CN114746545A

CN114746545A - Engineered erythrocytes using artificial MHC to present specific cancer neoantigens

Info

Publication number: CN114746545A
Application number: CN202080072937.3A
Authority: CN
Inventors: 高晓飞; 黄彦杰; 聂小千; 赵雨佳
Original assignee: West Lake Biomedical Technology Hangzhou Co ltd
Current assignee: West Lake Biomedical Technology Hangzhou Co ltd
Priority date: 2019-10-18
Filing date: 2020-10-16
Publication date: 2022-07-12
Also published as: WO2021073613A1

Abstract

The present invention provides methods for producing red blood cells or engineered red blood cells, and more particularly, engineered red blood cells (eRBCs) having artificial MHC molecules. These methods would make eRBCs a novel antigen presenting cell useful for modulating immune responses in cancer or immune diseases.

Description

Engineered erythrocytes using artificial MHC to present specific cancer neoantigens

Technical Field

The present disclosure relates to methods for producing red blood cells or engineered red blood cells, and more particularly, to engineered red blood cells with artificial MHC molecules.

Background

Effective anti-tumor immunity in humans has been associated with the presence of T cells against cancer neoantigens, a class of HLA-bound peptides caused by tumor-specific mutations, and recent data suggest that the recognition of such neoantigens is a major factor affecting the activity of clinical immunotherapy. Massively parallel whole exome sequencing has been used to detect all mutations in tumors to predict neoantigens. Vaccination with neoantigens can expand pre-existing populations of neoantigen-specific T cells and induce new cancer-specific T cells. Although neoantigens have become a potentially desirable target for anti-tumor immune responses, many questions remain to be answered prior to clinical use.

T cell activation requires MHC molecules to present MHC restricted peptides to specific T cell receptors. The lack of a specific antigen presentation system is one of the problems of neoantigen vaccination.

Red Blood Cells (RBCs) are the most abundant cell type in blood, accounting for one-fourth of the total number of human cells. Red blood cells have many unique properties that make them attractive tools for the in vivo delivery of natural and synthetic cargo (payload): the circulation range is wide (red blood cells pass through the whole blood circulation system of the body); good biocompatibility; long circulation half-life (about 120 days in humans); a large surface area to volume ratio; no nuclei and mitochondria (no ability to synthesize, proliferate, mutate protein).

Engineered red blood cells (eRBCs) are attractive vehicles for introducing new therapeutic agents, immunomodulators and diagnostic imaging probes into the human body. Human red blood cells can be produced from hematopoietic stem cell cultures, but the source of hematopoietic stem cells limits clinical applications.

Summary of The Invention

In one aspect, the present disclosure provides a method of producing Red Blood Cells (RBCs), comprising:

1) collection of lineage negative and CD34 negative cells (lin) from blood samples^-CD34^-A cell),

2) amplifying the lin^-CD34^-A cell; and

3) amplified-induced lin^-CD34^-The cells differentiate into mature red blood cells.

In some embodiments, the blood sample is a peripheral blood sample, an umbilical cord blood sample, or a fetal blood sample.

In some embodiments, the blood sample is a human peripheral blood sample.

In some embodiments, step 1) comprises isolating Peripheral Blood Mononuclear Cells (PBMC) from a peripheral blood sample and isolating lin from PBMC^-CD34^-A cell.

In some embodiments, step 1) comprises removing lineage positive (lin) from a blood sample by using a lineage cell removal kit⁺) A cell.

In some embodiments, step 2) comprises culturing the lin in hematopoietic stem cell expansion medium supplemented with a combination of cytokines^-CD34^-A cell, wherein the cytokine combination comprises fms-like tyrosine kinase 3 ligand (Flt3L), Stem Cell Factor (SCF), interleukin 3(IL-3), and interleukin 6 (IL-6).

In some embodiments, the cytokine combination comprises 50ng/mL human Flt3L, 50ng/mL human SCF, 10ng/mL human IL-3, and 10ng/mL human IL-6.

In some embodiments, the hematopoietic stem cell expansion medium is StemSpan^TMSFEM serum-free amplification medium.

In some embodiments, step 2) comprises 5% CO at 37 ℃₂Bottom culture lin^-CD34^-Cells were cultured for about 2-5 days.

In some embodiments, step 3) comprises:

i) culturing the amplified lin^-CD34^-Inducing the cells to differentiate into erythroid cells (erythroid cells); and

ii) culturing the erythroid cells to induce enucleation.

In some embodiments, step i) comprises culturing the expanded lin in a first differentiation medium supplemented with a cytokine associated with erythroid development^-CD34^-A cell.

In some embodiments, the cytokines associated with erythroid development include IL-3 and SCF.

In some embodiments, the first differentiation medium is Iscove Modified Dulbecco Medium (IMDM) containing Fetal Bovine Serum (FBS), human plasma, glutamine, BSA, transferrin, insulin, penicillin-streptomycin, IL-3, EPO, and SCF.

In some embodiments, the first differentiation medium is Iscove's Modified Dulbecco's Medium (IMDM) containing 10-15% FBS, 5-10% human plasma, 1-4mM glutamine, 1-2% BSA, 300-600. mu.g/mL human transferrin, 8-13. mu.g/mL human insulin, 2% penicillin-streptomycin, 3-5ng/mL human IL-3, 4-7U/mL human EPO, and 100ng/mL human SCF.

In some embodiments, step i) comprises 5% CO at 37 ℃₂Bottom culture of amplified lin^-CD34^-Cells were left for about 9 days.

In some embodiments, step ii) comprises culturing the erythroid cells in a second differentiation medium, wherein the second differentiation medium lacks cytokines associated with erythroid development compared to the first differentiation medium.

In some embodiments, the second differentiation medium is Iscove Modified Dulbecco Medium (IMDM) containing FBS, human plasma, glutamine, BSA, transferrin, insulin, penicillin-streptomycin, and EPO.

In some embodiments, the second differentiation medium is Iscove's Modified Dulbecco's Medium (IMDM) containing 15% FBS, 5-10% human plasma, 1-4mM glutamine, 1-2% BSA, 300-600. mu.g/mL human transferrin, 8-13. mu.g/mL human insulin, 2% penicillin-streptomycin, and 1-5U/mL human EPO.

In some embodiments, step ii) comprises 5% CO at 37 ℃₂Erythroid cells were cultured for about 7 days.

In another aspect, the present disclosure provides red blood cells produced by the above-described methods.

In another aspect, the present disclosure provides methods for producing engineered red blood cells (edrbs), comprising:

1) collection of lineage negative cells (lin) from blood or bone marrow samples^-Cells);

2) amplifying the lin^-A cell;

3) culturing the amplified lin^-Inducing the cells to differentiate into erythroid cells; and introducing a foreign nucleic acid into the amplified lin prior to or simultaneously with differentiation^-A cell; and

4) the erythroid cells are cultured to induce enucleation.

In some embodiments, the blood sample is a human peripheral blood sample.

In some embodiments, the lin^-The cell is lin^-CD34^-A cell.

In some embodiments, step 1) comprises isolating PBMCs from a peripheral blood sample and isolating lin from PBMCs^-CD34^-A cell.

In some embodiments, step 1) comprises removing lineage positive (lin) from a peripheral blood sample by using a lineage cell removal kit⁺) Thin and thinAnd (4) cells.

In some embodiments, step 2) comprises culturing the lin in hematopoietic stem cell expansion medium supplemented with a combination of cytokines^-A cell, wherein said cytokine combination comprises Flt3L, SCF, IL-3, and IL-6.

In some embodiments, step 2) comprises 5% CO at 37 ℃₂Bottom culture lin^-Cells were cultured for about 2-5 days.

In some embodiments, step 3) comprises culturing the expanded lin in a first differentiation medium supplemented with a cytokine associated with erythroid development^-A cell.

In some embodiments, the first differentiation medium is Iscove's Modified Dulbecco's Medium (IMDM) containing FBS, human plasma, glutamine, BSA, transferrin, insulin, penicillin-streptomycin, IL-3, EPO, and SCF.

In some embodiments, step 3) comprises 5% CO at 37 ℃₂Bottom culture of amplified lin^-Cells were left for about 9 days.

In some embodiments, step 4) comprises culturing the erythroid cells in a second differentiation medium, wherein the second differentiation medium lacks cytokines associated with erythroid development compared to the first differentiation medium.

In some embodiments, step 4) comprises 5% CO at 37 ℃₂Erythroid cells were cultured for about 7 days.

In some embodiments, in step 3), the amplified lin is cultured in a first differentiation medium^-The first day of cell, exogenous nucleic acid was introduced into the amplified lin^-In the cell.

In some embodiments, the exogenous nucleic acid is a nucleic acid that carries a lin intended for amplification^-An expression vector for a gene of interest to be expressed in a cell.

In some embodiments, the expression vector is a lentiviral expression vector.

In some embodiments, the gene of interest encodes a fusion protein.

In some embodiments, the fusion protein is a cell surface membrane protein comprising an anchor moiety, wherein the anchor moiety comprises at least the transmembrane region of CD235 a.

In some embodiments, the fusion protein comprises an artificial MHC single chain molecule and comprises, from N-terminus to C-terminus, an antigenic peptide, a first peptide linker, a β 2-microglobulin, a second peptide linker, and an MHC class I heavy chain that lacks a transmembrane region and a cytoplasmic region.

In some embodiments, the artificial MHC single chain molecule is fused at its C-terminus to the N-terminus of the anchor moiety, optionally via a third peptide linker.

In some embodiments, the fusion protein further comprises a signal peptide selected from the group consisting of a β 2-microglobulin signal peptide, a CD235a signal peptide, or a combination thereof.

In some embodiments, the first and second peptide linkers are Gly and Ser rich.

In some embodiments, the antigenic peptide is associated with a disorder and is capable of activating CD8 when presented by an MHC class I molecule⁺T cells.

In some embodiments, the antigenic peptide is a cancer neoantigen, or derived from an oncoprotein or a viral protein.

In some embodiments, the antigenic peptide is 8, 9, 10, or 11 amino acids in length.

In another aspect, the present disclosure provides edrbs produced by the above-described methods.

In another aspect, the present disclosure provides edrcs comprising a fusion protein comprising, from N-terminus to C-terminus, an antigenic peptide, a first peptide linker, a β 2-microglobulin, a second peptide linker, a heavy chain of an MHC class I molecule lacking a transmembrane region and a cytoplasmic region, a third peptide linker, and an anchor moiety, wherein the antigenic peptide is associated with a disorder and is capable of activating CD8 when presented by the MHC class I molecule⁺A T cell, and wherein the anchoring moiety comprises at least the transmembrane region of CD235 a.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the edrcs of the present disclosure and a physiologically acceptable excipient.

In another aspect, the present disclosure provides for the use of the edrcs of the present disclosure in the manufacture of a medicament for treating a disorder associated with an antigenic peptide.

In another aspect, the present disclosure provides a method for treating a disorder associated with an antigenic peptide in a subject, comprising:

a) collecting a blood sample or a bone marrow sample from a subject,

b) production of eRBCs and Using the methods of the present disclosure

c) Infusing a therapeutically effective amount of the eRBCs into the subject.

In some embodiments, the antigenic peptide is a fragment of HPV E6 or E7 protein.

In another aspect, the disclosure provides murine eRBCs comprising a peptide having the sequence of SEQ ID NO. 2 or 4.

Brief Description of Drawings

FIG. 1: design of artificial eRBCs antigen presentation system.

(A) The artificial eRBCs antigen presentation system utilizes engineered erythrocytes to express chimeric MHC class I molecules linked to specific neoantigenic peptides and fused to erythroid cell membrane proteins such as CD235a or other proteins. These engineered eRBCs can present neoantigens to neoantigen-specific memory T cells and activate the function of the T cells.

(B) Construction of the OT-1 peptide-microglobulin-MHC-CD 235a construct (upper and middle panels) and proposed conformation (lower panel) (the nucleic acid and amino acid sequences of which are shown in SEQ ID NOS: 1-4). The chimeric MHC molecule designed consisted of a membrane localization signal peptide (upper panel: b2m signal peptide; middle panel: CD235a signal peptide linked to b2m signal peptide), a specific peptide for T cell recognition (such as neoantigen) linked by a glycine/serine linker, beta 2-microglobulin and MHC heavy chain region. Since the MHC heavy chain lacks a transmembrane domain, CD235a fusion ensures transfer of the protein complex to the cell membrane. (OT-1: ovalbumin peptide residue 257-264(OVA 257-264); b2m, beta-2 microglobulin; Pep, peptide; T2A, 2A self-cleaving peptide; copGFP, green fluorescent protein cloned from the copepodia animal Pontellina plumata; beta: using the beta 2-microglobulin signal peptide; alpha. beta: using CD235a and the beta 2-microglobulin signal peptide).

(C) Workflow of ebbc treatment. A peripheral blood sample is taken from an individual. Separated LIN^-CD34⁺HSCs or LIN^-CD34^-PBMCs transduced with lentiviruses encoding chimeric MHC antigen presenting molecules. The transduced HSCs or PBMCs are cultured under conditions that induce erythroid proliferation and differentiation. Terminally differentiated (enucleated) eRBCs are returned to the patient for therapeutic purposes.

FIG. 2. Generation and characterization of eRBCs expressing chimeric MHC molecules.

(A) Display LIN^-CD34^-Microscopy images of transduction efficiency of PBMCs. Transduction of LIN with lentiviral vectors on day 5^-CD34^-PBMCs, the lentiviral vector encoding the MSCV promoter alone (control) or encoding MSCV-MHC-I OT1 β. The efficiency of viral infection was checked 48 hours after transduction. GFP signal indicates positively transduced cells. LIN in alpha beta transduction with MSCV-MHC-I OT1^-CD34⁺HSCs, LIN transduced with MSCV-MHC-I OT1 beta^-CD34⁺HSC, LIN transduced with MSCV-MHC-I OT1 alpha beta^-CD34^-Similar results can be obtained in PBMCs.

(B) Cell proliferation assay. During the erythroid culture period, LIN with or without lentiviral transduction was evaluated every three days using Invitrogen Countess II^-Cell number of PBMC.

(C) Erythroid differentiation assay. Flow cytometry was used to examine the expression levels of cell surface markers every two days during the culture of the erythroid lines to indicate the progress of erythroid differentiation. Cells were stained with antibodies against human CD235a, CD71, and CD 117.

(D) Enucleation analysis of eRBCs at the end of red line culture. The DNA dye Hoechst33342 was used to stain DNA. Enucleated eRBCs stained positive for CD235a and negative for Hoechst 33342. Approximately 80% of enucleated eRBCs are GFP positive, indicating that the engineered protein remains expressed until the terminal differentiation and enucleation stage.

(E) Benzidine-giemsa staining showed cell morphology of the edrbs on day 21 of culture.

FIG. 3: in vivo distribution of eRBCs expressing chimeric MHC molecules. eRBCs were derived from LIN transduced with MSCV-MHC-I OT1 beta^-CD34^-PBMC. 1X 10 prestained with DiR⁸edrcs, injected intravenously into MC38 tumor-loaded NSG mice. In vivo fluorescence imaging analysis was performed 7 days after the injection of the eRBCs. (left panel): representative in vivo imaging of the abdomen, back and sides of 1 mouse. (right panel): eRBCs distribution in each organ. LIN in alpha beta transduction with MSCV-MHC-I OT1^-CD34⁺HSCs, LIN transduced with MSCV-MHC-I OT1 beta^-CD34⁺HSCs, LIN transduced with MSCV-MHC-I OT1 alpha beta^-CD34^-In PBMCs, similar results can be obtained.

FIG. 4: evaluation of antigen presenting Capacity of eRBCs in vitro

(A) Experimental protocol for in vitro T cell activation. Mouse erythroid progenitors (BFU-Es/CFU-Es) were isolated from E13.5-14.5 fetal livers. These mouse erythroid progenitors were transduced with lentivirus encoding MSCV-MHC I α β OT1 and induced to differentiate into edrcs-MHC I α β OT 1. Mouse CD8 from OT1 (C57BL/6-Tg (TcraTcrb)1100Mjb/J⁺T cells recognize primarily OVA presented by MHC I_257-264) Separating CD8⁺T cells. C of eRBCs-MHC I alpha beta OT1 and OT1 miceD8⁺T cells were co-cultured for 2 days and both CD8 were analyzed by FACS⁺T cell activation markers, CD107a (B) and CD44 (C). (D) OT1 CD8⁺T cells alone (upper left) or OT1 CD8⁺Morphology of T cells and edrbs co-cultures under bright field microscopy. White arrow: activated T cell clusters. eRBC CD8⁺The cell number ratio of T cells was 1: 10.

Detailed Description

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, an element refers to one element or more than one element.

As used herein, the term "lineage negative cell" or "Lin^-By cell "is meant a cell that is substantially free of lineage markers. Lineage markers are characteristic of the cell lineage. Exemplary lineage markers are CD1c, CD3, CD11c, CD14, CD15, CD16, CD20, CD41, CD56, CD203c, CD235a, and/or BDCA 2. In fact, lineage negative cells are essentially not stained by lineage antibodies. Lineage negative cells include stem cells and progenitor cells. Thus, lineage negative cells also show stem and progenitor cell activity. Lin negative cells or a blood cell population enriched for lineage negative cells can be purified by enriching the blood cell population substantially free of lineage markers. For example, lineage negative cells can be purified by removing cells positive for at least one lineage marker selected from: CD1c, CD3, CD11c, CD14, CD15, CD16, CD20, CD41, CD56, CD203c, CD235a, and/or BDCA 2. In some cases, the lineage cell removal kit can be used for purification. In contrast, lineage positive (Lin)⁺) The cells are a mixture of all cells expressing markers of the mature cell lineage. Examples of lineage Positive cellsIncluding T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, erythroid cells and their committed precursors.

As used herein, the term "antigenic peptide" refers to a peptide that is capable of binding to the peptide-binding domain of an MHC molecule (particularly an MHC class I molecule) and thereby forming an MHC complex with the MHC molecule. As is well known in the art, presentation of antigenic peptides in MHC complexes on the surface of APCs does not typically involve, for example, intact proteins. In contrast, antigenic peptides located in the binding domain are typically small fragments of the entire protein. In some embodiments, the antigenic peptide is derived from a pathogen protein, such as a viral protein. In some embodiments, the antigenic peptide is a cancer neoantigen.

As used herein, the term "artificial MHC single chain molecule" or "artificial MHC" refers to a fusion protein comprising an antigenic peptide, a β 2 microglobulin, and an MHC class I heavy chain. Such fusion proteins are also referred to in some literature as "single chain trimers". Typically, there is a peptide linker between the antigenic peptide and the beta-2 microglobulin, and a peptide linker between the beta-2 microglobulin and the heavy chain of the MHC class I molecule. Thus, an artificial MHC single chain molecule may comprise, from N-terminus to C-terminus, an antigenic peptide, a first peptide linker, a β -2 microglobulin, a second peptide linker and an MHC class I molecule heavy chain. In some embodiments, the artificial MHC single chain molecule may further comprise a signal peptide at the N-terminus. In some embodiments, the MHC class I heavy chain portion of the artificial MHC single chain molecule is a truncated form, lacking a transmembrane region and a cytoplasmic region. In some embodiments of the disclosure, the artificial MHC single chain molecule is fused C-terminally to a CD235a molecule or a fragment thereof comprising at least a transmembrane region. In this protocol, once in the host cell (e.g., Lin)^-CD34^-PBMCs), artificial MHC single-chain molecules can be anchored to the cell membrane by CD235a molecules or fragments thereof.

As used herein, the term "culturing" refers to maintaining cells in culture for any period of time, whether or not the cells expand or differentiate.

As used herein, the term "differentiation" refers to the process by which less specialized cells (e.g., stem cells) develop or mature to have a more unique form and function with concomitant loss of potential. Cells of lower degree of specialization can be differentiated into cells of higher degree of specialization by culturing the cells in specific conditions or specific media known in the art.

As used herein, the term "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition, or carrier, e.g., a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium), solvent, or encapsulating material, that is involved in carrying or transporting a therapeutic compound for administration to a subject. Each excipient should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

CD235a, also known as "glycophorin a", is a single transmembrane glycoprotein expressed in mature red blood cells and erythroid precursor cells, a specific marker protein on the surface of red blood cells. Expression of CD235a indicates differentiation of the cells into erythroid cells.

CD117, also known as "c-kit", is a receptor for SCF stem cell growth factor expressed on the surface of hematopoietic stem cells and other cells. SCFs play an important role in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, cell development, migration, and function.

CD71, also known as "transferrin receptor 1", is a transmembrane glycoprotein composed of two monomers linked by two disulfide bonds. Each monomer binds to the intact transferrin molecule, producing an iron-transferrin receptor complex which enters the cell by endocytosis for the production of cellular hemoglobin during erythroid development.

In this study, we generated a novel series of methods that utilized lineage negative CD34 negative peripheral blood mononuclear cells (Lin)^-CD34^-PBMCs) or lineage negative CD34 positive hematopoietic stem cells (Lin)^-CD34⁺HSCs) to induce erythroid cell proliferation and differentiation, thereby laying the foundation for treatment of the edrcs. Genes encoding cell surface proteins fused to various engineered proteins are introduced into these hematopoietic stem and progenitor cells by lentiviral transduction. Then inducing erythroid differentiation and enucleationCulturing the cell under the conditions of (1). These enucleated reticulocytes or RBCs carrying the expressed therapeutic proteins can be used as long-term drug carriers for a variety of therapeutic purposes.

Classical antigen presentation utilizes APC to display antigenic peptides. The acquisition of the antigen is equivalent to the production of the wrong protein, which is a mutation of the protein caused by a non-synonymous mutation. Thus, to generate a personalized edrc-antigen presentation system, we performed Whole Exome Sequencing (WES) and RNA-seq to identify and predict tumor-specific neo-antigens. For the generation of antigen presentation systems, MHC molecules are essential components; therefore, we designed an artificial MHC, which is a single-chain unit composed of neoantigenic peptide, β 2 microglobulin, and the heavy chain of an MHC class I molecule (fig. 1B). This recombinant peptide, consisting of a single neoantigenic peptide- β 2-microglobulin-MHC I chain, can be recognized by peptide-specific T cells (fig. 1A). Under normal conditions, MHC molecules will be removed when reticulocytes differentiate into mature erythrocytes. To avoid removal of MHC molecules during differentiation, we chose to fuse an engineered peptide containing MHC with the mature red blood cell surface membrane protein CD235 a. This means that in our system only the specific neo-antigen is presented on the surface, a property that will greatly improve the efficiency of T cell recognition and activation.

Several publications have demonstrated that mature red blood cells can be derived from hematopoietic stem cells (LIN)^-CD34⁺HSCs) differentiate with varying efficiencies.

Example 1 from Lin^-CD34^-PBMCs or Lin^-CD34⁺Preparation of HSCs and characterization of RBCs or eRBCs

Lentiviral vector construction

Gene synthesis to obtain the designed MHC I OT1 gene. These sequences were constructed on lentiviral vector MSCV to give the sequence vector MSCV-MHC-I OT1 α β or MSCV-MHC-I OT1 β for viral packaging.

Virus package

Viral packaging was performed using MHC-I OT1: pSPAX2: VSVG ═ 2:1:1 ratio packaging. Take a 6-well plate as an example. According to MHC-I OT1: pSPAX2: VSVG 2 μ g 1 μ g by calcium phosphate transfection method. The plasmids were transfected into HEK 293T cells for viral packaging. 12 hours after transfection, calcium phosphate was removed by changing the medium, supernatants were collected at 48 hours and 72 hours, and cell culture supernatants were filtered through 0.45 μm filters.

Concentration of virus

The collected and filtered cell culture supernatant was ultracentrifuged and concentrated at 70000RCF at a temperature of 4 ℃ for 2 hours. After centrifugation, the supernatant was removed, the pellet resuspended in differentiation stage 1 medium, and the viral titer quantified by ELISA, stored at-80 ℃.

Lin^-CD34^-PBMCs isolation

Lin separation from human peripheral blood^-CD34^-PBMCs. Whole blood was diluted 1:1 with phosphate buffer solution, and lymphocyte separation solution (Lymphoprep) was used^TMStem cell Technologies) and lymphocyte separation tubes, and Peripheral Blood Mononuclear Cells (PBMCs) were isolated by centrifugation at 1200 × g for 15 minutes.

The purpose of the step is to realize the density gradient centrifugation of cell components through lymphocyte separation liquid, and separate PBMCs from different cells such as red blood cells, platelets and the like so as to ensure Lin^-CD34^-Subsequent enrichment of the cells. Lin isolation Using human lineage cell depletion kit (BD Biosciences)^-CD34^-A cell.

Lin^-CD34⁺Separation of HSCs

Whole blood was diluted 1:1 with phosphate buffer, and pre-enriched HSC were isolated by centrifugation at 1200 Xg for 15 minutes in lymphocyte separation tubes using lymphocyte separation solution (LymphoprpTM, STEMCELL Technologies).

The purpose of this step is to achieve density gradient centrifugation of the cellular components through the lymphocyte separation solution, separating the pre-enriched HSCs from other cell types such as red blood cells and platelets, to ensure Lin^-CD34⁺Subsequent enrichment of HSCs. Application of EasySep^TMHuman cord blood CD34 positive selection kit II (stemcell technologies) for CD34 isolation from pre-enriched HSC⁺HSCs. Will CD34⁺Cells were incubated with biotin-labeled antibodies against lineage specific antigens. Removal of spectra with specificityMarker-linked CD34⁺Cells and Lin was obtained by negative selection using streptavidin conjugated magnetic beads^-CD34⁺HSCs。

Purification of fetal liver erythroid progenitor cells from mouse embryos

1. Timed matings were set to obtain female mice 13.5-14.5 days of gestation.

2. Fetal Liver Cells (FLC) were isolated from each individual embryo on ice and placed in 1mL PBS.

3. Pipette up and down to obtain a single cell suspension, and pass through a25 μm filter (BD Falcon 35-2235). The screen was rinsed with 1ml PBS. Transudates (1 ml per embryo) were pooled.

4. The cells were pelleted by centrifugation at 1.5k RPM for 5 minutes, resuspended in red blood cell lysis buffer (ammonium chloride solution from Stemcell), and placed on ice for 10 minutes.

5. Cells were pelleted by centrifugation at 1.5k RPM for 5 minutes, lysis buffer removed, and resuspended in 10mL PBS-2% FBS.

6. Cells were counted by adding Chrompure rat IgG (Jackson ImmunoResearch, #012-⁴Cell number/mL).

7. Adding 1. mu.L/1X 10⁶Biotinylated anti-mouse TER119(BD Pharmingen, #553672) from individual cells was incubated for 15 minutes at 4 ℃.

8. Ms Lineage Panel (Fisher Scientific (Thermo Fisher Scientific) # BDB559971) was added to the cells (2. mu.L/1X 10)⁶Individual cells), incubated at 4 ℃ for 15 minutes.

9. Wash once with 10 volumes of PBS and centrifuge the cells at 1.5k RPM for 5 minutes at 4 ℃.

10. Streptavidin Particles Plus-DM (magnetic beads) (BD Pharmingen, #557812) (5. mu.L/1X 10) were added⁶Individual cells), incubated at 4 ℃ for 30 minutes.

11. 2-4 FACS tubes were prepared on magnetic scaffolds, paired on both sides.

12. 2mL of cells (4 mL total) were packed in each tube, and the cells were carefully removed from the tube and placed in another tube on the other side, avoiding disruption of the magnetically adhered beads. The same procedure was repeated, and Ter 119-negative and lineage-negative cells were transferred to new tubes.

13. Viable cells were counted and concentrated, and the cells were resuspended in 50-100. mu.L PBS (2% FBS).

Lentiviral transduction of LIN^-CD34⁺HSCs or LIN^-CD34^-PBMCs

LIN was transduced 5 days after amplification using MSCV-MHC-I OT1 virus^-CD34⁺HSCs or LIN^-CD34^-PBMCs. Differentiation phase 1 Medium was used at 1X 10⁶Resuspend the cells at the density of (c). The infection volume and virus dose were calculated. According to the final concentration of 5X 10⁷TU/ml to 5X 10⁸TU/mL, calculating the virus dosage; the mixture was incubated with Polybrene in concentrated virus solution for 5 min at 10 μ g/mL, depending on the volume of infection. The concentrated virus solution after incubation was added to the cells and mixed. The infection was performed by centrifugation using a horizontal rotor centrifuge at a speed of 500 Xg, a temperature of 32 ℃ and a time of 90 minutes. After centrifugation, the cells were incubated at 37 ℃ with 5% CO₂And (5) culturing. After two days, the virus infection efficiency was examined under a fluorescence microscope. The percentage of GFP positive cells indicates positively transduced cells.

Culture of eRBCs

Culturing the isolated Lin on day 0^-CD34^-Or Lin^-CD34⁺Culturing the cells in hematopoietic stem cell expansion medium (StemBan)^TMSFEM, stem cell Technologies) and add cytokine combinations and penicillin-streptomycin (Gibco). Cells were incubated at 37 ℃ with 5% CO₂And (4) culturing. Cultured under these culture conditions until day 5.

The purpose of this step is to perform the step at StemBan^TMAddition of cytokine combinations to SFEM serum-free expansion medium to expand and maintain Lin prior to induction of erythroid differentiation^-CD34^-Or Lin^-CD34⁺A cell. The cytokine combination comprises 50ng/mL of recombinant human fms-like tyrosine kinase 3 ligand (Flt3L), 50ng/mL of recombinant human Stem Cell Factor (SCF), 10ng/mL of recombinant human interleukin 3(IL-3), 10ng/mL of recombinant human interleukin 6(IL-6) and the like.

Culture change on day 5The culture system is characterized in that a culture medium is replaced by a differentiation stage 1 culture medium, and the culture medium consists of the following components: IMDM (Iscove modified Dulbecco's medium, Sigma-Aldrich), 10-15% fetal bovine serum (FBS, Gibco), 5-10% human Plasma (Plasma), 1-4mM glutamine, 1-2% BSA (bovine serum albumin), 300-600. mu.g/mL human transferrin (Holo human transferrin, Sigma-Aldrich), 8-13. mu.g/mL recombinant human insulin (Sigma-Aldrich), 2% penicillin-streptomycin (Gibco), 3-5ng/mL recombinant human interleukin III (rhIL-3, Peprotech), 4-7U/mL recombinant human erythropoietin (rhEpo, Amgen), 100ng/mL recombinant human stem cell factor (rhSCF, Peprotech). Cells were incubated at 37 ℃ with 5% CO₂And (5) culturing. Cultured under these culture conditions until day 14. The purpose of this experimental procedure was to induce Lin in the presence of sufficient erythroid-associated cytokines^-CD34^-Or Lin^-CD34⁺The cells differentiate into erythroid cells and substantial expansion is achieved. In this culture system, only cytokines relevant to erythroid development are provided, and cell proliferation and differentiation into erythroid cells are ensured. At the same time, viral infection occurs at the fastest stage of differentiation, ensuring target gene insertion and expression on the cell membrane.

On day 14, the culture system was changed and the medium was changed to differentiation phase 2 medium consisting of IMDM (Iscove modified Dulbecco's medium, Sigma-Aldrich), 15% fetal bovine serum (FBS, Gibco), 5-10% human Plasma (Plasma), 1-4mM glutamine, 1-2% BSA, 300-600. mu.g/mL human transferrin (Sigma-Aldrich), 8-13. mu.g/mL recombinant human insulin (Sigma-Aldrich), 2% penicillin-streptomycin (Gibco), 1-5U/mL recombinant human erythropoietin (rhEpo, Amgen). Cells were incubated at 37 ℃ with 5% CO₂And (5) culturing. Cultured under these culture conditions until day 21. The purpose of this experimental procedure is to reduce or eliminate some of the cytokines used under previous culture system conditions to promote enucleation, which is the final step in red blood cell maturation.

Benzidine-giemsa staining

1. After cytospin, cells were fixed with-20 ℃ methanol for 2 min at RT.

2. Washed with water and air dried. (slides can be stored at RT for later staining.)

3.<Preparation of benzidine staining solution>1 piece of benzidine (Sigma # D5905) was dissolved in 10mL of PBS and 10. mu. L H was added₂O₂And filtering by using a syringe.

4. The cell spots were covered with 300-500. mu.L of benzidine solution. RT incubation was 1 hour.

5. And (4) washing with water.

< giemsa stain > giemsa stain (Sigma # GS500) was diluted with water 1: 20.

RT staining for 35-40 min.

8. Washed with water and air dried.

9. And (6) sealing the sheet.

10. And (4) microscopic imaging.

Flow cytometer

Cells were stained with 1. mu.L of mouse anti-human CD235a APC antibody (BD Biosciences), 1. mu.L of mouse anti-human CD71 PerCpCy5.5 antibody (Biolegend), and 0.25. mu.L of mouse anti-human CD117 PE Cy7 antibody (eBioscience) 1. mu. L, hoechst33342 (Thermofeisher) in a 200. mu.L assay system. Data collection was performed on the CytoFLEX LX platform (Beckman Coulter). The results were analyzed using FlowJo software.

Reagent

Recombinant human fms-like tyrosine kinase 3 ligand (rhFlt 3L): FMS-like tyrosine kinase 3 ligand (Flt-3 ligand), also known as FL, Flt3L, and Flt3LG, promote differentiation of HSCs into cells of various hematopoietic lineages. FLT3LG is structurally homologous to Stem Cell Factor (SCF) and colony stimulating factor 1 (CSF-1). FLT3LG increases cell number by activating hematopoietic progenitor cells.

Recombinant human stem cell factor (rhSCF): kit ligand (KITLG), also known as Stem Cell Factor (SCF), belongs to the class I transmembrane glycoprotein of the SCF family. KITLG is a ligand for the receptor-type protein tyrosine kinase KIT. SCFs play an important role in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, cell development, migration, and function.

Recombinant human interleukin 3 (rhIL-3): is a glycoprotein belonging to the hematopoietic growth factor family, and shows multi-lineage activity in preclinical in vitro and in vivo studies. Hematopoietic progenitor cells can proliferate and differentiate into mature erythrocytes, mast cells, megakaryocytes, and granulocytes with the aid of IL-3 protein.

Recombinant human interleukin 6 (rhIL-6): is a multifunctional cytokine, and can regulate immune response, hematopoietic function, acute phase reaction and inflammatory reaction. And IL-3 synergistically promote hematopoietic cell proliferation.

Recombinant human erythropoietin (rhEpo): is a major erythropoietin, which interacts with a variety of other growth factors (IL-3, IL-6, glucocorticoids and SCF) to develop erythroid lineages from pluripotent progenitor cells. Erythroid burst forming unit (BFU-E) cells begin to express erythropoietin receptor and are sensitive to erythropoietin. It is an important erythroid hematopoietic cytokine.

Halo human transferrin: is the major ferritin in plasma, forms a complex with ferric ions, and is used for hemoglobin production in red blood cells.

Hoechst 33342: is a fluorescent dye used for dyeing DNA. The dye can pass through the cell membrane to bind to the DNA.

Results

We performed a series of experiments rigorously to characterize the efficiency of red blood cell proliferation/differentiation and the function of the edrbc. LIN^-CD34⁺HSC or LIN^-CD34^-PBMCs were transduced with lentiviruses encoding the MHC I OT1 α β/MHC I OT1 β genes. More than 95% of the cells were positively transduced (indicated by GFP signal, fig. 2A). We have found that engineering LIN^-CD34⁺HSC or LIN^-CD34^-PBMCs did not affect cell proliferation and differentiation during 21 days of erythroid culture (fig. 2B-D). At the end of the culture, most mature eRBCs still expressed the engineered protein. Furthermore, transduction did not alter enucleation efficiency and the morphology of the edrbc (fig. 2D and 2E).

Flow cytometry analysis showed that cells do not express CD235a during SFEM (serum free expansion medium) (expansion), whereas cells begin to express CD235a during the differentiation stage and CD235a positive cells increase as differentiation progresses. Near late stage differentiation (DIF2), almost all cells expressed CD235a, indicating that almost all cells were erythroid. This demonstrates that our differentiation system efficiently induces erythroid differentiation in vitro.

Changes in the expression level of CD117(SCF receptor) reflect differential utilization of SCF during erythroid differentiation. SCF is critical in regulating stem cell survival, maintenance, and proliferation. CD117 is not expressed under SFEM conditions, but CD117 levels rise rapidly during the early stages of differentiation (DIF1) and then decrease during the later stages of differentiation.

CD71 is the transferrin receptor, the expression of which is critical for erythroid cell function. CD71 not being in LIN under SFEM conditions^-Expressed in the cells, but the level of CD71 rises rapidly early in differentiation to ensure adequate hemoglobin synthesis. Then CD71 levels decreased during the late stage of differentiation. LIN transduced during in vitro erythroid culture^-Cell surface marker expression profiles of PBMCs indicated that erythroid differentiation proceeded normally (fig. 2C).

Example 2 in vivo tissue distribution of eRBCs in mice

In establishing cell therapy for clinical applications, the in vivo tissue distribution of cells is important. We next evaluated whether the edrc-antigen presentation system exhibited any preferential tissue distribution sites and dynamics. We used MC38 colorectal tumor-loaded NSG mice as a disease model. MC38 cells were seeded at the subcutaneous site and DiR pre-stained edrbs were injected intravenously into mice 1 week later. In vivo fluorescence imaging 7 days after irbcs injection showed that irbcs displayed strong signals throughout the body, except for the brain (probably due to the blood brain barrier) and the heart (probably due to the lack of capillaries). The signals of the eRBCs were more pronounced in tumor tissues, indicating that the eRBCs can be effectively distributed in tumor tissues, suggesting that antigen-presenting eRBCs can effectively interact with local lymphocytes in tumors and trigger T cell activation (FIG. 3).

Example 3 evaluation of the in vitro antigen presenting Capacity of eRBCs

To assess the antigen presenting capacity of the eRBCs system, we used OT1 mice, CD8, thereof⁺T cells predominantly recognize OVA presented by MHC I molecules_257-264A peptide. For concept verification, we are specific OVA_257-264The (MHC-OT1) peptide designed the edrbc antigen presentation system. eRBCs-MHC-OT1 and CD8 from OT1 mice⁺T cell co-culture showed that edrc-MHC-OT 1 has strong specific T cell activation function (fig. 4).

In vitro experiments show that our eRBCs system can efficiently present specific antigens. We next designed an in vivo model using tumor-laden mice. MC38 and CT26 are two colorectal cancer cell lines with many non-synonymous mutations. For these non-synonymous mutations, we predicted and designed specific neoantigens. We believe that these antigens can be presented using our system to activate T cells and promote eradication of tumors.

The system is also suitable for use in humans. For example, HPV E6 and E7 oncoproteins are essential for the development and maintenance of malignancies; thus, it is unlikely that certain cancer cells escape the immune response by mutations E6 and E7. E6 and E7 are often constitutively expressed at high levels and therefore may be ideal targets for developing vaccines against established HPV infections and lesions. We designed a new antigen for the E6/E7 protein of HPV type 16/18/52/59. In addition, activation of specific T cells against HPV E6/7 may be a potential therapeutic strategy for HPV-positive cervical cancer.

In the present disclosure, we generated edrcs with an artificial chimeric MHC pattern to present specific antigens to activate T cells, and the edrcs antigen presentation system elicited a strong T cell response. This technology will make eRBCs a novel antigen presenting cell for modulating immune responses in cancer or immune diseases.

Some of the amino acid sequences mentioned herein are listed below.

MHC I OT1 beta CD235a nucleotide sequence (SEQ ID NO:1)

MHC I OT1 β CD235a protein sequence: the mouse β 2-microglobulin signal peptide is indicated by dot underlining, the OT-1 peptide is indicated by solid underlining, the three peptide linkers are shown in bold, the mouse β 2-microglobulin is shown in italics, the H2-Kb (Y84C) heavy chain is indicated by double underlining, and CD235a is indicated by wavy underlining. (SEQ ID NO:2)

MHC I OT1 alpha beta CD235a nucleotide sequence (SEQ ID NO:3)

MHC I OT1 α β CD235a protein sequence: the CD235a signal peptide is indicated by short underlined lines, the mouse β 2-microglobulin signal peptide is indicated by dot underlined lines, the OT-1 peptide is indicated by solid underlined lines, the three peptide linkers are shown in bold, the mouse β 2-microglobulin is shown in italics, the H2-b (Y84C) heavy chain is indicated by double underlined lines, and CD235a is indicated by wavy underlined lines. (SEQ ID NO:4)

Claims

1. A method of producing engineered red blood cells (edrbs), comprising:

1) collection of lineage negative cells (lin) from blood or bone marrow samples^-A cell),

2) amplifying the lin^-A cell;

3) culturing the amplified lin^-Cells to induce their differentiation into erythroid cells; and, prior to or simultaneously with differentiation, introducing exogenous nucleic acid into the amplified lin^-A cell; and

4) the erythroid cells are cultured to induce enucleation.

2. The method of claim 1, wherein the blood sample is a peripheral blood sample, an umbilical cord blood sample, or a fetal blood sample.

3. The method of claim 1 or 2, wherein the blood sample is a human peripheral blood sample.

4. The method according to any of claims 1-3, wherein the lin^-The cell is a lin^-CD34^-A cell.

5. The method according to any one of claims 1-4, wherein step 1) comprises isolating PBMCs from a peripheral blood sample and isolating lin from PBMCs^-CD34^-A cell.

6. According to any of claims 1-5The method of one item, wherein step 1) comprises removing lineage positive (lin) from the peripheral blood sample using a lineage cell removal kit⁺) A cell.

7. The method according to any of claims 1-6, wherein step 2) comprises culturing the lin in hematopoietic stem cell expansion medium supplemented with a combination of cytokines^-A cell, wherein said cytokine combination comprises Flt3L, SCF, IL-3, and IL-6.

8. The method of any one of claims 1-7, wherein the hematopoietic stem cell expansion medium is StemSpan^TMSFEM serum-free amplification medium.

9. The method of any one of claims 1-8, wherein step 2) comprises 5% CO at 37 ℃₂Culturing the lin^-Cells were cultured for about 2-5 days.

10. The method according to any of claims 1 to 9, wherein step 3) comprises culturing the expanded lin in a first differentiation medium supplemented with a cytokine associated with erythroid development^-A cell.

11. The method of any one of claims 1-10, wherein the erythroid development-related cytokines comprise IL-3 and SCF.

12. The method of any one of claims 1-11, wherein the first differentiation medium is Iscove Modified Dulbecco Medium (IMDM) containing FBS, human plasma, glutamine, BSA, transferrin, insulin, penicillin-streptomycin, IL-3, EPO, and SCF.

13. The method of any one of claims 1-12, wherein the first differentiation medium is Iscove Modified Dulbecco Medium (IMDM) containing 10-15% FBS, 5-10% human plasma, 1-4mM glutamine, 1-2% BSA, 300-600 μ g/mL human transferrin, 8-13 μ g/mL human insulin, 2% penicillin-streptomycin, 3-5ng/mL human IL-3, 4-7U/mL human EPO, and 100ng/mL human SCF.

14. The method of any one of claims 1-13, wherein step 3) comprises 5% CO at 37 ℃₂Bottom culture of amplified lin^-Cells were left for about 9 days.

15. The method of any one of claims 1-14, wherein step 4) comprises culturing the erythroid cells in a second differentiation medium, wherein the second differentiation medium lacks cytokines associated with erythroid development compared to the first differentiation medium.

16. The method of any one of claims 1-15, wherein the second differentiation medium is Iscove Modified Dulbecco Medium (IMDM) containing FBS, human plasma, glutamine, BSA, transferrin, insulin, penicillin-streptomycin, and EPO.

17. The method of any one of claims 1-16, wherein the second differentiation medium is Iscove Modified Dulbecco Medium (IMDM) containing 15% FBS, 5-10% human plasma, 1-4mM glutamine, 1-2% BSA, 300-600 μ g/mL human transferrin, 8-13 μ g/mL human insulin, 2% penicillin-streptomycin, and 1-5U/mL human EPO.

18. The method of any one of claims 1-17, wherein step 4) comprises 5% CO at 37 ℃₂Erythroid cells were cultured under the conditions for about 7 days.

19. The method according to any one of claims 1 to 18, wherein in step 3), the amplified lin is cultured in a first differentiation medium^-On day one of the cells, exogenous nucleic acid was introduced into the amplified lin^-A cell.

20. Root of herbaceous plantThe method of any one of claims 1-19, wherein said exogenous nucleic acid is an expression vector carrying a lin intended for amplification^-A gene of interest expressed in a cell.

21. The method of any one of claims 1-20, wherein the expression vector is a lentiviral expression vector.

22. The method of any one of claims 1-21, wherein the gene of interest encodes a fusion protein.

23. The method according to any one of claims 1-22, wherein the fusion protein is a cell surface membrane protein comprising an anchor portion comprising at least the transmembrane region of CD235 a.

24. The method of any one of claims 1-23, wherein the fusion protein comprises an artificial MHC single chain molecule and comprises, from N-terminus to C-terminus, an antigenic peptide, a first peptide linker, β 2-microglobulin, a second peptide linker, and an MHC class I heavy chain that lacks a transmembrane region and a cytoplasmic region.

25. The method according to any one of claims 1-24, wherein the artificial MHC single chain molecule is fused at its C-terminus to the N-terminus of the anchor moiety, optimally with a third peptide linker.

26. The method of any one of claims 1-25, wherein the fusion protein further comprises a signal peptide selected from the group consisting of a β 2-microglobulin signal peptide or a CD235a signal peptide, or a combination thereof.

27. The method of any one of claims 1-26, wherein the first peptide linker and the second peptide linker are Gly and Ser rich.

28. The method of any one of claims 1-27, wherein the antigenic peptide is associated with a diseaseAre associated with disorders and are capable of activating CD8 when presented by MHC class I molecules⁺T cells.

29. The method of any one of claims 1-28, wherein the antigenic peptide is a cancer neoantigen or is derived from an oncoprotein or a viral protein.

30. The method of any one of claims 1-29, wherein the antigenic peptide is 8, 9, 10, or 11 amino acids in length.

31. eRBCs produced by the method of any one of claims 1-30.

32. eRBCs comprising a fusion protein, wherein the fusion protein comprises, from N-terminus to C-terminus, an antigenic peptide, a first peptide linker, a β 2-microglobulin, a second peptide linker, a heavy chain of an MHC class I molecule lacking a transmembrane region and a cytoplasmic region, a third peptide linker, and an anchor moiety, wherein the antigenic peptide is associated with a disorder and is capable of activating CD8 when presented by the MHC class I molecule⁺T cells, and wherein the anchoring moiety comprises at least the transmembrane region of CD235 a.

33. A pharmaceutical composition comprising the edrbs of claim 31 or 32 and a physiologically acceptable excipient.

34. Use of the eRBCs of claim 31 or 32 in the manufacture of a medicament for treating a condition associated with an antigenic peptide.

35. A method for treating a disorder associated with an antigenic peptide in a subject, comprising:

a) collecting a blood sample or a bone marrow sample from a subject,

b) generating eRBCs by using the method of any one of claims 1-30;

c) infusing a therapeutically effective amount of the eRBCs into the subject.

36. The method of claim 35, wherein the antigenic peptide is a fragment of HPV E6 or E7 protein.

37. A murine edrbs comprising a peptide having the sequence of SEQ ID No. 2 or 4.