CN114746545B

CN114746545B - Engineered erythrocytes presenting specific cancer neoantigens using artificial MHC

Info

Publication number: CN114746545B
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: 2024-08-06
Anticipated expiration: 2040-10-16
Also published as: WO2021073613A1; CN114746545A

Abstract

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

Description

Engineered erythrocytes presenting specific cancer neoantigens using artificial MHC

Technical Field

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

Background

Effective anti-tumor immunity in humans has been linked to the presence of T cells directed against cancer neoantigens, a class of HLA-binding peptides caused by tumor-specific mutations, and recent data indicate that recognition of such neoantigens is a major factor affecting the activity of clinical immunotherapy. Large-scale parallel whole exome sequencing has been used to detect all mutations in tumors to predict neoantigens. Vaccination with neoantigen can expand the pre-existing population of neoantigen-specific T cells and induce new cancer-specific T cells. Although neoantigens have become potential ideal targets 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. Lack of a specific antigen presentation system is one of the problems of neoantigen vaccination.

Red Blood Cells (RBCs) are the most abundant cell types in blood, accounting for one quarter of the total number of human cells. Erythrocytes have a number of unique properties that make them an attractive tool for in vivo delivery of natural and synthetic cargo substances (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 (lifetime in humans of about 120 days); a large surface area to volume ratio; no nuclei and mitochondria (no ability to protein synthesis, proliferation, mutation).

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

Summary of The Invention

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

1) Lineage negative and CD34 negative cells (lin ^-CD34^- cells) were collected from the blood samples,

2) Amplifying the lin ^-CD34^- cells; and

3) The expanded lin ^-CD34^- cells were induced to differentiate into mature erythrocytes.

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 (PBMCs) from the peripheral blood sample and isolating lin ^-CD34^- cells from the PBMCs.

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

In some embodiments, step 2) comprises culturing the lin ^-CD34^- cells in hematopoietic stem cell expansion medium supplemented with a cytokine combination, wherein the cytokine combination comprises fms-like tyrosine kinase 3 ligand (Flt 3L), 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 a StemSpan ^TM SFEM serum-free expansion medium.

In some embodiments, step 2) comprises culturing the lin ^-CD34^- cells at 37℃for about 2-5 days with 5% CO ₂.

In some embodiments, step 3) comprises:

i) Culturing the expanded lin ^-CD34^- cells to induce differentiation into erythroid cells (erythroid cells); and

Ii) culturing the erythroid cells to induce enucleation.

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

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

In some embodiments, the first differentiation medium is Iscove's Modified Dulbecco's 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 μg/mL human transferrin, 8-13 μg/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 culturing the expanded lin ^-CD34^- cells at 37 ℃ for about 9 days at 5% co ₂.

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 μg/mL human transferrin, 8-13 μg/mL human insulin, 2% penicillin-streptomycin, and 1-5U/mL human EPO.

In some embodiments, step ii) comprises culturing the erythroid cells at 37 ℃ for about 7 days at 5% co ₂.

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

In another aspect, the present disclosure provides a method for producing engineered red blood cells (eRBCs), comprising:

1) Collecting lineage negative cells (lin ^- cells) from a blood sample or a bone marrow sample;

2) Amplifying the lin ^- cells;

3) Culturing the expanded lin ^- cells to induce differentiation into erythroid cells; and introducing exogenous nucleic acid into the expanded lin ^- cells prior to or concurrent with differentiation; 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 ^- cell is a lin ^-CD34^- cell.

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

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

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

In some embodiments, step 2) comprises culturing the lin ^- cells at 37℃for about 2-5 days with 5% CO ₂.

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

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 culturing the expanded lin ^- cells at 37 ℃ for about 9 days at 5% co ₂.

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 culturing the erythroid cells at 37 ℃ for about 7 days at 5% co ₂.

In some embodiments, in step 3), exogenous nucleic acid is introduced into the expanded lin ^- cells on the first day of culturing the expanded lin ^- cells in a first differentiation medium.

In some embodiments, the exogenous nucleic acid is an expression vector carrying a gene of interest intended for expression in the amplified lin ^- cells.

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 a 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 β2-microglobulin, a second peptide linker, and an MHC class I molecule heavy chain lacking 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 anchoring 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 β2-microglobulin signal peptide, a CD235a signal peptide, or a combination thereof.

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

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

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 eRBCs produced by the above method.

In another aspect, the disclosure provides eRBCs comprising a fusion protein comprising, from N-terminus to C-terminus, an antigenic peptide, a first peptide linker, a β2-microglobulin, a second peptide linker, an MHC class I molecule heavy chain lacking a transmembrane region and a cytoplasmic region, a third peptide linker, and an anchor, wherein the antigenic peptide is associated with a disorder and is capable of activating CD8 ⁺ T cells when presented by an MHC class I molecule, and wherein the anchor comprises at least the transmembrane region of CD235 a.

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

In another aspect, the present disclosure provides the use of eRBCs of the present disclosure in the manufacture of a medicament for the treatment of 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) A blood sample or a bone marrow sample is collected from a subject,

B) eRBCs and producing using the methods of the present disclosure

C) A therapeutically effective amount eRBCs is infused into a subject.

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

In another aspect, the disclosure provides a mouse 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 panels) (the nucleic acid and amino acid sequences of which are shown in SEQ ID NOS: 1-4). The chimeric MHC molecules designed consisted of a membrane-localized signal peptide (upper panel: b2m signal peptide; middle panel: CD235a signal peptide linked to b2m signal peptide), a specific peptide for T cell recognition (e.g., neoantigen) linked via a glycine/serine linker, beta 2-microglobulin, and an MHC heavy chain region. Because the MHC heavy chain lacks a transmembrane domain, CD235a fusion ensures that the protein complex is transferred to the cell membrane. (OT-1: ovalbumin peptide residues 257-264 (OVA 257-264); b2m, beta-2 microglobulin; pep, peptide; T2A,2A self-cleaving peptide; copGGFP, green fluorescent protein cloned from copepoda Pontellina plumata; beta: beta 2-microglobulin signal peptide; alpha beta: CD235a and beta 2-microglobulin signal peptide).

(C) eRBC workflow of treatment. Peripheral blood samples are collected from individuals. LIN ^-CD34⁺ HSCs or LIN ^-CD34^- PBMCs were isolated and transduced with lentiviruses encoding chimeric MHC antigen presenting molecules. Transduced HSCs or PBMCs are cultured under conditions that induce erythroid proliferation and differentiation. The terminally differentiated (enucleated) eRBCs is returned to the patient for therapeutic purposes.

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

(A) Microscopic images showing the transduction efficiency of LIN ^-CD34^- PBMCs. LIN ^-CD34^- PBMCs were transduced on day 5 with a lentiviral vector encoding the MSCV promoter alone (control) or encoding MSCV-MHC-I OT1 beta. Viral infection efficiency was checked 48 hours after transduction. GFP signal indicates positively transduced cells. Similar results can be obtained in LIN ^-CD34⁺ HSCs transduced with MSCV-MHC-IOT1αβ, LIN ^-CD34⁺ HSCs transduced with MSCV-MHC-IOT1β, LIN ^-CD34^- PBMCs transduced with MSCV-MHC-IOT1αβ.

(B) Cell proliferation assay. During erythroid culture, invitrogen Countess II was used to evaluate cell numbers of LIN ^- PBMCs with or without lentivirus transduction every three days.

(C) Red differentiation analysis. Flow cytometry was used to examine the expression level of cell surface markers every two days during erythroid culture to indicate erythroid differentiation progression. Cells were stained with antibodies to human CD235a, CD71 and CD 117.

(D) Enucleation analysis of eRBCs at the end of red line culture. DNA dye Hoechst 33342 was used to stain DNA. Enucleated eRBCs staining was CD235a positive and Hoechst 33342 negative. About 80% of the enucleated eRBCs was GFP positive, indicating that the engineered protein remained expressed until the final differentiation and enucleation stage.

(E) Benzidine-giemsa staining showed cell morphology at day eRBCs of culture.

Fig. 3: in vivo distribution of eRBCs expressing chimeric MHC molecules. eRBCs were derived from LIN ^-CD34^- PBMC transduced with MSCV-MHC-IOT1β. 1X 10 ⁸ eRBCs pre-stained with DiR, injected intravenously into NSG mice bearing MC38 tumors. In vivo fluorescence imaging analysis was performed 7 days after eRBCs injection. (left panel): representative in vivo imaging of the abdomen, back and sides of 1 mouse. (right panel): eRBCs distributions for each organ. Similar results can be obtained in LIN ^-CD34⁺ HSCs transduced with MSCV-MHC-IOT1αβ, LIN ^-CD34⁺ HSCs transduced with MSCV-MHC-IOT1β, LIN ^-CD34^- PBMCs transduced with MSCV-MHC-IOT1αβ.

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

(A) Experimental protocol for T cell activation in vitro. Mouse erythroid progenitors (BFU-Es/CFU-Es) were isolated from E13.5-14.5 fetal liver. These mouse erythroid progenitors were transduced with a lentivirus encoding MSCV-MHC iαβOT1 and induced to differentiate into eRBCs-MHC iαβOT1. CD8 ⁺ T cells were isolated from OT1 mice (C57 BL/6-Tg (TcraTcrb) 1100Mjb/J; CD8 ⁺ T cells of this mice predominantly recognize OVA _257-264 presented by MHC I). CD8 ⁺ T cells from eRBCs-MHC iαβot1 and OT1 mice were co-cultured for 2 days and analyzed by FACS for two CD8 ⁺ T cell activation markers, CD107a (B) and CD44 (C). (D) Morphology of OT1 CD8 ⁺ T cells alone (top left) or OT1 CD8 ⁺ T cells and eRBCs co-cultures under bright field microscopy. White arrow: activated T cell clusters. eRBC cell count ratio of CD8 ⁺ T cells = 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 cells" or "Lin ^- cells" refers to cells that are 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 BDCA2. In fact, lineage negative cells are not substantially 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 lineage negative cell enriched blood cell populations can be purified by enriching blood cell populations that are 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 BDCA2. In some cases, purification can be performed using lineage cell removal kits. In contrast, lineage positive (Lin ⁺) cells are a mixture of all cells expressing mature cell lineage markers. Examples of lineage positive cells include T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, erythroid cells and their committed precursor cells.

As used herein, the term "antigenic peptide" refers to a peptide capable of binding to the peptide binding domain of an MHC molecule (particularly an MHC class I molecule) to form an MHC complex with the MHC molecule. As is well known in the art, presentation of antigen peptides in MHC complexes on the surface of APCs typically does not involve, for example, intact proteins. In contrast, an antigenic peptide located in a binding domain is typically a small fragment of an intact 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 β2 microglobulin and an MHC class I molecule heavy chain. Such fusion proteins are also referred to in some documents 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 an 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 beta-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 heavy chain portion of the MHC class I molecule in the artificial MHC single chain molecule is in 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 at the C-terminus to a CD235a molecule or a fragment thereof comprising at least a transmembrane region. In this approach, once expressed in host cells (e.g., lin ^-CD34^- PBMCs), the artificial MHC single chain molecule can be anchored to the cell membrane by a CD235a molecule or fragment thereof.

As used herein, the term "culturing" refers to the maintenance of cells in a medium for any period of time, whether or not there is cell expansion or differentiation.

As used herein, the term "differentiation" refers to the process by which cells of a lesser degree of specialization (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 specificity may be differentiated into cells of higher degree of specificity by culturing the cells in specific conditions or in specific media as known in the art.

As used herein, the term "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium), solvent or encapsulating material, that participates in carrying or transporting the 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 deleterious to the subject.

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

CD117, also known as "c-kit", is a SCF stem cell growth factor receptor expressed on the surface of hematopoietic stem cells and other cells. SCF plays 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 an intact transferrin molecule, producing an iron-transferrin receptor complex that enters the cell by endocytosis for production of cellular hemoglobin during erythroid development.

In this study, we generated a series of new methods to induce erythroid proliferation and differentiation using lineage negative CD34 negative peripheral blood mononuclear cells (Lin ^-CD34^- PBMCs) or lineage negative CD34 positive hematopoietic stem cells (Lin ^-CD34⁺ HSCs), thus laying the foundation for eRBCs treatment. Genes encoding cell surface proteins fused to various engineered proteins are introduced into these hematopoietic stem and progenitor cells by lentiviral transduction. The cells are then cultured under conditions that induce erythroid differentiation and enucleation. 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 APCs to display antigenic peptides. The acquisition of antigen is equivalent to the production of a protein with errors, which is a mutation of the protein caused by a non-synonymous mutation. Thus, to generate a personalized eRBC-antigen presentation system, we performed Whole Exome Sequencing (WES) and RNA-seq to recognize and predict tumor-specific neoantigens. To create an antigen presentation system, MHC molecules are an essential component; thus, we designed an artificial MHC which is a single chain unit consisting of a neoantigenic peptide, β2 microglobulin and MHC class I molecule heavy chain (fig. 1B). This recombinant peptide consisting of the neoantigenic peptide-beta 2-microglobulin-MHC I single chain can be recognized by peptide-specific T cells (FIG. 1A). Normally, 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 mature erythrocyte surface membrane protein CD235 a. This means that in our system, only this specific neoantigen can be presented on the surface, a property that will greatly increase the efficiency of T cell recognition and activation.

Several publications have demonstrated that mature erythrocytes can differentiate with different efficiency from early erythroid progenitors derived from hematopoietic stem cells (LIN ^-CD34⁺ HSCs).

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

Lentiviral vector construction

Gene synthesis to obtain the designed MHC I OT1 gene. These sequences were constructed on lentiviral vector MSCV to give sequence vectors MSCV-MHC-IOT1αβ or MSCV-MHC-IOT1β for virus packaging.

Virus package

Viral packaging is performed using MHC-I OT1: pSPAX 2:VSVG=2:1:1 ratio packaging. Take a 6-well plate as an example. According to MHC-I OT1: pSPAX2: vsvg=2 μg:1 μg using the calcium phosphate transfection method. The plasmids were transfected into HEK 293T cells for viral packaging. After 12 hours of transfection, the calcium phosphate was removed by pipetting, and the supernatants were collected at 48 hours and 72 hours and the cell culture supernatants were filtered with 0.45 μm filters.

Virus concentration

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

Lin ^-CD34^- PBMCs isolation

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

The aim of this step is to achieve density gradient centrifugation of the cellular components by lymphocyte separation, separating the PBMCs from the different cells such as erythrocytes, platelets, etc., to ensure subsequent enrichment of Lin ^-CD34^- cells. Lin ^-CD34^- cells were isolated using the human lineage cell removal kit (BD Biosciences).

Lin ^-CD34⁺ HSCs isolation

Whole blood was diluted 1:1 with phosphate buffer and pre-enriched HSCs were isolated using lymphocyte separation solutions (LymphoprepTM, STEMCELL Technologies) in lymphocyte separation tubes by centrifugation at 1200 x g for 15 minutes.

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 erythrocytes and platelets to ensure subsequent enrichment of Lin ^-CD34⁺ HSCs. CD34 ⁺ HSCs were isolated from pre-enriched HSCs using the EasySep ^TM human cord blood CD34 positive selection kit II (Stemcell Technologies). CD34 ⁺ cells were incubated with biotin-labeled antibodies against lineage specific antigens. CD34 ⁺ cells with specific lineage markers were removed and Lin ^-CD34⁺ HSCs were obtained by negative selection using magnetic beads conjugated to streptavidin.

Purification of fetal liver erythroid progenitor cells from mouse embryos

1. Timed mating was set to obtain female mice 13.5-14.5 days gestation.

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

3. The single cell suspension was aspirated up and down with a pipette and passed through a 25 μm filter (BD Falcon 35-2235). The screen was rinsed with 1ml PBS. The flow-through (1 ml per embryo) was pooled.

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

5. The pelleted cells were centrifuged 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, # 012-000-003) at 50 μl/mouse, incubating at 4 ℃ for 5 minutes (wells×25×10 ⁴ =cell number/mL).

7. Biotinylated anti-mouse TER119 (BD Pharmingen, # 553672) was added at 1. Mu.L/1X 10 ⁶ cells and incubated at 4℃for 15 min.

8. MS LINEAGE PANEL (FISHER SCIENTIFIC (Thermo FISHER SCIENTIFIC) # BDB 559971) was added to the cells (2. Mu.L/1X 10 ⁶ cells) and incubated at 4℃for 15 min.

9. The cells were washed once with 10 volumes of PBS and centrifuged at 1.5 kRPM for 5 minutes at 4 ℃.

10. STREPTAVIDIN PARTICLES Plus-DM (magnetic beads) (BD Pharmigen, # 557812) (5. Mu.L/1X 10 ⁶ cells) was added and incubated at 4℃for 30 minutes.

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

12. 2ML of cells (4 mL total) were dispensed into each tube, and the cells were carefully removed from the tube and placed into another tube on the other side, avoiding disruption of magnetically attached beads. The same procedure was repeated and Ter119 negative and lineage negative cells were transferred to new tubes.

13. Viable cells were counted and concentrated, and cells were resuspended with 50-100 μl PBS (2% fbs).

Lentivirus transduced LIN ^-CD34⁺ HSCs or LIN ^-CD34^- PBMCs

After 5 days of amplification, LIN ^-CD34⁺ HSCs or LIN ^-CD34^- PBMCs were transduced with MSCV-MHC-I OT1 virus. Cells were resuspended at a density of 1×10 ⁶ using differentiation phase 1 medium. The infection volume and virus dose were calculated. The virus dose is calculated according to the final concentration of 5X10 ⁷ TU/mL to 5X10 ⁸ TU/mL; the mixture was incubated with Polybrene in concentrated virus solution for 5 minutes at 10 μg/mL, depending on the infectious volume. The concentrated virus solution after incubation was added to the cells and mixed. Centrifugal infection was performed using a horizontal rotor centrifuge at a speed of 500×g, a temperature of 32 ℃ and a time of 90 minutes. After centrifugation, the cells were cultured at 37℃under 5% CO ₂. Two days later, the virus infection efficiency was examined under a fluorescence microscope. The percentage of GFP positive cells indicates positively transduced cells.

ERBCs culture

Isolated Lin ^-CD34^- or Lin ^-CD34⁺ cells were cultured on day 0, cells were cultured in hematopoietic stem cell expansion medium (Stemspan ^TM SFEM, STEMCELL Technologies), and cytokine combinations and penicillin-streptomycin (Gibco) were added. Cells were cultured at 37℃under 5% CO ₂. Cultures were grown to day 5 under these culture conditions.

The objective of this step was to add a cytokine combination to the StemSpan ^TM SFEM serum-free expansion medium to expand and maintain Lin ^-CD34^- or Lin ^-CD34⁺ cells prior to inducing erythroid differentiation. The cytokine combination comprises 50ng/mL recombinant human fms-like tyrosine kinase 3 ligand (Flt 3L), 50ng/mL recombinant human Stem Cell Factor (SCF), 10ng/mL recombinant human interleukin 3 (IL-3), 10ng/mL recombinant human interleukin 6 (IL-6), etc.

The culture system was changed on day 5, and the medium was changed to differentiation stage 1 medium consisting 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 μg/mL human transferrin (Holo human transferrin, sigma-Aldrich), 8-13 μg/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 cultured at 37℃under 5% CO ₂. Cultures were grown under these culture conditions to day 14. The purpose of this experimental procedure is to induce differentiation of Lin ^-CD34^- or Lin ^-CD34⁺ cells into erythroid cells and achieve substantial expansion in the presence of sufficient erythroid-related cytokines. In this culture system, only cytokines associated with erythroid development are provided, and proliferation and differentiation of cells into erythroid cells are ensured. At the same time, viral infection occurs at the stage of the fastest differentiation, ensuring that the target gene is inserted and expressed on the cell membrane.

The culture system was changed on day 14, and the medium was changed to differentiation stage 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 cultured at 37℃under 5% CO ₂. Cultures were grown under these culture conditions to day 21. The purpose of this experimental procedure is to reduce or eliminate some of the cytokines used under the previous culture system conditions to promote enucleation, which is the last step in erythrocyte maturation.

Benzidine-giemsa staining

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

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

< Preparation of benzidine staining solution > 1 piece of benzidine sheet (Sigma #d5905) was dissolved in 10mL of PBS, added to 10 μ L H ₂O₂, and filtered with a syringe.

4. The cell sites were covered with 300-500. Mu.L of benzidine solution. The RT was incubated for 1 hour.

5. And (5) washing with water.

< Giemsa staining > 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 (5) sealing the piece.

10. And (5) 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 PerCp Cy5.5 antibody (Biolegend), 0.25. Mu.L of mouse anti-human CD117 PE Cy7 antibody (eBioscisence) 1. Mu. L, hoechst 33342 (Thermofisher) in a 200. Mu.L assay system. Data acquisition was performed on a CytoFLEX LX platform (Beckman Coulter). Results were analyzed using FlowJo software.

Reagent(s)

Recombinant human fms-like tyrosine kinase 3 ligand (rhFlt L): FMS-like tyrosine kinase 3 ligands (Flt-3 ligands), 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), a type I transmembrane glycoprotein belonging to the SCF family. KITLG is a ligand for the receptor type protein tyrosine kinase KIT. SCF plays 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 exhibiting multiple 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 response and inflammatory response. Promote hematopoietic cell proliferation in conjunction with IL-3.

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

Holo human transferrin: is the major ferritin in plasma, forming a complex with iron ions for hemoglobin production in erythrocytes.

Hoechst33342: is a fluorescent dye for staining DNA. The dye can penetrate the cell membrane and bind to DNA.

Results

We have strictly performed a series of experiments to characterize the efficiency of erythrocyte proliferation/differentiation and the function of eRBC. LIN ^-CD34⁺ HSC or LIN ^-CD34^- PBMC were transduced with lentiviruses encoding MHC IOT1α β/MHC IOT1β genes. More than 95% of the cells were transduced positively (indicated by GFP signal, fig. 2A). We found that the engineered LIN ^-CD34⁺ HSC or LIN ^-CD34^- PBMC did not affect proliferation and differentiation of cells during 21 days erythroid culture (FIGS. 2B-D). At the end of the culture, most of the maturation eRBCs still expresses the engineered protein. In addition, transduction did not alter the enucleation efficiency and morphology of eRBC (fig. 2D and 2E).

Flow cytometry analysis showed that cells did not express CD235a during the SFEM (serum-free expansion medium) (expansion) phase, whereas cells began to express CD235a during the differentiation phase, and CD235a positive cells increased as differentiation progressed. Near the late differentiation stage (DIF 2), almost all cells expressed CD235a, indicating that almost all cells are erythroid cells. This suggests that our differentiation system induces erythroid differentiation efficiently in vitro.

Changes in CD117 (SCF receptor) expression levels 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 rapidly increase at the early stage of differentiation (DIF 1) and then decrease at the later stage of differentiation.

CD71 is a transferrin receptor whose expression is critical for erythroid cell function. CD71 is not expressed in LIN ^- cells under SFEM conditions, but the level of CD71 rises rapidly at early stages of differentiation to ensure adequate synthesis of hemoglobin. CD71 levels then decreased in the late stage of differentiation. Cell surface marker expression profile of LIN ^- PBMCs transduced during in vitro erythroid culture 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 assessed whether eRBC-antigen presentation systems exhibited any preferential tissue distribution sites and dynamics. We used NSG mice loaded with MC38 colorectal tumors as disease models. MC38 cells were inoculated into subcutaneous sites and DiR pre-stained eRBCs was injected intravenously into mice after 1 week. In vivo fluorescence imaging after eRBCs days of injection showed that eRBCs showed strong signals throughout the body, except for the brain (possibly due to blood brain barrier) and the heart (possibly due to lack of capillaries). The eRBCs signal was more pronounced in tumor tissue, indicating that eRBCs could be efficiently distributed into tumor tissue, suggesting that eRBCs presenting antigen could efficiently interact with local lymphocytes in the tumor and trigger T cell activation (fig. 3).

EXAMPLE 3 evaluation of eRBCs in vitro antigen presenting Capacity

To assess the antigen presenting capacity of the eRBCs system we used OT1 mice whose CD8 ⁺ T cells predominantly recognized the OVA _257-264 peptide presented by MHC I molecules. For the purpose of proof of concept, we designed eRBC antigen presentation system for specific OVA _257-264 (MHC-OT 1) peptides. Co-culture of eRBCs-MHC-OT1 with CD8 ⁺ T cells from OT1 mice showed that eRBC-MHC-OT1 had a 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-loaded mice. MC38 and CT26 are two colorectal cancer cell lines with many non-synonymous mutations. For these nonsensical mutations, we predicted and designed specific neoantigens. It is believed 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 malignancy; thus, it is unlikely that some cancer cells will escape the immune response by mutating E6 and E7. E6 and E7 are often constitutively expressed at high levels and thus may be ideal targets for developing vaccines against established HPV infections and lesions. We designed a novel antigen for the E6/E7 protein of 16/18/52/59-type HPV. Furthermore, activation of specific T cells against HPV E6/7 may be a potential therapeutic strategy for HPV-positive cervical cancer.

In this disclosure, we generated eRBCs with an artificial chimeric MHC pattern to present specific antigen-activated T cells, and the eRBCs 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 OT 1. Beta. CD235a nucleotide sequence (SEQ ID NO: 1)

MHC I OT1 beta CD235a protein sequence: mouse β2-microglobulin signal peptide is indicated with dot underline, OT-1 peptide is indicated with solid line underline, three peptide linkers are shown in bold, mouse β2-microglobulin is shown in italics, H2-Kb (Y84C) heavy chain is indicated with double underline, CD235a is indicated with wave underline. (SEQ ID NO: 2)

MHC IOT 1. Alpha. Beta. CD235a nucleotide sequence (SEQ ID NO: 3)

MHC I OT1 αβcd235a protein sequence: CD235a signal peptide is underlined with a short line, mouse β2-microglobulin signal peptide is underlined with a dot, OT-1 peptide is underlined with a solid line, three peptide linkers are shown in bold, mouse β2-microglobulin is shown in italics, H2-b (Y84C) heavy chain is underlined with a double underline, and CD235a is underlined with a wave. (SEQ ID NO: 4)

Claims

1. A method for producing engineered erythrocytes (eRBCs) with artificial MHC molecules, comprising:

1) Lineage negative cells (lin ^- cells) were collected from blood samples or bone marrow samples,

2) Amplifying the lin ^- cells;

3) Culturing the expanded lin ^- cells to induce differentiation into erythroid cells; and, introducing exogenous nucleic acid into the expanded lin ^- cells prior to or concurrent with differentiation; and

4) Culturing the erythroid cells to induce enucleation,

Wherein the exogenous nucleic acid encodes a fusion protein, wherein the fusion protein comprises an artificial MHC single chain molecule and is N-terminal to C-terminal to a signal peptide, an antigenic peptide, a first peptide linker, a beta 2-microglobulin, a second peptide linker, an MHC class I heavy chain 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+ T cells when presented by an MHC class I molecule, and

Wherein:

The signal peptide is selected from beta 2-microglobulin signal peptide or CD235a signal peptide or a combination thereof;

the antigenic peptide is 8, 9, 10 or 11 amino acids in length;

the first peptide linker is GCGASGGGGSGGGGS;

The second peptide linker is GGGGSGGGGSGGGGSGGGGS;

The third peptide linker is AAALEVS;

And wherein the first peptide linker to the anchor moiety consists of amino acid residues 48 to 599 in the amino acid sequence of SEQ ID NO. 4.

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 2, wherein the blood sample is a human peripheral blood sample.

4. The method of any one of claims 1-3, wherein the lin ^- cells are lin ^-CD34^- cells.

5. The method of claim 1, wherein step 1) comprises isolating PBMCs from a peripheral blood sample and isolating lin ^-CD34^- cells from the PBMCs.

6. The method of claim 1, wherein step 1) comprises removing lineage positive (lin ⁺) cells from the peripheral blood sample using a lineage cell removal kit.

7. The method of claim 1, wherein step 2) comprises culturing the lin ^- cells in hematopoietic stem cell expansion medium supplemented with a cytokine combination comprising Flt3L, SCF, IL-3, and IL-6.

8. The method of claim 7, wherein the hematopoietic stem cell expansion medium is a StemSpan ^TM SFEM serum-free expansion medium.

9. The method of claim 1, wherein step 2) comprises culturing the lin ^- cells at 37 ℃ for about 2-5 days with 5% co ₂.

10. The method of claim 1, wherein step 3) comprises culturing the expanded lin ^- cells in a first differentiation medium supplemented with a cytokine associated with erythroid development.

11. The method of claim 10, wherein the erythroid developmental related cytokines include IL-3 and SCF.

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

13. The method of claim 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 μg/mL human transferrin, 8-13 μg/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 claim 1, wherein step 3) comprises culturing the expanded lin ^- cells at 37 ℃ for about 9 days at 5% co ₂.

15. The method of claim 1, 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 claim 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 claim 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 μg/mL human transferrin, 8-13 μg/mL human insulin, 2% penicillin-streptomycin, and 1-5U/mL human EPO.

18. The method of claim 1, wherein step 4) comprises culturing erythroid cells at 37 ℃ for about 7 days under 5% co ₂.

19. The method of claim 1, wherein in step 3), exogenous nucleic acid is introduced into the expanded lin ^- cells on the first day of culturing the expanded lin ^- cells in a first differentiation medium.

20. The method of claim 1, wherein the exogenous nucleic acid is an expression vector carrying a gene of interest intended for expression in an amplified lin ^- cell.

21. The method of claim 1, wherein the expression vector is a lentiviral expression vector.

22. The method of claim 1, wherein the antigenic peptide is a cancer neoantigen or derived from a oncoprotein or a viral protein.

23. ERBCs produced by the method of any one of claims 1-22.

24. ERBCs comprising a fusion protein, wherein the eRBC is a Lin ^- cell, and wherein the fusion protein is from N-terminus to C-terminus: a signal peptide, an antigenic peptide, a first peptide linker, a beta 2-microglobulin, a second peptide linker, an MHC class I heavy chain 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 ⁺ T cells when presented by an MHC class I molecule, and wherein the anchor moiety comprises at least the transmembrane region of CD235a

Wherein:

the antigenic peptide is 8, 9, 10 or 11 amino acids in length;

the first peptide linker is GCGASGGGGSGGGGS;

The second peptide linker is GGGGSGGGGSGGGGSGGGGS;

The third peptide linker is AAALEVS;

25. A pharmaceutical composition comprising eRBCs of claim 23 or 24 and a physiologically acceptable excipient.

26. Use of eRBCs according to claim 23 or 24 in the manufacture of a medicament for the treatment of a condition associated with an antigenic peptide.

27. The use of claim 26, wherein the antigenic peptide is a fragment of HPV E6 or E7 protein.

28. A mouse eRBCs comprising an artificial MHC single chain molecule consisting of the amino acid sequence of SEQ ID No.2 or 4.