cells (TILs).
  • TILs tumor infiltrating cells
  • the immune cells can be from the subject (eg, a patient) to be treated.
  • the subject can be a mammal, such as a mouse, monkey or human.
  • the immune cells are isolated from
  • T cells can be obtained from blood collected from a subject using any technique, such as Ficoll separation.
  • Cells from the subject's circulating blood can be obtained by apheresis or leukocytosis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated white blood cells, as well as red blood cells and platelets.
  • Cells collected by apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium, such as phosphate buffered saline (PBS), for subsequent processing steps. After washing, cells can be resuspended in various biocompatible buffers, such as PBS without Ca, Mg.
  • PBS phosphate buffered saline
  • unwanted components of the apheresis sample can be removed and the cells resuspended directly in culture medium.
  • the sample may be provided directly by the subject, or indirectly through one or more intermediaries, such as a sample collection service provider or a medical provider (eg, a doctor or nurse).
  • isolating T cells from peripheral blood leukocytes can include lysing red blood cells and separating peripheral blood leukocytes from monocytes by, eg, PERCOL TM gradient centrifugation.
  • immune cells such as T cells can be further isolated by positive or negative selection techniques, eg, the immune cells of the present invention are PD-1 positive T cell subsets.
  • PD-1 positive cells likely represent a cell population that has undergone interaction with the target cell, and thus has the ability to identify the target cell.
  • immune cells obtained from peripheral blood, such as PBMCs are sorted with PD-1 as a marker.
  • PD-1 + T cells can be sorted by magnetic sorting (eg, using magnetic beads) or flow cytometry using PD-1 antibodies, preferably using magnetic beads.
  • the present invention considers that the sorted PD-1-positive T cell subsets are T cell subsets that have already interacted with the harmful cells to be targeted, so the T cells contained therein have the ability to specifically recognize harmful cells.
  • the TCR repertoire or TCR profile of the antigen In order to enable T lymphocytes used in cell therapy to target and recognize harmful cells such as tumor cells, one approach in the prior art is to obtain TCRs that can target harmful cells such as tumor cells by screening TCR libraries and obtain their genetic information , whereby patient-derived T cells are genetically engineered to give them the ability to recognize antigens on harmful cells.
  • the present invention can not perform TCR screening, nor additionally introduce exogenous TCR into T cells used for treatment, but directly select autologous cells from patients.
  • Those T cells that have a high probability of having TCRs specific for harmful cells themselves ensure the targeting and safety of cells used for treatment.
  • the present invention does not require T cells to possess one or more specific TCRs, as the TCR repertoire will vary widely among patients and their disease states.
  • the present invention only requires the existence of discrimination between the T cell population and harmful cells, and this discrimination is mainly obtained by sorting PD-1 positive T cells in the present invention.
  • the methodology of the present invention can be used to target a variety of deleterious cells and treat a variety of diseases.
  • “harmful cells” refer to cells that are targeted and attacked by immune cells, in particular tumor cells, cancer cells, virus-infected cells.
  • the T cells of the present invention may also be CD3 positive, CD4 positive, CD4 negative or CD8 positive T cell subsets.
  • Subpopulations of cells with or without a particular marker can be sorted using any technique in the art. For example, sorting can be performed by matrices such as magnetic beads coupled with antibodies to the relevant markers, such as CD3 + T cells by CD3 magnetic beads or CD3/CD28 magnetic beads. Maximal contact with the substrate surface, eg, the magnetic bead surface, can be facilitated by varying the cell concentration. In addition, negative selection of cell populations can also be achieved with combinations of antibodies directed against unwanted cell-specific surface markers.
  • the monoclonal antibody cocktail can include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • the immune cells are cells in an enriched population of cells.
  • One or more desired cell types can be enriched by any suitable method, for example, by treating the cell population to trigger expansion and/or differentiation into desired cell types, or to prevent the growth of unwanted cell types, Either kill or lyse the unwanted cell type, or purify the desired cell type (eg, on an affinity column to retain the desired or unwanted cell type based on one or more cell surface markers).
  • PD-1 + T cells are depleted, "passivated”, and diminished in potency and function as a result of their interactions with target cells.
  • the present invention restores and improves the efficacy of PD-1 + T cells by modifying them.
  • the PD-1 expression of the modified immune cells of the present invention is knocked out or knocked down.
  • knock-out of gene expression herein refers to substantially eliminating the expression of the gene.
  • knock-down of gene expression herein refers to a reduction in the expression of that gene.
  • the modified immune cells of the invention are knocked out of endogenous PD-1 expression.
  • Knockdown or knockdown of PD-1 can be achieved using any method known in the art, including but not limited to: RNAi (including the use of siRNA or shRNA), CRISPR-Cas methods, transcription activator-like effector nucleic acids Enzyme (TALEN) technology, site-directed mutagenesis, zinc finger nuclease (ZFN) technology, or a combination thereof.
  • RNAi including the use of siRNA or shRNA
  • CRISPR-Cas methods CRISPR-Cas methods
  • transcription activator-like effector nucleic acids Enzyme (TALEN) technology transcription activator-like effector nucleic acids Enzyme
  • ZFN zinc finger nuclease
  • a CRISPR/Cas approach is used to knock out endogenous PD-1 in immune cells, the method comprising using a guide RNA (gRNA) capable of hybridizing to the target gene PD-1 and a Cas protein or its encoding nucleic acid molecular.
  • gRNA guide RNA
  • an sgRNA targeting PD-1 is used to knock out endogenous PD-1 in immune cells, preferably the sgRNA has a nucleotide sequence as set forth in SEQ ID NO:4.
  • the Cas9 protein or its encoding nucleic acid molecule is used to knock down PD-1.
  • the Cas9 protein can be derived from Streptococcus pyogenes, Streptococcus thermophilus, or other species.
  • Cas9 can include spCas9, Cpfl, CasY, CasX or saCas9.
  • the modified immune cells of the invention comprise enhancer receptors (ERs).
  • ERs enhancer receptors
  • Enhanced receptors can be used to provide further control over immune cell activity, such as, but not limited to, immune cell activation and expansion.
  • Enhanced receptor-ligand binding can generate immune cell activation signals rather than immune cell inactivation signals in modified immune cells.
  • Initiating immune cell activation signals rather than immune cell inactivation signals in modified immune cells can minimize immunosuppressive effects in immune cells. Minimizing immunosuppressive effects in immune cells can increase the effectiveness of immune cells in an immune response, eg, by increasing immune cell cytotoxicity against target cells (eg, tumor cells or infected cells).
  • the enhancer receptor may comprise the extracellular domain (ECD) of the protein.
  • the protein may be a signaling receptor or any functional fragment, derivative or variant thereof.
  • the signaling receptor can be a membrane-bound receptor.
  • a signaling receptor can induce one or more signaling pathways in a cell.
  • the signaling receptor can be a non-membrane-bound receptor.
  • Enhancer receptors may comprise fragments (eg, extracellular domains) of receptors selected from the group consisting of receptors for G protein-coupled receptors (GPCRs), integrin receptors, cadherin receptors, catalytic receptors (eg, kinases) ), death receptors, checkpoint receptors, cytokine receptors, chemokine receptors, growth factor receptors, hormone receptors and fragments of immune receptors such as extracellular domains.
  • GPCRs G protein-coupled receptors
  • integrin receptors e.g, integrin receptors
  • cadherin receptors eg, kinases
  • catalytic receptors eg, kinases
  • the enhancer receptors comprise fragments of immune checkpoint receptors, which can be involved in the regulation of the immune system.
  • immune checkpoint receptors include, but are not limited to, PD-1, CTLA-4, BTLA), KIR, IDO, LAG3, TIM-3, TIGIT, SIRPa, NKG2D, preferably PD-1.
  • Enhancer receptors may comprise fragments that bind any suitable immune checkpoint receptor ligand.
  • suitable immune checkpoint receptor ligands include, but are not limited to, B7-1, B7-H3, B7-H4, HVEM (Herpesvirus Entry Mediator), AP2M1, CD80, CD86, SHP-2, PPP2R5A, MHC (e.g., I class, class II), CD47, CD70, PD-L1 (or PDL1), and PD-L2.
  • the region that enhances the binding of receptors to such ligands can be either natural receptors for such ligands or monoclonal antibodies to such ligands.
  • Enhancer receptors also comprise an intracellular domain (ICD), which can be a costimulatory molecule or a fragment, variant, derivative thereof that elicits immune cell activation signals.
  • costimulatory molecules can be used to modulate proliferation and/or survival signals in immune cells.
  • the ICD is the intracellular domain of a costimulatory molecule selected from the group consisting of MHC class I proteins, MHC class II proteins, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, Integrins, SLAM proteins, activated NK cell receptors, BTLA or Toll ligand receptors.
  • the costimulatory molecule is a T cell costimulatory molecule, preferably a T cell positive costimulatory molecule, preferably CD28.
  • the ECD and ICD of the enhancer receptor can be linked by a transmembrane domain.
  • the transmembrane domain comprises a polypeptide.
  • a transmembrane polypeptide can have any suitable polypeptide sequence.
  • the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein or a variant thereof having at least one amino acid change.
  • the transmembrane polypeptide comprises a non-native polypeptide sequence, eg, a sequence of a polypeptide linker.
  • Polypeptide linkers can be flexible or rigid. Polypeptide linkers can be structured or unstructured.
  • the transmembrane polypeptide transmits a signal from the ECD to the ICD, eg, a signal indicative of ligand binding.
  • the ECD comprises a transmembrane domain.
  • the ICD comprises a transmembrane domain.
  • the means of transfecting the enhancer receptor gene can be electrotransfected into PD-1 + T cells with the enhancer receptor mRNA, or by lentivirus, adeno-associated virus, or non-viral vector, the enhancer receptor gene can be transfected into PD-1 + T cells in cells.
  • the enhancing receptor is a transmembrane protein expressed on the surface of immune cells, and consists of an extracellular domain (ECD) and an intracellular domain (ICD), wherein the ICD contains a costimulatory molecule that triggers immune cell activation signals, Can trigger immune cell activation signal, ECD can bind to the target cell of the immune cell, is the receptor, ligand, antibody of the membrane protein of the target cell of the PD-1 + T cell, or any target cell binding The complete sequence structure of a substance or a portion containing its binding domain.
  • ECD extracellular domain
  • ICD intracellular domain
  • the enhancing receptor is V1E or V2E
  • the ECD comprises a fragment of PD-1 (eg, a fragment of the extracellular domain of PD-1) or the scFv sequence of an anti-PD-L1 monoclonal antibody, respectively
  • the ICD of the enhancer receptor comprises the intracellular signaling domain of CD28. More preferably, the enhancing receptor comprises the transmembrane region of CD28 or the transmembrane region of CD8.
  • the enhancer receptor comprises a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, or has at least 75%, 80%, 85%, 90%, 95%, Sequences that are 97% or 99% homologous, or consist of such sequences.
  • the modified immune cells of the invention comprise a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • immune cells modified with enhanced receptors such as T cells, have stronger immune cell activation function than cells without enhanced receptor modification.
  • CARs can aid in the expansion of modified immune cells.
  • the CAR specifically recognizes another antigen that is different from the natural TCR of the immune cell.
  • the CAR comprises an antigen interacting domain (antigen binding domain), a transmembrane domain and an intracellular signaling domain capable of binding to a B cell surface protein.
  • the B cell surface protein can be any protein that can be found on the surface of B cells, preferably CD19.
  • the antigen-interacting domain of the CAR is capable of binding to surface proteins on non-B cells, so long as the binding to the surface proteins does not significantly impair the general health or immune system of the host.
  • antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies or functional derivatives, variants or fragments thereof, including but not limited to Fab , Fab', F(ab')2, Fv, single chain Fv (scFv), minibodies, diabodies and single domain antibodies such as the heavy chain variable domains (VH) of camelid-derived Nanobodies, light Chain variable domain (VL) and variable domain (VHH).
  • the antigen binding domain comprises at least one of Fab, Fab', F(ab')2, Fv and scFv, preferably scFv.
  • the antigen binding domain comprises an antibody mimetic.
  • An antibody mimetic refers to a molecule capable of binding a target molecule with an affinity comparable to that of an antibody.
  • the antigen binding domain comprises a transmembrane receptor or any derivative, variant or fragment thereof.
  • the antigen binding domain may comprise at least the ligand binding domain of a transmembrane receptor.
  • the antigen-interacting domain of the CAR is capable of binding a surface protein or fragment thereof of a B cell coupled (eg, by covalent and/or non-covalent bonds) to a particle (eg, a nanoparticle).
  • the particles can be any particulate material comprising organic and/or inorganic materials. Particles can have various shapes and sizes.
  • the particles can be about 1 nanometer (nm) to about 50 micrometers ( ⁇ m) in at least one dimension.
  • the particles can be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 ⁇ m, 5 ⁇ m, 10 ⁇ m, 50 ⁇ m or larger in at least one dimension.
  • the particles may be up to about 50 ⁇ m, 10 ⁇ m, 5 ⁇ m, 1 ⁇ m, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or less in at least one dimension.
  • the particles can be nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanoribbons, and the like.
  • particles examples include metal nanoparticles (eg, gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic nanosemiconductor nanoparticles, core-shell nanoparticles, particles with an inorganic core with a polymer shell, particles with a polymer shell particles of organic nuclei and their mixtures.
  • metal nanoparticles eg, gold nanoparticles, silver nanoparticles, and iron nanoparticles
  • intermetallic nanosemiconductor nanoparticles eg., gold nanoparticles, silver nanoparticles, and iron nanoparticles
  • core-shell nanoparticles examples include particles with an inorganic core with a polymer shell particles with a polymer shell particles of organic nuclei and their mixtures.
  • the particles may be organic nanoparticles such as cross-linked polymers, hydrogel polymers, biodegradable polymers, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL) , copolymers, polysaccharides, starch, cellulose, chitosan, polyhydroxyalkanoates (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (eg, proteins), or combinations thereof.
  • Particles that present B cell surface proteins on their surface can be introduced in vitro into immune cells containing CARs that bind B cell surface proteins.
  • particles presenting B cell surface proteins can be introduced in vivo (eg, by local or systemic injection) with the CAR-containing immune cells. These particles can be used to expand CAR-containing immune cell populations in vitro or in vivo.
  • the antigen-interacting domain of the CAR is capable of binding B cell surface proteins or fragments thereof on dead B cells.
  • B cell apoptosis can occur before or after the development of an immune response.
  • dead B cells or their fragments can still present B cell surface proteins or fragments thereof on the surface.
  • the ability of a CAR to target live and dead B cells can increase the probability that modified immune cells comprising the CAR bind to B cell surface proteins and initiate signaling from intracellular signaling domains.
  • signaling by the intracellular signaling domain can promote the expansion (proliferation) of immune cells comprising the CAR.
  • the intracellular signaling domain of CAR can induce the activity of modified immune cells. Intracellular signaling domains can transduce effector function signals and direct cells to perform specialized functions.
  • a signaling domain may comprise signaling domains of other molecules. In some cases, a truncated portion of the signaling domain is used for the CAR.
  • An intracellular signaling domain comprises a plurality of signaling domains involved in immune cell signaling, or any derivative, variant or fragment thereof.
  • the intracellular signaling domain of a CAR can include a costimulatory domain, eg, from a costimulatory molecule.
  • the CAR of the present application comprises the sequence of SEQ ID NO: 3 or has at least 75%, 80%, 85%, 90%, 95%, 97% or 99% homology to the sequence sequence, or consists of such a sequence.
  • modified immune cells can enhance immune cell proliferation through the binding of CAR to B cell surface proteins compared to immune cells lacking CAR.
  • Proliferation of immune cells can refer to expansion or phenotypic changes of immune cells.
  • Modified immune cells of the invention comprising a CAR can bind to B cell surface proteins and can proliferate from about 5 times to about 10 times, from about 10 times to about 50 times, about 50 times the proliferation of corresponding immune cells lacking the CAR to about 100 times, about 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, about 600 times to about about 700 times.
  • Proliferation can be determined at least about 12, 24, 36, 48, 60, 72, 84, or 96 hours after contacting the B cells with the modified immune cells comprising the CAR.
  • Proliferation can be determined in vitro or in vivo, eg, by measuring the number of immune cells. Methods for determining the number of immune cells may include flow cytometry, trypan blue exclusion, and/or blood cell counting. Proliferation can also be determined by phenotypic analysis of immune cells.
  • the various domains of the enhancing receptors and CARs provided herein can be linked by chemical bonds, such as amide or disulfide bonds; small organic molecules (eg, hydrocarbon chains); amino acid sequences, such as peptide linkers (eg, about 3-200 in length) amino acid sequence of amino acids), or a combination thereof.
  • Peptide linkers can provide the flexibility needed to enable proper expression, activity and/or conformational positioning of the chimeric polypeptide.
  • the peptide linker can be of any suitable length to link the at least two domains of interest, and is preferably designed to be flexible enough to allow one or both domains to which it is linked to fold properly and/or function and/or be active.
  • the peptide linker is about 0 to 200 amino acids, about 10 to 190 amino acids, about 20 to 180 amino acids, about 30 to 170 amino acids, about 40 to 160 amino acids, about 50 to 150 amino acids in length amino acids, about 60 to 140 amino acids, about 70 to 130 amino acids, about 80 to 120 amino acids, about 90 to 110 amino acids.
  • the linker sequence may comprise an endogenous protein sequence.
  • the linker sequence comprises glycine, alanine and/or serine amino acid residues.
  • the linker may have a GS, GGS, GGGGS, GGSG, or SGGG motif and contain multiple such motifs, eg, multiple repetitions of the same motif.
  • the linker sequence can include any naturally occurring amino acid, non-naturally occurring amino acid, or a combination thereof.
  • the modified immune cells of the invention further comprise modifications that regulate immune cell death, such as suicide switches.
  • a suicide switch can be activated to eliminate immune cells.
  • the suicide switch can be a drug-induced suicide switch.
  • the suicide switch may comprise inducible caspase 9.
  • the modified immune cells of the invention do not comprise exogenously introduced TCRs.
  • Modification can be accomplished by introducing desired compositions and molecules, such as polypeptides and/or nucleic acids, such as nucleic acids encoding polypeptides, into immune cells using any suitable delivery method.
  • the various components can be delivered simultaneously or separately.
  • the delivery method may comprise introducing into immune cells one or more nucleic acids comprising a nucleotide sequence encoding a composition of the invention, eg, it may be an expression vector comprising a nucleotide sequence encoding a product of interest, such as recombinant expression vector.
  • Any suitable vector compatible with the host cell can be used in the present invention.
  • Non-limiting examples of delivery or transformation methods include, but are not limited to, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) mediated Transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.
  • PKI polyethyleneimine
  • Non-viral vector delivery systems can include DNA plasmids, RNA (eg, transcripts), naked nucleic acids, and nucleic acids such as liposomes complexed with delivery vehicles.
  • Viral vector delivery systems may include DNA and RNA viruses, which may or may not integrate with the cell's genome after delivery to the cell.
  • RNA or DNA virus-based systems can be used to target specific cells in the body and transport the viral load to the nucleus.
  • Viral vectors can be administered directly (in vivo), or used to treat cells in vitro, and can optionally be administered to cells (ex vivo).
  • Virus-based systems can include retroviral, lentiviral, adenovirus, adeno-associated virus, and herpes simplex virus vectors for gene transfer. Integration into the host genome can be performed using retroviral, lentiviral and adeno-associated virus gene transfer methods, which can result in long-term expression of the inserted transgene. High transduction efficiencies can be observed in many different cell types and target tissues.
  • the present invention uses lentiviral vectors.
  • the invention provides methods of modifying immune cells comprising adding one or more polynucleotides, or one or more vectors as described herein, or one or more transcripts thereof, and/or Methods of delivering one or more proteins transcribed therefrom to immune cells.
  • the present disclosure also provides modified immune cells produced by these methods, and comprising these immune cells.
  • the modified immune cells of the present invention can target and act on a variety of target cells in vitro, in vivo or ex vivo.
  • Target cells can be isolated cells.
  • the target cells can be cells in an organism.
  • the target cell can be an organism.
  • the target cells can be cells in cell culture.
  • the target cells may be mammalian cells or derived from mammalian cells, which may be rodents, primates such as humans.
  • the target cells may be or may be derived from prokaryotic cells, such as bacterial cells, archaeal cells, or may be or derived from eukaryotic cells.
  • a target cell can be a cell infected with a pathogen, which can be a microorganism, including, but not limited to, bacteria, fungi, or viruses.
  • the target cell may be a host cell, such as a host cell of the pathogen.
  • the target cell is, for example, a cell infected with a hepatitis virus, preferably a hepatitis B virus, or a cell infected with a human papilloma virus.
  • Target cells can be derived from a specific organ or tissue.
  • the target cell can be a unicellular organism.
  • Target cells can be stem cells or progenitor cells, including but not limited to adult stem cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), and progenitor cells such as cardiac progenitor cells, neural progenitor cells, and the like.
  • the target cells can be pluripotent stem cells.
  • Target cells can be genetically modified cells.
  • the target cell can contain the target nucleic acid.
  • the target cells can be diseased cells, such as cells from a diseased subject.
  • Diseased cells can have altered metabolism, gene expression and/or morphological characteristics.
  • a diseased cell can be a cancer cell or a virus-infected cell.
  • the target cells are tumor cells, especially cancer cells.
  • the tumors include solid tumors and hematological tumors, preferably solid tumors.
  • the modified immune cells of the present invention can be used to treat tumors, in other words to target tumor cells.
  • the target cells form tumors.
  • Treatment with the modified immune cells of the present invention results in stable, non-progressive and/or non-metastatic tumor growth.
  • stable growth means that one or more tumors do not increase in volume by more than 1%, 5%, 10%, 15%, or 20% for a period of time following treatment.
  • the period of time is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks, preferably at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, more preferably at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years .
  • treatment with the modified immune cells of the present invention reduces the size of a tumor or the number of tumor cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
  • the tumor is completely eliminated, or reduced to below detection levels.
  • the subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks after treatment, preferably at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, more preferably at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
  • the amount of modified immune cells of the invention administered to the subject is between 10 4 and 10 10 , between 10 4 and 10 9 , between 10 5 and 10 8 , and between 10 6 and 10 . Between 10 and 7 .
  • the PD-1 + T cells of the present invention contain expansion factor modifications such as CD19 CAR, the total amount of reinfusion can be appropriately lower than the PD-1 + T cells of the present invention that do not contain the modification, because the CD19 CAR It is helpful for the expansion of the reinfused immune cells in the patient, and this effect is described in detail in Example 5.
  • the total amount of reinfused cells can be between 10 4 to 10 9 , 10 5 to 10 8 , 10 6 to 10 between 7 .
  • the modified immune cells of the present invention can be reinfused to the subject in one or more times, such as one, two, three, or four times.
  • the amount of immune cells reinfused each time can be the same or different.
  • the modified immune cells of the present invention can be used to treat diseases or conditions associated with viral infection, in other words targeting cells infected with the virus.
  • the immune cells of the present invention can clear virus in a patient while treating cancer in a patient, eg, residual virus after antiviral therapy.
  • the modified immune cells and methods of the present invention are particularly suitable for patients known to have been infected with the virus.
  • the viral infection may or may not be associated with cancer.
  • the immune cells of the invention are specialized for targeting virus-infected cells, treating virus-related diseases, and/or clearing viruses.
  • the viral infection is a hepatitis B virus infection.
  • the viral infection is a chronic hepatitis B virus infection, eg, the liver is infected with the hepatitis B virus for more than six months.
  • Diseases associated with chronic HBV infection include liver cirrhosis and hepatocytoma.
  • the viral infection is a human papillomavirus (HPV) infection. More than 100 human papillomaviruses have been identified so far. According to whether it is related to cancer, it is generally divided into high-risk types (including but not limited to types 16, 18, 31, 45, 52, and 84) and non-carcinogenic low-risk types (such as type 6 causing condyloma acuminatum) , 11, 42, 43, 44). HPV can cause the proliferation of squamous epithelium of human skin and mucous membranes, resulting in diseases of the skin, respiratory tract, oral cavity, digestive tract, eye, anus and genitourinary system.
  • HPV can cause the proliferation of squamous epithelium of human skin and mucous membranes, resulting in diseases of the skin, respiratory tract, oral cavity, digestive tract, eye, anus and genitourinary system.
  • Warts such as common warts, flat warts, condyloma acuminatum, tumors or cancers such as squamous cell carcinoma, cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer.
  • Death of target cells can be determined by any suitable method including, but not limited to, counting cells before and after treatment, or measuring levels of markers associated with live or dead cells.
  • the degree of cell death can be determined by any suitable method. In some embodiments, the degree of cell death is determined relative to starting conditions. For example, an individual may have a known starting amount of target cells, such as a known size of a starting cell mass or a known concentration of circulating target cells. In this case, the degree of cell death can be expressed as the ratio of surviving cells to the starting cell population after treatment.
  • Various cell death assays are available, and a variety of detection methods are available. Examples of detection methods include, but are not limited to, cell staining, microscopy, flow cytometry, cell sorting, and combinations thereof.
  • the efficacy of the treatment in reducing tumor size can be determined by measuring the percentage of resected tissue that is necrotic (ie, dead). In some embodiments, the treatment is effective if the percentage of necrosis of the resected tissue is greater than about 20%, eg, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the percent necrosis of the resected tissue is 100%, ie, no viable tumor tissue is present or detectable.
  • Exposure of target cells to the modified immune cells of the present invention can be performed in vitro or in vivo. Exposing a target cell to a modified immune cell generally refers to bringing the target cell into contact with the modified immune cell and/or in sufficient proximity such that the target cell's antigen (eg, membrane-bound or non-membrane-bound antigen) can interact with a receptor expressed on the immune cell. Including enhanced receptor and/or CAR binding.
  • the modified immune cells can be exposed to the target cells in vitro by co-culturing the target cells and the modified immune cells. Co-cultivation can be performed as adherent cells or in suspension.
  • Target cells and modified immune cells can be co-cultured in various suitable types of cell culture media, eg, with supplements, growth factors, ions, and the like.
  • Target cells can be exposed to modified immune cells in vivo, eg, by administering the modified immune cells to a subject, eg, a human, and allowing the modified immune cells to localize to the target cells through the circulatory system.
  • the modified immune cells can be delivered to the region where the target cells are located, eg, by direct injection.
  • the exposure can be for any suitable length of time, such as at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours.
  • hours at least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, At least 2 weeks, at least 3 weeks, at least 1 month or more.
  • the tumor tissue from the same patient was processed to construct a human tumor xenograft (Patient-Derived tumor Xenograft, PDX) mouse model.
  • PDX Human tumor xenograft
  • the tumor samples were cut with a sterilized surgical blade to a size of 3 mm ⁇ 3 mm ⁇ 3 mm, and subcutaneously transplanted into 6-8 week old NPI (genetic background NOD-Prkdcem1Idmo-il2rgem2Idmo) male mice.
  • mice Before transplantation, inject anesthesia into the NPI mice intraperitoneally. After the mice are anesthetized, use sterile ophthalmic scissors to cut an opening of about 1 cm in the outer and upper skin of the right hindlimb of the mouse, and use ophthalmic forceps to push the tumor mass to the outer side of the right hindlimb subcutaneously and then close it. surgical incision.
  • the tumor after tumor formation is the P0 generation
  • the long diameter a and the short diameter b of the tumor are measured with a vernier caliper
  • the tumor grows to a size of 800-1000mm 3
  • the tumor is harvested, and the necrotic tissue is removed, and each piece is cut again according to the size of 3mm ⁇ 3mm ⁇ 3mm, and the above steps are repeated and transplanted into NPI mice subcutaneously for tissue passage.
  • the tumor was marked as the P1 generation (the tumor bearing situation of mice is shown in Figure 1).
  • This step was repeated, and when it was passed to the P3 generation, tumor tissues were extracted from 3 mice respectively for subsequent in vitro functional experiments (the dissected tumor tissues are shown in Figure 2).
  • the tumor samples obtained in each generation can be used for genomics, proteomics and pathological analysis, etc.
  • the frozen samples can be stored and resuscitated for tumor-bearing use when there is a need for subsequent experiments.
  • peripheral blood PD-1 + T cells and PD-1 ⁇ T cells from the same patient were sorted.
  • step 2 Repeat step 2 and wash with normal saline for the second time to obtain cell pellets;
  • step 8 Mix the resuspended cells in step 8 and add them to the LS-type column. After passing through the column, rinse the column with 3ml buffer, wash 3 times, collect the unadsorbed cells, namely PD-1 - PBMC cells, and count them. For subsequent sorting of PD-1 - T cells;
  • the resuspended PD-1 + T cells were added to CD3/CD28 DynaBeads (Gibco, Cat#40203D) for activation, and subcultured.
  • the proportion of T cells in PD-1 - PBMC was detected by CD3 flow cytometry, and the total number of T cells was calculated according to the cell count;
  • T cells According to the number of T cells, according to the ratio of T cells to CD3/CD28 DynaBeads is 1:3, take the corresponding number of CD3/CD28 DynaBeads in a 15ml centrifuge tube, and remove the preservation solution on the magnetic stand;
  • step 3 Repeat step 3 twice, and use 1-5ml medium to resuspend CD3/CD28 DynaBeads for the last time;
  • Tumor tissues were isolated from the 3 P3-generation mice obtained in the above section II, cut into pieces, prepared into single cell suspensions by enzymatic digestion, and named as 1# tumor tissue, 2# tumor tissue, 3# tumor tissue, respectively organize.
  • the PD-1 ⁇ T cells and PD-1 + T cells prepared in Section III above were co-cultured with the three tumor tissue cells to form six cultures, which were cultured at 24 hours and 48 hours, respectively.
  • the ELISPOT experiment was carried out on the substances, and the activation of different T cells was investigated by detecting IFN- ⁇ .
  • the IFN- ⁇ release from PD-1 + T cells was significantly higher than that of PD- 1- T cells, implying that PD-1 + T cells were activated by tumor tissue, while PD- 1- T cells were cannot.
  • the results show that the PD-1 + T cells obtained from the peripheral blood of patients by the above-mentioned sorting method can effectively recognize tumor cells, generate an immune response to them, and initiate killing, indicating that there are certain cells in the peripheral blood of patients that can exert an immune function on tumor cells. T cells (tumor-specific T cells), and these cells are mainly concentrated in the PD-1 + fraction.
  • the experiments in this example were carried out.
  • the inventors constructed an in vitro model using tumor cells characterized by NY-ESO-1 expression and constructed PD-1 + TCR- containing a corresponding TCR capable of binding NY-ESO-1 T cells.
  • the PD-1 gene in T cells was knocked out using CRISPR -Cas9 technology to observe the changes in its effect on tumor cells.
  • CD3/CD28 DynaBeads were then added to the sorted PD-1 + T cells and cultured overnight.
  • T cells were cultured and expanded, and the expression rate of this TCR was 40.1% by flow cytometry (Fig. 4B).
  • the CD3/CD28 DynaBeads were removed on the 4th day after adding the lentivirus, and the PD-1 sgRNA and Cas9 protein were transferred into PD-1 + NOTD-T cells and PD-1 + TCR-T by electroporation on the 5th day. Endogenously expressed PD-1, and the untreated group and the empty shock group were used as controls. As shown in Figure 4C, the expression of PD-1 was detected by flow cytometry on the 7th day after electroporation. The positive proportions of PD-1 in the NOTD-T cell untreated group and the empty electric shock group were 39.6% and 34.8%, respectively.
  • PD-1 knockout The proportion of PD-1 positive after PD-1 was reduced to 3.6%; the proportion of PD-1 positive in TCR-T cell untreated group and empty electric shock group was 40.1% and 37.9%, respectively, and the proportion of PD-1 positive after PD-1 was knocked out decreased to 4.0% , the knockout effect is better, and follow-up experiments can be carried out.
  • ELISA experiments were performed. Different groups of T cells were co-cultured with bladder cancer cell line J82 or J82-NY-ESO1 (J82-NY) tumor cells overexpressing NY-ESO-1 protein for 24 hours, and ELISA was used to detect the secretion of IFN- ⁇ and IL-2. Condition.
  • PD-1 knockdown significantly promoted cytokine secretion from PD-1 + TCR-T cells, after the same transfection of TCR-expressing viral vector and co-culture with J82-NY-ESO1 tumor cells , PD-1 + T cells with PD-1 knockout increased IFN- ⁇ secretion by nearly 50% and IL-2 secretion compared to two controls (untreated control and control with null motor). More than 80%, indicating that knocking out PD-1 can effectively enhance the immune efficacy of PD-1 + T cells.
  • enhancer receptors for T cells used for treatment which contain The extracellular domain of the target cell that binds the immune cells, and the intracellular domain of the T cell costimulatory molecule, as described in the applicant's prior application PCT/CN2020/073017.
  • T cells In order to verify whether the enhanced receptor has an effect on the immune efficacy of PD-1 + T cells obtained by the method of the present invention, the inventors constructed the following three PD-1 + TCRs containing TCR targeting the tumor target NY-ESO-1 as follows T cells:
  • V1E-TCR-T cells containing the enhancer receptor V1E and a TCR targeting NY-ESO-1, where V1E contains a partial sequence of PD1 as an extracellular domain, and contains the transmembrane and cellular transmembrane of CD28. Inner domain, the sequence of V1E is shown in SEQ ID NO:1;
  • V2E-TCR-T cells containing the enhancer receptor V2E and a TCR targeting NY-ESO-1, wherein V2E contains an scFv antibody sequence capable of binding PD-L1 protein as an extracellular domain, and contains The transmembrane and intracellular domains of CD28, the sequence of V2E is shown in SEQ ID NO: 2;
  • PD-1 + NOTD-T cells without TCR targeting NY-ESO-1 and any enhancer receptors were used as controls.
  • the specific construction process is as follows.
  • the patient's leukocytes were aspirated, and PD-1 monoclonal antibody was added to separate PD-1 + T cells.
  • the sorting efficiency is shown in Figure 5A.
  • CD3/CD28 DynaBeads were subsequently added to PD-1 + T cells and cultured overnight.
  • the PD-1 + T cells were divided into 4 groups on the second day, namely:
  • Group 1 NOTD-T, without virus transfection
  • Group 2 TCR-T, adding a lentiviral vector expressing TCR that recognizes NY-ESO-1;
  • V1E-TCR-T adding a lentiviral vector expressing V1E-TCR, the structure of the vector is shown in Figure 13;
  • Group 4 V2E-TCR-T, adding a lentiviral vector expressing V2E-TCR, the structure of the vector is shown in Figure 14.
  • enhanced receptor V1E can significantly promote the secretion of IFN- ⁇ from PD-1 + TCR-T cells; enhanced receptor V1E and V2E can significantly promote the secretion of IL-2 from PD-1 + TCR-T cells , indicating that both enhancer receptors can effectively enhance PD-1 + T cell function.
  • the above four groups of T cells were added to 96-well plates that had been coated with OKT3 (CD3 antibody), PD-L1 protein or OKT3+PD-L1 protein.
  • OKT3 CD3 antibody
  • PD-L1 protein or OKT3+PD-L1 protein.
  • the supernatant was collected, and the secretion of IFN- ⁇ was detected by ELISA.
  • OKT3 could effectively activate T cells to secrete IFN- ⁇ .
  • OKT3 was co-stimulated with PD-L1 protein, the amount of IFN- ⁇ secreted by cells in TCR-T and NOTD groups decreased. This is because PD-L1 protein binds to PD-1 protein on the surface of T cells and inhibits T cells.
  • T cell function by PD-L1 on target cells This also mimics the inhibition of T cell function by PD-L1 on target cells.
  • the function of T cells expressing the enhancer receptor was not only not inhibited by PD-L1 protein, but the T cells expressing the enhancer receptor treated with both OKT3 and PD-L1 had significantly higher relative to treatment with OKT3 alone IFN- ⁇ secretion, indicating that the binding of PD-L1 protein to V1E or V2E enhancing receptors will not only contact the inhibition of PD-L1 itself on T cells, but also significantly promote the secretion of cytokines by T cells.
  • the PD-1 + T cells obtained by the present invention can effectively convert the inhibitory signal of PD-L1 into an activating signal for T cells by expressing the enhanced receptor, thereby enhancing the function of PD-1 + T cells.
  • mice engrafted with a tumor cell line and T cells expressing a TCR that recognizes the tumor cell line and enhancer receptors, and the other includes tumor cells from a patient with a solid tumor and T cells from the same patient.
  • mice Twenty NSG mice were first inoculated with J82-NY-ESO1 tumor cells. When the mean tumor volume reached 100 mm, 20 mice were randomly divided into 5 groups of 4 mice each.
  • PD-1 + T cells were sorted from the peripheral blood of tumor patients, and the sorted negative components were defined as PD-1 ⁇ T cells.
  • PD-1 - TCR-T, PD-1 + TCR-T and PD-1 + V1E-TCR-T cells were prepared as described in Example 3, and PD-1 + NOTD cells (PD-1 + NOTD cells without virus transfection) were prepared as described in Example 3. -1+T cells) as controls.
  • mice After the cells were prepared, they were re-infused to 4 groups of mice, while 1 group of mice was re-infused with PBS as a blank control.
  • the tumor volume in mice was calculated by measuring the length, width and height of the subcutaneous tumor at different time points after reinfusion.
  • Tumor volume mean curves for each group are shown in Figure 6F.
  • the mean tumor volume of mice in each group reinfused with PD-1 - TCR-T, PD-1 + TCR-T and PD-1 + V1E-TCR-T cells was 25.43 mm 3 , 102.4 mm 3 and 0mm 3 , all showed good tumor inhibition effect.
  • PD-1 + V1E-TCR-T cells had the strongest tumor-suppressive effect, and the tumors of all 4 mice were completely cleared, achieving a 100% CR curative effect.
  • This therapeutic effect is better than that of PD-1 - TCR-T, indicating that the enhanced receptor loaded in PD-1 + T can effectively improve the function of PD-1 + T cells, making the original tumor inhibition effect inferior to PD-1.
  • the exhausted state of PD-1 + T cells by T cells was reversed, resulting in a better effect than PD- 1- T cells that did not express the enhancer receptor.
  • mice transplanted with tumor tissue from patients and T cells from the same patients Similar experiments were performed using mice transplanted with tumor tissue from patients and T cells from the same patients.
  • the inventors constructed the tumor tissue from solid tumor patients into a PDX (Patient-Derived Tumor Xenograft) model.
  • PDX Principal-Derived Tumor Xenograft
  • 20 mice were selected.
  • the average tumor volume reached 100 mm 3
  • the 20 mice were randomly divided into 4 groups with 5 mice in each group.
  • PD-1 + T cells were sorted from peripheral blood from the same patient, and the sorted negative fraction was defined as PD-1 ⁇ T cells.
  • mice After the cells were prepared, they were re-infused to 3 groups of mice, while 1 group of mice was re-infused with PBS as a blank control. The tumor volume in mice was measured at different time points after reinfusion.
  • PD-1 + T cells in the peripheral blood of patients with advanced tumors are considered to be T cells that once infiltrated the tumor and returned to the peripheral blood after being suppressed by the tumor microenvironment, and are referred to as cTILs (circulating tumor infiltrating lymphocytes).
  • cTILs circulating tumor infiltrating lymphocytes
  • PBMCs of patients will be collected, PD-1 + T cells (cTIL) will be isolated, and they will express V1E-enhancing receptors and gene modification of the amplification factor (CD19CAR, sequence shown in SEQ ID NO: 3), prepared into super cTIL (ScTIL), and then infused back to the patient.
  • V1E-enhancing receptors and gene modification of the amplification factor CD19CAR, sequence shown in SEQ ID NO: 3
  • ScTIL super cTIL
  • TMB Tumer Mutation Burden, which is estimated as the number of mutations per megabase in the genome, with less than 5 being low mutation burden, 5-10 being medium mutation burden, and more than 10 being high Mutation load. It is generally accepted in the art that a higher TMB is more suitable for immunotherapy, and a lower TMB is less suitable for this type of therapy.
  • the selected patients are solid tumor patients with eight different cancer types (gallbladder cancer, ovarian cancer, lung cancer, colon cancer, pancreatic neuroendocrine tumor, renal cancer, mucosal melanoma, pancreatic cancer), all of which are advanced and Traditional multi-line therapy is ineffective, and there are multiple distant metastatic lesions in the body, and most patients do not have high levels of TMB.
  • cancer types gallbladder cancer, ovarian cancer, lung cancer, colon cancer, pancreatic neuroendocrine tumor, renal cancer, mucosal melanoma, pancreatic cancer
  • Super cTILs were prepared by transfecting cTILs cells with combined lentiviruses simultaneously loaded with V1E-CD19 CAR.
  • the lentiviral transfection process was the same as described in Example 3.
  • flow cytometry was used to detect the proportion of CAR + cells (considered as effective cells) in the cells, which showed that the proportion of effective cells was 10%-40%.
  • the translucent pale-yellow ScTIL cell suspension for intravenous infusion was revived at the bedside with a 38.5°C water bath, and then rapidly intravenously infused.
  • the volume of reinfusion is determined according to the total amount of cells, the total amount of cells is 6.9 ⁇ 10 5 -4.5 ⁇ 10 8 and the volume of reinfusion is 20-40ml, the cell density is about 3 ⁇ 10 6 -3 ⁇ 10 7 /ml, and the total effective cells ( The amount of CAR positive cells) is shown in Table 2. After the reinfusion, the patient continued to be hospitalized for observation.
  • the treatment plan of the present invention which modifies the cTIL cells with the amplification factor CD19 CAR and the enhancing receptor, and sees that it is returned to the subject, does not produce uncontrollable serious side effects, and has ideal safety.
  • the results showed that the disease was effectively controlled in all subjects during the six-week observation period after the reinfusion of the cells, indicating that the The disease control rate of ScTIL for advanced solid tumors was about 100% (9/9), that is, the response rate of patients to treatment was 100%, and three patients had significant tumor shrinkage or loss of activity, that is, the objective response rate was 33% (3/9).
  • the efficacy assessments in the far right column of Table 2 all use the RECIST criteria.
  • the imaging data of patients 1-4 are shown in Figure 16, and the lesions are indicated by arrows or scales. As shown, the tumor lesions shrank significantly after treatment.
  • the PD-1 + T cells modified with the amplification factor CD19 CAR and the enhanced receptor of the present invention can effectively control or even treat a variety of different solid tumors, and have a wide range of potential applications, and its effect is not related to the patient's tumor mutation load. association, so its application is not limited to patients with high TMB.
  • Peripheral blood was collected at different time points (30 minutes, 24 hours, 4 days, 7 days, 10 days, 14 days, 28 days, 71 days and 91 days) after reinfusion for flow cytometry to detect the number of CAR-positive cells and The ratio of the number of lymphocytes to the number of lymphocytes, thus dynamically monitoring the proportion of CAR-positive cells (ie ScTIL cells) in the peripheral blood of patients 1 and 2, and calculating the absolute value of its number in the patient.
  • the fold expansion of ScTIL cells in peripheral blood was calculated by the following formula:
  • Amplification multiple [number concentration of lymphocytes (number/L) ⁇ circulating blood volume (L) ⁇ ratio of ScTIL to lymphocytes] / number of ScTIL cells returned
  • the number and concentration of lymphocytes can be obtained by blood routine detection; the proportion of ScTIL in T cells can be obtained by flow cytometry to detect the proportion of CAR+ cells in CD3 + cells.
  • the total circulating blood volume was converted according to the weight in kilograms before the cell reinfusion treatment.
  • the total blood volume (L/L) was calculated as 80ml/kg body weight for men and 75ml/kg body weight for women.
  • the cTIL cells modified with the expansion factor CD19 CAR in the present invention can effectively use the recognition of the B cells by the CD19 CAR to help them expand in vivo.
  • the ScTIL in the present invention is collected from peripheral blood-derived TIL (cTIL), and these T cells have the property of naturally recognizing tumors, that is, the first recognition property of T cells.
  • cTIL peripheral blood-derived TIL
  • the introduction of the design of CD19 CAR targeting B cells can achieve a large expansion of ScTIL in vivo by means of the stimulation of B cells to its target CD19.
  • the cTIL cells with the expansion factor CD19 CAR of the present invention can not only use the CD19 CAR to recognize B cells to cause the large-scale expansion of TIL or tumor-recognizing T cells in vivo, but also use the cells themselves to naturally stimulate tumor cells. Identify attributes to further identify and kill tumor cells in tumor patients. This dual recognition of the immune cells modified in the present invention was successfully demonstrated by the above-mentioned clinical trials.
  • the disease control rate of ScTIL therapy is 100%, but if only natural TIL cells are reinfused, it is not enough to produce such a curative effect, and the reasons are as follows:
  • the number of reinfused cells is not sufficient to produce such a therapeutic effect. Even on the basis of the number of T cells detected in peripheral blood after expansion (10 5 -10 6 /kg body weight), combined with the relevant literature reports on cell therapy in the past (such as Steven Rosenberg's report on neoantigen-reactive T cells or TIL therapy). According to reports, the amount of cells reinfused is generally around 10 to the 11th power), such a dose is lower than the conventional reinfusion dose, so it is not enough to achieve the therapeutic effect observed in this example in the treatment of solid tumors.
  • TIL The source of ScTIL is TIL, which is usually exhausted T cells. Without genetic modification, the effect of TIL in killing tumors is very limited, and the return of such a low dose of exhausted T cells is not enough to obtain this example. Efficacy.
  • PD-1 + T cells can be obtained from easily obtained peripheral blood through simple sorting, so tumor-recognizing T cells can be quickly obtained and the preparation cycle is shortened;
  • the design of enhancer receptors can make super cTIL (ScTIL) cells change from an inhibitory state to an activated state, thereby enhancing the tumor-killing efficiency.
  • CAR targeting B cells CD19
  • CD19 The design of CAR targeting B cells (CD19) can greatly expand ScTIL cells in vivo, so only a few ScTIL cells need to be prepared, and there is no need for large expansion in vitro.
  • the cell preparation process is simple and fast, and does not require tedious steps such as gene sequencing, screening, and synthesis of traditional neoantigen therapy technology, thereby greatly shortening the preparation cycle and further greatly reducing the preparation cost.
  • the ScTIL therapy of the present invention shows very good curative effect on solid tumor patients of different cancer types and ineffective after traditional multi-line treatment, and has potential wide application.
  • the ScTIL therapy of the present invention has ideal safety: a) there is no serious off-target or autoimmune disease and other toxic side effects; b) only a slight cytokine storm (CRS) occurs, which is much lower than CAR-T treatment of B cells CRS grade of hematological tumors; c) Although it kills normal B cells, its side effects will not be stronger than the application of CAR-T in the treatment of B cell hematological tumors. This is because after CD19 CAR-T treatment of B-line hematological malignancies, the absence of B cells usually lasts for no less than 18 months, and some patients even require alternative immunoglobulin therapy for life.
  • CRS cytokine storm
  • B cells recovered and stabilized at levels close to or at the lower limit of the normal physiological range within 1-3 months after ScTIL treatment, while no immunoglobulin levels were found to fall below the normal physiological range in all subjects during this period.
  • the lower limit of normal physiological level The lower limit of normal physiological level.
  • Example 6 Enhanced receptor-modified PD-1 + T cells in the treatment of malignant solid tumors with incidental clearance of residual virus from previous HBV infection
  • the inventors In a clinical trial of using ScTIL cells to treat patients with solid tumors, the inventors surprisingly observed that the patients' hepatitis B markers and liver enzymes were abnormal after treatment with the modified immune cells (ScTIL) of the present invention. Based on this observation, the inventors speculate and design experiments to verify, and then propose that the immune cells of the present invention can be used to treat diseases associated with HBV infection and to treat residual HBV virus in the body.
  • ScTIL modified immune cells
  • ALT/AST is higher than the upper limit of the normal range
  • HBsAg is transiently positive, HBsAb and/or other antibodies then turn positive, while HBV DNA copy number and HBcAb IgM remain negative all the time;
  • the cell products were tested for the presence of HBV and the presence of HBV in the peripheral blood of the subjects, and the results were all negative. Therefore, it can be considered that the cell products were not contaminated with HBV, and the subjects did not have exogenous HBV infection during the clinical trial. .
  • ScTIL is not chemically toxic, and its molecular mechanism of action ensures that it does not damage autologous healthy tissue, so this possibility can be ruled out.
  • Treatment cell preparation ScTIL cells 70ml ⁇ 2 bags + 30mL ⁇ 1 bag, the total number of effective cells is 2.5 ⁇ 10E9.
  • the cells were reinfused in two times, on December 13, 2019 (70ml, effective cell number 1.03x10E9) and December 18, 2019 (70+30ml, effective cell number 1.47x10E9), and the two reinfusion processes went smoothly.
  • the body temperature increased 6 hours after the completion of the reinfusion, and the self-reported chills, headache, mild nausea, no vomiting and other special discomforts. The fever continued for 3 days and then returned to normal levels.
  • liver enzyme spectrum was re-examined and found to be elevated: ALT 65U/L (normal range 0-50U/L), AST 61U/L (normal range 0-40U/L) L), five items of hepatitis B were not checked at that time;
  • liver function and HBV-related indicators are shown in Figure 8A.
  • Hepatitis B related indicators detection information is a prefix of Hepatitis B related indicators detection information
  • liver enzyme spectrum was found to be elevated: ALT 163U/L (normal range 0-50U/L), AST 113U/L (normal range 0-40U/L) ), five items of hepatitis B were not checked at that time;
  • ALT 743U/L normal range 0-50U/L
  • AST 243U/L normal range 0-40U/L
  • five hepatitis B findings Hepatitis B surface antigen positive, and core antibody positive, but core antibody IgM negative;
  • liver function and HBV-related indicators are shown in Figure 8B.
  • liver enzymes ALT, AST
  • HBsAb/HBcAb surface antibody/core antibody
  • liver enzymes decreased to normal levels.
  • peripheral blood and cell products were negative for hepatitis B virus (HBV) DNA copy number and HBcAb IgM, and plasma immunoglobulins (IgG, IgM, IgA) remained within the normal range.
  • HBV protein presented by the MHC mechanism of hepatocytes can be recognized by immune lymphocytes, but the attack of target cells by immune lymphocytes can be affected by the immune killing escape mechanism of virus-infected hepatocytes (PD-L1 of hepatocytes).
  • the monocytes collected in the examples of the present application and used for ScTIL preparation were prepared by sorting using PD-1 antibody magnetic beads and obtaining PD-1 positive (PD-1 + ) lymphocytes.
  • PD-1 + cells contain a subset of CTLs that specifically recognize HBV-presented membrane proteins, but whose killing activity on target cells is inhibited by PD-L1 expressed by hepatocytes. These cells were loaded with enhanced receptors via lentiviral transfection thereby reactivating their cell killing capacity.
  • these modified cells will recognize target cells expressing HBV proteins via specific TCRs and react with target cells via the cell membrane enhancer, namely the extracellular domain of PD-1, with highly expressed PD- Combined with L1 to inversely trigger further activation of ScTIL and enhanced killing effect on target cells.
  • the disintegrated hepatocytes challenged by CTL will result in the release of HBV components into the peripheral bloodstream and trigger humoral immunity and elevated antibody titers, manifesting as transient HBsAg positivity and secondary antibody positivity (Figure 11).
  • ScTIL can play an anti-malignant solid tumor effect and simultaneously clear previous infections. Then, the viral components remaining in some target cells are hidden, thereby eliminating the potential risk of recurrence of previous viral infections in the future when the immune system is weakened.
  • the modified immune cells of the present invention can eliminate viruses in the body and prevent the recurrence of virus-related diseases while treating tumor diseases in tumor patients.
  • HBV DNA copy number is lower than the positive threshold
  • HBV antibodies appeared relatively late and remained positive for a long time.
  • liver enzymes The abnormality of liver enzymes is usually positively correlated with the quantitative abnormality of HBV antigens.
  • HBV components in the process of replication remain in some hepatocytes, which may lead to the recurrence of infection when the body's immunity is weakened.
  • Peripheral blood PD-1-positive lymphocytes contain specific subsets that can specifically recognize and express HBV membrane protein components that are presented to hepatocyte membranes, but cannot kill residual viral components due to inhibition by secondary PD-L1 expression. target cells.
  • the enhanced receptor molecule of ScTIL can overcome the immune escape caused by PD-L1, clear the residual HBV viral components in the body, and eliminate the hidden danger of future infection recurrence.
  • Example 7 Enhanced receptor-modified PD-1 + T cells in the treatment of diseases associated with HPV infection
  • pro-PD-1 + T cells were sorted based on peripheral blood samples from patients with HPV positive tumors and ScTIL cells expressing enhancing receptors and optional CD19 CAR were then prepared.
  • the prepared ScTIL cells are returned to the patient for tumor treatment and clearance of HPV infection.

Abstract

修饰的免疫细胞及其在免疫治疗中的用途，所述免疫细胞为来自外周血的PD-1 +T细胞。所述免疫治疗用于肿瘤治疗或与病毒感染相关的疾病的治疗和预防。

Description

修饰的免疫细胞及其用途 技术领域
本发明属于生物医药技术领域，具体而言属于免疫细胞治疗领域。
背景技术
T细胞是人体最重要的特异性免疫细胞之一，近年来在备受瞩目的细胞治疗领域中扮演着重要的作用，例如可以通过修饰并回输患者的自体T细胞进行细胞治疗。然而，鉴于肿瘤的异质性，患者MHC的个体性，以及T细胞受体(TCR)产生过程(VDJ重排)的随机性，患者体内特异识别有害细胞的T细胞群各不相同，换言之是一个包含高度个体化的TCR队列的T细胞群体。如何从患者自体快速获得具有特异识别有害细胞(如癌细胞或病毒宿主细胞)的能力的T细胞群，并且在此基础上加以修饰，进而更高效、安全地应用于细胞治疗，是一个技术难题。
发明内容
本发明的发明人提出，PD-1阳性T细胞(PD-1 +T细胞)是一组处于被抑制状态的细胞，这类细胞很可能是能够识别靶细胞并且经历过与靶细胞的PD-L1的相互作用的T细胞。在个体没有自身免疫病史的情况下，可将PD-1 +T细胞视为攻击过有害细胞的T细胞的集合。正因为该集合中的T细胞已经与有害细胞有过接触，因此可以认为这些T细胞拥有靶向有害细胞的能力。经过加工的外周血中的PD-1 +T细胞有望用于治疗各类疾病，尤其是肿瘤和感染性疾病。PD-1 +T细胞的优点是有较好的对有害细胞的识别特性，但缺憾是PD-1的高表达使得这些细胞处于耗竭状态，功能被抑制，难以起到杀伤效果。为了使PD-1 +T细胞兼具识别特性和免疫活性，本发明提出了克服PD-1 +T细胞抑制状态的几种修饰方法，由此完成了本发明。
因此，在第一方面，本申请涉及一种修饰的免疫细胞，所述免疫细胞是来源于外周血中的PD-1阳性T(PD-1 +T)细胞。优选地，所述PD-1 +T细胞来源于外周血单个核细胞(PBMC)。例如，所述PD-1 +T细胞是通过分选PBMC获得的细胞，例如通过PD-1和CD3作为标志物进行分选。
在优选的实施方案中，所述修饰是能够减弱或消除PD-1 +T细胞的抑制状态的修饰。 例如，所述修饰可以通过对所述免疫细胞进行基因改造和/或蛋白质修饰进行。
在一个具体的实施方案中，所述修饰是对PD1 +T细胞的遗传修饰，其使得所述PD1 +T细胞的PD-1表达缺失或降低。所述遗传修饰可以在体内或体外进行。在一个实施方案中，所述遗传修饰在体内通过基因编辑或基因治疗进行。在另一个实施方案中，所述遗传修饰在体外进行，例如在所述PD-1 +T细胞的制备过程中进行。所述遗传修饰可以是对所述PD-1 +T细胞的内源PD-1的敲除或敲低。敲除内源PD-1基因的手段包括但不限于CRISPR/Cas方法，TALEN等。
在一个具体的实施方案中，所述修饰是对PD-1 +T细胞表面的PD-1的修饰，其使得PD-1 +T细胞表面的PD-1与其配体的结合能力相对于未修饰之前有所下降。在具体的实施方案中，通过将PD-1抗体与所述PD-1 +T细胞混合，使得PD-1抗体竞争性结合PD-1 +T细胞表面的PD-1，从而降低PD-1与其配体的结合能力，由此完成修饰。在一个实施方案中，所述修饰在体内通过与PD-1抗体联合给药进行。在另一个实施方案中，所述修饰在体外进行，如通过在PD-1 +T细胞制备的过程中加入PD-1抗体进行。所述PD-1抗体优选是PD-1单克隆抗体或单链抗体。
在进一步的实施方案中，所述经修饰的PD-1 +T细胞具有增强受体(ER)，所述增强受体包含：细胞外结构域(ECD)和细胞内结构域(ICD)，所述ICD包含引发免疫细胞激活信号的共刺激分子，所述ECD包含特异性结合所述免疫细胞的靶细胞的部分。在具体的实施方案中，所述特异性结合免疫细胞的靶细胞的部分选自所述靶细胞的膜蛋白的受体、配体和抗体，或其具有与靶细胞结合功能的部分或片段。优选地，所述ECD包含PD1的部分序列或抗PD-L1抗体优选抗PD-L1scFv，和/或所述ICD源自CD28。
优选地，所述修饰的免疫细胞对受试者中的靶细胞具有靶向性。在一些实施方案中，所述靶细胞是肿瘤细胞，特别是癌细胞。在一些实施方案中，所述靶细胞是受病毒感染的宿主细胞，所述病毒如肝炎病毒，优选乙型肝炎病毒、丙型肝炎病毒、丁型肝炎病毒，或人乳头瘤病毒(HPV)。在一些实施方案中，所述修饰的免疫细胞的靶细胞选自下组中的一种或多种：肿瘤细胞、癌细胞、受病毒感染的细胞。
在进一步的实施方案中，所述经修饰的PD-1 +T细胞表达嵌合抗原受体(CAR)，所述CAR特异性识别与所述免疫细胞的天然TCR所识别的不同的另一种抗原。优选地，所述另一种抗原是CD19。
在进一步的实施方案中，所述经修饰的PD-1 +T细胞包含其他修饰，例如自杀开关。
第二方面，本申请提供包含第一方面的修饰的免疫细胞的细胞群体。
第三方面，本申请提供一种制备修饰的免疫细胞的方法，所述方法包括：(a)从外周血分选PD-1 +的T细胞；和(b)对所述步骤(a)分选的PD-1 +T细胞进行如下一种或多种处理：i.敲除或敲低PD-1的表达；ii.与PD-1抗体混合；iii.使其表达增强受体(ER)；iv.使其表达嵌合抗原受体(CAR)；v.使其表达控制细胞死亡的修饰，如自杀开关。在优选的实施方案中，所述步骤(b)的处理至少包括使其表达增强受体(ER)。进一步地，所述步骤(b)的处理包括使其表达嵌合抗原受体(CAR)。在另一种实施方案中，或在此基础上的实施方案中，所述步骤(b)的处理包括敲除或敲低PD-1的表达和/或与PD-1抗体混合。额外地，所述步骤(b)的处理包括使其表达自杀开关。所述处理可以在体内或体外进行。所述处理可以在将所述修饰的免疫细胞回输至受试者之前、之后或同时进行。在具体的实施方案中，所述方法的步骤(a)的分选包括从外周血单个核细胞以任意顺序进行PD-1 +细胞分选和CD3 +细胞分选。在优选的方案中，所述处理至少包括i.敲除或敲低PD-1的表达。
第四方面，本申请涉及通过第三方面的方法制备的修饰的免疫细胞。
第五方面，本申请涉及一种药物组合物，其包含第一方面或第四方面的修饰的免疫细胞，或第二方面的细胞群体。
第六方面，本申请涉及第一方面或第四方面的免疫细胞或第二方面的细胞群体在治疗中的用途，或在制备用于药物中的用途。所述治疗或药物用于(1)治疗肿瘤，和/或(2)治疗或预防与病毒感染相关的疾病或症状，或防止与病毒感染相关的疾病或症状的复发。优选地，所述病毒感染为慢性病毒感染。在具体的实施方案中，所述病毒为肝炎病毒，优选乙型肝炎病毒。在另一个具体的实施方案中，所述病毒为人乳头瘤病毒。本发明的免疫细胞可以用于在受试者中清除治疗后的残余病毒成分，预防病毒感染的复发。
附图说明
图1是展现实施例1中的PDX小鼠荷瘤情况的照片。
图2是展现实施例1中为了分离肿瘤组织解剖后的荷瘤小鼠的照片。
图3A-B展示了使用来自患者外周血的PD-1 +T细胞或PD-1 -T细胞与患者的肿瘤组织共培养，进行ELISPOT实验以检测IFNγ分泌的结果，其显示了两种T细胞对肿瘤的识别性和响应性的差异；(A)共培养24小时的ELISPOT染色照片和ELISPOT统计结果；(B)共培养48小时的ELISPOT染色照片和ELISPOT统计结果。
图4A-E显示了检测PD-1敲除对PD-1 +T细胞功能影响的体外实验结果。(A)流式检测PD-1的分选效率；(B)流式检测TCR的表达情况，以未用表达TCR的病毒转染的细胞作为对 照(PD-1 +NOTD)；(C)在表达和不表达TCR的PD-1 +T细胞中流式检测PD-1的敲除效果，两种细胞均以未经处理和进行空电击的细胞作为对照；(D-E)ELISA实验检测T细胞与J82-NY肿瘤细胞共培养后，T细胞的IFNγ(D)和IL-2(E)的分泌。
图5A-D显示了检测增强受体对PD-1 +T细胞功能影响的体外实验结果。(A)流式检测PD-1的分选效率；(B-C)ELISA实验检测表达和不表达增强受体的T细胞与J82-NY肿瘤细胞共培养后，T细胞的IFNγ(B)和IL-2(C)的分泌；(D)ELISA实验检测表达和不表达增强受体的T细胞在包被了OKT3和/或PD-L1蛋白的96孔板中的IFNγ分泌。
图6A-F显示实施例4中在接种了J82-NY-ESO1肿瘤细胞的小鼠中进行的体内实验的结果，证明了增强受体对PD-1 +T细胞的抑瘤功能的影响。(A)PBS对照；(B)PD-1 +NOTD；(C)PD-1 -TCR-T细胞；(D)PD-1 +TCR-T细胞；(E)PD-1 +-V1E-TCR-T细胞；(F)各组平均值的整合图。
图7A-C显示实施例4中在接种了来自结肠癌患者的肿瘤组织的小鼠中进行的体内实验的结果，证明了增强受体对PD-1 +T细胞的抑瘤功能的影响。(A)PBS对照；(B)PD-1-T细胞；(C)PD-1 +T细胞；(D)PD-1 +-V2E-T细胞。
图8显示实施例6中的患者(A-B)和对照受试者(C)的肝功和HBV相关指标。
图9显示HBV在宿主肝细胞内的生物学过程。
图10显示细胞毒性T淋巴细胞(CTL)攻击与靶细胞免疫逃逸。
图11显示ScTIL清除HBV在肝细胞中残余成分(不包括活性病毒)。
图12是用于引入识别NY-ESO-1的TCR的慢病毒载体构建体pLenti-TCR-NY ESO1的质粒图。
图13是用于引入增强受体V1E的慢病毒载体构建体pLenti-V1E-T2A-TCR的质粒图。
图14是用于引入增强受体V2E的慢病毒载体构建体pLenti-V2E-T2A-TCR的质粒图。
图15是作为扩增因子使用的CD19 CAR的结构示意图。
图16是实施例5中患者1-4在治疗前后的影像学检查结果。
具体实施方式
定义
除非另有说明，否则本文公开的一些方法的实践采用免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA的常规技术，这些技术在本领域的技术范围内。
术语“约”意指在本领域普通技术人员确定的特定值的可接受误差范围内，这将部分 取决于如何测量或确定该值，即，测量系统的局限性。例如，根据本领域的实践，“约”可以表示在1或大于1的标准偏差内。或者，“约”可表示给定值的最多20％，最多10％，最多5％或最多1％的范围。或者，特别是对于生物系统或过程，该术语可以表示数值的一个数量级，优选地在5倍内，更优选地在2倍内。在申请和权利要求中描述特定值的情况下，除非另有说明，否则应当假定术语“约”意味着在特定值的可接受误差范围内。
术语“基因”在本文指核酸(例如DNA，例如基因组DNA和cDNA)及其相应的编码RNA转录物的核苷酸序列。基因组DNA在本文包括插入的非编码区以及调节区，并且可包括5'和3'末端。在一些用途中，该术语包括转录序列，包括5'和3'非翻译区(5'-UTR和3'-UTR)、外显子和内含子。在一些基因中，转录区将包含编码多肽的“开放阅读框”。在该术语的一些用途中，“基因”仅包含编码多肽所必需的编码序列如“开放阅读框”或“编码区”。在一些情况下，基因不编码多肽，例如核糖体RNA基因(rRNA)和转移RNA(tRNA)基因。在一些情况下，术语“基因”不仅包括转录序列，而且还包括非转录区域，包括上游和下游调节区、增强子和启动子。基因可以指生物基因组中其天然位置中的“内源基因”或天然基因。基因可以指“外源基因”或非天然基因。非天然基因可以指通常不在宿主生物体中发现但通过基因转移引入宿主生物体的基因。非天然基因也可以指不在生物体基因组中的天然位置的基因。非天然基因还可以指天然存在的核酸或多肽序列，其包含突变、插入和/或缺失(例如，非天然序列)。
术语“核苷酸”通常是指碱-糖-磷酸盐组合。核苷酸可包含核苷酸的类似物或衍生物以及合成核苷酸。核苷酸可以通过已知的技术进行标记以便检测。可检测标记可包括例如放射性同位素、荧光标记、化学发光标记、生物发光标记和酶标记。
术语“多核苷酸”、“寡核苷酸”和“核酸”可互换使用，指任何长度的聚合形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，或其类似物，其可以是单股、双股或多股形式。多核苷酸对细胞可以是外源的或内源的。多核苷酸可以是基因或其片段。多核苷酸可以是DNA或RNA。多核苷酸可以具有任何三维结构，并且可以执行已知或未知的任何功能。多核苷酸可包含一种或多种类似物(例如改变的主链，糖或核碱基)。
术语“表达”是指多核苷酸从DNA模板转录(例如转录成mRNA或其他RNA转录物)和/或转录的mRNA随后翻译成肽、多肽或蛋白质的一个或多个过程。转录物和编码的多肽可统称为“基因产物”。如果多核苷酸衍生自基因组DNA，则表达可包括在真核细胞中剪接mRNA。表达的“上调”或“下调”通常是指相对于其在野生型状态下的表达水平，多核苷酸(例如RNA，例如mRNA)和/或多肽序列的表达水平增加或降低。
涉及表达或活性的术语“调节”是指改变表达或活性水平。调节可以在转录水平和/或翻译水平发生。
术语“肽”、“多肽”和“蛋白质”在本文中可互换使用，是指通过肽键连接的至少两个氨基酸残基的聚合物。术语“肽”，“多肽”和“蛋白质”在本文中可互换使用，是指通过肽键连接的至少两个氨基酸残基的聚合物。该术语不意味着特定长度的聚合物，也不意味着该聚合物是天然的、经修饰的或合成的。在某些情况下，聚合物可被非氨基酸中断。该术语包括任何长度的氨基酸链，包括全长蛋白质，和具有或不具有二级和/或三级结构的蛋白质(例如，结构域)。该术语还包括已经被修饰的氨基酸聚合物，例如，通过二硫键形成、糖基化、脂化、乙酰化、磷酸化、氧化和任何其他操作，例如与标记组分的缀合。本文所用的术语“氨基酸”通常是指天然和非天然氨基酸，包括但不限于修饰的氨基酸和氨基酸类似物。术语“氨基酸”包括D-氨基酸和L-氨基酸。
术语“融合物”可以指包含一个或多个非天然序列(例如，部分)的蛋白质和/或核酸。融合物可包含一种或多种相同或不同的非天然序列。融合物可以是嵌合物。融合物可包含核酸亲和标签、条形码(barcode)或肽亲和标签。融合物可以提供使多肽定位于亚细胞部位的信号，例如，用于靶向细胞核的核定位信号(NLS)，用于靶向线粒体的线粒体定位信号，用于靶向叶绿体的叶绿体定位信号，内质网(ER)保留信号等。融合物可以提供可以用于跟踪或纯化的非天然序列(例如，亲和标签)。融合物可以包含小分子，例如生物素或染料，例如Alexa氟染料，Cyanine3染料，Cyanine5染料。
如本文所用的术语“抗原”是指能够被选择性结合剂结合的分子或其片段。例如，抗原可以是可以被选择性结合剂如受体结合的配体。作为另一个例子，抗原可以是抗原分子，其可以被选择性结合剂如免疫蛋白(例如抗体)结合。抗原还可以指能够在动物中使用以产生能够结合该抗原的抗体的分子或其片段。
如本文所用的术语“抗体”是指具有免疫球蛋白样功能的蛋白质结合分子。术语抗体包括抗体(例如，单克隆和多克隆抗体)及其衍生物、变体和片段。抗体包括但不限于不同类别(即IgA、IgG、IgM、IgD和IgE)和亚类(例如IgG1、IgG2等)的免疫球蛋白(Ig)。其衍生物、变体或片段可以指保留相应抗体的结合特异性(例如完整和/或部分)的功能性衍生物、变体或片段。抗原结合片段包括Fab、Fab'、F(ab') 2、可变片段(Fv)、单链可变片段(scFv)、微抗体、双抗体和单结构域抗体(“sdAb”或“纳米抗体”或“骆驼抗体”)。术语抗体包括已经优化、工程化或化学缀合的抗体和抗体的抗原结合片段。已经优化的抗体的实例包括亲和力成熟的抗体。已经改造的抗体的实例包括Fc优化的抗体(例如，在片段可结晶区域中优化的抗体) 和多特异性抗体(例如，双特异性抗体)。
术语“TCR”或“T细胞受体”具有本领域通常理解的含义。TCR是T细胞介导的抗原识别的基础，是免疫系统中的关键一环。TCR具有高度多样性，这种多样性与疾病之间的关系是免疫学领域的研究热点。T细胞在某个条件下或某个时间点包含的TCR的集合可被称为TCR组库(TCR repertoire)或TCR谱(TCR profile)，其随着疾病的发生和发展可能发生很大的变化。
术语“受试者”、“个体”和“患者”在本文中可互换使用，指脊椎动物，优选哺乳动物，例如人。哺乳动物包括但不限于鼠类、猿猴、人类、农场动物、运动动物和宠物。还包括体内获得的或体外培养的生物实体的组织，细胞及其后代。
术语“治疗”在本文指用于获得有益或所需结果(包括但不限于治疗益处和/或预防益处)的方法。例如，治疗可包括施用本发明的修饰的免疫细胞或包含该免疫细胞的细胞群。治疗益处是指被治疗的一种或多种疾病或症状存在任何与治疗相关的改善。对于预防益处，本发明修饰的免疫细胞可以施用于有风险形成特定疾病或症状的受试者，或施用于存在疾病的一种或多种生理征兆的受试者，即使疾病或症状可能还没有表现出来。
术语“有效量”或“治疗有效量”是指组合物的量，例如包含本发明的修饰的免疫细胞如淋巴细胞(例如，T淋巴细胞和/或NK细胞)的组合物的量，在以该量给予有需要的受试者时足以产生所需的活性。
免疫细胞
本发明的免疫细胞来自于外周血，如外周血单个核细胞(PBMC)，外周血淋巴细胞(PBL)和其他血细胞亚群，包括但不限于T细胞、自然杀伤细胞、单核细胞、单核细胞前体细胞、造血干细胞或非多能干细胞。在一些情况下，本发明的免疫细胞可以是任何免疫细胞，包括任何T细胞，例如肿瘤浸润细胞(TIL)。免疫细胞可以来自待治疗的受试者(例如，患者)。所述受试者可以是哺乳动物，如小鼠、猴或人。在具体的实施方案中，所述免疫细胞分离自受试者，如人类受试者的外周血单核细胞(PBMC)。
可以使用任何技术，例如Ficoll分离，从受试者收集的血液中获得T细胞。来自受试者的循环血液的细胞可以通过单采血液成分术或白细胞去除术获得。单采血液成分产品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞以及其他有核白细胞，还包括红细胞和血小板。可以洗涤通过单采血液成分术收集的细胞以除去血浆部分，并将细胞置于合适的缓冲液或培养基中，例如磷酸盐缓冲盐水(PBS)，用于随后的处理步骤。洗涤后，可 将细胞重悬于各种生物相容性缓冲液中，例如不含Ca、Mg的PBS。或者，可以除去单采血液成分样品中不需要的组分，并将细胞直接重悬于培养基中。样品可以由受试者直接提供，或间接通过一个或多个居间者提供，例如样品采集服务提供者或医学提供者(例如医生或护士)。在一些实施方案中，从外周血白细胞中分离T细胞可包括裂解红细胞并通过例如PERCOL TM梯度离心将外周血白细胞与单核细胞分离。
可以通过正向或负向的选择技术进一步分离免疫细胞如T细胞的特定亚群，如本发明的免疫细胞是PD-1阳性的T细胞亚群。不受限于任何理论，认为PD-1阳性细胞很可能代表了经历过与靶细胞的相互作用的细胞群体，因此该细胞群体具有对靶细胞的识别性。在具体的实施方案中，以PD-1为标志物对获得自外周血的免疫细胞如PBMC进行分选。例如，可以使用PD-1抗体通过磁力分选(如使用磁珠)或流式细胞术来分选PD-1 +T细胞，优选使用磁珠进行分选。
本发明认为分选出的PD-1阳性的T细胞亚群是与希望靶向的有害细胞已经进行过相互作用的T细胞亚群，因此其中包含的T细胞具有能够特异性识别有害细胞上的抗原的TCR组库或TCR谱。为了使细胞治疗中使用的T淋巴细胞能够靶向识别有害细胞如肿瘤细胞，现有技术中的一种做法是通过筛选TCR库获得能够靶向该有害细胞如肿瘤细胞的TCR并获得其遗传信息，据此对来自患者的T细胞进行遗传改造，赋予其识别有害细胞上抗原的能力。与这种通过外源修饰赋予T细胞识别性的技术不同，本发明可以不进行TCR筛选，也不向用于治疗的T细胞中额外引入外源TCR，而是通过直接从患者自体细胞分选那些大概率本身具有特异性针对有害细胞的TCR的T细胞，来保证用于治疗的细胞的靶向性和安全性。因此，本领域技术人员能够理解，本发明不要求T细胞具有一种或多种特定的TCR，因为TCR组库会因患者及其疾病状态而具有巨大差异。本发明要求T细胞群体与有害细胞之间存在识别性即可，而这种识别性在本发明中主要通过分选PD-1阳性的T细胞来获得。也正因如此，本发明的方法学可以用于针对多种有害细胞并治疗多种疾病。
在本发明的上下文中，“有害细胞”指被免疫细胞靶向并攻击的细胞，特别是肿瘤细胞、癌症细胞、受病毒感染的细胞。
本发明的T细胞还可以是CD3阳性、CD4阳性、CD4阴性或CD8阳性的T细胞亚群。可以使用任何本领域的技术来分选具有或不具有特定标志物的细胞亚群。例如，可以通过偶联有针对相关标志物的抗体的基质如磁珠进行分选，如通过CD3磁珠或CD3/CD28磁珠来分选CD3 +T细胞。可以通过改变细胞浓度来促成和基质表面例如磁珠表面的最大接触。另外，也可以用针对不想要的细胞特有的表面标志物的抗体组合实现对细胞群体的负选择。 一种合适的技术包括通过负磁性免疫粘附进行细胞分选，其利用针对不想要的细胞上存在的细胞表面标志物的单克隆抗体混合物。例如，为了分离CD4 +细胞，单克隆抗体混合物可包括针对CD14、CD20、CD11b、CD16、HLA-DR和CD8的抗体。
在一些实施方案中，免疫细胞是经富集的细胞群中的细胞。可通过任何合适的方法富集一种或多种所需的细胞类型，例如，对细胞群进行处理以触发扩增和/或分化成所需细胞类型，或阻止不需要的细胞类型的生长，或杀死或裂解不需要的细胞类型，或纯化所需细胞类型(例如在亲和柱上纯化以基于一种或多种细胞表面标志物保留所需或不需要的细胞类型)。
修饰
PD-1 +T细胞由于经历过与靶细胞的相互作用而被消耗、“钝化”，效力和功能有所削弱。本发明通过对PD-1 +T细胞进行修饰来恢复、提升其功效。
本发明的修饰的免疫细胞的PD-1表达被敲除或敲低。基因表达的“敲除(knock-out)”在本文指基本上消除该基因的表达。基因表达的“敲低(knock-down)”在本文指该基因的表达的降低。在优选的实施方案中，本发明的修饰的免疫细胞的内源PD-1表达被敲除。可以使用本领域已知的任何方法来实现对PD-1的敲除或敲低，所述方法包括但不限于：RNAi(包括使用siRNA或shRNA)、CRISPR-Cas方法、转录激活样效应因子核酸酶(TALEN)技术、定点诱变、锌指核酸酶(ZFN)技术或其组合。
在优选的实施方案中，使用CRISPR/Cas方法来敲除免疫细胞的内源PD-1，所述方法包括使用能够与目标基因PD-1杂交的引导RNA(gRNA)以及Cas蛋白或其编码核酸分子。在具体的实施方案中，使用靶向PD-1的sgRNA来敲除免疫细胞的内源PD-1，优选地所述sgRNA具有如SEQ ID NO:4所述的核苷酸序列。在具体的实施方案中，使用Cas9蛋白或其编码核酸分子来敲除PD-1。所述Cas9蛋白可以源自酿脓链球菌(Streptococcus pyogenes)、嗜热链球菌(Streptococcus thermophilus)或其他物种。例如，Cas9可以包括spCas9、Cpfl、CasY、CasX或saCas9。
在一个方面，本发明的修饰的免疫细胞包含增强受体(ER)。增强受体可用于提供对免疫细胞活性的进一步控制，例如但不限于免疫细胞活化和扩增。增强受体与配体的结合可以在修饰的免疫细胞中产生免疫细胞活化信号而不是免疫细胞失活信号。在修饰的免疫细胞中引发免疫细胞活化信号而不是免疫细胞失活信号可以使免疫细胞中的免疫抑制作 用最小化。最小化免疫细胞中的免疫抑制作用可以增加免疫细胞在免疫应答中的有效性，例如通过增加针对靶细胞(例如肿瘤细胞或受感染细胞)的免疫细胞细胞毒性。
增强受体可以包含蛋白质的胞外结构域(ECD)。所述蛋白质可以是信号传导受体或其任何功能片段、衍生物或变体。在一些情况下，所述信号传导受体可以是膜结合受体。响应于配体结合，信号传导受体可以诱导细胞中的一种或多种信号传导途径。在一些情况下，信号传导受体可以是非膜结合受体。增强受体可包含选自如下受体的片段(如细胞外结构域)：G蛋白偶联受体(GPCR)的受体、整合素受体、钙粘蛋白受体、催化受体(例如激酶)、死亡受体、检查点受体、细胞因子受体、趋化因子受体、生长因子受体、激素受体和免疫受体的片段，例如细胞外结构域。
在一些实施方案中，增强受体包含免疫检查点受体的片段，其可以参与免疫系统的调节。此类受体的非限制性实例包括但不限于PD-1、CTLA-4、BTLA)、KIR、IDO、LAG3、TIM-3、TIGIT、SIRPα、NKG2D，优选PD-1。
增强受体可以包含结合任何合适的免疫检查点受体配体的片段。此类配体的非限制性实例包括但不限于B7-1、B7-H3、B7-H4、HVEM(疱疹病毒进入介质)、AP2M1、CD80、CD86、SHP-2、PPP2R5A、MHC(例如，I类、II类)、CD47、CD70、PD-L1(或PDL1)和PD-L2。增强受体与此类配体结合的区域，可以是此类配体的天然受体，也可以是此类配体的单克隆抗体。
增强受体还包含胞内结构域(ICD)，其可以是引发免疫细胞激活信号的共刺激分子或其片段、变体、衍生物。在一些实施方案中，共刺激分子可用于调节免疫细胞中的增殖和/或存活信号。在一些实施方案中，ICD是共刺激分子的细胞内结构域，所述共刺激分子选自MHC I类蛋白、MHC II类蛋白、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、SLAM蛋白、活化NK细胞受体、BTLA或Toll配体受体。在优选的实施方案中，所述共刺激分子是T细胞共刺激分子，优选T细胞正共刺激分子，优选CD28。
增强受体的ECD和ICD可以通过跨膜结构域连接。在一些实施方案中，所述跨膜结构域包含多肽。跨膜多肽可具有任何合适的多肽序列。在一些情况下，跨膜多肽包含内源或野生型跨膜蛋白的跨膜部分的多肽序列或其具有至少一个氨基酸变化的变体。在一些实施方案中，跨膜多肽包含非天然多肽序列，例如多肽接头的序列。多肽接头可以是柔性的或刚性的。多肽接头可以是结构化的或非结构化的。在一些实施方案中，跨膜多肽将来自ECD的信号传递至ICD，例如指示配体结合的信号。在一些实施方案中，ECD包含跨膜结构域。在一些实施方案中，ICD包含跨膜结构域。
转染增强受体基因的手段可以向PD-1 +T细胞电转增强受体的mRNA，或通过慢病毒、腺相关病毒、或非病毒载体，将增强受体基因转染到PD-1 +T细胞中。
所述增强受体是是一种表达于免疫细胞表面的跨膜蛋白，由细胞外结构域(ECD)与细胞内结构域(ICD)组成，其中ICD包含引发免疫细胞激活信号的共刺激分子，可引发免疫细胞活化信号，ECD可以结合所述免疫细胞的靶细胞，是所述PD-1 +T细胞的靶细胞的膜蛋白的受体、配体、抗体，或可与靶细胞结合的任意一种物质的完整序列结构或包含其结合域的部分。
在具体的实施方案中，所述增强受体为V1E或V2E，ECD分别包含PD-1的片段(例如PD-1胞外结构域的片段)或抗PD-L1单克隆抗体的scFv序列，且所述增强受体的ICD包含CD28的胞内信号传导结构域。更优选地，所述增强受体包含CD28的跨膜区或CD8的跨膜区。在最优选的实施方案中，所述增强受体包含选自SEQ ID NO:1或SEQ ID NO:2的序列，或与序列具有至少75％、80％、85％、90％、95％、97％或99％同源性的序列，或由这样的序列组成。
在一些实施方案中，本发明的修饰的免疫细胞包含嵌合抗原受体(CAR)。当CAR能识别靶细胞时，经增强受体修饰的免疫细胞如T细胞比未经增强受体修饰的细胞有更强的免疫细胞活化功能。在一些情况下，CAR可以帮助修饰的免疫细胞进行扩增。
在优选的实施方案中，所述CAR特异性识别与所述免疫细胞的天然TCR不同的另一种抗原。优选地，所述CAR包含能够结合B细胞表面蛋白的抗原相互作用结构域(抗原结合结构域)、跨膜结构域和细胞内信号传导结构域。B细胞表面蛋白可以是可以在B细胞表面上发现的任何蛋白质，优选CD19。在一些实施方案中，CAR的抗原相互作用结构域能够结合非B细胞上的表面蛋白，只要与表面蛋白的结合不显著损害宿主的一般健康状态或免疫系统。
抗原结合结构域的非限制性实例包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠抗体或其功能衍生物、变体或片段，包括但不限于Fab、Fab'、F(ab')2、Fv、单链Fv(scFv)、微抗体、双抗体和单结构域抗体如骆驼科动物衍生的纳米抗体的重链可变结构域(VH)、轻链可变结构域(VL)和可变结构域(VHH)。在一些实施方案中，抗原结合结构域包含Fab、Fab'、F(ab')2、Fv和scFv中的至少一种，优选scFv。在一些实施方案中，抗原结合结构域包含抗体模拟物。抗体模拟物是指能够以与抗体相当的亲和力结合靶分子的分子。在一些实施方案中，抗原结合结构域包含跨膜受体或其任何衍生物，变体或片段。例如，抗原结合结构域可以包含至少跨膜受体的配体结合结构域。
在一些实施方案中，CAR的抗原相互作用结构域能够结合与颗粒(例如，纳米颗粒)偶联(例如，通过共价和/或非共价键)的B细胞的表面蛋白或其片段。颗粒可以是包含有机和/或无机材料的任何颗粒材料。颗粒可具有各种形状和尺寸。颗粒在至少一个维度上可以是约1纳米(nm)至约50微米(μm)。颗粒在至少一个维度上可以是至少约1nm、5nm、10nm、50nm、100nm、500nm、1μm、5μm、10μm、50μm或更大。颗粒在至少一个维度上可以是至多约50μm、10μm、5μm、1μm、500nm、100nm、50nm、10nm、5nm、1nm或更小。颗粒可以是纳米颗粒、微颗粒、纳米球、微球、纳米棒、微米棒、纳米纤维、纳米带等。颗粒的实例包括金属纳米颗粒(例如，金纳米颗粒，银纳米颗粒和铁纳米颗粒)、金属间纳米半导体纳米颗粒、核-壳纳米颗粒、具有聚合物壳的无机核的颗粒、具有聚合物壳的有机核的颗粒以及它们的混合物。或者，颗粒可以是有机纳米颗粒，例如交联聚合物、水凝胶聚合物、可生物降解的聚合物、聚丙交酯(PLA)、聚乙交酯(PGA)、聚己内酯(PCL)、共聚物、多糖、淀粉、纤维素、壳聚糖、聚羟基链烷酸酯(PHA)、PHB、PHV、脂质、肽、肽两亲物、多肽(例如蛋白质)或其组合。在表面上呈递B细胞表面蛋白的颗粒可以在体外引入包含结合B细胞表面蛋白的CAR的免疫细胞。作为另外一种选择或除此之外，呈递B细胞表面蛋白的颗粒可以与包含CAR的免疫细胞一起体内引入(例如局部或全身注射)。这些颗粒可用于在体外或体内扩增包含CAR的免疫细胞群。
在一些实施方案中，CAR的抗原相互作用结构域能够在死B细胞上结合B细胞表面蛋白或其片段。B细胞凋亡可以在免疫应答发展之前或之后发生。因此，死B细胞或其碎片仍然可以在表面上呈现B细胞表面蛋白或其片段。CAR靶向活B细胞和死B细胞的能力可以增加包含所述CAR的修饰的免疫细胞结合B细胞表面蛋白和启动细胞内信号传导结构域的信号传导的概率。在一些情况下，细胞内信号传导结构域的信号传导可促进包含CAR的免疫细胞的扩增(增殖)。
CAR的胞内信号传导结构域可以诱导修饰的免疫细胞的活性。胞内信号传导结构域可以转导效应子功能信号并指导细胞执行特化功能。信号传导结构域可包含其他分子的信号传导结构域。在一些情况下，将信号域的截短部分用于CAR。胞内信号传导结构域包含参与免疫细胞信号传导的多个信号传导结构域，或其任何衍生物、变体或片段。CAR的胞内信号传导结构域可包括共刺激结构域，例如来自共刺激分子。
在一个具体的实施方案中，本申请的CAR包含SEQ ID NO:3的序列或与该序列具有至少75％、80％、85％、90％、95％、97％或99％同源性的序列，或由这样的序列组成。
与缺乏CAR的免疫细胞相比，包含的修饰的免疫细胞可通过CAR与B细胞表面蛋白 的结合可以增强免疫细胞增殖。免疫细胞的增殖可以指免疫细胞的扩增或表型变化。包含CAR的本发明的修饰的免疫细胞可与B细胞表面蛋白结合，其增殖可以是缺乏CAR的相应免疫细胞的增殖的约5倍至约10倍，约10倍至约50倍，约50倍至约100倍，约100倍至约200倍，约为200倍至约300倍，约300倍至约400倍，约400倍至约500倍，约500倍至约600倍，大约600倍至约700倍。增殖可以在使B细胞与包含CAR的修饰的免疫细胞接触后至少约12、24、36、48、60、72、84或96小时确定。可以在体外或体内确定增殖，例如通过测定免疫细胞的数量。测定免疫细胞的数量的方法可包括流式细胞术、台盼蓝排除法和/或血细胞计数法。也可以通过免疫细胞的表型分析来确定增殖。
本文提供的增强受体和CAR的各种结构域可通过化学键连接，例如酰胺键或二硫键；小有机分子(例如烃链)；氨基酸序列，例如肽接头(例如，长度约3-200个氨基酸的氨基酸序列)，或其组合。肽接头可提供所需的灵活性以使嵌合多肽能够具有恰当的表达、活性和/或构象定位。肽接头可以具有任何合适的长度以连接至少两个目标结构域，并且优选设计为有足够的柔性以使其连接的一个或两个结构域能够正确折叠和/或发挥功能和/或具有活性。在一些实施方案中，肽接头的长度为约0至200个氨基酸，约10至190个氨基酸，约20至180个氨基酸，约30至170个氨基酸，约40至160个氨基酸，约50至150个氨基酸，约60至140个氨基酸，约70至130个氨基酸，约80至120个氨基酸，约90至110个氨基酸。在一些实施方案中，接头序列可包含内源蛋白质序列。在一些实施方案中，接头序列包含甘氨酸，丙氨酸和/或丝氨酸氨基酸残基。在一些实施方案中，接头可以以GS、GGS、GGGGS、GGSG或SGGG为基序并含有多个这样的基序，例如多个重复的相同基序。接头序列可包括任何天然存在的氨基酸，非天然存在的氨基酸或其组合。
在一些实施方案中，本发明的修饰的免疫细胞还包含调控免疫细胞死亡的修饰，如自杀开关。在严重毒性的情况下，例如高细胞因子血症出现时，可以激活自杀开关以消除免疫细胞。当免疫系统具有过于强烈的反应以致许多炎性细胞因子被释放时，会引发轻微至严重的症状，包括发烧、头痛、皮疹、心跳加速、低血压和呼吸困难，此时可以启动自杀开关。自杀开关可以是药物诱导自杀开关。自杀开关可包含诱导型半胱天冬酶9。
在优选的实施方案中，本发明的修饰的免疫细胞不包含外源引入的TCR。
可以使用任何合适的递送方法将需要的组合物和分子如多肽和/或核酸如编码多肽的核酸引入免疫细胞以完成修饰。各种组分可以同时或分开递送。递送方法可包括将一种或多种核酸引入免疫细胞，所述核酸包含编码本发明组合物的核苷酸序列，例如其可以是包含编码目标产物的核苷酸序列的表达载体，如重组表达载体。与宿主细胞相容的任何合 适的载体可用于本发明。
递送或转化方法的非限制性实例包括但不限于病毒或噬菌体感染、转染、缀合、原生质体融合、脂质转染、电穿孔、磷酸钙沉淀、聚乙烯亚胺(PEI)介导的转染、DEAE-葡聚糖介导的转染、脂质体介导的转染、粒子枪技术、磷酸钙沉淀、直接微注射和纳米颗粒介导的核酸递送。
基于病毒和非病毒的基因转移方法可用于将核酸引入哺乳动物细胞或组织中，因此在本发明中可用于将目标核酸引入培养中的细胞中。非病毒载体递送系统可包括DNA质粒、RNA(例如转录物)、裸核酸和与递送载体复合的核酸例如脂质体。病毒载体递送系统可包括DNA和RNA病毒，其在递送至细胞后可与细胞的基因组整合或不整合。
基于RNA或DNA病毒的体系可用于靶向体内的特定细胞并将病毒的负载运输至细胞核。病毒载体可以直接(体内)给药，或用于体外处理细胞，并且可以任选地(离体)施用至细胞。基于病毒的系统可包括用于基因转移的逆转录病毒、慢病毒、腺病毒、腺伴随病毒和单纯疱疹病毒载体。利用逆转录病毒、慢病毒和腺伴随病毒基因转移方法可以在宿主基因组中进行整合，这可以产生插入的转基因的长期表达。在许多不同的细胞类型和靶组织中可以观察到高转导效率。优选地，本发明使用慢病毒载体。
在一些方面，本发明提供修饰免疫细胞的方法，包括将一种或多种多核苷酸，或如本文所述的一种或多种载体，或其一种或多种转录物，和/或由其转录的一种或多种蛋白质递送至免疫细胞的方法。在一些方面，本公开还提供由这些方法产生的修饰的免疫细胞，以及包含这些免疫细胞。
靶细胞
本发明的修饰的免疫细胞可以靶向并作用于体外、体内或离体的多种靶细胞。靶细胞可以是分离的细胞。靶细胞可以是生物体内的细胞。靶细胞可以是生物体。靶细胞可以是细胞培养物中的细胞。
靶细胞可以是哺乳动物细胞或源自哺乳动物细胞，所述哺乳动物可以是啮齿动物、灵长类如人。靶细胞可以是或可以源自原核细胞，如细菌细胞、古细菌细胞，或者可以是或源自真核细胞。
靶细胞可以是被病原体感染的细胞，所述病原体可以是微生物，包括但不限于细菌、真菌或病毒。靶细胞可以是宿主细胞，如所述病原体的宿主细胞。在具体的实施方案中，所述靶细胞是例如受到肝炎病毒感染的细胞或受到人乳头瘤病毒感染的细胞，所述肝炎病 毒优选乙型肝炎病毒。
靶细胞可以来自特定器官或组织。靶细胞可以是单细胞生物。
靶细胞可以是干细胞或祖细胞，包括但不限于成体干细胞、胚胎干细胞、诱导多能干细胞(iPSCs)和祖细胞如心脏祖细胞、神经祖细胞等。靶细胞可以是多能干细胞。
靶细胞可以是经遗传修饰的细胞。靶细胞可包含靶核酸。
靶细胞可以是患病细胞，如来自患病受试者的细胞。患病细胞可具有改变的代谢，基因表达和/或形态学特征。患病细胞可以是癌细胞或被病毒感染的细胞。
在优选的实施方案中，所述靶细胞是肿瘤细胞，特别是癌细胞。所述肿瘤包括实体瘤和血液肿瘤，优选实体瘤。
治疗用途
本发明的修饰的免疫细胞可以用于治疗肿瘤，换言之以肿瘤细胞为靶细胞。在一些实施方案中，靶细胞形成肿瘤。用本发明的修饰的免疫细胞治疗可使肿瘤的生长稳定、不进展和/或不转移。例如，所述“生长稳定”意味着在治疗之后的一段时间内，一个或多个肿瘤的体积增加不超过1％、5％、10％、15％或20％。在一些实施方案中，所述一段时间为至少约1、2、3、4、5、6、7、8、9、10、11、12或更多周，优选至少约1、2、3、4、5、6、7、8、9、10、11、12或更多个月，更优选至少约1、2、3、4、5、6、7、8、9、10或更多年。在一些实施方案中，用本发明的修饰的免疫细胞治疗可使肿瘤的大小或肿瘤细胞的数量减少至少约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或更多。在一些实施方案中，肿瘤被完全消除，或降低至低于检测水平。在一些实施方案中，受试者在治疗后保持无肿瘤至少约1、2、3、4、5、6、7、8、9、10、11、12或更多周，优选至少约1、2、3、4、5、6、7、8、9、10、11、12或更多个月，更优选至少约1、2、3、4、5、6、7、8、9、10或更多年。
在用于治疗肿瘤时，施用给受试者的本发明的修饰的免疫细胞的量是10 4至10 10之间，10 4至10 9之间，10 5至10 8之间，10 6至10 7之间。在本发明的PD-1 +T细胞包含扩增因子修饰如CD19 CAR时，回输的总量可以适当低于不包含所述修饰的本发明的PD-1 +T细胞，这是因为CD19 CAR有助于回输的免疫细胞在患者体内的扩增，这一效果在实施例5中有详细的记载。例如，在本发明的PD-1 +T细胞包含扩增因子修饰如CD19 CAR时，回输细胞的总量可以在10 4至10 9之间，10 5至10 8之间，10 6至10 7之间。本发明的修饰的免疫细胞可以分为一次或多次回输给受试者，例如一次、两次、三次、四次。每次回输的免疫细胞的量可以相同或不同。
本发明的修饰的免疫细胞可以用于治疗与病毒感染相关的疾病或症状，换言之以受病毒感染的细胞为靶细胞。在一些实施方案中，本发明的免疫细胞在治疗患者中癌症的同时可以清除患者体内的病毒，例如抗病毒治疗后的残余病毒。因此，本发明的经修饰的免疫细胞和方法特别适合于已知感染过病毒的患者。所述病毒感染可以与癌症有关，也可以与癌症无关。在另一些实施方案中，本发明的免疫细胞被专门用于针对受病毒感染的细胞、治疗与病毒相关的疾病和/或清除病毒。
在优选的实施方案中，所述病毒感染是乙肝病毒感染。例如，所述病毒感染是慢性乙肝病毒感染，如肝脏被乙肝病毒感染的时间持续超过六个月。与慢性乙肝病毒感染相关的疾病包括肝硬化、肝细胞瘤。
在另一个优选的实施方案中，所述病毒感染是人乳头瘤病毒(HPV)感染。迄今为止已经发现了超过100种人乳头瘤病毒。按照是否与癌症相关一般分为致癌的高危型(包括但不限于16型、18型、31型、45型、52型、84型)和不致癌的低危型(如引发尖锐湿疣的6型、11型、42型、43型、44型)。HPV可以引起人皮肤黏膜的鳞状上皮增殖，导致皮肤、呼吸道、口腔、消化道、眼部、肛门和泌尿生殖系统的疾病。与HPV相关的疾病包括但不限于疣如寻常疣、扁平疣、尖锐湿疣，肿瘤或癌症如鳞状细胞癌、宫颈癌、外阴癌、阴道癌、阴茎癌、肛门癌、口咽癌。
靶细胞的死亡可以通过任何合适的方法确定，包括但不限于在处理之前和之后计数细胞，或测量与活细胞或死细胞相关的标记物的水平。细胞死亡程度可通过任何合适的方法确定。在一些实施方案中，相对于起始条件确定细胞死亡程度。例如，个体可具有已知起始量的靶细胞，例如已知大小的起始细胞团或已知浓度的循环靶细胞。在这种情况下，细胞死亡程度可表示为治疗后存活细胞与起始细胞群的比率。可以使用各种细胞死亡分析，并且可以使用多种检测方法。检测方法的实例包括但不限于细胞染色、显微术、流式细胞术、细胞分选及其组合。
当在治疗期结束后对肿瘤进行手术切除时，可以通过测量坏死(即，死亡)的切除组织的百分比来确定治疗在减小肿瘤大小方面的功效。在一些实施方案中，如果切除组织的坏死百分比大于约20％则治疗有效，例如至少约30％、40％、50％、60％、70％、80％、90％或100％。在一些实施方案中，切除组织的坏死百分比是100％，即，没有活肿瘤组织存在或可检测到。
将靶细胞暴露于本发明的修饰的免疫细胞可以在体外或体内进行。将靶细胞暴露于修饰的免疫细胞通常是指使靶细胞与修饰的免疫细胞接触和/或足够接近使得靶细胞的 抗原(例如，膜结合或非膜结合抗原)可以与免疫细胞中表达的受体包括增强受体和/或CAR结合。通过共培养靶细胞和修饰的免疫细胞，可以在体外将修饰的免疫细胞暴露于靶细胞。共培养可以作为贴壁细胞或者悬浮液进行。靶细胞和修饰的免疫细胞可以在各种合适类型的细胞培养基中共培养，例如与补充物、生长因子、离子等共培养。可以在体内将靶细胞暴露于修饰的免疫细胞，例如，通过将修饰的免疫细胞施用于受试者，例如人，并允许所述修饰的免疫细胞通过循环系统定位于靶细胞来实现。在一些情况下，可以将修饰的免疫细胞递送至靶细胞所在区域，例如通过直接注射。所述暴露可以进行任何合适的时间长度，例如至少1分钟、至少5分钟、至少10分钟、至少30分钟、至少1小时、至少2小时、至少3小时、至少4小时、至少5小时、至少6小时、至少7小时、至少8小时、至少12小时、至少16小时、至少20小时、至少24小时、至少2天、至少3天、至少4天、至少5天、至少6天、至少1周、至少2周、至少3周、至少1个月或更长时间。
实施例
为了更全面地理解和应用本发明，下文将参考实施例和附图详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例1.验证癌症患者的PD-1 +T细胞对肿瘤的识别特性
为了验证癌症患者外周血PD-1 +T细胞对肿瘤的识别特性，将来源于同一患者的肿瘤组织进行处理，构建人源肿瘤异种移植(Patient-Derived tumor Xenograft,PDX)小鼠模型，具体流程如下。
I.肿瘤移植
1.将手术中从结肠癌患者取得的新鲜肿瘤样本转至60mm无菌培养皿中，加入适量含1％青霉素链霉素的PBS对肿瘤进行清洗。
2.用灭菌手术刀片将肿瘤样本按3mm×3mm×3mm大小进行切割，并对6-8周龄NPI(遗传背景NOD-Prkdcem1Idmo-il2rgem2Idmo)雄性小鼠进行皮下移植。
3.移植前对NPI小鼠腹腔注射麻醉剂，待小鼠麻醉后用灭菌眼科剪在小鼠右后肢外上方皮肤剪开约1cm开口，用眼科镊将肿瘤块推送至右后肢外侧皮下后闭合手术切口。
II.肿瘤观察
1.成瘤后的肿瘤为P0代次，使用游标卡尺量取肿瘤长径a与短径b，并以公式(V)＝1/2×a×b 2进行肿瘤体积计算。
2.在P0肿瘤长至800-1000mm 3大小时摘取肿瘤，剔除坏死组织后按每块3mm×3mm×3mm大小再次进行切割，并重复上述步骤移植到NPI小鼠皮下进行组织传代，再次成瘤后将肿瘤标记为P1代次(小鼠荷瘤情况如图1所示)。重复此步骤，传至P3代次时，分别从3只小鼠身上摘取肿瘤组织，进行后续体外功能实验(解剖的肿瘤组织如图2所示)。
3.每代次获得的肿瘤样本均可用于基因组学、蛋白组学及病理学分析等使用，同时可留存缓冻样本，在后续有实验需求时复苏以进行荷瘤使用。
III.PD-1 +T和PD-1 -T细胞的分选
在PDX小鼠模型传至第3代次时，分选来自同一患者的外周血PD-1 +T细胞和PD-1 -T细胞。
IIIa.PD-1 +T细胞分选
1.根据预计分选PD-1 +细胞数和PD-1 +细胞比例，取出相应数量的PBMC细胞于离心管中；
2.加入足量生理盐水重悬洗涤细胞，600g离心10min，弃掉上清；
3.重复步骤2生理盐水洗涤第2遍，获得细胞沉淀；
4.每1×10 7个总细胞加入40μl缓冲液重悬；每1×10 6个有效细胞加2μl抗-PD-1-Biotin抗体(Biolegend，Cat#329934)，混匀后4℃避光孵育10-15min；
5.每1×10 7个总细胞加0.5-1ml缓冲液清洗，600g离心10min，吸弃上清；
6.每1×10 7个总细胞加80μl缓冲液重悬，每1×10 7个有效细胞加20ul抗-Biotin MicroBeads(Meltenyi，Cat#130-090-485)，混匀后4℃避光孵育15min；
7.每1×10 7个总细胞加入1-2ml缓冲液清洗，600g离心10min，吸弃上清；
8.每1×10 8个总细胞加入1ml缓冲液重悬细胞；
9.将LS型分选柱放在磁力架上，下方放置15ml离心管，用3ml缓冲液润洗柱子1次，然后替换为新的50ml离心管作收集管；
10.将步骤8中重悬的细胞混匀加入LS型柱内，过柱完成后，用3ml缓冲液冲洗柱子，洗3次，收集未吸附的细胞，即PD-1 -PBMC细胞，计数，用于后续分选PD-1 -T细胞；
11.将LS型分选柱从磁力架上取下，放入一新的15ml离心管上；
12.加入5ml缓冲液于柱内，迅速用活塞将柱内液体推出，收集到磁珠标记的细胞即为PD-1 +细胞，计数，备用；
13.分别取步骤10和步骤12分选出的PD-1 -和PD-1 +细胞各5×10 5个并标记CD3-APC的抗体，进行流式检测；
14.根据实验需要，取相应数量的PD-1 +细胞，600g离心10min，用完全培养基重悬细胞；
15.重悬的PD-1 +T细胞加入CD3/CD28 DynaBeads(Gibco，Cat#40203D)进行激活，传代培养。
IIIb.PD-1-T细胞分选
1.CD3流式检测PD-1 -PBMC中T细胞的比例，并根据细胞计数计算出T细胞的总数目；
2.根据T细胞数目，按照T细胞与CD3/CD28 DynaBeads比例为1:3，取相应数量的CD3/CD28 DynaBeads于15ml的离心管中，磁力架上去除保存液；
3.将离心管从磁力架上取下，加入培养基重悬CD3/CD28 DynaBeads(10倍体积)，置于磁力架上去除培养基；
4.重复步骤3两次，最后一次使用1-5ml培养基重悬CD3/CD28 DynaBeads；
5.将1/2的重悬CD3/CD28 DynaBeads加入悬浮细胞液中，调整细胞浓度3.35-15×10 6/ml于新的离心管中(50ml离心管15-50ml，15ml离心管2-15ml)；
6.将离心管置于杂交炉上(1rpm/min)，室温18-20℃孵育30min；
7.小心的将离心管置于磁力架上，静置2min，收集与CD3/CD28 DynaBeads结合的T细胞，并用适量的培养基重悬，计数；
8.未吸附的细胞悬液重复步骤5-7，第二次收集与CD3/CD28 DynaBeads结合的T细胞，并用适量的培养基重悬，计数；
9.未吸附的细胞悬液计数，冻存；
10.将步骤7、8两部分收集的与CD3/CD28 DynaBeads结合的T细胞合并，调整细胞密度到1.2×10 6/ml，加入Tscm(T150 100-120ml，T75 50-60ml，T25 15-29ml)；
11.混匀放入CO2培养箱，传代培养。
IV.检测T细胞的 激活
从上述第II部分获得的3只P3代次小鼠身上分离肿瘤组织并剪碎，通过酶消化 法制备成单细胞悬液，并分别命名为1#肿瘤组织、2#肿瘤组织、3#肿瘤组织。将上述第III部分制备的PD-1 -T细胞和PD-1 +T细胞分别与所述3个肿瘤组织细胞共培养，形成六种培养物，在24小时和48小时分别对这六种培养物进行ELISPOT实验，通过检测IFN-γ来考察不同T细胞的激活情况。
如图3A-B所示，PD-1 +T 细胞的IFN-γ释放显著高于PD-1 -T细胞，意味着PD-1 +T细胞被肿瘤组织激活，而PD-1 -T细胞则不能。该结果表明通过上述分选方法从患者外周血中获得的PD-1 +T细胞能够有效识别肿瘤细胞并对其产生免疫应答、启动杀伤，说明患者外周血中存在能够对肿瘤细胞发挥免疫功能的T细胞(肿瘤特异性T细胞)，并且这些细胞主要集中在PD-1 +组分中。
实施例2.敲除PD-1对PD-1 +T细胞功能的提升
为了验证PD-1的敲除是否能够通过消除PD-1 +T细胞的“抑制状态”来有效提升PD-1 +T细胞的功能，进行了本实施例中的实验。发明人构建了一种体外模型，其中使用了以NY-ESO-1表达为特征的肿瘤细胞，并构建了包含与之相对应的能够结合NY-ESO-1的TCR的PD-1 +TCR-T细胞。在此基础上使用 CRISPR-Cas9技术对T细胞中的PD-1基因进行了敲除，以观察其对肿瘤细胞的效果变化。
首先，单采患者白细胞，加入PD-1单抗分选PD-1 +T细胞，分选效率如图4A所示。
随后在分选出的PD-1 +T细胞中加入CD3/CD28 DynaBeads，培养过夜。第二天将PD-1 +T细胞分为两组，一组为NOTD-T，不加病毒 转染；一组为TCR-T，加入表达识别NY-ESO-1的TCR的慢病毒载体(pLenti-TCR-NY ESO1，质粒结构如图12所示)，按照MOI(慢病毒数目/细胞数)＝2加入慢病毒。培养及扩增T细胞，流式检测该TCR的表达率为40.1％(图4B)。
加入慢病毒后第4天去除CD3/CD28 DynaBeads，第5天通过电转的方式将PD-1sgRNA和Cas9蛋白转入至PD-1 +NOTD-T细胞和PD-1 +TCR-T中，敲除内源表达的PD-1，并以不处理组和空电击组作为对照。如图4C所示，电转后第7天流式检测PD-1的表达情况，NOTD-T细胞不处理组和空电击组PD-1阳性比例分别为39.6％和34.8％，敲除PD-1后PD-1阳性比例降至3.6％；TCR-T细胞不处理组和空电击组PD-1阳性比例分别为40.1％和37.9％，敲除PD-1后PD-1阳性比例降至4.0％，敲除效果较好，可进行后续实验。
为了验证敲除PD-1对PD-1 +T细胞功能的影响，进行了ELISA实验。将不同组的T细胞与膀胱癌细胞系J82或过表达NY-ESO-1蛋白的J82-NY-ESO1(J82-NY)肿瘤细胞共培养24小时，ELISA检测IFN-γ和IL-2分泌的情况。如图4D和4E所示，敲除PD-1能显著促进PD-1 +TCR-T细胞分泌细胞因子，在同样转染了表达TCR的病毒载体并与J82-NY-ESO1肿瘤细 胞共培养之后，与两种对照(未经处理的对照和进行了空电机的对照)相比，敲除了PD-1的PD-1 +T细胞的IFN-γ分泌增加了将近50％，IL-2分泌增加了80％以上，表明敲除PD-1能有效提升PD-1 +T细胞的免疫效力。
实施例3.增强受体对PD-1 +T细胞功能的提升(体外实验)
在实体瘤的细胞治疗中，为了解除抑制性受体如PD-1对免疫细胞的抑制作用，甚至进一步提高T细胞的效力，提出可以为用于治疗的T细胞引入增强受体，其包含能够结合所述免疫细胞的靶细胞的胞外结构域，和T细胞共刺激分子的胞内结构域，如申请人的在先申请PCT/CN2020/073017中所述。
为了验证增强受体对通过本发明方法获得的PD-1 +T细胞的免疫效力是否有影响，发明人构建了如下三种包含靶向肿瘤靶点NY-ESO-1的TCR的PD-1 +T细胞：
1.PD-1 +TCR-T细胞，包含靶向NY-ESO-1的TCR；
2.PD-1 +V1E-TCR-T细胞，包含增强受体V1E和靶向NY-ESO-1的TCR，其中V1E包含PD1的部分序列作为胞外结构域，并包含CD28的跨膜和胞内结构域，V1E的序列如SEQ ID NO:1所示；
3.PD-1 +V2E-TCR-T细胞，包含增强受体V2E和靶向NY-ESO-1的TCR，其中V2E包含能够结合PD-L1蛋白的scFv抗体序列作为胞外结构域，并包含CD28的跨膜和胞内结构域，V2E的序列如SEQ ID NO:2所示；
将不含靶向NY-ESO-1的TCR和任何增强受体的PD-1 +NOTD-T细胞作为对照。具体构建流程如下。
首先，单采患者白细胞，加入PD-1单抗分选PD-1 +T细胞，分选效率如图5A所示。随后在PD-1 +T细胞中加入CD3/CD28 DynaBeads，培养过夜。第二天将PD-1 +T细胞分为4组，分别为：
第1组：NOTD-T，不加病毒转染；
第2组：TCR-T，加入表达识别NY-ESO-1的TCR的慢病毒载体；
第3组：V1E-TCR-T，加入表达V1E-TCR的慢病毒载体，载体结构如图13所示；
第4组：V2E-TCR-T，加入表达V2E-TCR的慢病毒载体，载体结构如图14所示。
对于第2-4组，按照MOI(慢病毒数目/细胞数)＝2加入慢病毒，培养及扩增T细胞。
为了验证增强受体对PD-1 +T细胞功能的影响，进行了ELISA实验。发明人将不同组的T细胞与J82肿瘤细胞或过表达NY-ESO1的J82-NY-ESO1肿瘤细胞共培养24小时，ELISA检测IFN-γ和IL-2分泌的情况。如图5B和5C所示，增强受体V1E能显著促进PD-1 +TCR-T细 胞分泌IFN-γ；增强受体V1E和V2E均能显著促进PD-1 +TCR-T细胞分泌IL-2，表明两种增强受体均能有效提升PD-1 +T细胞功能。
为了进一步验证增强受体对PD-1 +T细胞功能的影响，将上述4组T细胞分别加入已经包被了OKT3(CD3抗体)、PD-L1蛋白或OKT3+PD-L1蛋白的96孔板中，培养48小时取上清，ELISA检测IFN-γ分泌的情况。如图5D所示，OKT3能够有效激活T细胞分泌IFN-γ。OKT3联合PD-L1蛋白共同刺激时，TCR-T和NOTD组细胞分泌的IFN-γ的量有所下降，这是由于PD-L1蛋白结合到T细胞表面的PD-1蛋白，抑制了T细胞的功能所导致的，这也模拟了靶细胞上的PD-L1对T细胞功能的抑制。与之相反，表达增强受体的T细胞功能不仅没有被PD-L1蛋白所抑制，相对于仅使用OKT3的处理，同时使用OKT3和PD-L1处理的表达增强受体的T细胞具有显著更高的IFN-γ分泌，说明PD-L1蛋白结合到V1E或V2E增强受体上不仅会接触PD-L1本身对T细胞的抑制，还会显著促进T细胞分泌细胞因子。这个结果表明通过使本发明获得的PD-1 +T细胞表达增强受体能够有效将PD-L1的抑制信号转变成对T细胞的激活信号，增强PD-1 +T细胞的功能。
实施例4.增强受体对PD-1 +T细胞功能的提升(体内实验)
为了进一步验证增强受体对PD-1 +T细胞体内抑瘤功能的影响，构建了两种体系。一种包括移植有肿瘤细胞系的小鼠和表达能够识别该肿瘤细胞系的TCR以及增强受体的T细胞，另一种包括来自实体瘤患者的肿瘤细胞和来自同一患者的T细胞。
1.使用接种了J82-NY-ESO1肿瘤细胞系的小鼠的测试
首先为20只NSG小鼠接种了J82-NY-ESO1肿瘤细胞。当平均肿瘤体积达到100mm 3时，将20只小鼠随机分为5组，每组4只。
与此同时，从肿瘤患者外周血中分选PD-1 +T细胞，同时将分选后的阴性组分定义为PD-1 -T细胞。对分选后的PD-1 +T和PD-1 -T细胞进行慢病毒转染，以MOI＝2的剂量分别加入表达TCR(识别NY-ESO1的TCR)和表达V1E-TCR的慢病毒，如实施例3中所述制备PD-1 -TCR-T、PD-1 +TCR-T和PD-1 +V1E-TCR-T细胞，以PD-1 +NOTD细胞(未经病毒转染的PD-1+T细胞)为对照。
细胞制备完成后，分别回输给4组小鼠，同时1组小鼠回输PBS作为空白对照。回输后在不同的时间点通过测量皮下肿瘤的长、宽、高来计算小鼠体内肿瘤的体积。
结果如图6所示，回输PBS和PD-1 +NOTD细胞的小鼠肿瘤持续增殖(图6A、6B)，在T细胞回输后第44天肿瘤体积达到500mm 3左右。而另外3组小鼠回输了PD-1 -TCR-T、PD- 1 +TCR-T和PD-1 +V1E-TCR-T细胞，肿瘤体积均有所减小(图6C-E)，且PD-1 +V1E-TCR-T细胞的效果最为明显，肿瘤基本上完全被去除。
在图6F中显示了各组的肿瘤体积平均值曲线。回输了PD-1 -TCR-T、PD-1 +TCR-T和PD-1 +V1E-TCR-T细胞的各个组内小鼠的肿瘤体积平均值分别为25.43mm 3、102.4mm 3和0mm 3，均显示出了较好的抑瘤效果。
如上所述，PD-1 +V1E-TCR-T细胞的抑瘤效应最强，4只小鼠的肿瘤均被完全清除，实现100％CR的疗效。该疗效比PD-1 -TCR-T的抑瘤效果更好，表明PD-1 +T中负载的增强受体能够有效提升PD-1 +T细胞的功能，使得原本肿瘤抑制效果不如PD-1 -T细胞的PD-1 +T细胞的耗竭状态被逆转，产生了更优于不表达增强受体的PD-1 -T细胞的效果。
2.使用移植了患者肿瘤组织的小鼠的测试
使用移植了来自患者的肿瘤组织的小鼠和来自同一患者的T细胞进行了类似的实验。
发明人将来自实体瘤患者的肿瘤组织构建成PDX(Patient-Derived Tumor Xenograft)模型。PDX模型传至第5代时，选取20只小鼠，在平均肿瘤体积达到100mm 3时，将20只小鼠随机分为4组，每组5只。
与此同时，从来自于同一患者的外周血中分选PD-1 +T细胞，同时将分选后的阴性组分定义为PD-1 -T细胞。对分选后的一部分PD-1 +T细胞进行慢病毒转染，以MOI＝2的剂量加入表达增强受体V2E的慢病毒(该构建体与实施例3中表达V2E-TCR的慢病毒载体相同，只是不包括编码TCR的片段)，并制备PD-1 --T、PD-1 +-T和PD-1 +V2E-T细胞。
细胞制备完成后，分别回输给3组小鼠，同时1组小鼠回输PBS作为空白对照。回输后在不同的时间点测定小鼠体内肿瘤的体积。
比较各组不同小鼠的肿瘤增殖情况，结果如图7A-C所示。由于使用的是来自患者的肿瘤组织直接进行移植，相较于上文使用的细胞系而言，移植到每只小鼠体内的肿瘤组织在细胞构成上可能具有较大的异质性，使得同组中不同小鼠之间的结果也存在一些区别。因此，本实验中没有如前一实验中那样计算各组的平均值并进行比较。
尽管如此，从图7中也可以看出，回输PBS和PD-1 --T、PD-1 +-T细胞的所有小鼠肿瘤均持续增殖，且增殖速率较快，未见任何抑瘤效应。而回输PD-1 +V2E-T细胞的小鼠，5只小鼠中有3只出现肿瘤体积缩小，其中两只小鼠的肿瘤体积由最初的94mm 3和85mm 3分别减小到21mm 3和52mm 3(图7D)。
该结果表明PD-1 +T细胞中负载增强受体能够有效提升PD-1 +T细胞的功能，甚至逆 转PD-1 +T细胞的状态，促进其在体内的抑瘤能力。
实施例5.经增强受体和CAR修饰的PD-1 +T细胞的首批临床试验
在本申请的背景中，认为晚期肿瘤患者外周血中的PD-1 +T细胞是曾经在肿瘤中浸润过，并且在被肿瘤微环境抑制后又返回外周血的T细胞，将其称为cTIL(circulating tumor infiltrating lymphocyte)。
将对传统治疗无效的9位晚期实体瘤患者(或称受试者)纳入临床试验研究，采集患者PBMC，分离出其中的PD-1 +T细胞(cTIL)，对其进行表达V1E增强受体和扩增因子(CD19CAR，序列如SEQ ID NO:3所示)的基因修饰，制备成超级cTIL(ScTIL)，然后回输给患者。具体的试验如下。
1.受试者的选择
患者治疗前的临床情况如下下表所示。
表1.患者临床情况
序号 性别 年龄 肿瘤类型 临床分期 TMB
1 男 54 胆囊癌 IV期 中
2 女 54 卵巢癌 IV期 中
3 女 50 肺癌 IV期 低
4 男 73 结肠癌 IV期 高
5 女 53 胰腺神经内分泌瘤 IV期 中
6 男 63 肺癌 IV期 低
7 男 52 肾癌 IV期 中
8 女 61 粘膜型黑色素瘤 IV期 低
9 男 57 胰腺癌 IV期 中
在上表中，TMB代表肿瘤突变负荷(Tumer Mutation Burden)，以基因组中每百万碱基的突变数量来评估，低于5为低突变负荷，5-10为中突变负荷，10以上为高突变负荷。本领域通常认为TMB越高越适合免疫治疗，越低越不适合这类疗法。
从上表可知，入选患者为八种不同癌种(胆囊癌、卵巢癌、肺癌、结肠癌、胰腺神经内分泌瘤、肾癌、粘膜型黑色素瘤、胰腺癌)的实体瘤患者，均为晚期且经传统多线治疗无效，体内拥有多个远端转移病灶，并且大部分患者TMB不是高水平。
2.治疗前的细胞制备
2.1PD-1 +T(cTIL)细胞的分离：
(1)单采患者自体外周血单个核细胞，分离PBMC。用流式细胞仪检测PD-1 +T细胞(PD-1 +CD3 +双阳性细胞)占总T细胞(CD3 +细胞)的比例(见下表)。经鉴定发现PD-1 +T细胞在总T细胞中比例为28.5％(患者1)和14.3％(患者2)(表2)，将如此高比例的PD-1 +T细胞视为来自肿瘤组织的TIL，其从肿瘤组织进入了外周血，因此称为cTIL(circulating TIL)。
(2)用前述方法分选PD-1 +T细胞。
2.2ScTIL的制备：
用同时装载V1E-CD19 CAR的联合慢病毒转染cTILs细胞，从而制备超级cTIL(ScTIL)。慢病毒转染过程同实施例3所述。经过8-10天的自然扩增过程，使用流式细胞术检测细胞中的CAR +细胞(视为有效细胞)比例，显示有效细胞比例为10％-40％。经过质控放行检验后，装入输液袋。整个制备过程仅为8-10天(不含新抗原成分识别性T细胞鉴定过程)。
3.回输治疗及监测：
所有患者均静脉回输ScTIL。将用于静脉回输的半透明淡黄色ScTIL细胞悬液用38.5℃水浴在床旁复苏后，快速经静脉输注。回输体积根据细胞总量确定，细胞总量为6.9×10 5-4.5×10 8回输体积为20-40ml，细胞密度约为3×10 6-3×10 7/ml，总有效细胞(CAR阳性细胞)回输量见表2。回输后的患者持续住院观察。
3.1安全性评估
所有9名受试者中，只有3人出现高热，其中1人自行缓解，另2人使用托珠单抗治疗后症状缓解，细胞因子风暴(CRS)发生率为33％(3/9)，且均为1级CRS，CRS风险远低于CD19 CAR-T治疗血液肿瘤；所有患者，无一在治疗后出现自身免疫病。
可见，本发明用扩增因子CD19 CAR和增强受体修饰cTIL细胞并见其回输到受试者的治疗方案没有产生不可控的严重副作用，具有理想的安全性。
3.2疗效评估
根据回输前的基线和回输细胞后的两个月的影像学检查，结果显示，所有受试者在回输细胞后六周的观察期内，疾病总体都得到有效控制，说明本申请的ScTIL对晚期实体瘤的疾病控制率约为100％(9/9)，即患者对治疗的响应率为100％，其中三例患者的肿瘤出现 明显缩小或活性消失，即客观缓解率为33％(3/9)。
表2.患者治疗结果
Figure PCTCN2021126480-appb-000001
表2中最右栏的疗效评估均采用RECIST标准。患者1-4的影像学资料见图16，病灶处以箭头或标尺示出。如图所示，在治疗后肿瘤病灶显著缩小。
可见，本发明用扩增因子CD19 CAR和增强受体修饰的PD-1 +T细胞可以有效控制甚至治疗多种不同的实体瘤，有着广泛的潜在应用谱系，且其效果与患者肿瘤突变负荷没有关联性，因此其应用不局限于TMB高的患者。
3.3 CAR助力ScTIL在体内扩增特性的验证
在回输后的不同时间点(30分钟，24小时，4天，7天，10天，14天，28天，71天和91天)分别采取外周血用于流式检测CAR阳性细胞数量及与淋巴细胞数量的比值，由此动态监测患者1和2的外周血CAR阳性细胞(即ScTIL细胞)比例，并推算其在患者体内的数量绝对值。通过如下公式计算ScTIL细胞在外周血中扩增的倍数：
扩增倍数＝[淋巴细胞数浓度(个/L)×循环血量(L)×ScTIL占淋巴细胞比例]/回输ScTIL细胞数量
数据显示，细胞回输后30分钟外周血内CAR拷贝数即显著高于回输的有效细胞量，提示细胞回输后即刻开始快速扩增。因此，将回输细胞量直接可以作为以上计算公式中的基数，回输后30分钟采血检测的外周血CAR数值已不能作为未经扩增的基线数值。
其中，淋巴细胞数浓度可由血常规检测得到；ScTIL占T细胞比例可由流式细胞检测CAR+细胞占CD3 +细胞比例得到。总循环血量按细胞回输治疗前的公斤体重值换算，男性按80ml/公斤体重，女性按75ml/公斤体重计算出总血容量(升/L)。
经计算，在回输ScTIL后第14天(D14)，随机抽取患者1和2检测并计算ScTIL细胞在外周血中分别扩增倍数，发现分别为140倍和754倍。同时发现外周血B细胞数量大幅减少至回输前的8％-25％(数量减少75％-92％)。
表3.回输后14天ScTIL细胞和B细胞的数量变化
Figure PCTCN2021126480-appb-000002
即便在观察期内患者均未施用外源性的免疫球蛋白，所有患者均未出现免疫缺陷。
可见，本发明中具有扩增因子CD19 CAR修饰的cTIL细胞可以有效借助CD19 CAR对B细胞的识别来帮助其在体内扩增。
3.4 ScTIL双重识别性的验证
本发明中的ScTIL采集自外周血来源的TIL(cTIL)，这些T细胞具有天然识别肿瘤的属性，即T细胞的第一识别性。此外，在ScTIL基础上，引入靶向B细胞的CD19 CAR的设计，可借助B细胞对其靶标CD19的刺激，实现ScTIL在体内的大幅度扩增。
随机抽取患者1和患者2(表1和表2)，动态监测其外周血循环肿瘤细胞数(CTC)，比较回输当天的基线值，和回输后两个月内的复查的数值。结果显示，这两位受试者在回输ScTIL细胞后两个月，每5毫升外周血中的CTC数量均大幅下降，见下表。
表4.治疗后CTC的数量变化
Figure PCTCN2021126480-appb-000003
Figure PCTCN2021126480-appb-000004
可见，本发明带有扩增因子CD19 CAR的cTIL细胞，不仅可以借助CD19 CAR识别B细胞来引起TIL或肿瘤识别性T细胞在体内的大规模扩增，还可借助细胞本身对肿瘤细胞的天然识别属性，进一步识别并杀灭肿瘤患者体内的肿瘤细胞。本发明中修饰的免疫细胞的这种双重识别性通过上述临床试验成功得以证实。
4.疗效证据和分析
本实施例中ScTIL疗法的疾病控制率为100％，但如果仅回输天然TIL细胞，不足以产生这样的疗效，理由如下：
1)回输细胞的数量不足以产生这样的疗效。即便以扩增之后外周血中检测到的T细胞数量(10 5-10 6/kg体重)为基础，结合过去细胞治疗的相关文献报道(如Steven Rosenberg关于新抗原反应性T细胞或TIL治疗的报道，回输的细胞量大体在10的10-11次方左右)来看，这样的剂量低于常规回输剂量，因此在治疗实体瘤时不足以取得本实施例中观察到的疗效。
2)ScTIL的来源是TIL，通常为耗竭性T细胞，在不加以基因改造的情况下，TIL杀伤肿瘤的作用是非常有限的，回输如此低剂量的耗竭性T细胞不足以取得本实施例的疗效。
因此，根据本实施例的疗效数据可从侧面证明，本发明将增强受体应用于PD-1 +T细胞可以增强这些来自外周血的免疫细胞抑制肿瘤的能力。
5.总结
综合本实施例的多项实验结果，可以总结出本发明ScTIL疗法具有以下特点：
1)从易于获得的外周血通过简单分选即可获得PD-1 +T细胞(cTIL)，因此可快速得到肿瘤识别性T细胞，缩短了制备周期；
2)增强受体的设计可使超级cTIL(ScTIL)细胞从抑制状态变为激活状态，从而增强对肿瘤的杀伤效率。
3)靶向B细胞(CD19)的CAR的设计可使ScTIL细胞在体内得以大幅扩增，因此仅需制备很少的ScTIL细胞，且无需体外大量扩增。
4)细胞制备过程简单快速，无需传统新抗原治疗技术的基因测序、筛选、合成等繁琐步骤，从而大幅缩短了制备周期，并进一步大幅降低了制备成本。
5)本发明的ScTIL疗法对于不同癌种，且经传统多线治疗无效的实体瘤患者都显示出非常好的疗效，具有潜在的广泛应用。
6)本发明的ScTIL疗法具有理想的安全性：a)没有严重的脱靶或自身免疫病等 毒副作用；b)仅有轻微的细胞因子风暴(CRS)出现，远低于CAR-T治疗B细胞血液肿瘤的CRS级别；c)尽管对正常B细胞有所杀伤，但其副作用不会强于CAR-T治疗B细胞血液肿瘤的应用。这是因为CD19 CAR-T治疗B系血液恶性肿瘤后，B细胞缺如的状况通常持续不少于18个月，部分患者甚至终生需要替代性免疫球蛋白疗法。相比之下，ScTIL治疗后B细胞均于1-3个月内恢复并稳定于接近或达到正常生理范围下限的水平，而在此期间，所有受试者的免疫球蛋白均未发现低于正常生理水平下限。
实施例6.增强受体修饰的PD-1 +T细胞治疗恶性实体肿瘤附带清除既往HBV感染残留病毒
发明人在使用ScTIL细胞治疗实体瘤患者的临床试验中，惊讶地观察到在使用本发明的修饰的免疫细胞(ScTIL)治疗后患者的乙肝标志物和肝酶出现了异常。基于这种观察结果，发明人作出了推测并设计了实验进行验证，继而提出本发明的免疫细胞可用于治疗与HBV感染相关的疾病，以及用于治疗体内残留的HBV病毒。
1.ScTIL细胞治疗后出现的乙肝标志物与肝酶异常——性质与诱因
在临床试验中，使用ScTIL细胞治疗后在两例组外患者中观察到了以下现象。
基线处检测肝功能及HBV标志物正常，细胞治疗后出现：
1)ALT/AST高于正常范围上限；
2)HBsAg一过性阳性，HBsAb和/或其它抗体随后转为阳性，而HBV DNA拷贝数及HBcAb IgM始终保持阴性；
3)受试者无主观急性肝炎临床特征(食欲差，黄疸，肝大及肝区胀痛等)；
4)用于回输的细胞产品HBV检测阴性，血浆免疫球蛋白保持正常生理水平。
为了确定导致上述现象的原因，设计并进行了多项实验，由此通过回答以下几个问题来分析导致这些现象的诱因。
1)是否存在HBV对细胞产品的污染或外源性HBV感染？
检测了细胞产品中是否含有HBV以及受试者外周血中是否存在HBV，结果均为阴性，因此可以认为细胞产品没有受到HBV的污染，受试者在临床试验期间也没有外源性HBV感染发生。
2)是否由于继发于B细胞减少的免疫球蛋白降低，导致HBV病毒易感性增高而继发感染？
患者接受ScTIL的细胞治疗后观察到了外周血淋巴细胞计数较治疗前基线水平降低，但血浆免疫球蛋白浓度始终保持在正常生理范围，因此可以排除这种可能。
3)肝功异常是否源于细胞治疗相关不良反应或自体免疫性肝损伤？
ScTIL无化学毒性，其分子作用机制可以确保不伤及自体健康组织，因此可以排除这种可能。
2.患者基本情况
病例-1(WJ)
女，36岁，胃印戒细胞癌并宫颈转移2年。测序检测结果TMB 1.03，新抗原16，HLA全杂合。2019年11月15日实施单个核细胞单采，采集细胞总数为5.5x10E9/62ml。本实施例使用的ScTIL细胞的制备与实施例5中的基本相同，但用V2E增强受体(SEQ ID NO:2)替代了V1E增强受体，与CD19-CAR一起用于转染TIL细胞以获得ScTIL。治疗细胞制备：ScTIL细胞70ml×2袋+30mL×1袋，总有效细胞数量2.5x10E9。细胞分为两次回输，日期分别为2019年12月13日(70ml，有效细胞数量1.03x10E9)和2019年12月18日(70+30ml，有效细胞数量1.47x10E9)，两次回输过程顺利。两次均于完成回输后6小时出现体温升高，自述畏寒，头疼，与轻度恶心，无呕吐及其它特殊不适，发热均持续3天后降至正常水平。
乙肝相关指标检测情况：
1)患者于2019/11/18(治疗前)和2019/12/16(第1次细胞回输当天)检查肝酶谱与乙肝五项均未见异常；
2)第2次回输后21天(2020/01/09)复查肝酶谱正常；
3)第2次回输后64天(2020/02/20)再次复查发现肝酶谱升高：ALT 65U/L(正常范围0-50U/L)，AST 61U/L(正常范围0-40U/L)，当时未查乙肝五项；
4)第2次回输后115天(2020/04/15)复查，发现肝酶学已降至正常范围，但乙肝五项发现乙肝表面抗体阳性，和核心抗体阳性(次日复查为阴性)，而送检HBV DNA拷贝数结果为阴性。为排除细胞产品HBV污染，于当日外送第三方检测“细胞制备用单采血样本”和“成品细胞样本”，乙肝病毒检测结果均为阴性。
以上肝酶与乙肝五项异常的时间段内，患者自述有轻度纳差，但无恶心，呕吐，肝区涨痛不适，未出现肝大，黄疸等急性传染性感染常见表现。患者肝功与HBV相关指标见图8A。
病例-2(SSY)
男，46岁，乙状结肠癌合并肠周淋巴结转移。测序检测结果TMB 2.3，新抗原7，HLA全杂合。2019年11月15日实施单个核细胞单采，采集细胞总数为4.66x10E9/54ml(制备完成后目前剩余细胞量为1.8x10E9)；治疗细胞制备:ScTIL细胞50ml×2袋，总有效细胞数量9.91x10E8。细胞分两次回输，日期分别为2020/01/02和2020/01/06，两次回输量各为4.955x10E8/50ml，过程顺利。首次回输后7小时出现畏寒、体温升高最高达39.6℃，口服扑热息痛+物理降温后体温降至正常。发热持续3天，自感除乏力外余无不适，此后体温自行降至正常水平。第2次回输后未见明显体温改变与不适。
乙肝相关指标检测信息：
1)患者于2019/12/09(治疗前)，2020/01/06(第2次细胞回输当天)，和2020/01/16(第2次细胞回输后第10天)共3次查肝酶谱均未见异常；治疗前查乙肝五项为阴性。
2)第2次回输后77天(2020/03/23)复查发现肝酶谱升高:ALT 163U/L(正常范围0-50U/L)，AST 113U/L(正常范围0-40U/L)，当时未查乙肝五项；
3)第2次回输后85天(2020/04/01)复查，ALT 743U/L(正常范围0-50U/L)，AST 243U/L(正常范围0-40U/L)，乙肝五项发现乙肝表面抗原阳性，和核心抗体阳性，但核心抗体IgM为阴性；
4)第2次回输后93天(2020/04/09)复查，ALT 301U/L(正常范围0-50U/L)，AST 76U/L(正常范围0-40U/L)，乙肝五项乙肝表面抗原转阴，核心抗体仍为阳性；
5)第2次回输后132天(2020/04/09)复查，ALT与AST均降低至正常范围，未查乙肝五项。
以上肝酶与乙肝五项异常的时间段内，患者自述有轻度纳差，但无恶心，呕吐，肝区涨痛不适，也未出现肝大，黄疸等急性传染性感染常见表现。患者肝功与HBV相关指标见图8B。
对照病例-真实HBV感染(CY)
女，26岁，结肠癌术后伴多发肺部转移，于2019年5月9日接受ScTIL细胞(有效细胞量6.4x10E8)回输治疗。患者于2019年8月1日检测乙肝标志物，结果为阴性；于2019年9月13日有高度可疑血源性HBV接触事件；2019年10月12日检测发现乙肝表面抗原与e抗原阳性，抗体阴性，检测乙肝病毒DNA拷贝数为阳性；给予保肝治疗后于2019年11月12日复查，结果为乙肝抗原与乙肝病毒DNA拷贝数均转阴，而乙肝表面抗体与核心抗体转为阳性。患者肝功与HBV相关指标见图8C。
3.乙肝标志物与肝酶异常与细胞治疗的相关性机理
以上所述两例患者的特点在于，在观察到免疫细胞抗肿瘤疗效的同时，在细胞回输后6-8周检测到肝酶(ALT，AST)升高，一过性HBsAg(表面抗原)转为阳性(持续一周后转阴)，继之以HBsAb/HBcAb(表面抗体/核心抗体)由阴性转为阳性和肝酶降低至正常水平。在此期间，外周血与细胞产品的乙型肝炎病毒(HBV)DNA拷贝数和HBcAb IgM检测均呈阴性，血浆免疫球蛋白(IgG，IgM，IgA)均保持在正常范围内。
发明人认为这种现象与既往HBV感染后少量病毒成分驻留于肝细胞内(图9)，其病毒DNA的复制/组装/出胞程序被宿主增强的免疫机能所静默化有关。HBV感染后，被肝细胞MHC机制提呈的HBV蛋白可以被免疫淋巴细胞所识别，但免疫淋巴细胞对靶细胞的攻击可受到病毒感染肝细胞的免疫杀伤逃逸性机制(肝细胞的PD-L1表达)的抑制，从而阻止了对包含HBV成分的肝细胞的免疫性毁损和HBV清除(图10)，并因此产生了机体免疫机能显著降低时乙肝感染复发的风险。
本申请实施例中采集并用于ScTIL制备的单核细胞通过使用PD-1抗体磁珠进行分选并获得PD-1阳性(PD-1 +)淋巴细胞来制备。如前文所述，这些PD-1 +细胞中包含了特异性识别HBV提呈膜蛋白、但对靶细胞的杀伤活性受到肝细胞所表达PD-L1抑制的CTL亚群。将这些细胞经由慢病毒转染而负载了增强受体由此重新激活其细胞杀伤能力。在并回输后，这些经修饰的细胞(ScTIL)会经由特异性TCR来识别表达HBV蛋白的靶细胞，并且经由细胞膜增强因子即PD-1的胞外域与靶细胞反应性高表达的PD-L1相结合来反向触发ScTIL的进一步活化和对靶细胞的强化杀伤效应。遭CTL攻击而崩解的肝细胞将导致HBV成分释放入外周血流并激发体液免疫和抗体滴度升高，表现为一过性HBsAg阳性和继发性抗体阳性(图11)。由于在既往乙肝病毒感染时，在宿主细胞内已完成组装的活性病毒颗粒不会受到干扰素抑制病毒复制的影响，不会滞留于宿主细胞内，而是经由主动出胞机制释放入血，因此在肝细胞崩解时不会发生活性HBV病毒颗粒被释放入血并发生感染的播散。这一推测已并被HBV DNA和HBcAb IgM的阴性检测结果所证实。
综上所述，发明人根据实验结果推测如下作用机制，ScTIL作为具有特异性抗原识别能力的异质性T淋巴细胞亚群组合，在发挥抗恶性实体肿瘤效应的同时，也可以附带清除既往感染后隐匿残留于部分靶细胞内的病毒成分，从而消除既往病毒感染在未来免疫力低下时复发的潜在风险。
因此，本发明的经修饰的免疫细胞可以在治疗肿瘤患者的肿瘤疾病的同时，清除体内病毒、防止与病毒相关的疾病的复发。
4.用途
基于上述实验发现，以及ScTIL细胞治疗后出现假性HBV感染的临床意义，提出涉及本发明的ScTIL细胞治疗的如下场景和用途。
4.1细胞治疗后假性HBV感染与真实HBV感染(见对照病例)的鉴别诊断
ScTIL细胞治疗后假性HBV感染的特点：
(1)HBV DNA拷贝数低于阳性阈值；
(2)肝酶谱与HBV抗原异常先期出现，持续1-2个月后恢复正常；
(3)HBV抗体相对滞后出现并在较长时间内维持阳性。
真实HBV感染的特点：
(1)HBV DNA拷贝数升高并发于抗原阳性，且呈现高滴度；
(2)肝酶异常通常与HBV抗原的定量异常呈正相关。
4.2使用ScTIL疗法清除残留HBV病毒成分
既往HBV感染痊愈后，部分肝细胞内残留复制过程中的HBV成分，有可能在机体免疫力降低时导致感染复发。
外周血PD-1阳性淋巴细胞中包含特定亚群，可特异性识别表达被提呈到肝细胞膜的HBV膜蛋白成分，但由于被继发性表达PD-L1所抑制而不能杀伤残留病毒成分的靶细胞。
ScTIL的增强受体分子能够克服PD-L1导致的免疫逃逸，清除体内残留的HBV病毒成分，消除了未来感染复发的隐患。
实施例7.增强受体修饰的PD-1 +T细胞治疗与HPV病毒感染相关的疾病
使用如实施例5中所述的方法，基于来自HPV阳性肿瘤患者的外周血样品分选促PD-1 +T细胞并进而制备表达增强受体和任选的CD19 CAR的ScTIL细胞。将制备好的ScTIL细胞回输给患者用于治疗肿瘤并清除HPV感染。

Claims (19)

  1. 一种修饰的免疫细胞，所述免疫细胞是来源于外周血中的PD-1阳性T(PD-1 +T)细胞。
  2. 如权利要求1所述的修饰的免疫细胞，所述PD-1 +T细胞来源于外周血单个核细胞(PBMC)。
  3. 如权利要求1或2所述的修饰的免疫细胞，所述修饰包括基因改造或细胞表面的蛋白修饰。
  4. 如权利要求3所述的修饰的免疫细胞，所述修饰使所述PD-1 +T细胞的PD-1表达缺失或降低，或使PD-1的功能受到抑制。
  5. 如权利要求4所述的修饰的免疫细胞，PD-1表达的所述缺失或降低通过敲除或敲低PD-1基因进行。
  6. 如权利要求4所述的修饰的免疫细胞，对PD-1功能的抑制通过将所述免疫细胞与PD-1抗体在体内或在体外接触进行，所述接触导致所述PD-1抗体结合所述免疫细胞表面的PD-1。
  7. 如权利要求1至6中任一项所述的修饰的免疫细胞，所述修饰的免疫细胞包含增强受体(ER)，所述增强受体包含细胞外结构域(ECD)和细胞内结构域(ICD)，其中所述ECD能够结合所述免疫细胞的靶细胞，所述ICD包含源自引发免疫细胞激活信号的共刺激分子或其片段。
  8. 如权利要求7所述的修饰的免疫细胞，所述ECD包含所述靶细胞的膜蛋白的受体、配体和抗体，或所述靶细胞的膜蛋白的受体、配体和抗体具有与靶细胞结合的功能的部分或片段。
  9. 如权利要求8所述的修饰的免疫细胞，所述ECD包含PD1的部分序列或抗PD-L1抗体优选抗PD-L1 scFv，和/或所述ICD源自CD28。
  10. 如权利要求1至9中任一项的修饰的免疫细胞，其对受试者中的靶细胞具有靶向性。
  11. 如权利要求10所述的修饰的免疫细胞，所述靶细胞选自下组中的一种或多种：肿瘤细胞、癌细胞、受病毒感染的细胞。
  12. 如权利要求11所述的修饰的免疫细胞，所述病毒如肝炎病毒，优选乙型肝炎病毒，或人乳头瘤病毒。
  13. 如权利要求1至12中任一项所述的修饰的免疫细胞，其进一步表达嵌合抗原受体 (CAR)，所述CAR特异性识别与所述免疫细胞的天然TCR不同的另一种抗原，优选CD19。
  14. 如权利要求1至13中任一项所述的修饰的免疫细胞，其进一步包含其他调控免疫细胞死亡的修饰，如自杀开关。
  15. 包含如权利要求1至14中任一项所述的修饰的免疫细胞的细胞群体。
  16. 一种制备治疗性免疫细胞的方法，所述方法包括：
    (a)从外周血分选PD-1 +的T细胞；和
    (b)对所述步骤(a)分选的PD-1 +T细胞进行如下一种或多种处理：
    i.敲除或敲低PD-1的表达；
    ii.与PD-1抗体混合；
    iii.使其表达增强受体(ER)；
    iv.使其表达嵌合抗原受体(CAR)；
    v.使其表达自杀开关。
  17. 通过权利要求19的方法获得的修饰的免疫细胞。
  18. 一种组合物，包含权利要求1-14或17中任一项所述的免疫细胞或权利要求15的细胞群体。
  19. 如权利要求18的组合物，其用于
    (1)治疗肿瘤，和/或
    (2)治疗或预防与病毒感染相关的疾病或症状，或防止与病毒感染相关的疾病或症状的复发。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107249606A (zh) * 2014-10-31 2017-10-13 宾夕法尼亚大学董事会 改变修饰的t细胞中的基因表达及其用途
US20180044424A1 (en) * 2015-02-02 2018-02-15 Novartis Ag Car-expressing cells against multiple tumor antigens and uses thereof
CN110016465A (zh) * 2018-01-09 2019-07-16 北京卡替医疗技术有限公司 一种包含b细胞及肿瘤双识别性t细胞的免疫细胞药物
CN110452870A (zh) * 2019-05-20 2019-11-15 河南省肿瘤医院 一种肿瘤特异性t细胞的分离培养方法及由其获得的产品
CN111542595A (zh) * 2017-11-10 2020-08-14 北京卡替医疗技术有限公司 经修饰的免疫细胞及其用途
CN112266899A (zh) * 2020-10-26 2021-01-26 北京卡替医疗技术有限公司 修饰的免疫细胞及其用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105153315B (zh) * 2015-10-09 2019-04-02 重庆精准生物技术有限公司 免疫抑制受体联合肿瘤抗原嵌合受体及其应用
JP2020503015A (ja) * 2016-12-13 2020-01-30 カースゲン セラピューティクス リミテッドＣａｒｓｇｅｎ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｌｉｍｉｔｅｄ 抗ｃｄ１９のヒト化抗体及びｃｄ１９を標的とする免疫エフェクター細胞
CN108085340B (zh) * 2017-12-07 2020-07-07 北京市肿瘤防治研究所 一种同时表达靶向cd19和cd20的car与pd1-cd28嵌合受体的慢病毒载体
CN110079502B (zh) * 2018-02-07 2020-11-06 阿思科力(苏州)生物科技有限公司 Pd-l1 car-nk细胞及其制备和应用
JP7170737B2 (ja) * 2018-02-27 2022-11-14 レイドス， インコーポレイテッド Ｐｄ－１ペプチド阻害剤
CN111686128A (zh) * 2019-03-12 2020-09-22 重庆精准生物技术有限公司 一种缺氧可调控的启动子在car-t中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107249606A (zh) * 2014-10-31 2017-10-13 宾夕法尼亚大学董事会 改变修饰的t细胞中的基因表达及其用途
US20180044424A1 (en) * 2015-02-02 2018-02-15 Novartis Ag Car-expressing cells against multiple tumor antigens and uses thereof
CN111542595A (zh) * 2017-11-10 2020-08-14 北京卡替医疗技术有限公司 经修饰的免疫细胞及其用途
CN110016465A (zh) * 2018-01-09 2019-07-16 北京卡替医疗技术有限公司 一种包含b细胞及肿瘤双识别性t细胞的免疫细胞药物
CN110452870A (zh) * 2019-05-20 2019-11-15 河南省肿瘤医院 一种肿瘤特异性t细胞的分离培养方法及由其获得的产品
CN112266899A (zh) * 2020-10-26 2021-01-26 北京卡替医疗技术有限公司 修饰的免疫细胞及其用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
QING LI, HONG-MEI WANG, ZHANG YU-JUAN, AN-WEN LIU, WEI-PING MIN: "The Role of Exhausted T Cells in Tumor", PROGRESS IN MODERN BIOMEDICINE, XIAN DAI SHENG WU YI XUE JIN ZHAN BIAN JI BU, CN, vol. 17, no. 9, 31 March 2017 (2017-03-31), CN , pages 1788 - 1792, XP055751398, ISSN: 1673-6273, DOI: 10.13241/j.cnki.pmb.2017.09.051 *
SUGITA SUNAO, USUI YOSHIHIKO, HORIE SHINTARO, FUTAGAMI YURI, ABURATANI HIROYUKI, OKAZAKI TAKU, HONJO TASUKU, TAKEUCHI MASARU, MOCH: "T-Cell Suppression by Programmed Cell Death 1 Ligand 1 on Retinal Pigment Epithelium during Inflammatory Conditions", INVESTIGATIVE OPTHALMOLOGY & VISUAL SCIENCE, ASSOCIATION FOR RESEARCH IN VISION AND OPHTHALMOLOGY, US, vol. 50, no. 6, 1 June 2009 (2009-06-01), US , pages 2862, XP055925195, ISSN: 1552-5783, DOI: 10.1167/iovs.08-2846 *
XIE, LUHONG: "Research Progress in Immune Checkpoint PD-1 and Antibodies for Same in Tumors", JIANYAN YIXUE YU LINCHUANG = LABORATORY MEDICINE AND CLINIC, CHONGQING SHI WEISHENG XINXI ZHONGXIN, CN, vol. 14, no. 9, 31 May 2017 (2017-05-31), CN , pages 1353 - 1355, 1362, XP009536062, ISSN: 1672-9455 *

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