TW201923073A - Modified K562 cell - Google Patents

Abstract

Modified K562 cells having reduced expression of MHC class I as compared to wildtype K562 cells are disclosed. Also disclosed are articles and methods for producing such modified K562 cells, and methods using such modified K562 cells.

Description

Modified K562 cells

FIELD OF THE INVENTION The present invention relates to the fields of cell biology, genetic engineering, and medical treatment and prevention methods.

BACKGROUND OF THE INVENTION Cell-based adoptive cancer immunotherapy uses autologous or allogeneic immune cells to treat malignancies. Cytotoxic T lymphocytes engineered with antigen-specific T cell receptor (TCR) or chimeric antigen receptor (CAR) have been studied and have shown promising therapeutic effects in a variety of cancers (1-3). Many methods have been studied for T cell expansion in vitro in the presence or absence of feeder cells (4-7). Dynamic feeder cells engineered with Fc receptors, CD32 or CD64, costimulatory molecules, CD40 ligands, B7-2 (CD86) or 4-1BB ligands (CD137L) and membrane-bound interleukins have shown significant improvement Proliferation and maturation of cytotoxic T lymphocytes (8-11). Feeder cells can also be genetically modified for use as artificial antigen-presenting cells (aAPC) to express molecules that stimulate antigen-specific T cells (12). K562 cells have been used as aAPC in many studies to promote the expansion of cytotoxic T lymphocytes (13-15) and CAR-T cells (16-21).

K562 cell line is a human myeloid leukemia cell line derived from a patient with blast crisis (22). This cell line is a mixture of erythrocytes, a community-forming unit equivalent to erythrocytes with nearly triploid karyotypes (22, 23). K562 cells are superior to other cell types as feeder cells because they are cultured and propagated in serum-free media, are suitable for transfection, and are easily modified for use as artificial antigen-presenting cells. K562 cells also exhibit molecules such as ICAM (CD54) and LFA-3 (CD58), through which K562 cells can interact with T cells (8, 9). After being engineered to express costimulatory molecules and tumor-associated antigens, a562C-based AAPCs can support antigen-independent and antigen-dependent T cell expansion and specific CAR-T cell expansion.

The lack of expression of major histocompatibility complex (MHC) molecules in K562 cells has been widely reported (24, 25), which supports the use of K562 cells as feeder cells in a method of expanding T cells in vitro. However, a recent study showed that K562 cells can up-regulate MHC class I expression during co-culture with immune cells, and the authors have also detected and killed one of the immune cell populations expanded from HLA class I mismatched donors. Presence of Allo-Reactive Cytotoxic T Cells in K562 Feeder Cells (26).

SUMMARY OF THE INVENTION The present invention relates to a modified K562 cell, a method for producing the same, a preparation for producing a modified K562 cell, and the use of the K562 cell. In particular, modified K562 cells can be used to generate / expand a population of immune cells that exhibit reduced allogeneic reactivity compared to immune cell populations generated / expanded using wild-type K562 cells. This is achieved through the inhibition of MHC class I expression by modified K562 cells.

In one aspect, the invention provides modified K562 cells with reduced MHC class I performance compared to wild-type K562 cells. In some embodiments, the modified K562 cells comprise a modification of a gene encoding a MHC class I polypeptide relative to a wild-type K562 cell.

In some embodiments, the modification reduces or prevents the performance of a polypeptide encoded by a gene encoding a MHC class I polypeptide. In some embodiments, the gene encoding a MHC class I polypeptide is B 2 M.

In some embodiments, the modified K562 cells comprise modifications that increase the performance of one or more factors that increase the activation or proliferation of immune cells. In some embodiments, the modified K562 cells comprise a nucleic acid encoding one or more factors capable of increasing the activation / proliferation of immune cells. In some embodiments, one or more factors capable of increasing immune cell activation / proliferation are selected from the group consisting of a costimulatory molecule, cytokine, or antigen. In some embodiments, the costimulatory molecule is selected from the group consisting of CD40L, CD86, CD137L, CD80, or CD83. In some embodiments, the interleukin is selected from the group consisting of IL-21, IL-15, membrane-bound IL-21, and membrane-bound IL-15.

In some embodiments, the modified K562 cells comprise modifications that increase the performance of one or more Fc receptors.

In another aspect, the invention provides a modified K562 cell comprising a modification to reduce or prevent the performance of a polypeptide encoded by B 2 M.

In some embodiments, the modified K562 cells comprise modifications that increase the performance of one or more of the following: CD64, CD86, CD137L, and membrane-bound IL-21. In some embodiments, the modified K562 cells comprise modifications that increase antigenic performance. In some embodiments, the modified K562 cells comprise a modification that increases CD19 performance.

In another aspect, the present invention provides a method for generating modified K562 cells that has reduced MHC class I performance compared to wild-type K562 cells, the method comprising modifying K562 cells to reduce or prevent MHC I Class performance.

In some embodiments, the modification reduces or prevents the performance of a polypeptide encoded by a gene encoding a MHC class I polypeptide. In some embodiments, the gene encoding a MHC class I polypeptide is B 2 M.

In some embodiments, the method comprises modifying K562 cells to increase the performance of one or more factors capable of increasing the activation or proliferation of immune cells. In some embodiments, the method comprises introducing into a K562 cell a nucleic acid encoding one or more factors capable of increasing immune cell activation / proliferation. In some embodiments, one or more factors capable of increasing immune cell activation / proliferation are selected from the group consisting of a costimulatory molecule, cytokine, or antigen. In some embodiments, the costimulatory molecule is selected from the group consisting of CD40L, CD86, CD137L, CD80, or CD83. In some embodiments, the interleukin is selected from the group consisting of IL-21, IL-15, membrane-bound IL-21, and membrane-bound IL-15.

In some embodiments, the method comprises introducing into a K562 cell a nucleic acid encoding one or more Fc receptors.

In some embodiments, the method comprises modifying K562 cells to increase antigen performance.

In another aspect, the invention provides a modified K562 cell obtained or obtainable by a method according to the invention.

In another aspect, the invention provides a method for generating or expanding an immune cell population comprising contacting immune cells in vitro, in vivo or ex vivo with a modified K562 cell according to the invention.

In some embodiments, the method is a method for generating or expanding an antigen-specific immune cell population, wherein the method comprises culturing immune cells in the presence of a modified K562 cell according to the invention comprising or expressing an antigen. In some embodiments, the antigen-specific immune cells are CAR-modified immune cells, and wherein the modified K562 cells comprise or express a CAR-specific antigen.

In another aspect, the invention provides a population of immune cells produced or expanded by a method according to the invention.

In another aspect, the invention provides an immune cell population according to the invention, which is provided in a method of medical treatment or prevention of a disease or condition.

In another aspect, the invention provides the use of an immune cell population according to the invention in the manufacture of a medicament for use in a method of medical treatment or prevention of a disease or condition.

In another aspect, the invention provides a method of treating or preventing a disease or condition in an individual, comprising administering to the individual an immune cell population according to the invention.

In another aspect, the invention provides a method of treating or preventing a disease or condition in an individual, comprising:
(a) isolating immune cells from the individual;
(b) generating or expanding an immune cell population by culturing the immune cells isolated in step (a) in the presence of the modified K562 cells according to the present invention; and
(c) administering to the individual the population of immune cells produced or expanded in step (b).

In some embodiments, the disease or condition is a T cell dysfunction disorder, cancer or infectious disease. In some embodiments, the cancer is selected from the group consisting of colon cancer, colon cancer, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular carcinoma , Head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urethral epithelial cancer, melanoma, advanced melanoma, Renal cell carcinoma, ovarian cancer, or mesothelioma.

In another aspect, the invention provides a nucleic acid or a plurality of nucleic acids encoding a site-specific nuclease (SSN) system that targets B 2 M.

In some embodiments, the nucleic acid or nucleic acids encode a CRISPR / Cas9 system. In some embodiments, the nucleic acid or nucleic acids encode a CRISPR RNA (crRNA) that targets B 2 M exons. In some embodiments, the nucleic acid or nucleic acids encode a crRNA that targets B2M exon 1 and / or a crRNA that targets exon 2.

In another aspect, the invention provides a vector or a plurality of vectors encoding a nucleic acid or a plurality of nucleic acids according to the invention.

In another aspect, the invention provides a method for producing a modified cell that has a reduced MHC class I performance compared to a comparable unmodified cell, the method comprising A modification is introduced with a nucleic acid, a plurality of nucleic acids, a vector, or a plurality of vectors as in the present invention. In some embodiments, the modified cells are modified K562 cells.

Description The present invention is directed to a modified K562 cell that can be used to generate / expand a population of immune cells for use in methods such as adoptive cell transfer, wherein the generated / expanded population contains less specificity for MHC class I Immune cells.
K562 cells

K562 cell line is a human myeloid leukemia cell line derived from the pleural exudate of a 53-year-old woman with chronic acute myelogenous leukemia and described in Klein et al., International Journal of Cancer (1976) 18, 421. K562 cells are available from various commercial and non-commercial sources, such as the American Type Culture Collection (ATCC) and Sigma (Sigma catalog number 89121407). K562 cell lines were obtained by methods that did not involve the destruction of human embryos.

In some embodiments according to various aspects of the invention, K562 cells (also referred to herein as "wild-type" or "WT" K562 cells or "unmodified" K562 cells) are deposited with ATCC accession number CCL-243 Deposited cells of cells. In some embodiments, the K562 cell line is a human myeloid leukemia cell, which comprises the following DNA profile: STR-PCR data: enamel protein: X; CSF1PO: 9,10; D13S317: 8; D16S539: 11,12; D5S818: 11,12; D7S820: 9,11; THO1: 9.3; TPOX: 8,9; vWA: 16. In some embodiments, K562 cells are human myeloid leukemia cells, which comprise the genome of a cell line cell deposited under ATCC accession number CCL-243.

The aspect of the present invention relates to modified K562 cells. As used herein, a "modified" K562 cell is a K562 cell that has been altered in such a way that the modified K562 cell is different from a wild-type K562 cell. This change can alter one or more of the structural and / or functional characteristics of K562 cells.
Reduced performance of MHC Class I

In some embodiments, the performance of MHC class I of the modified K562 cells according to the invention is reduced compared to wild-type K562 cells. Performance may refer to gene performance and / or protein performance.

MHC class I molecules are heterodimers of alpha chains and beta2-microglobulin (B2M). The α-chain has three domains named α1, α2, and α3. The α1 and α2 domains together form a peptide-binding groove presented by MHC class I molecules to form a peptide-MHC complex. MHC class I α-chains are polymorphic, and different α-chains are capable of binding and presenting different peptides. In humans, MHC class I alpha-chains are encoded by the human leukocyte antigen (HLA) gene. B2M is necessary for the cell surface appearance of MHC class I molecules; its defects can destroy the functional structure of MHC class I complexes and reduce the surface performance of MHC class I molecules (27, 28). Because the B2M gene in the human genome is extremely conserved, the destruction of B2M can result in low immunogenic cells that lack the performance of MHC class I molecules.

Reduced MHC Class I performance may refer to reduced performance of one or more polypeptides of the MHC Class I molecule. In some embodiments, the performance of β2 microglobulin (B2M) polypeptide in modified K562 cells is reduced compared to wild-type K562 cells. In some embodiments, the performance of MHC class I alpha chain polypeptides of modified K562 cells is reduced compared to wild-type K562 cells. In some embodiments, the performance of MHC class I complexes of modified K562 cells is reduced compared to wild-type K562 cells.

The expression of a polypeptide or a polypeptide complex by a cell can be determined by analysis according to various methods well known to the skilled person. Such methods include analysis using antigen-binding molecules (such as antibodies or aptamers) specific for the polypeptide / polypeptide complex of interest, such as Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

In some embodiments, compared to wild-type K562 cells, the surface performance of one or more polypeptides of the MHC class I complex of modified K562 cells is reduced, such as B2M, MHC class I alpha chain polypeptides, and / or MHC class I The surface appearance of the composite is reduced. Surface expression refers to the expression of related polypeptide / polypeptide complexes that can be detected on the cell surface (ie, in or at the cell membrane). When the polypeptide / polypeptide complex is expressed on the cell surface, the surface expression of a given polypeptide or polypeptide complex can be analyzed, for example, on intact cells using antigen-binding molecules specific to the extracellular polypeptide / polypeptide complex region of the cell.

In some embodiments, the gene expression of one or more polypeptides of the MHC class I molecule of the modified K562 cells is reduced compared to wild-type K562 cells. The genetic expression of a cell can be determined, such as quantitatively encoding mRNAs of one or more polypeptides, such as by quantitative real-time PCR (qRT-PCR) or by reporter-based methods. For example, a gene for one or more polypeptides of a MHC class I molecule can be identified by detecting a decrease in the level of a nucleic acid encoding a B2M polypeptide (e.g., a wild-type B2M polypeptide) and / or a decrease in the level of a nucleic acid encoding a MHC class I alpha chain polypeptide. Reduced performance.

As used herein, "MHC class I polypeptide" refers to a constituent polypeptide of a MHC class I molecule (ie, a polypeptide complex of a MHC class I alpha chain polypeptide and a B2M polypeptide).

In some embodiments, the cell's MHC class I performance is analyzed under certain environmental conditions, such as in response to treating the cell with an agent capable of up-regulating MHC class I gene or protein expression. In some embodiments, the modified K562 cells of the invention, upon stimulation with IFNγ, show one or more polypeptides of MHC class I molecules compared to the level of gene / protein performance of wild-type K562 cells in response to similar stimuli (e.g., B2M or MHC class I alpha chain) decreased gene expression or protein expression. In some embodiments, the modified K562 cells respond to stimuli-like genes with wild-type K562 cells after stimulation with a cell culture supernatant of a co-culture of K562 cells and immune cells (eg, PBMC or T cells). Compared to the level of protein expression, it shows a decrease in gene expression or protein expression of one or more polypeptides of MHC class I molecules (eg, B2M or MHC class I alpha chain).

Relative to the performance level of wild-type K562 cells, the level of gene expression or protein expression of a given factor can be "reduced" by measuring the performance level of factors in modified K562 cells and measuring the performance of factors in wild-type K562 cells Level, and by comparing these values to determine if the performance level of the modified K562 cells is reduced relative to the performance level of wild-type K562 cells. In some embodiments, the reduced performance level may be less than 1 times the performance level of wild-type K562 cells under the same conditions, such as less than 0.99 times, 0.98 times, 0.97 times, 0.96 times, 0.95 times, 0.94 times, 0.93 times, 0.92 times, 0.91 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times or less than 0.1 times.

In some embodiments, the modified K562 cells according to the invention have substantially no MHC class I, B2M or MHC class I alpha chain gene / protein expression. In some embodiments, the modified K562 cells have undetectable levels of gene / protein performance of MHC class I, B2M, or MHC class I alpha chains (e.g., by standard methods for detecting gene and / or protein performance Determination).

In some embodiments, the modified K562 cells do not substantially show surface manifestations of MHC class I, for example, as determined by flow cytometry analysis using antibodies capable of binding to MHC class I. In some embodiments, the modified K562 cells do not substantially show surface appearance of B2M, for example, as determined by flow cytometry analysis using antibodies capable of binding to B2M. In this type of analysis, the staining level of the modified antibody to K562 cells by the relevant antibody may not be significantly different from the staining level of the appropriate isotype control antibody of the same type.

In some embodiments, the modified K562 cells may be referred to as MHC class I negative or MHC class I knockout K562 cells. In some embodiments, the modified K562 cells may be referred to as B2M negative or B2M gene knockout K562 cells.

The modified K562 cells of the invention may have reduced MHC I, for example, as a result of treatment with an agent that reduces gene and / or protein performance of one or more polypeptides of the MHC class I molecule (e.g., B2M polypeptide or MHC class I alpha chain polypeptide). Class performance. Agents capable of preventing or reducing the expression of one or more polypeptides of the MHC class I molecule can inhibit, for example, the RNA encoding the B2M polypeptide or the MHC class I alpha chain polypeptide by inhibiting the transcription of the gene encoding the B2M polypeptide or the MHC class I alpha chain polypeptide. Post-transcriptional processing reduces the stability of RNA encoding B2M polypeptide or MHC class I alpha chain polypeptide, inhibits translation of RNA encoding B2M polypeptide or MHC class I alpha chain polypeptide, and promotes RNA encoding B2M polypeptide or MHC class I alpha chain polypeptide Degradation of B2M polypeptides or MHC class I alpha chain polypeptides, post-translational processing, inhibition of the association of B2M polypeptides and MHC class I alpha chain polypeptides, inhibition of the formation of MHC class I peptide complexes, and reduction of B2M peptides and MHC class I alphas Stability of chain polypeptides or MHC class I polypeptide complexes, or promote degradation of B2M polypeptides, MHC class I alpha chain polypeptides or MHC class I polypeptide complexes to prevent or reduce the performance of one or more polypeptides of MHC class I molecules. In some embodiments, the agent can inhibit gene expression or protein expression of MHC class I via RNA interference (RNAi). In some embodiments, the agent may be or may encode a shRNA or siRNA that targets a nucleic acid encoding a B2M or MHC class I alpha chain.

In some preferred embodiments, the modified K562 cells of the invention comprise modifications to a nucleic acid encoding a MHC class I polypeptide. Compared to wild-type K562 cells, the modification reduces the level of genes and / or proteins of one or more polypeptides of the cell's MHC class I molecules (eg, B2M or MHC class I alpha chains).

In some embodiments, the modified K562 cells comprise modifications to a gene encoding a MHC class I polypeptide. In some embodiments, the modified K562 cells comprise a modification of a gene encoding a B2M polypeptide relative to a wild-type K562 cell. In some embodiments, the modified K562 cells comprise modifications to genes encoding MHC class I alpha chains relative to wild-type K562 cells.

The nucleotide sequence of the gene encoding human B2M (NCBI reference sequence: NG_012920.1) is shown in SEQ ID NO: 1. The mRNA sequence transcribed from NG_012920.1 is shown in SEQ ID NO: 2 (NCBI reference sequence: XM_005254549.3), and its protein coding sequence is shown in SEQ ID NO: 3.

In some embodiments, the modified K562 cells comprise a non-wild-type B 2 M allele. That is, in some embodiments, the modified K562 cells comprise a B 2 M allele, which includes modifications relative to the B 2 M alleles possessed by wild-type K562 cells.

The K562 cell genome contains three alleles of the B 2 M gene (45). For this reason, generating B2M gene knockout K562 cells is challenging because K562 cells must be modified in such a way that all three copies of B2M are destroyed.

In some embodiments, the modified K562 cells comprise more than one non-wild-type B2M allele. In some embodiments, the modified K562 cells comprise modifications to each B2M allele. In some embodiments, the modified K562 cells lack the B2M alleles possessed by wild-type K562 cells.

The human B2M polypeptide was translated into a 119 amino acid polypeptide having the amino acid sequence shown in SEQ ID NO: 4 (UniProt: P61769-1, v1). After processing to remove 20 amino acid signal peptides, mature B2M has the amino acid sequence shown in SEQ ID NO: 5.

In some embodiments, the modification to the B2M allele comprises an insertion, substitution, or deletion in a nucleic acid sequence encoding a B2M polypeptide. In some embodiments, the modification reduces or prevents performance of a polypeptide according to SEQ ID NO: 4 or SEQ ID NO: 5 from a modified nucleic acid sequence. In some embodiments, the modified K562 cells comprise a B2M allele, which does not encode an amino acid sequence according to SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the modified K562 cells lack a nucleic acid encoding a polypeptide according to SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments, the modification introduces a premature stop codon in a sequence transcribed from a non-wild-type B2M allele. In some embodiments, the non-wild-type B2M allele encodes a truncated and / or non-functional B2M polypeptide. In some embodiments, the non-wild-type B2M allele encodes a misfolded and / or degraded B2M polypeptide. In some embodiments, the non-wild-type B2M allele encodes a B2M polypeptide that is unable to participate in a functional MHC class I polypeptide complex. In some embodiments, the non-wild-type B2M allele encodes a B2M polypeptide that cannot associate with a MHC class I alpha chain.

In some embodiments, the non-wild-type B2M allele comprises a modification to a nucleic acid encoding an exon of B2M . In some embodiments, the non-wild-type B2M allele comprises a modification to a nucleic acid sequence encoding exon 1 of B2M . In some embodiments, the non-wild-type B2M allele comprises a modification to a nucleic acid sequence encoding exon 2 of B2M .

In some embodiments, the non-wild-type B2M allele comprises an insertion, deletion, or substitution of a nucleic acid sequence encoding exon 1 of B2M . In some embodiments, the non-wild-type B2M allele comprises an insertion, deletion, or substitution of a nucleic acid sequence encoding exon 2 of B2M .

In some embodiments, the non-wild-type B2M allele comprises an insertion of a nucleotide (eg, thymidine (T)) between positions corresponding to 70 and 71 of SEQ ID NO: 1. In some embodiments, the non-wild-type B2M allele comprises a deletion corresponding to positions 51 to 69 of SEQ ID NO: 1.

In some embodiments, the non-wild-type B2M allele comprises a nucleic acid sequence encoding a marker (eg, a detectable marker and / or a selectable marker). In some embodiments, the label is a fluorescent protein, an enzyme, or an enzyme substrate. In some embodiments, the non-wild-type B2M allele comprises a nucleic acid sequence encoding an antibiotic resistance (eg, a nucleic acid sequence encoding a neomycin resistance gene neo ).

The skilled person can determine whether a given cell contains a wild-type B2M allele or a non-wild-type B2M allele, and can also determine the nucleotide sequence of the B2M allele by methods well known to the skilled person, including by the classical Termination method sequencing or by Next Generation Sequencing (NGS) reviewed by, for example, Metzker, ML, Nat Rev Genet January 2010; 11 (1): 31-46 (incorporated by reference herein).
Increased expression of factors and / or Fc receptors that can increase immune cell activation / proliferation

In some embodiments, the performance of one or more of the modified K562 cells according to the present invention capable of increasing immune cell activation / proliferation is increased compared to wild-type K562 cells. In some embodiments, the performance of one or more Fc receptors of the modified K562 cells according to the invention is increased compared to wild-type K562 cells.

The performance may be a genetic and / or protein performance. Gene performance can be measured by various means known to those skilled in the art, such as by using quantitative real-time PCR (qRT-PCR) or by reporter-based methods to measure mRNA levels. Protein performance can be measured by various methods well known in the art, such as by antibody-based methods, such as by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or Reporter-based approach.

Relative to the performance level of wild-type K562 cells, the level of gene expression or protein expression of a given factor can be increased by measuring the level of factor performance in modified K562 cells and measuring the level of factor performance in wild-type K562 cells. And by comparing these values to determine whether the performance level of the modified K562 cells increased relative to the performance level of wild-type K562 cells. In some embodiments, the increased performance level may be 1 times greater than that of wild-type K562 cells under the same conditions, such as greater than 1.01 times, 1.02 times, 1.03 times, 1.04 times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times , 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more than 100 times.

Factors capable of increasing immune cell activation can be identified by contacting immune cells or a population of immune cells with the factor and subsequently analyzing one or more immune cell activation markers of the immune cells. Factors that increase immune cell activation can be detected by increasing the level of performance of one or more immune cell activation markers and / or manifesting one or more immune cell activations after treatment with related factors (compared to appropriate control conditions) An increase in the proportion of labeled cells was identified. T-cell activation markers include, for example, CD69 and CD45R0. Markers for NK cell activation include, for example, CD69, CD107a, and KLRG1.

Similarly, a factor capable of increasing immune cell proliferation can be identified by contacting an immune cell or a population of immune cells with the factor and subsequently analyzing the immune cell proliferation. Factors that increase immune cell proliferation can be identified by detecting increased levels of immune cell proliferation after treatment with related factors (compared to appropriate control conditions). Cell proliferation levels can be determined by analyzing cell division over time. Cell division can be analyzed, for example, by in vitro analysis of 3H-thymidine incorporation or by CFSE dilution analysis, as described in Fulcher and Wong, Immunol Cell Biol (1999) 77 (6): 559-564, which The entire citation is incorporated herein.

In some embodiments, the factor capable of increasing the activation / proliferation of immune cells is selected from the group consisting of a co-stimulatory molecule, a cytokine, or an antigen.

K562 cells are usually engineered to increase the performance of costimulatory molecules; see, for example, Suhoski et al., Mol Ther (2007) 15 (5): 981-988, Turtle and Riddell Cancer J. (2010) 16 (4): 374- 381, and Butler and Hirano, Immunol Rev. (2014) Jan; 257 (1): 10, all of which are incorporated herein by reference in their entirety. K562 cells with increased costimulatory molecules promote the activation and proliferation of immune cells that display costimulatory ligands.

In some embodiments, the performance of one or more of the following costimulatory molecules of modified K562 cells according to the present invention is increased compared to wild-type K562 cells: CD70, CD40, LFA3, ICAM1, CD80, CD86, CD137L, OX40L, ICOSL, LIGHT, LTb and GITRL. In some embodiments, the performance of one or more of the following costimulatory molecules of modified K562 cells is increased compared to wild-type K562 cells: CD40L, CD70, CD80, CD83, CD86, ICOSL, GITRL, CD137L, and OX40L. In some embodiments, the performance of one or more of the following costimulatory molecules of modified K562 cells is increased compared to wild-type K562 cells: CD40L, CD86, and CD137L.

K562 cells are also engineered to increase the performance of other molecules that can stimulate the activation / proliferation of immune cells, such as cytokines. For example, Wang et al., Clin Exp Immunol. (2013) 172 (1): 104-12 (incorporated herein by reference in its entirety) describe IL-21 that has been engineered to exhibit membrane binding on the cell surface (mbIL-21) The use of K562 cells (which also modify the cell surface appearance of CD137L) to expand NK cells in the PBMC population, and Denman et al., PLoS One (2012) 7 (1): e30264, described as engineered Use of K562 cells expressing membrane-bound membrane-bound IL-15 (mbIL-15) to expand NK cells.

In some embodiments, compared to wild-type K562 cells, the performance of one or more of the following molecules of modified K562 cells according to the invention is increased: IL-21, membrane-bound IL-21, IL-15 And membrane-bound IL-15. In some embodiments, the performance of membrane-bound IL-21 and / or membrane-bound IL-15 of modified K562 cells is increased compared to wild-type K562 cells.

In some embodiments, the performance of one or more antigens of the modified K562 cells according to the invention is increased compared to wild-type K562 cells. As used herein, "antigen" refers to a molecule capable of activating an immune cell that exhibits a receptor specifically directed against an antigen.

In some embodiments, the antigen is a cancer cell antigen. Cancer cell antigens are antigens expressed or overexpressed by cancer cells. The cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or a fragment thereof. Cancer cell antigens can be expressed abnormally by cancer cells (for example, cancer cell antigens can be expressed abnormally), or they can be expressed by cancer cells with abnormal structures. In some embodiments, the antigen is expressed on the cell surface of a cancer cell (ie, the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the cancer cell antigen is an antigen whose performance correlates with the development, progression, or severity of symptoms of the cancer. Cancer-associated antigens may be related to the etiology or pathology of cancer, or they may behave abnormally due to cancer. In some embodiments, the cancer cell antigen line is, for example, upregulated by cancer cells (e.g., in RNA and / or protein) compared to the performance level of comparable non-cancer cells (e.g., non-cancer cells derived from the same tissue / cell type) Substandard). In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not by comparable non-cancer cells (eg, non-cancer cells derived from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be a product of a mutant oncogene or a mutant tumor suppressor gene. In some embodiments, the cancer-associated antigen may be an overexpressing cellular protein, a cancer antigen produced by an oncovirus, a carcinoembryonic antigen, or a cell surface glycolipid or glycoprotein product.

In some embodiments, the antigen is CD19. CD19 disease associations are reviewed, for example, in Wang et al., Exp Hematol Oncol. (2012) 1:36. CD19 is highly conserved on most B-cell tumors and is present in most acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma (Cooper et al. Blood Cells Mol Dis. (2004) ) 33 (1): 83-9). Most B-cell malignancies show CD19 at normal to high levels (80% of ALL, 88% of B-cell lymphoma, and 100% of B-cell leukemia). CD19 was also observed in cases of bone marrow malignancies, including 2% of AML cases.

In some embodiments, the performance of one or more Fc receptors of the modified K562 cells is increased compared to wild-type K562 cells. Feeder cells and artificial antigen-presenting cells are usually engineered to increase the expression of Fc receptors such as CD64, CD32, and CD16, and to improve the presentation of, for example, agonist anti-CD3 antibodies (such as pure OKT3) and / or agonist anti-CD28 antibodies, Thus activating immune cells expressing CD3-see, for example, Turtle and Riddell Cancer J. (2010) 16 (4): 374-381, which is incorporated herein by reference. Cells expressing increased levels of Fc receptors are particularly suitable for use in methods of generating / expanding immune cell populations using agonist anti-CD3 and / or anti-CD28 antibodies for antigen-independent T cell activation.

Thus, in some embodiments, the performance of one or more Fc receptors of the modified K562 cells according to the invention is increased compared to wild-type K562 cells. In some embodiments, the Fc receptor is a receptor for Fcy. In some embodiments, the Fc receptor is selected from the group consisting of CD64, CD32, and CD16.

In some embodiments, compared to wild-type K562 cells, modified K562 cells according to the present invention have increased performance of one or more of the following factors: CD19, CD40L, CD86, CD137L, mbIL-21, and CD64.

Compared with wild-type K562 cells, the modified K562 cells according to the present invention can be engineered to increase the performance of one or more factors, and therefore can increase the performance of one or more factors capable of increasing the activation / proliferation of immune cells and / or Increase the performance of one or more Fc receptors.

In some embodiments, the modified K562 cells comprise an exogenous nucleic acid encoding one or more factors capable of increasing the activation / proliferation of immune cells and / or one or more Fc receptors. As used herein, "exogenous" nucleic acid refers to a nucleic acid that is not endogenous to wild-type K562 cells; that is, a nucleic acid that is not included in the genome of wild-type K562 cells.

In some embodiments, the modified K562 cells contain one or more factors that encode to increase the activation / proliferation of immune cells and / Or exogenous nucleic acid of one or more Fc receptors.

In some embodiments, the modified K562 cells stably express an exogenous nucleic acid encoding one or more factors capable of increasing the activation / proliferation of immune cells and / or one or more Fc receptors. In some embodiments, the exogenous nucleic acid can be integrated into the genome of a modified K562 cell.

In some embodiments, the exogenous nucleic acid can be integrated into the genome of the modified K562 cells at a particular locus. In some embodiments, the exogenous nucleic acid can be integrated into the genome of a modified K562 cell at a genomic safe harbor (GSH). GSH is a site that supports the stable integration and expression of exogenous nucleic acids, while minimizing the risk of unwanted interactions with the host cell genome (see, eg, Sadelain et al., Nat Rev Cancer. (2011) 12 (1): 51-8).

Several safe GSHs have been identified for the stable integration of foreign nucleic acids in human cells, including AAVS1 , the naturally occurring site of AAV virus integration on chromosome 19; the CCR5 gene, which is the chemokine receptor gene, Also known as the HIV-1 co-receptor; and human orthologs of the mouse Rosa26 locus (see, eg, Papapetrou and Schambach Mol Ther. (2016) 24 (4): 678-684). In some embodiments, the exogenous nucleic acid is integrated at AAVS1 .

In some embodiments, the modified K562 cells according to the invention may be treated to inhibit / prevent cell proliferation (ie, the cells may be "inactive"). Inactivated cells may lack the ability to undergo cell division (and may therefore lack the ability to proliferate). In some embodiments, K562 cells can be inactivated by treatment with mitomycin C or cyclosporine A or exposure to ionizing radiation (eg, gamma radiation, X-rays, or UV light). Conditions suitable for such treatment / exposure are known to the skilled person and can be determined, for example, with reference to Roy et al., J Hematother Stem Cell Res (2001) 10 (6): 873-80.

In some embodiments, a modified K562 cell or a modified K562 cell line according to the invention is provided in an isolated form or a substantially purified form. Modified K562 cells can be isolated / purified, for example, from one or more other cell types, such as wild-type K562 cells.

In specific embodiments, the modified K562 cells of the invention may include one of the following phenotypes (e.g., as determined by flow cytometry using antibodies specific for related factors): MHC class I negative / B2M negative ; MHC class I / B2M negative, CD40L positive, CD86 positive, CD137L positive, and mbIL-2 positive; MHC class I negative / B2M negative, CD40L positive, CD86 positive, CD137L positive, mbIL-2 positive, and CD64 positive; MHC I Class Negative / B2M negative, CD40L positive, CD86 positive, CD137L positive, mbIL-2 positive, and CD19 positive; MHC Class I negative / B2M negative, CD40L positive, CD86 positive, CD137L positive, mbIL-2 positive, CD64 positive, and CD19 positive .
Functional characteristics of modified K562 cells

The modified K562 cells of the invention can be characterized by reference to one or more functional properties.

In some embodiments, the modified K562 cells according to the present invention have one or more of the following characteristics:
Compared with wild-type K562 cells, the surface expression of B2M and / or MHC class I is reduced after stimulation with an agent that up-regulates the B2M / MHC class I surface expression of wild-type K562 cells;
Compared with wild-type K562 cells, the lysis by NK cells is increased;
Expansion of T cells showing reduced cytotoxicity to cells expressing HLA-C * 03 or HLA-C * 05 compared to T cells expanded using wild-type K562 cells;
Compared with T cells expanded using wild-type K562 cells, the expansion is less specific for T cells expressing HLA-C * 03 or HLA-C * 05 cells;

In some embodiments, after stimulation with an agent that up-regulates the B2M / MHC class I surface expression of wild-type K562 cells, compared to wild-type K562 cells, the B2M and / or MHC class I surface expression of modified K562 cells reduce. In some embodiments, the agent is IFNγ. In some embodiments, the agent is a co-culture supernatant layer of T cells and wild-type K562 cells. The surface performance of B2M / MHC class I can be determined in vitro, for example, by flow cytometry using antibodies specific for B2M / MHC class I. In some embodiments, as described in the examples of this disclosure, after stimulation with an agent that up-regulates the surface performance of B2M / MHC Class I, compared to wild-type K562 cells, the B2M and And / or MHC Class I surface appearance. In some embodiments, in a comparable analysis, modified K562 cells show less than 1-fold compared to wild-type K562 cells after stimulation with an agent that up-regulates the B2M / MHC Class I surface expression of wild-type K562 cells, eg, Less than 0.99 times, 0.98 times, 0.97 times, 0.96 times, 0.95 times, 0.94 times, 0.93 times, 0.92 times, 0.91 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 B2M and / or MHC Class I surface performance at or below 0.1 times.

In some embodiments, modified K562 cells are easier to lyse by NK cells than wild-type K562 cells. Sensitivity to lysis by NK cells can be determined in vitro using, for example, NK cell cytotoxicity assays. Cytotoxicity analysis is known to the skilled person and is reviewed, for example, in Zaritskaya et al., Expert Rev Vaccines (2011), 9 (6): 601-616, which is incorporated herein by reference in its entirety. In some embodiments, as described in the examples of this disclosure, analysis is performed by NK cell lysing lines using a DELFIA EuTDA cytotoxicity assay. In some embodiments, the level of cytotoxicity (e.g., determined to be a level of cytolysis) exhibited by NK cells to modified K562 cells is greater than 1-fold, such as greater than 1.01-fold, 1.02-fold, 1.03-fold, 1.04 Times, 1.05 times, 1.06 times, 1.07 times, 1.08 times, 1.09 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or more than 10-fold cytotoxicity levels (e.g., cytolytic) percentage).

It should be understood that the modified K562 cells of the present invention can be used to generate / expand an immune cell population with reduced allogeneic reactivity compared to an immune cell population generated / expanded using wild-type K562 cells. In particular, the modified K562 cells of the present invention can be used to generate / expand an immune cell population that specifically targets a reduced number of cells expressing HLA-C * 03 or HLA-C * 05 cells.

In some embodiments, the modified K562 cells expand T cells that exhibit reduced cytotoxicity to cells expressing HLA-C * 03 or HLA-C * 05 compared to T cells expanded using wild-type K562 cells . The cytotoxicity of the expanded T cell population to cells expressing HLA-C * 03 or HLA-C * 05 can be analyzed, for example, using a cytotoxicity assay, such as Zaritskaya et al., Expert Rev Vaccines (2011), 9 (6): 601- The analysis described in 616 was performed in vitro. In some embodiments, the cytotoxicity of T cells to cells expressing HLA-C * 03 or HLA-C * 05 is analyzed using a DELFIA EuTDA cytotoxicity assay as described in the examples of this disclosure. In some embodiments, in comparable analysis, the level of cytotoxicity exhibited by cultured expanded T cells in the presence of modified K562 cells to cells expressing HLA-C * 03 or HLA-C * 05 is less than 1 times, such as less than 0.99 times, 0.98 times, 0.97 times, 0.96 times, 0.95 times, 0.94 times, 0.93 times, 0.92 times, 0.91 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, or less than 0.1 times the level of cytotoxicity exhibited by cultured expanded T cells in the presence of wild-type K562 cells.

In some embodiments, compared to T cells expanded using wild-type K562 cells, modified K562 cell expansion is less specific to cells expressing HLA-C * 03 or HLA-C * 05 (i.e., to It is reactive, for example, T cells which show cytotoxicity). In some embodiments, in a comparable analysis, the modified K562 cells expand less than 1-fold, such as less than 0.99-fold, 0.98-fold, 0.97-fold, 0.96-fold, 0.95-fold, 0.94-fold, 0.93-fold, 0.92-fold, 0.91 times, 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, 0.4 times, 0.3 times, 0.2 times, or less than 0.1 times The specificity of expansion by wild-type K562 cells is directed against the expression of HLA-C * 03 or HLA -T cells of C * 05 cells.
Method for generating modified K562 cells

The invention also provides a method for generating a modified K562 cell according to the invention.

In some embodiments, the method comprises modifying K562 cells (eg, wild-type K562 cells) to reduce or prevent the performance of MHC class I. In some embodiments, the method comprises modifying K562 cells to reduce or prevent MHC class I performance on the cell surface. In some embodiments, the method comprises modifying K562 cells to reduce or prevent the performance of B2M and / or MHC class I alpha chain polypeptides.

In some embodiments, the method comprises modifying K562 cells to reduce or prevent the performance of a polypeptide encoded by a gene encoding a MHC class I polypeptide. In some embodiments, the method comprises modifying K562 cells to reduce or prevent the performance of a polypeptide encoded by B2M. In some embodiments, the method comprises modifying K562 cells to reduce or prevent the performance of a polypeptide encoded by a gene (eg, an HLA gene) encoding a MHC class I alpha chain polypeptide.

In some embodiments, the modification comprises treating K562 cells with an agent capable of reducing gene and / or protein performance of one or more polypeptides of the MHC class I molecule (eg, a B2M polypeptide or an MHC class I alpha chain polypeptide). In some embodiments, the agent may be capable of inhibiting the transcription of genes encoding B2M polypeptides or MHC class I alpha chain polypeptides, inhibiting post-transcriptional processing of RNA encoding B2M polypeptides or MHC class I alpha chain polypeptides, and reducing B2M polypeptide or MHC encoding Stability of RNA of type I α chain polypeptide, inhibiting translation of RNA encoding B2M polypeptide or MHC type I α chain polypeptide, promoting degradation of RNA encoding B2M polypeptide or MHC type I α chain polypeptide, and inhibiting B2M polypeptide or MHC type I Post-translational processing of α-chain polypeptides, inhibits the association of B2M polypeptides and MHC class I α-chain polypeptides, inhibits the formation of MHC class I polypeptide complexes, and reduces B2M polypeptides, MHC class I α-chain polypeptides, or MHC class I polypeptide complexes Stability, or promote degradation of B2M polypeptides, MHC class I alpha chain polypeptides or MHC class I polypeptide complexes. In some embodiments, the agent can inhibit gene expression or protein expression of MHC class I via RNA interference (RNAi). In some embodiments, the agent may be or may encode a shRNA or siRNA that targets a nucleic acid encoding a B2M or MHC class I alpha chain.

In some embodiments, the method comprises modifying a nucleic acid encoding a MHC class I polypeptide. Compared with wild-type K562 cells, the modification reduces the level of genes and / or proteins of one or more polypeptides of the cell's MHC class I molecules (eg, B2M polypeptide or MHC class I alpha chain polypeptide).

In some embodiments, the method comprises modifying a gene encoding a MHC class I polypeptide. In some embodiments, the method comprises modifying a gene encoding a B2M polypeptide. In some embodiments, the method comprises modifying a gene encoding a MHC class I alpha chain.

In some embodiments, the method comprises modifying one or more alleles of a B2M gene. In some embodiments, the method comprises modifying each B2M allele.

In some embodiments, the method comprises introducing an insertion, substitution, or deletion into a nucleic acid sequence encoding a B2M polypeptide.

In some embodiments, the method comprises introducing a modification that reduces or prevents performance of a polypeptide according to SEQ ID NO: 4 or SEQ ID NO: 5 from a modified nucleic acid sequence. In some embodiments, the method comprises modifying K562 cells to include a B2M allele that does not encode an amino acid sequence according to SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the method comprises modifying K562 cells to delete a nucleic acid encoding a polypeptide according to SEQ ID NO: 4 or SEQ ID NO: 5.

In some embodiments, the method comprises modifying the B2M allele to introduce a premature stop codon in a sequence transcribed from the B2M allele. In some embodiments, the method comprises modifying a B2M allele to encode a truncated and / or non-functional B2M polypeptide. In some embodiments, the method comprises modifying a B2M allele to encode a misfolded and / or degraded B2M polypeptide. In some embodiments, the method comprises modifying a B2M allele to encode a B2M polypeptide that is unable to participate in a functional MHC class I polypeptide complex. In some, the method comprises modifying the B2M allele to encode a B2M polypeptide that is unable to associate with a MHC class I alpha chain.

In some embodiments, the method comprises modifying a nucleic acid encoding a B2M exon. In some embodiments, the method comprises modifying a nucleic acid sequence encoding exon 1 of B2M . In some embodiments, the method comprises modifying a nucleic acid sequence encoding B2M exon 2.

In some embodiments, the method comprises introducing an insertion, deletion, or substitution into a nucleic acid sequence encoding B2M exon 1. In some embodiments, the method comprises introducing an insertion, deletion, or substitution into a nucleic acid sequence encoding B2M exon 2.

In some embodiments, the method includes inserting a nucleotide (eg, thymidine (T)) between positions corresponding to 70 and 71 of SEQ ID NO: 1. In some embodiments, the method deletions correspond to positions 51 to 69 of SEQ ID NO: 1.

In some embodiments, the method includes inserting a nucleic acid sequence encoding a marker (eg, a detectable marker and / or a selectable marker) into a nucleic acid sequence encoding a B2M polypeptide. In some embodiments, the label is a fluorescent protein, an enzyme, or an enzyme substrate. In some embodiments, the method comprises inserting a nucleic acid sequence encoding an antibiotic resistance (eg, a nucleic acid sequence encoding a neomycin resistance gene neo ) into a nucleic acid sequence encoding a B2M polypeptide.

Modification of a nucleic acid encoding an MHC class I polypeptide (such as a B2M polypeptide or an MHC class I alpha chain polypeptide) according to the method of the present invention can be accomplished in a variety of ways known to the skilled person, including modification of the target nucleic acid by homologous recombination and use of site specificity Nuclease (SSN) performs target nucleic acid editing. Previous studies have used conventional gene targeting mediated by adeno-associated virus (AAV) vectors or B2M destruction of human embryonic stem cells using transcriptional activator-like effector nucleases (TALEN) (29, 30).

In some embodiments, the methods employ targeting by homologous recombination, such as in Mortensen Curr Protoc Neurosci. (2007) Chapter 4: Unit 4.29 and Vasquez et al., PNAS 2001, 98 (15): 8403-8410 In the review, the two documents are incorporated herein by reference in their entirety. Targeting by homologous recombination involves the exchange of nucleic acid sequences through an exchange event directed by a homologous sequence.

In some embodiments, the methods employ target nucleic acid editing using SSN. Gene editing using SSN is reviewed, for example, in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48 (10): e265, which is incorporated herein by reference in its entirety. Enzymes capable of generating site-specific double-strand breaks (DSBs) can be engineered to introduce DSBs into a target nucleic acid sequence of interest. DSB can be repaired by error-prone non-homologous end joining (NHEJ), in which the two ends of the break are reconnected, usually with nucleotides inserted or deleted. Alternatively, DSB can be repaired by highly homologous directed repair (HDR), in which a DNA template whose ends are homologous to the break site is provided and introduced at the DSB site.

SSNs that can be engineered to produce target-specific nucleic acid sequence-specific DSBs include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly spaced palindromic repeats / CRISPR-related 9 (CRISPR / Cas9) system.

The ZFN system is reviewed, for example, in Umov et al., Nat Rev Genet. (2010) 11 (9): 636-46, which is incorporated herein by reference in its entirety. ZFNs include a programmable zinc finger DNA binding domain and a DNA cleavage domain (eg, the Fok I endonuclease domain). DNA binding domains can be identified by screening zinc finger arrays capable of binding to a target nucleic acid sequence.

The TALEN system is reviewed, for example, in Mahfouz et al., Plant Biotechnol J. (2014) 12 (8): 1006-14, which is incorporated herein by reference in its entirety. TALEN contains a programmable DNA-binding TALE domain and a DNA cleavage domain (such as the Fok I endonuclease domain). TALE contains a repeating domain consisting of 33-39 amino acid repeats, except that the two residues at positions 12 and 13 of each repeat are repeatable variable two residues (RVD). For the same. Each RVD determines the binding of repeats to nucleotides in the target DNA sequence based on the following relationships: "HD" binds to C, "NI" binds to A, "NG" binds to T, and "NN" or "NK" binds to G (Moscou and Bogdanove, Science (2009) 326 (5959): 1501.).

CRISPR / Cas9 and related systems, such as CRISPR / Cpf1, CRISPR / C2c1, CRISPR / C2c2, and CRISPR / C2c3 are reviewed, for example, in Nakade et al., Bioengineered (2017) 8 (3): 265-273, which is incorporated by reference in its entirety Incorporated herein. These systems include endonucleases (such as Cas9, Cpf1, etc.) and single guide RNA (sgRNA) molecules. sgRNA can be engineered to target endonuclease activity to a nucleic acid sequence of interest.

In some embodiments, modifying a nucleic acid encoding a MHC class I polypeptide (eg, a B2M polypeptide or a MHC class I alpha chain polypeptide) according to the methods of the present invention comprises modification using a CRISPR / Cas9 system.

In some embodiments, the methods include introducing into a K562 cell a nucleic acid or plurality of nucleic acids that target a nucleic acid encoding a MHC class I polypeptide (e.g., a nucleic acid encoding a B2M polypeptide or a MHC class I alpha chain polypeptide). RNA (crRNA) and Cas9 endonucleases. In some embodiments, the crRNA targets a nucleic acid encoding a B2M polypeptide. In some embodiments, the crRNA targets B2M . In some embodiments, the crRNA targets B2M exons. In some embodiments, crRNA targets B2M exon 1. In some embodiments, the crRNA targets B2M exon 2. The nucleic acid or plurality of nucleic acids may be contained in one or more vectors.

Nucleic acids / vectors can be introduced into K562 cells by any suitable means, such as by transformation, transfection, electroporation, or transduction. In some embodiments, the methods include introducing the nucleic acid / vector into K562 cells by electroporation, such as Delgado-Cañedo et al., Cytotechnology. (2006) 51 (3): 141-8 (incorporated by reference in its entirety and (Incorporated herein) or as described in Example 1 of this disclosure. In some embodiments, the methods include introducing a nucleic acid / vector into K562 cells by transduction, as described in Example 1 of the present disclosure.

In some embodiments, the methods of the invention comprise modifying K562 cells to increase the performance of one or more factors (such as costimulatory molecules, cytokines, or antigens) capable of increasing immune cell activation / proliferation. In some embodiments, the methods comprise modifying K562 cells to increase the performance of one or more Fc receptors.

In some embodiments, the methods of the invention comprise modifying K562 cells to increase performance of one or more of: CD70, CD40, LFA3, ICAM1, CD80, CD86, CD137L, OX40L, ICOSL, LIGHT, LTb, and GITRL. In some embodiments, the methods comprise modifying K562 cells to increase performance of one or more of: CD40L, CD70, CD80, CD83, CD86, ICOSL, GITRL, CD137L, and OX40L. In some embodiments, the methods comprise modifying K562 cells to increase performance of one or more of the following: CD40L, CD86, and CD137L. In some embodiments, the methods comprise modifying K562 cells to increase the performance of CD86 and / or CD137L.

In some embodiments, the methods of the invention comprise modifying K562 cells to increase the performance of one or more of: IL-21, membrane-bound IL-21, IL-15, and membrane-bound IL-15. In some embodiments, the methods comprise modifying K562 cells to increase the performance of membrane-bound IL-21 and / or membrane-bound IL-15.

In some embodiments, the methods of the invention comprise modifying K562 cells to increase the performance of one or more antigens. In some embodiments, the methods comprise modifying K562 cells to increase the performance of immune cells of interest, the immune cells comprising a specific receptor (eg, TCR or CAR). In some embodiments, the methods include modifying K562 cells to increase the performance of cancer cell antigens, such as the cancer cell antigens described herein. In some embodiments, the methods comprise modifying K562 cells to increase the performance of CD19.

In some embodiments, the methods of the invention comprise modifying K562 cells to increase the performance of one or more Fc receptors. In some embodiments, the methods comprise modifying K562 cells to increase the performance of the Fcy receptor. In some embodiments, the methods comprise modifying K562 cells to increase the performance of an Fc receptor selected from the group consisting of CD64, CD32, and CD16.

In some embodiments, the methods of the invention comprise modifying K562 cells to increase performance of one or more of the following: CD19, CD40L, CD86, CD137L, mbIL-21, and CD64.

In some embodiments, modifying K562 cells to increase the performance of one or more factors capable of increasing the activation / proliferation of immune cells and / or the performance of one or more Fc receptors comprises encoding one or more encodings capable of increasing the activation / proliferation of immune cells. Factors and / or nucleic acids of one or more Fc receptors are introduced into K562 cells.

In some embodiments, modifying K562 cells to increase performance of one or more factors capable of increasing immune cell activation / proliferation and / or one or more Fc receptors comprises introducing one or more nucleic acids or vectors encoding related proteins into the cell. Nucleic acids / vectors can be introduced into K562 cells by any suitable means, such as by transformation, transfection, electroporation, or transduction.

In some embodiments, one or more nucleic acids encoding one or more factors capable of increasing the activation / proliferation of immune cells and / or one or more Fc receptors are integrated into the genome of K562 cells. In some embodiments, one or more nucleic acids encoding one or more factors capable of increasing the activation / proliferation of immune cells and / or one or more Fc receptors are integrated at a genomic safe harbor (GSH), such as the GSH described herein, In the genome of K562 cells. In some embodiments, GSH is AAVS1 . In some embodiments, the method comprises introducing into a K562 cell a nucleic acid encoding one or more SSNs that target AAVS1 . In some embodiments, the method comprises introducing into a K562 cell a nucleic acid encoding a ZFN targeted to AAVS1 .

In some embodiments, one or more nucleic acids / vectors encoding one or more factors capable of increasing the activation / proliferation of immune cells and / or one or more Fc receptors further comprise sequences homologous to the sequence of AAVS1 , for example, for use AAVS1- targeting SSN (eg, AAVS1- targeting ZFN) cuts AAVS1 and performs site-specific integration of nucleic acids encoding one or more factors capable of increasing immune cell activation / proliferation and / or one or more Fc receptors.

The modified K562 cells containing the desired modification can be cultured / expanded, for example, from a single cell pure line. Such methods may include culturing in the presence of a selection agent corresponding to a selectable marker in order to successfully introduce a nucleic acid of interest into a cell.

In some embodiments, the methods include treating the modified K562 cells to inhibit / prevent cell proliferation (ie, "inactivate" the modified K562 cells). In some embodiments, the methods include treating the cells with mitomycin C or cyclosporine A, or exposing the cells to ionizing radiation (eg, gamma radiation, X-rays, or UV light).

In some embodiments, the methods include isolating a modified K562 cell or a population of modified K562 cells, for example, from one or more other cell types (eg, wild-type K562 cells).
Nucleic acid / vector

The invention also provides a nucleic acid or a plurality of nucleic acids for generating a modified K562 cell according to the invention. The nucleic acid encodes a site-specific nuclease (SSN) system that targets B2M . In some embodiments, the SSN system is a ZFN system, a TALEN system, a CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system, or a CRISPR / C2c3 system.

In some embodiments, the nucleic acid encodes a CRISPR / Cas9 system. In some embodiments, the nucleic acid encodes a CRISPR RNA (crRNA) that targets B2M exons. In some embodiments, the nucleic acid encodes a crRNA that targets B2M exon 1. In some embodiments, the nucleic acid encodes a crRNA that targets B2M exon 2. In some embodiments, the nucleic acid encodes a crRNA that targets B2M exon 1 and exon 2. The CRISPR / Cas9 system also contains trans-activated crRNA (tracrRNA) for processing crRNA into its mature form. Thus, in some embodiments, the nucleic acid encodes a tracrRNA of crRNA.

In some embodiments, the crRNA targeted to B2M exon 1 comprises or consists of the nucleic acid of SEQ ID NO: 26. In some embodiments, the crRNA targeted to B2M exon 2 comprises or consists of the nucleic acid of SEQ ID NO: 27. Positions 21 to 42 of SEQ ID NOs: 26 and 27 provide the backbone sequence derived from plastid pX260 (Addgene # 42229; pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK -puro).

In some embodiments, the tracrRNA comprises or consists of the nucleic acid of SEQ ID NO: 28. SEQ ID NO: 28 is derived from plastid pX260 (Addgene # 42229; pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro).

Nucleic acids can be provided in one or more vectors. The invention also provides a vector or a plurality of vectors comprising a nucleic acid or a plurality of nucleic acids according to the invention. As used herein, a "vector" is a nucleic acid molecule that is used as a vehicle for transferring exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a cell. Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding a sequence to be expressed. The vector may also include a stop codon and a performance enhancer.

In some embodiments, a crRNA-encoding nucleic acid is operably linked to a ubiquitin 6 (U6) promoter. In some embodiments, a nucleic acid encoding Cas9 is operably linked to a chicken β-actin promoter (CBh) promoter. In some embodiments, a tracRNA-encoding nucleic acid is operably linked to the H1 promoter.

Any suitable vector can be used, including, for example, the pX260 plastid containing the CRISPR / Cas9 system described in Cong et al., Science 339, 819 and the pFastBac plastid described in Zeng et al., Stem Cells (2007) 25, 1055 . Multiple nuclear polyhedrosis virus (the AcMNPV) derived from the insect Autographa californica (Autographa californica) Baculovirus vectors have previously been used to efficiently transduce human pluripotent stem cells and other cell lines (37-40). This vector is reported to be capable of carrying multiple large DNA cassettes, has low cytotoxicity and is very safe because it is non-integrated.

Other suitable vectors include plastids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gamma retrovirus vectors (e.g. murine leukemia virus (MLV) derived vectors), lentiviral vectors, adenoviral vectors, adenoviruses, Related viral vectors, acne virus vectors and herpes virus vectors), transposon-based vectors and artificial chromosomes (such as yeast artificial chromosomes), such as Maus et al., Annu Rev Immunol (2014) 32: 189-225 or Morgan and Boyerinas, Biomedicines As mentioned in 2016 4, 9, these documents are incorporated herein by reference in their entirety.
Method for using modified K562 cells

The modified K562 cells of the present invention can be used in a method for generating / expanding an immune cell population. In particular, the modified K562 cells can be used to generate / expand an allogeneic immune cell population compared to an immune cell population generated / expanded using wild-type K562 cells. In some embodiments, modified K562 cells can be used to generate / expand immune cells with reduced reactivity to HLA molecules (e.g., HLA-C * 03 and / or HLA-C * 05) expressed by wild-type K562 cells group.

Therefore, the present invention provides a method for generating / expanding a population of immune cells, the method comprising contacting the immune cells with the modified K562 cells according to the present invention.

Immune cells can be contacted with modified K562 cells in vitro, ex vivo or in vivo. In some embodiments, the methods comprise culturing immune cells in vitro or ex vivo. In some embodiments, the methods comprise co-culturing a population of immune cells with a modified K562 cell according to the invention.

The cell culture system according to the method of the present invention uses a suitable cell culture medium and is performed under environmental conditions (such as temperature, pH, humidity, atmospheric conditions, stirring, etc.) suitable for culturing immune cells in vitro. It is a person skilled in cell culture techniques Well known.

Conveniently, the cell culture can be maintained in a humid atmosphere containing 5% CO ₂ at 37 ° C. The culture can be performed in any container suitable for the volume of the culture, for example, in a well of a cell culture plate, a cell culture flask, a bioreactor, or the like. It can be easily determined by the skilled person that the cell culture can be established and / or maintained at any suitable density. For example, the culture may be ~ 0.5 × 10 ⁶ to ~ 5 × 10 ⁶ cells / ml in culture (e.g., ~ 1 × 10 ⁶ cells / ml) to establish initial density. Cells can be cultured in any suitable cell culture vessel. In some embodiments, the cells are cultured in a bioreactor. In some embodiments, the cells are cultured in a bioreactor as described in Somerville and Dudley, Oncoimmunology (2012) 1 (8): 1435-1437, which is incorporated herein by reference in its entirety. In some embodiments, the cells are cultured in a GRex cell culture vessel, such as a GRex flask or a GRex 100 bioreactor.

Reference can be made to Example 1 of this disclosure to determine suitable conditions for co-culture of modified K562 cells and immune cells. Suitable conditions for using modified K562 cells in a method for generating / expanding an immune cell population (e.g., period of cell culture, ratio of modified K562 cells to immune cells, culture medium, etc.) can also be referred by the skilled person, e.g., this reference The invention examples are easily determined.

In some embodiments, the modified K562 cells are used as feeder cells to support, for example, the growth and / or survival of cells of an immune cell population in a culture that produces / expands immune cells. In some embodiments, the modified K562 cells are used as a factor to increase the activation / proliferation of the immune cell population being generated / expanded.

In some embodiments, the immune cell population generated / expanded according to the present invention is generated / expanded in an immune cell population, such as a peripheral blood mononuclear cell (PBMC) population or a peripheral blood lymphocyte (PBL) population. The immune cells to be generated / expanded may be present in the initial population of immune cells (such as PBMC or PBL) at a low frequency, and the initial population of the immune cells according to the present invention is preferably cultured such that The increase in the number of cells and / or results in an increase in the proportion of such cells in the cell population at the end of the culture.

For example, where the method is used to generate / expand a T cell population, the T cell population can be generated / expanded from the PBMC population, and these methods can increase the number of T cells and / or cause the The proportion of T cells in the cell population increased.

Immune cells (eg, PBMC, PBL) that generate / expand an immune cell population according to the method of the present invention can be freshly obtained or can be thawed from previously obtained and frozen immune cell samples.

In embodiments of the methods disclosed herein, the generation / amplification of an immune cell population may involve culturing a PBMC population. In some embodiments, the population of immune cells can be generated / expanded from a population of T cells (eg, a heterogeneous and / or specific T cell population), which can be obtained from a blood sample or a PBMC population.

The population of immune cells produced / expanded according to the method of the invention can be any desired population of immune cells. In some embodiments, the immune cell population expanded / produced according to the method of the invention is a population of one of the following cell types: neutrophils, eosinophils, basophils, dendritic cells, lymphocytes , Monocytes, T cells, B cells, NK cells, NKT cells, congenital lymphoid cells (ILC), antigen-specific immune cells (i.e., cells that display receptors specific for the antigen; for example, antigen-specific T Cells and / or antigen-specific NK cells), cells expressing TCR, cells expressing CAR (e.g. CAR-T cells and / or CAR-NK cells), CD4 + T cells, CD8 + T cells (e.g. CD8 + cytotoxic T cells) .

In some embodiments, a population of immune cell population T cells (eg, CD4 + T cells, CD8 + T cells, CD8 + cytotoxic T cells, or antigen-specific T cells) expanded / produced according to the methods of the invention. In some embodiments, a population of immune cell lineage NK cells (eg, antigen-specific NK cells) expanded / produced according to the methods of the invention.

In some embodiments, the method is used to generate / expand immune cells (eg, T cells, NK cells) in an antigen-independent manner. In some embodiments, T cells can be activated in an antigen-independent manner, such as via CD3 stimulation, optionally in combination with stimulation via CD28. In some embodiments, T cells can be activated by treatment with a agonist anti-CD3 antibody (eg, pure OKT3), optionally in combination with stimulation with a agonist anti-CD28 antibody. In embodiments where anti-CD3 and / or anti-CD28 are used for antigen-independent activation of T cells, modified K562 cells can be modified to increase one or more Fc receptors (e.g., one or more Fcγ receptors, such as CD64 , CD32 and / or CD16). The Fc receptor promotes the presentation of antibodies and therefore T cell activation.

In some embodiments, the methods comprise a modification that is modified to increase the performance of one or more Fc receptors (eg, one or more Fcγ receptors, such as one or more of CD64, CD32, and / or CD16) A population of immune cells (such as PBMC or PBL) is cultured in the presence of K562 cells and in the presence of a agonist anti-CD3 antibody (eg, pure OKT3), optionally in the presence of a agonist anti-CD28 antibody.

In some embodiments, the immune cell population expanded / produced in accordance with these methods exhibits a population of cells (eg, cells expressing TCR or cells expressing CAR) that are specific for receptors against the antigen. In some embodiments, the binding of an antigen or a fragment thereof to an immune cell that exhibits a receptor specific to the antigen results in one or more immune receptor tyrosine-based immune cells that exhibit a receptor specific to the antigen. Phosphorylation of its activation motif (ITAM). In some embodiments, ITAM is an ITAM of a CD3 polypeptide.

In some embodiments, the immune cell population expanded / produced according to the methods is a T cell population expressing TCR or a T cell population expressing CAR.

In some embodiments, the receptor chimeric antigen receptor (CAR) is specific for an antigen. CAR is a recombinant receptor that provides antigen-binding and T-cell activation functions. CAR structure and engineering modifications are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257 (1), which is incorporated herein by reference in its entirety. The CAR comprises an antigen-binding region connected to the cell membrane anchoring region and the signaling region. The optional hinge region can provide separation between the antigen-binding region and the cell membrane anchoring region and can serve as a flexible linker. The signaling region of CAR allows T cell activation. The CAR signaling region may comprise an amino acid sequence of the intracellular domain of CD3-zeta, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of cells expressing CAR. Signaling regions containing sequences of other ITAM-containing proteins such as FcyRI have also been used in CAR (Haynes et al., 2001 J Immunol 166 (1): 182-187). The signal transduction region of the CAR may also include a costimulation sequence derived from the signal transduction region of the costimulatory molecule to promote the activation of T cells that exhibit CAR after binding to the target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27.

In some embodiments, the antigen-specific receptor is a CAR comprising a CD19 binding domain.

In some embodiments, the antigen-specific receptor is a T cell receptor (TCR). TCR is a heterodimer antigen-binding molecule that typically contains alpha- and beta-chains. In nature, α-chains and β-chains appear as complexes with unchanged CD3 polypeptides on the cell surface of T cells (αβ T cells). Alternative TCRs containing γ and δ chains are expressed on T cell subsets (γδ T cells).

In some embodiments, the modified K562 cells can be used as antigen-presenting cells (APC) in a method for antigen-dependent expansion of immune cells. The modified K562 cells can be used as antigen-presenting APCs to generate / expand immune cells specific to the antigen. The modified K562 cells may contain or express antigens.

In an embodiment of the method of modifying K562 cells for use in generating / expanding immune cells expressing receptors specific for an antigen, the modified K562 cells may be modified to increase the receptor-specific antigen of K562 cells. which performed. For example, in a method in which modified K562 cells are used to generate / expand immune cells that express receptors specific for cancer cell antigens, modified K562 cells can be modified to increase cancer cell antigens of K562 cells Performance.

By way of illustration, an example of this disclosure describes the expansion of a T cell population expressing a CAR comprising a CD19 binding domain by culture in the presence of K562 cells modified to express CD19.

In some embodiments, the methods comprise culturing a population of immune cells (eg, PBMC or PBL) in the presence of modified K562 cells modified to increase antigen performance. In some embodiments, the methods comprise culturing immune cells (e.g., T cells) modified to express a receptor specific for an antigen (e.g., CAR) in the presence of modified K562 cells that are modified to increase antigenic performance group.

In some embodiments, the modified K562 cells can be used as artificial costimulatory factors, such as providing a costimulatory signal to immune cells to be produced / expanded. In some embodiments, the methods comprise culturing a population of immune cells (eg, PBMC or PBL) in the presence of modified K562 cells modified to increase the performance of one or more factors capable of increasing immune cell activation / proliferation.
Use of an immune cell population generated / expanded by culture in the presence of modified K562 cells

The population of immune cells expanded by culture in the presence of modified K562 cells according to the present invention can be used, for example, in the treatment or prevention of diseases / conditions. The disease / condition may be any disease / condition that will obtain a therapeutic or preventive benefit from an increase in the number of immune cells produced / expanded according to the methods described herein. In some embodiments, the disease / condition is a T cell dysfunction disorder, an infectious disease, or cancer.

T cell dysfunction disorders can be diseases / conditions in which normal T cell dysfunction results in an individual's immune response to pathogenic antigens being down-regulated, such as being infected by exogenous media such as microorganisms, bacteria, and viruses Produced, or produced by the host in some disease states, such as in some forms of cancer (eg, in the form of tumor-associated antigens). T cell dysfunction disorders may include T cell depletion or T cell responsive loss. T cell depletion includes states where CD8 + T cells are unable to proliferate in response to antigen stimulation or exert T cell effector functions such as cytotoxicity and secretion of cytokines (eg, IFNγ). Depleting T cells can also be characterized by the persistent manifestation of one or more T cell depletion markers, such as PD-1, CTLA-4, LAG-3, TIM-3. T cell dysfunction disorders can manifest as infections or fail to produce an effective immune response against the infection. Infection can be chronic, persistent, latent or slow, and can be the result of a bacterial, viral, fungal or parasitic infection. Thus, treatment can be provided to patients with bacterial, viral or fungal infections. Examples of bacterial infections include Helicobacter pylori infection. Examples of viral infections include HIV, hepatitis B or hepatitis C infection. T cell dysfunction disorders may be associated with cancer, such as tumor immune escape. Many human tumors display tumor-associated antigens recognized by T cells and capable of inducing an immune response.

The infectious disease may be, for example, a bacterial, viral, fungal or parasitic infection. In some embodiments, it may be particularly desirable to treat chronic / persistent infections, such as where such infections are associated with T cell dysfunction or T cell depletion. It is well known that T cell depletion is a state of T cell dysfunction that occurs during many chronic infections (including viruses, bacteria, and parasites) and in cancer (Wherry Nature Immunology Vol. 12, No. 6, pp. 492-499, 2011 June). Examples of treatable bacterial infections include Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio cholerae (Vibrio chloerae), Staphylococcus spp., Streptococcus spp., Escherichia, Klebsiella, Proteus, Yale Yersinia, Erwina, Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) tuberculosis)) and Pseudomonas aeruginosa infection. For example, a bacterial infection can be sepsis or tuberculosis. Examples of treatable viral infections include influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choroid plexus meningitis virus (LCMV), Herpes simplex virus and human papilloma virus (HPV) infection. Examples of treatable fungal infections include Alternaria sp, Aspergillus sp, Candida sp, and Histoplasma sp. Fungal infections can be fungal septicemia or histoplasmosis. Examples of treatable parasitic infections include Plasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium ovale, Plasmodium vivax or Plasmodium chabaudi). Parasitic infections can be diseases such as malaria, leishmaniasis, and toxoplasmosis.

In a specific embodiment, the disease / condition to be treated / prevented is cancer. Cancer can be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation itself), neoplasm or tumor or unwanted cell proliferation, increased risk or propensity for neoplasm or tumor. Cancer can be benign or malignant and can be primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissue include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or not including brain) cerebellum, cervix, colon, duodenum, endometrium , Epithelial cells (e.g. renal epithelial cells), gallbladder, esophagus, glia, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, uterine muscle Layer, nasopharynx, intestinal omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid, tongue, tonsils , Trachea, uterus, vulva, white blood cells. In some embodiments, the cancer to be treated may be a cancer selected from the group consisting of: colon, rectum, nasopharynx, cervix, oropharynx, stomach, liver, head and neck, oral cavity, esophagus, lips, oral cavity , Tongue, tonsils, nose, throat, salivary glands, sinuses, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovaries or mesothelium.

The tumor to be treated may be a tumor of the nervous or non-nervous system. Nervous system tumors can originate from the central or peripheral nervous system, such as gliomas, neuroblastomas, meningiomas, neurofibromas, ependymal tumors, schwannomas, neurofibrosarcomas, astrocytomas, and oligodendrocytes Glioma. Non-neurological cancers / tumours can originate from any other non-neurological tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Howe Chicking's lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), skin T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid Cancer, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer and sarcoma.

In some embodiments, the cancer is selected from the group consisting of: colon cancer, colon cancer, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, head and neck squamous Cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urinary tract epithelial cancer, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or interstitial cancer Dermatoma.

In some embodiments, the cancer to be treated / prevented is a virus-related cancer, such as an EBV-related cancer or an HPV-related cancer. "EBV-related" and "HPV-related" cancers can be cancers caused or exacerbated by corresponding viral infections, cancers where infection is a risk factor, and / or cancers that are positively related to the onset, development, progression, severity, or metastasis of the infection. EBV-related cancers that can be treated with cells generated by the methods of this disclosure include nasopharyngeal cancer (NPC) and gastric cancer (GC). HPV-related medical conditions that can be treated with cells produced by the methods disclosed in this disclosure include at least genital hypoplasia, cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, anal intraepithelial neoplasia, cervical Cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, genital cancer, oral papilloma, oropharyngeal cancer. In some embodiments, the cancers to be treated according to various aspects of the present disclosure are nasopharyngeal carcinoma (NPC; for example, Epstein-Barr Virus (EBV) positive NPC), cervical cancer (CC; For example, human papillomavirus (HPV) -positive CC), oropharyngeal cancer (OPC; for example, HPV-positive OPC), gastric cancer (GC; for example, EBV-positive GC), hepatocellular carcinoma (HCC; for example, hepatitis B virus (HBV) -positive HCC), lung cancer (e.g., non-small cell lung cancer (NSCLC)), and head and neck cancer (e.g., cancer derived from tissues of the lips, mouth, nose, sinuses, pharynx or throat, such as head and neck squamous cell carcinoma (HNSCC)) One or more.

A population of immune cells generated / expanded according to the methods described herein can also be used in methods that include adoptive cell transfer (ACT). In particular, modified K562 cells can be used to generate / expand a population of immune cells, which can then be administered to an individual to treat / prevent a disease / condition.

The present invention provides a method of treatment or prevention, which comprises adoptively transferring immune cells (eg, T cells, effector T cells, antigen-specific T cells, NK cells) produced (ie, produced or expanded) according to the methods of the present invention. Adoptive cell transfer generally refers to a method of obtaining immune cells from an individual, usually by taking a blood sample and isolating the immune cells therefrom. Immune cells are then usually processed or altered in some way, optionally expanded, and then administered to the same individual or to different individuals. Treatment is generally aimed at providing an individual with a population of immune cells with certain desired characteristics, or increasing the frequency of immune cells with such characteristics in the individual.

Adoptively transferred cells can be, for example, T cells, antigen-specific T cells (e.g., virus-specific T cells), antigen-specific CD4 T cells, antigen-specific CD8 T cells, effector memory CD4 T cells, effector memory CD8 T cells, central Memory CD4 T cells, central memory CD8 T cells, cytotoxic CD8 + T cells (ie, CTL) NK cells or antigen-specific NK cells.

In some cases, the immune cells are derived from the patient to whom they were introduced (autologous cell therapy). That is, cells can be obtained from a patient, generated according to the methods described herein, and then returned to the same patient.

The methods disclosed herein can also be used in allogeneic cell therapy, in which cells obtained from different individuals are introduced into a patient. Immune cell populations generated / expanded by a method comprising culturing in the presence of the modified K562 cells of the present invention are particularly suitable for allogeneic adoptive cell therapy because it is similar to the method by culturing in the presence of wild-type K562 cells The method of generation / amplification showed reduced allogeneic reactivity compared to immune cells.

Adoptive T cell transfer is described in, for example, Chia WK et al., Molecular Therapy (2014), 22 (1): 132-139, Kalos and June 2013, Immunity 39 (1): 49-60 and Cobbold et al., ( 2005) J. Exp. Med. 202: 379-386, which is incorporated herein by reference in its entirety.

In the present invention, the purpose of adoptive transfer is to introduce immune cells or increase the frequency of immune cells in an individual.

Accordingly, the present invention provides a method for treating or preventing a disease or condition in an individual, comprising:
(a) generating or expanding a population of immune cells by culturing a population of immune cells (such as PBMC or PBL) in the presence of modified K562 cells according to the present invention; and
(b) administering to the individual the population of immune cells produced or expanded in step (a).

The invention also provides a method for treating or preventing a disease or condition in an individual, comprising:
(a) isolating immune cells (e.g., PBMC or PBL) from an individual;
(b) generating or expanding a population of immune cells by culturing immune cells (such as PBMCs) in the presence of modified K562 cells according to the present invention, and;
(c) administering a population of immune cells produced / expanded to the individual.

In some embodiments, a system in which immune cells (e.g., PBMC or PBL) are isolated in step (a) is administered to an individual that produces / expands an immune cell population (i.e., adoptive transfer line autologous cells) in step (c) of). In some embodiments, the system of immune cells (e.g., PBMC or PBL) in step (a) is different from the individual (i.e., adoptive transfer lineage) in which the immune cell population generated / expanded in step (c) is administered Allogeneic).

In some embodiments, the method may include one or more of the following steps: collecting a blood sample from the individual; isolating PBMC or PBL from the blood sample; producing by culture in the presence of modified K562 cells according to the invention or Expansion of the immune cell population; collection of the generated or expanded immune cell population; mixing of the generated or expanded immune cell population with an adjuvant, diluent or carrier; administration of the generated or expanded immune cell population or combination to an individual Thing.

The skilled person can determine appropriate reagents and procedures for adoptive transfer of immune cells generated or expanded according to the method of the present invention, for example, refer to Chia WK et al., Molecular Therapy (2014), 22 (1): 132-139, Kalos and June 2013, Immunity 39 (1): 49-60 and Cobbold et al. (2005) J. Exp. Med. 202: 379-386.
Sequence identity

As used herein, "sequence identity" refers to nucleotide / amino acid residues in a subject sequence and a reference sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity between the sequences. Consistent percentage of nucleotide / amino acid residues. Pairings and multiple sequence alignments for the purpose of determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be accomplished in a variety of ways known to those skilled in the art, such as using publicly available Obtained computer software such as Clustal Omega (Söding, J. 2005, Bioinformatics 21, 951-960), T-coffee (Notredame et al. 2000, J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann And Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30 (4) 772-780 software. When using such software, it is preferred to use, for example, gap penalties and extensions Default parameters for penalties.
sequence
SEQ ID NO: description sequence 1 Human B2M gene sequence (NCBI reference sequence: NG_012920.1) 2 Human B2M mRNA (NCBI reference sequence: NM_004048.2) 3 Human B2M protein coding sequence ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAA 4 Human B2M (UniProt: P61769-1, v1) MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM 5 Mature human B2M (UniProt: P61769-1, residues 21-199 of v1) IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM 6 Forward B2M EX1 target AAACggccgagatgtctcgctccgGT 7 Reverse B2M EX1 target TAAAACcggagcgagacatctcggcc 8 Forward B2M EX2 target AAACgaagttgacttactgaagaaGT 9 Reverse B2M EX2 target TAAAACttcttcagtaagtcaacttc 10 Forward B2M EX1 WT TCTCGAATGAAAAATGCAGGTCCG 11 Reverse B2M EX1 WT TGACGCTTATCGACGCCCTAAACTT 12 Forward B2M EX1 HDR GACTCCACCACCACGAAATGGC 13 Reverse B2M EX1 HDR CCCCATCAAGCTGATCCGGA 14 Forward B2M EX2 WT GCTTGACACCAAGTTAGCCC 15 Reverse B2M EX2 WT TGGATGGGACTCATTCAGGGT 16 Forward AAVS1 WT CACTCGCTGGGTTCCCTTTTCCT 17 Reverse AAVS1 WT GGCTGGCTACTGGCCTTATCTCACA 18 AAVS1 CD64 HDR CTTTGGCAGCCTGTGCTGACCCAT 19 Reverse AAVS1 CD64 HDR TCGAGGTCTGAGTGGCTGTGCCAT 20 Forward AAVS1 EpCAM HDR CTTTGGCAGCCTGTGCTGACCCAT twenty one Reverse AAVS1 EpCAM HDR AAGAGCCCGCTCTCATCGCAGTCA twenty two HLA-C * 03 type CACAGACTGACCGAGTGAG twenty three Reverse HLA-C * 03 typing AGCGTCTCCTTCCCATTCTT twenty four Forward HLA-C * 05 typing CCGAGTGAACCTGCGGAAA 25 Reverse HLA-C * 05 typing CGCGCGCTGCAGCGTCTT 26 CrRNA targeting B2M EX1 ggccgagatgtctcgctccggttttagagctatgctgttttg 27 CrRNA targeting B2M EX2 gaagttgacttactgaagaagttttagagctatgctgttttg 28 tracrRNA ggaaccattcaaaacagcatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttt ***

The invention includes combinations of such aspects and preferred features, except where such combinations are clearly not allowed or explicitly avoided.

The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now be described by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

Throughout this specification, including the scope of subsequent patent applications, the word "comprise" and variations (such as "comprises / comprising") shall be understood to imply the inclusion of the integers, unless the context requires otherwise Or steps or integers or groups of steps but does not exclude any other integers or steps or steps or groups of integers or steps.

It must be noted that, unless the context clearly indicates otherwise, as used in this specification and the scope of the accompanying patent application, the singular forms "a (an / an)" and "the" include plural referents. Ranges may be expressed herein as "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when values are expressed as approximations by using the antecedent "about", it should be understood that a particular value forms another embodiment.

Where a nucleic acid sequence is disclosed herein, its reverse complement is also explicitly considered.

The methods described herein can preferably be performed in vitro. The term "in vitro" is intended to cover experiments using cells in culture, and the term "in vivo" is intended to cover experiments using whole multicellular organisms.

Examples <br/> In the following examples, the present inventors describe the use of a CRISPR / Cas9-based system gene to knock out B2M genes in K562 cells to generate MHC class I molecular-deficient K562 cells. The inventors have demonstrated that even after stimulation with a wild-type K562 cell / T cell co-culture supernatant or interferon-γ, B562M knockout K562 cells still do not express MHC class I molecules on the cell surface. The present inventors have shown that B2M / MHC class I molecularly deficient K562 cells can be used as a "skeleton" cells that produce aAPC, which can support the in vitro expansion of robust antigen-independent T cells and antigen-specific anti-CD19 CAR-T Cells are expanded in vitro. Importantly, it was confirmed that T cells expanded with B2M gene knockout of K562 cells aAPC showed reduced allogeneic reactivity compared with T cells expanded with comparable K562 cells without B2M gene knockout.
Example 1: Materials and Methods 1.1 Cell Culture

Human Myeloid Leukemia Cell Line K562 Cells (ATCC, Manassas, VA, USA) in Iskov's Modified Dulbecco's Medium with 10% Fetal Bovine Serum (FBS, Hyclone ™ GE Healthcare, Little Chalfont, UK) (Iscove's Modified Dulbecco's Medium, IMDM, Lonza, Basel, Switzerland). When used as aAPC co-culture feeder cells, the K562 cell line was inactivated by 100Gy (10,000 Rads) gamma-irradiation or treated with 20 μg / mL mitomycin C (Roche Diagnostics, Basel, Switzerland) for one hour, And then washed three times with phosphate buffered saline (PBS, Lonza) and transferred to co-culture medium. To induce B2M and human leukocyte antigen (HLA), K562 cells were collected with 500 IU / mL IFN-γ (PeproTech, Rocky Hill, NJ, USA) or from K562 and T cell co-cultures (1: 1 ratio for 48 hours) The supernatant layer was treated for 48 hours.

Fresh peripheral blood mononuclear cells (PBMCs) were isolated from the skin color hematopoietic layer of healthy donors by Ficoll®-Paque PREMIUM 1.084 (GE Healthcare) by density gradient centrifugation. PBMC and Dynabeads® Human T-Activator CD3 / CD28 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in AIM V® medium (Invitrogen, Thermo) with 5% human AB serum (Valley Biomedical, Winchester, VA, USA) (Fisher Scientific) for T cell activation. Primary activated CD3 + T cells were collected and the beads were depleted on day 7. For in vitro antigen-independent T cell expansion, T cells and aAPC based on B2M gene knockout K562 were used at a ratio of 1:50 in AIM V with 5% human AB serum and 300 IU / mL IL-2 (PeproTech) ® co-culture. Anti-CD3 (OKT-3) antibodies (60 ng / mL, eBioscience, San Diego, CA, USA) were added during the first week and the fresh medium was changed or supplemented accordingly during the co-culture. For in vitro antigen-dependent T cell expansion, activated T cells were transduced with a lentiviral vector encoding an anti-CD19 CAR and in 5% human AB serum, 300 IU / mL IL-2, 5 ng / mL IL- 7 (PeproTech) and 5 ng / mL IL-15 (PeproTech) in AIM V® medium were stimulated with aAPC expressing CD19 every week at a ratio of 1: 1. CD8 + T cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to isolate CD8 + T cells by magnetic beads. Magnetic sorting was performed according to the manufacturer's instructions.

The target tumor cell lines MCF-7 (ATCC) and FaDu (ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Lonza) with 10% FBS. MDA-MB-435 (ATCC) cells were cultured in DMEM with 10% FBS and 10 μg / ml bovine insulin (Sigma-Aldrich, St. Louis, MO, USA). SKOV-3 (ATCC) cells were cultured in McCoy's 5A (modified) medium (Gibco) with 10% FBS. Daudi (ATCC), Raji (ATCC) and A549 (ATCC) cells were cultured in RPMI 1640 medium (Lonza) with 10% FBS. Therefore, cells are passaged every other day.
1.2 Construction of plastids and baculovirus vectors

PX260 plastid (Addgene, Cambridge, MA, USA) containing the CRISPR / Cas9 system is described in Cong et al., Science 339, 819 (32); Addgene # 42229; pX260-U6-DR-BB-DR-Cbh-NLS- hSpCas9-NLS-H1-shorttracr-PGK-puro. The ZiFiT Targeter online software (41) was used to design and select a crRNA sequence targeting B2M gene exon 1 (EX1) and exon 2 (EX2) (Table 1, Example 1.8) and was cloned into pX260. Construction of a B2M EX1 homologous-direct integration donor plastid containing an EF1α (eukaryotic translation elongation factor 1α) promoter driving the expression of the EGFP gene and a PGK (small Murine phosphoglycerate kinase 1) promoter flanked by homologous DNA sequences from B2M EX1 locus (chromosome 15: nucleotides 44,710,501-44,711,401 and nucleotides 44,711,615-44,712,485, GRCh38.p2 primary assembly). Construct a donor plastid for B2M EX1-EX2 homology-direct integration, which contains the same cassette as B2M EX1 homology-direct integration, flanked by a homologous DNA sequence from the B2M EX1-EX2 locus (chromosome 15: Nucleotides 44,710,501-44,711,401 and Nucleotides 44,715,553-44,716,188, GRCh38.p2 primary assembly). ZFN-containing vectors are described in Tay et al., Journal of gene medicine 15, 384 (37). Construction of a homologous-direct integration donor plastid for AAVS1 locus, which contains a homologous DNA sequence flanking the AAVS1 locus (chromosome 19: nucleotides 55,116,611-55,115,767 and nucleotides 55,115,765-55,114,929, GRCh38.p2 (Primary assembly) costimulator performance cassette. CD64 (FcγRI, GenBank deposit number BC032634), CD86 (B7-2, GenBank deposit number NM_175862), 4-1BBL (CD137L, GenBank deposit number NM_003811) and membrane-bound IL-21 (mbIL21, GenBank deposit number NM_021803.3) The coding sequence was PCR amplified from human PBMC cDNA library and cloned into pFastBac ™ 1 (Invitrogen) vector as a performance cassette. The CD64-2A-CD137L-2A-CD86 expression cassette under the control of the Cytomegalovirus (CMV) promoter was sub-selected into a donor plastid, and the EpCAM-2A-mbIL21 expression under the control of the CMV promoter The cassette is sub-selected into another donor plastid. Donor plastids for CD19 expression were constructed using the CMV promoter to drive the expression of the CD19-IRES-Puro (puromycin resistance gene) expression cassette. The CMV promoter was used to construct plastids for anti-CD19 CAR expression to drive scFv (anti-CD19) -CD8TM (CD8 transmembrane domain) -CD28-CD3ζ expression cassette performance. The anti-CD19 CAR expression cassette was sub-selected into a lentiviral vector for lentivirus production.

For baculovirus vector construction, pFastBac ™ 1 (Invitrogen) plastids were previously described (39). The crRNAs targeting EX1 and EX2, the tracRNA with Cas9, and the homologous-direct integration donors for B2M EX1-EX2 were cloned into three pFastBac ™ 1 plastids, respectively. Three recombinant shuttle vectors were generated according to the protocol of the Bac-to-Bac® Baculovirus Expression System (Invitrogen). Insect Sf9 cells (ATCC) were cultured in Sf-900 ™ II SFM (Gibco) medium and transfected with their recombinant shuttle vector by Cellfectin® II reagent (Invitrogen) according to the protocol. Three recombinant baculovirus (BV) vectors, BV-crRNA-EX1, BV-crRNA-EX2-Cas9-tracRNA, and BV-donor-EX1-EX2, were generated, propagated, and collected from insect Sf9 cells.
1.3 Generating B2M gene knockout K562 pure lines

To generate B2M knockout cells by electroporation, 5 × 10 ⁶ K562 cells were treated with pX260-B2M-EX1 and donor vectors in Opti-MEM® I reducing serum medium (Gibco) with 2 μg DNA per vector via electroporation. A perforator (Nepa Gene, Chiba, Japan) was used for electroporation. The cells are then transferred to the culture medium. Four days after electroporation, cells were selected by culturing in the presence of 500 μg / mL Geneticin® (G418 sulfate, Gibco) for 2 weeks before single cell seeding. In order to generate B2M gene knockout pure lines by baculovirus transduction, each well of a 12-well plate was coated with 5 × 10 ⁶ K562 cells, and the infection rate was 500 Pfu per BV in each cell in Opti-MEM® (MOI) Transduced BV overnight. The cells are then transferred to the culture medium. Two weeks after transduction, cells were selected by culturing in the presence of 500 μg / mL Geneticin®. Two weeks after selection, fluorescence-activated cell sorting (FACS) was performed with a BD FACSAria ™ I flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) for single cell seeding. EGFP-positive cells were gated and seeded in 96-well plates with one cell per well. Single cell pure lines were subsequently cultured and expanded. Once 80-90% confluence was reached, the K562 single cell pure line in a 96-well plate was replicated for genotyping and subculture. One disk was used for genomic DNA extraction, followed by genotyping by PCR. For subsequent sequencing, the PCR products were purified and sub-selected into pGEM®-T Easy Vector Systems (Promega, Fitchburg, WI, USA). Individual vectors were collected for sequencing. The pure lines identified by subsequent amplification were used for further studies.
1.4 Generate aAPC based on K562 cells

To generate K562-based aAPCs for T cell expansion methods, WT K562 cells or B2M gene knockout pure line EX1EX2 # 5 K562 cells were electroporated with ZFN and donor plastids, and purified at 1 µg / ml puromycin (Gibco ). Cells were then stained with anti-CD64 APC (Miltenyi Biotec, Bergisch Gladbach, Germany) antibodies, and one cell per well was seeded in a 96-well plate with culture medium, and APC-positive populations were sorted by FACS. Single-cell colonies were performed and two pure lines with the highest performance of CD64, CD86, 4-1BBL, and mbIL21 were selected, namely K562A from WT K562 cells and K562B from EX1EX2 # 5. To expand anti-CD19 CAR-expressing T cells, the CD19 gene was introduced into K562B cells via transfection. After two weeks of puromycin selection, cells were stained with anti-CD19 APC (eBioscience) antibodies, and APC-positive cells were gated and seeded in 96-well plates with culture medium per cell. Single cell pure lines with high and stable CD19 performance were selected and expanded for further analysis.
1.6 Cytotoxicity analysis

Cytotoxicity was analyzed using a DELFIA® EuTDA cytotoxic reagent (PerkinElmer, Waltham, MA, USA) in a standard 2-hour tritium release assay. The analysis was performed according to the manufacturer's instructions. Target cells were first labeled with BATDA reagent for 5 to 15 minutes at 37 ° C, and then washed three times with PBS. Effector cells and labeled target cells were mixed in triplicate with different effector to target (E: T) ratios in AIM V® medium with 5% human AB serum. The mixed cells were incubated in a humidified incubator at 37 ° C for 2 hours. Spontaneous and maximum release were determined by culturing target cells in the absence of effector cells and with lysis buffer, respectively. After incubation, the supernatant layer was transferred to a solution mixed with rhenium and analyzed using a VICTOR ™ time-lapse fluorescence meter (PerkinElmer). The specific dissolution percentage is calculated as follows:% dissolution = [(experimental release-spontaneous release) / (maximum release-spontaneous release)] × 100.
1.7 Statistical analysis

Data were collected as described above and analyzed using Prism version 7 software (GraphPad). Data are expressed as mean (SD) and analyzed by two-way analysis of variance, Tukey's multiple comparisons test, and Student's t-test of independent samples. Representative histograms and graphs were selected from independent replicates based on the mean.
1.8 Oligonucleotides and primers Table 1 :
1.9 Antibody Table 2 :
1.5 Western blot method, flow cytometry analysis and intracellular staining

For Western blot analysis, sample proteins were extracted by lysing cells with radioimmunoprecipitation analysis (RIPA) buffer (Nacalai Tesque, Kyoto, Japan), analyzed in SDS-PAGE gel under reducing conditions, and then electrotransfected on Nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Rabbit anti-B2M antibody pure line EP2978Y (1: 5000 dilution, Abcam, Cambridge, UK) and mouse anti-β-actin antibody pure line GT5512 (1: 1000 dilution, Abcam) were used as primary antibodies. Goat anti-rabbit IgG-HRP (1: 5000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse IgG-HRP (1: 2000 dilution, Santa Cruz Biotechnology) were used as secondary antibodies. The membrane was developed and observed for chemiluminescence using a MYECL ™ imager (Thermo Fisher Scientific).

For flow cytometry analysis, cells were stained with antibodies in autoMACS® running buffer (Miltenyi Biotec) and analyzed by BD Accuri ™ C6 flow cytometer (BD Biosciences). Data were analyzed using CFlow® Sampler software (BD Biosciences). Multicolor staining was analyzed using a BD ™ LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). For anti-F (ab ') ₂ staining, T cells were first treated with biotin-conjugated AffiniPure F (ab') ₂ fragment (goat anti-mouse IgG, F (ab ') ₂ fragment specificity, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour and then restained with Streptevdin APC (BD Biosciences). For intracellular staining, K562 cells were first fixed with 0.01% paraformaldehyde (PFA, Thermo Fisher Scientific) and penetrated with 0.5% Tween-20 (Bio-Rad Laboratories), and then analyzed by flow cytometry.
Example 2: Factors in T-cell co-culture conditioned medium can induce the expression of B2M and MHC class I molecules on K562 cells

The present inventors first investigated whether soluble factors produced by co-cultures of T cells and K562 cells could stimulate upregulation of MHC class I molecules on wild-type K562 cells.

Wild-type K562 cells were co-cultured with human primary T cells for 48 hours, and the resulting cell culture supernatant was collected and used to stimulate K562 cells cultured in another cell culture plate. In another case, K562 cells were stimulated with IFNγ, while in another case, K562 cells were unstimulated.

In order to simulate the situation where inactivated K562 cells were used as aAPC feeder cells for immune cell expansion culture, K562 cells were deactivated by treatment with mitomycin C or by gamma irradiation. In another case, K562 cells were not inactive.

The performance of MHC class I molecules was analyzed by flow cytometry. Compared with the performance levels on non-activated K562 cells, a moderate increase in B2M and HLA-A, B, C performance levels was observed on K562 cells after mitomycin C treatment or gamma irradiation (Figures 1A and 1B) . Stimulated with a supernatant layer collected from a K562 / T cell co-culture, the performance level reached as high as 90% of the cells, and the level was comparable to that induced by treatment with IFN-γ (Figure 1A and B). Non-inactivated and non-activated K562 cells show similar up-regulation levels, suggesting that inhibition of K562 cell proliferation will not disrupt the underlying mechanisms of B2M and MHC class I molecular upregulation. The performance of MLA class II molecule HLA-DR was also analyzed, but it was not detected under any conditions (Figure 2B), consistent with previous studies (43, 44).
Example 3: B2M knockout K562 cells show MHC class I negative phenotype

The present inventors designed two different clustered regularly spaced short palindromic repeat (CRISPR) / Cas9 systems to genetically knock out B2M genes to generate MHC class I performance-deficient K562 cells.

For the first system, K562 cells were co-charged with a CRISPR / Cas9 construct targeting B2M gene exon 1 (EX1) and a donor cassette containing EGFP and a neomycin resistance gene flanked by homologous sequences Perforate to integrate the donor cassette sequence instead of EX1 (Figure 3A).

Because K562 cells contain three alleles of the B2M gene (45), it is challenging to genotype all alleles simultaneously in a single K562 cell. To solve this problem, the inventors further designed a second CRISPR / Cas9 system targeting B2M EX1 and exon 2 (EX2), the purpose of which is to achieve complete destruction of the B2M gene (Figure 3B). A baculovirus vector with excellent transduction efficiency in K562 cells was used to deliver the second CRISPR / Cas9 system.

Subsequently, by culturing for 2 weeks in the presence of geneticin, K562 cells electroporated with EX1 targeting system and K562 cells transduced with EX1EX2 targeting system were selected to enrich stable EGFP-positive cells. Single cells were then seeded into individual wells of a 96-well plate based on EGFP-positive cell sorting by FACS. Single cell pure lines were collected-78 from electroporated cells and 60 from transduced cells, and PCR genotyping was performed to select pure lines carrying B2M site-specific integration of selectable markers (Figure 4A). Ten representative positive pure lines were collected from each method and amplified for further analysis. To detect defects in B2M expression, single cell pure lines were stimulated with IFN-γ for 48 hours and subsequently analyzed by flow cytometry. Defective types were observed in one pure line (EX1 # 7) derived from EX1 electroporated K562 cells and four pure lines (EX1EX2 # 2, # 5, # 6, # 7) derived from EX1EX2 system transduced K562 cells. B2M performance (Figures 3C and 3D). B2M gene knockout was also confirmed in five pure lines by Western blot analysis (Figure 4D). Through DNA sequencing, the B2M site-specific integration of selectable markers into one allele and mutations or early termination codons in the other two alleles of the B2M gene were detected in five B2M-deficient pure lines. 4B and 4C).

MHC class I performance was further examined in five B2M knockout (B2MKO) K562 pure lines. K562 cells were deactivated by treatment with mitomycin C or gamma irradiation, or non-inactivated K562 were stimulated by treatment with IFN-γ or cell culture supernatant collected from K562 / T cell co-culture cell. In contrast to the up-regulation of B2M and HLA-A, B, and C molecules observed in wild-type K562 cells, the performance of these molecules in B2MKO K562 cells was almost undetectable by flow cytometry (Figures 5A, 5B And 4E).

Since cells without MHC class I expression are very sensitive to NK cell lysis (46), the inventors used EX1EX2 # 5 pure line to perform functional analysis based on NK cell-mediated lysis to further characterize MHC class I defects. WT and B2MKO K562 cells were stimulated with IFN-γ and subsequently co-cultured with human primary NK cells with different effect (E): target (T) cell ratios. As expected, the percentage of NK-specific lysis of B2MKO K562 cells was much higher compared to lysis of WT K562 cells (Figure 5C). These findings confirm that B2MKO K562 cells exhibit an MHC class I defective phenotype.
Example 4: T cells co-cultured with aAPC based on B2M knockout K562 cells show reduced allogeneic reactivity

K562 B2MKO pure line EX1EX2 # 5 is used to generate aAPC. Genes encoding Fc receptor CD64, necessary co-stimulatory molecules CD86 and CD137L, and membrane-bound IL-21 (mbIL21) were stably introduced into EX1EX2 # 5 cells via ZFN-mediated AAVS 1 site-specific integration (Figure 6A).

The modified cells were subjected to single-cell colony selection, and pure lines with high performance levels of four molecules were selected and named "K562B" (Figures 6B and 6C). The performance of MHC class I phenotypic defects and myeloid lineage markers in K562B was examined and confirmed (Figures 6D and 6E).

Wild-type K562 cells (ie, not B2M knockout) were also modified to express CD64, CD86, CD137L, and mbIL21. This cell line was named "K562A" (Figure 6B) and was used as a control aAPC expressing MHC I in the following experiments.

To assess the allogeneic reactivity of T cells, PBMCs were first primed with inactivated K562A cells, and then CD8 ⁺ T cells were isolated by negative selection of magnetic beads. The collected CD8 + T cells were then expanded by stimulation with K562A or K562B cells, once a week for four weeks (Figure 7A).

CD8 ⁺ T cells stimulated with K562A showed a much higher expansion factor than the counterpart using K562B (Figure 8C). Phenotyping was performed by flow cytometry to assess expanded T cells. Activated effect memory CD8 ⁺ T cells are dominant in both K562A stimulated and K562B stimulated T cell populations, with high-level performance of CD86 and HLA-DR (Figure 8D).

Cytotoxicity analysis was subsequently performed by mixing CD8 ⁺ T cells produced by expansion with K562A or K562B cells after stimulation of target cells with IFN-γ to up-regulate MHC class I performance (Figures 7B to 7D). CD8 ⁺ T cells isolated immediately after PBMC priming (i.e., without culture and expansion in the presence of K562A or K562B cells) can effectively kill IFN-γ stimulated wild-type K562 cells (Figure 7B, upper left, triangle) . After CD8 ⁺ T cells were expanded with K562A cells, increased cytotoxicity was observed against IFN-γ stimulated WT K562 cells (Figure 7B, top left panel, diamond). In contrast, the cytotoxicity of CD8 ⁺ T cells expanded with K562B cells against WT K562 cells did not increase significantly compared to the cytotoxicity shown by CD8 ⁺ T cells isolated immediately after PBMC priming (Figure 7B, top left, square) .

Since K562 cells can only up-regulate the expression of HLA-C molecules among the MHC class I molecules they encode (A * 11,31; B * 18,40; C * 03,05) (26), the inventors have studied The expanded T cells target tumor cells expressing the HLA-C allele (HLA-C * 03 or HLA-C * 05) expressed by K562 or not expressing the HLA-C allele HLA-C * 03 or HLA- Allogeneic reactivity of C * 05 tumor cells (Figures 8A and 8B).

With the culture of CD8 ⁺ T cell expansion in the presence as compared K562B, by CD8 ⁺ T cells cultured in the presence of cells amplified for expression K562A K562 cells by tumor expression of HLA-C allele of cells The cytotoxicity shown was much higher (Figures 7B to 7D). Regardless of whether T cells were expanded by culture in the presence of K562A or K562B cells, cytotoxicity was not observed when tumor cells that did not express the HLA-C allele expressed by K562 cells were used as target cells (Figures 7B to 7D ).

These results indicate that although wild-type K562 cells are used as feeder cells or artificial APC, allo-reactive cytotoxic T lymphocytes (CTLs) can expand and may be enriched, but B2MKO K562 cells do not stimulate allo-reactivity CTL. T cells expanded by culturing in the presence of B2M knockout K562 cells will contain fewer allo-reactive T cells and a larger proportion of T cells with the desired specificity.
Example 5: B2M gene knockout does not affect the ability of K562 cells to be used as aAPC for antigen-independent and antigen-dependent T cell expansion

K562 cells are commonly used as feeder cells for large-scale expansion of αβ-T cells in vitro. To evaluate the ability of aAPC based on B2MKO K562 to serve as a method for APCs to expand T cells in an antigen-independent manner, PBMCs were first activated by treatment with micro-Dynabeads coated with CD3 / CD28 antibodies for one week, and then T Cells were cultured in the presence of non-activated K562B or K562A cells at a ratio of 1:50 in the presence of anti-CD3 antibody OKT-3 for 2 weeks (Figure 9A). Applying this method to a representative study of PBMCs from three donors, 1000-2000-fold total cell expansion was achieved at the end of the 14-day culture (Figures 9B to 9D). K562B and K562A cells achieved comparable expansion folds among different donors. The inventors then analyzed the composition of the expanded immune cell population after 14 days of expansion (Figures 9E to 9H).

The CD8 ⁺ αβ-T cell population accounts for the major part (60% to 75%), followed by the CD4 ⁺ αβ-T cell population (20% to 40%). CD3 ^- CD56 ⁺ NK cells expanded from 3% to 13% by Dynabead stimulation, but a decrease in the proportion of NK cells to 3% to 5% was observed during the 14-day T cell / K562 cell co-culture. There was no significant expansion of the γδ-T cell population (1.5% to 3%). In CD8 ⁺ αβ-T cell proliferation of, more than 90% of effector memory T cells (CCR7 ^- CD45RA ^-) showed less than 3%, and T-cell depletion marker PDl (i.e., PD1 ⁺ CD8 ^+). The results obtained with K562B and K562A cells were comparable among different donors.

The results showed that the B2M knockout K562 cells could be used as aAPC for antigen-independent expansion of T cells.

In order to study the use of aAPC based on B2MKO K562 for the expansion of antigen-dependent T cells, CD19 antigen was introduced into K562B cells and a single cell pure line K562B + CD19 expressing CD19 was established (Figure 10A). Human primary T cells from two different donors were also transduced with a lentiviral vector encoding an anti-CD19 CAR to generate stable anti-CD19-CAR-T cells (Figure 10A).

Anti-CD19 CAR-T cells were cultured in the ratio of 1: 1 in the absence of activated K562B + CD19 cells or K562B cells, and restimulated weekly for T cell expansion (Figure 10B). When CAR-T cells were co-cultured with K562B + CD19 cells but not K562B cells, the T cells expanded steadily (Figures 10C and 10D).

When K562B + CD19 cells were used to stimulate CAR-T cells, the stable performance of anti-CD19 CAR increased from 9% of T cells on day 4 to 93% of expanded T cells on day 25, indicating that CAR-T cells after expansion Significant enrichment (Figures 10E and 10F). When T cells were stimulated with K562B cells, anti-CD19 CAR-positive T cells decreased slightly from the original 9% to 6% on day 25 (Figures 10E and 10F).

By coculture with K562B CD19 + amplified by the CAR-T cell phenotyping, and found mainly based CD8 ⁺ effector memory T cells (CCR7 ^- CD45RA ^-) (FIG. 10G).

To evaluate the cytotoxicity of the expanded CAR-T cells, an in vitro cytotoxicity analysis was performed using the DELFIA cytotoxicity kit. Dose-dependent cytolysis of CD19-positive tumor target cells was observed with anti-CD19-CAR-T cells expanded with K562B + CD19 cells, but these T cells did not show cytotoxicity against CD19-negative tumor cells (Figures 10H and 10I, (Circled), demonstrating that the CD19 antigen-specific tumor cell killer line is mediated by expanded CAR-T cells.

The results show that B2M gene knockout in K562 cells can be used as aAPC to expand antigen-specific T cells.
Example 6: Conclusion

K562 leukemia cell line is widely used to construct aAPC for in vitro immune cell expansion due to its characteristics without MHC class I expression. However, a recent study reported that its MHC class I molecules, at least HLA-C, can be invoked on NK cell expansion during co-culture with PBMC (26).

In this study, the inventors confirmed that B2M and MHC class I molecules are up-regulated on K562 cells under K562 / T cell co-culture conditions. K562-based aAPC up-regulated MHC class I molecules can be alloantigens of MHC mismatch donors, activating and triggering specific expansion of allo-reactive T cells.

To solve this problem, the inventors used the baculovirus CRISPR / Cas9 system to destroy the B2M gene in K562. A total of five single-cell-derived B2M gene knockout K562 pure lines were selected and characterized, and it was confirmed that MHC class I performance was not exhibited even after stimulation with IFN-γ or T cell / K562 co-culture supernatant.

K562-based aAPCs were generated from one of the B2M gene knockout pure lines by introducing the necessary costimulatory molecules using ZFN-mediated AAVS1 site-specific integration. AAPC co-cultured T cells based on the B2M knockout K562 showed reduced allogeneic reactivity against K562 cells and target cells expressing the HLA-C allele expressed by K562. AAPC based on B2MKO K562 has also been shown to support robust antigen-independent T cell expansion and antigen-specific anti-CD19 CAR-T cell expansion.

First, K562 cells were reported to lack MHC class I molecular manifestations and only carry trace levels of endogenous B2M manifestations (22, 47, 48). Based on these characteristics and other advantageous characteristics as feeder cells, K562 cells are widely used as feeder cells for in vitro expansion of immune cells.

Many studies claim that K562 cell lines are HLA-negative. However, reduced B2M expression is associated with t (15; 18) chromosomal translocation near the B2M gene (45), but the cell line still carries the wild-type human B2M allele, which means it can perform under certain conditions Or raise B2M.

Previous studies have shown that IFN-γ can up-regulate the expression of MHC class I molecules on K562 cells, mainly HLA-C (42, 48, 49). Lapteva et al. Also reported that MHC class I molecules are upregulated on K562 co-cultured with NK cells (26). The inventors have also observed that B2M and MHC class I molecules are up-regulated on K562 cells co-cultured with T cells. Such up-regulation can be induced by cytokines such as IFN-γ, which are secreted by T cells or other immune cells in a co-culture environment. The inventors have also shown that K562, which is not activated by treatment with mitomycin C or gamma-irradiation, can still up-regulate B2M and MHC class I performance in response to this stimulus (not related to proliferation inhibition).

It was also found that mitomycin C treatment and γ-irradiation induced moderate up-regulation of B2M and MHC class I performance on K562 cells, consistent with the results of previous studies (50, 51). This may be the result of DNA and cytoplasmic protein damage accumulated in K562 cells after mitomycin C treatment or γ-irradiation. Intracellular damage can trigger up-regulation of MHC complexes, thereby presenting abnormal peptides to circulating immune cells.

Since K562 cells can up-regulate MHC class I expression under co-culture conditions, aAPC based on K562 can induce the expansion of the same specific T cells during T cell expansion (specifically targeting MHC class I molecules expressed by K562 cells ). Allogeneic responses of T cells to K562 can occur directly or indirectly (52). As a direct allogeneic recognition, allo-reactive T cells can be activated when their TCR directly recognizes the allo-peptide-MHC complex on K562 cells. As indirect allogeneic recognition, dendritic cells and other monocytes in PBMC will first take up K562 cell alloantigens, and then help present K562 alloantigens to activate T cells. Due to the coexistence of alloantigens and costimulatory molecules on K562-based aAPCs in a T cell expansion scenario, alloactive T cells will subsequently be highly expanded. Lapteva et al. Detected allogenic reactive CD8 ⁺ T cells triggered and expanded by K562-based aAPC after K562 / PBMC co-culture, and even confirmed its allogeneic response to K562 HLA-C * 05 (26) . HLA-C allogeneic recognition is rare in MHC molecular mismatches, but it still occurs frequently (53).

The inventors also detected such allogeneic reactivity against K562 HLA-C after K562 / PBMC co-culture from at least three donors whose HLA-C did not match K562 HLA-C (Figures 8A and 8B). The present inventors used wild type (K562A) and a2PC based on B2MKO (K562B) K562 to expand alloactive T cell populations. Its research showed that aAPC based on the B2M gene knockout of K562 can reduce allogeneic reactivity during amplification. The inventors have observed that it is better to use aAPC based on wild-type K562 instead of B2M gene knockout aAPC to expand allo-reactive T cells. The expanded allo-reactive T cell lines are terminally differentiated and activated effector memory T cells with high-performing CD8, CD86, and HLA-DR. The cytotoxicity of the expanded T cells is at least limited by the HLA-C alleles HLA-C * 03 and HLA-C * 05 of K562. It was found that allo-reactive T cells do not target cell lines exhibiting, for example, HLA-C * 07, HLA-C * 12, or HLA-C * 16. Allo-reactive T cells may show some cross-reactivity to HLA-C * 02 or C * 04 because they exhibit a moderate lytic effect on MDA-MB-468 (HLA-C * 02, * 04) ( Information not shown). Therefore, B2M gene knockout is necessary for K562 cells to produce aAPC with minimal immunogenicity and to ensure the specificity of T cell expansion.

Many methods have been studied to reduce the immunogenicity of specific cells. HLA destruction is a direct method and it is performed on T cells, human embryonic stem cells (hESC) (54), and hematopoietic stem cells (55). However, due to the high level of polymorphisms in MHC class I molecules, this strategy is not universal. B2M gene knockout is an alternative because this microglobulin is conserved and essential for the formation of MHC class I molecules. After B2M knockout, reductions in MHC class I molecules and B2M performance have been achieved in hESC, and no abnormalities have been reported on their B2M knockout cells (29, 30).

In the examples of the present invention, the inventors produced B2M gene knockout K562 cells at a single cell level. In addition to disrupting the site-specific integration of the B2M gene, deletions and insertions are also introduced into the B2M EX1 coding sequence by CRISPR / Cas9, which will cause reading frame shifts and early termination codons in new reading frames. Therefore, the performance of B2M and MHC class I was completely exhausted in the B2M knockout K562 pure line. K562 cell lines have been reported to have two normal copies of human chromosome 15 and t (15; 18) chromosome translocation (45), which indicates that there are three copies of the B2M gene in K562 cells. In each of the knockout single-cell pure lines, in addition to the integration of selectable markers, at most two types of B2M mutant alleles were detected. This finding is consistent with the presence of three copies of the B2M gene in K562 cells, and all three copies were destroyed in B2M-deficient pure lines. In addition, compared with wild-type K562 cells, B2M knockout pure lines were cultured for more than sixty generations without abnormality, indicating that the B2M knockout lines are a safe and effective way to produce stable, low immunogenic cells. Due to the lack of inhibitory ligands against NK cells, B2M gene knockout can make cells more sensitive to NK cell lysis (46), but this is not an important consideration for feeder cells in T cell expansion methods, because Add a new aAPC. In addition, NK cells will be more easily activated by B2M gene knockout K562 cells, which means that aAPC based on B2M gene knockout K562 may be beneficial to NK cell expansion.

In the example of the present invention, two different strategies were used to gene knock out the B2M gene in K562. One strategy uses the CRSIPR / Cas9 system, which targets B2M EX1 introduced directly into K562 cells by electroporation, and the other uses the CRSIPR / Cas9 system to simultaneously target B2M EX1 and EX2, which are introduced by baculovirus transduction K562. Mutations were observed in the disrupted B2M allele only in the EX1 region (Figure 4B), which indicates that the disruptive efficacy of the EX1 target is higher than that of the EX2 target. Consistently, Mandal et al. Also reported that this EX1 target sequence is highly potent (34). CRISPR / nickase systems (56, 57), CRISPR / Cpf1 (58), or fusions with other DNA-binding domains (59) or FokI nucleases (60, 61) can be used to reduce random genome damage and facilitate precise gene knockout .

For genomic modification, some safe gene ports have been identified and used for foreign gene expression. The AAVS 1 locus has been studied, and stable AAVS 1 site-specific integration has been demonstrated in previous studies (36-38). In the example of the present invention, the AAVS 1 site is used for the stable performance of integrated costimulatory molecules, and this precise engineering does not interfere with the normal function of K562 cells. In addition, despite changes in the performance of some markers, it was confirmed that lineage-specific markers CD71 and CD235a were highly expressed on aAPC based on B2M gene knockout K562 after genomic modification (Figure 6D). It is reported that the K562 cell line is a mixture of colony-forming red blood cell progenitor cells and red blood cell mother cells (23). The aAPC of K562 was eliminated based on the B2M gene. Due to two rounds of single-cell selection, more mature red blood cell cells may be derived from K562 cell lines. This single cell selection can help change the expression pattern of some cell markers and reduce their spectrum to a certain level.

K562 cells have been widely used as aAPCs for T cell expansion in vitro (8-11). In the examples of the present invention, the present inventors have demonstrated that a2PCs based on B2MKO K562 and aAPCs based on wild type K562 can generally support robust T cell expansion in an antigen-independent or dependent manner. According to the methods described herein, in an antigen-independent context, T cells can be expanded thousands of times from PBMCs in vitro within three weeks. The expanded T cell line was activated by anti-CD3 antibody OKT-3 and costimulatory molecules on aAPC based on B2M gene knockout K562. CD64 as an Fc receptor facilitates the binding of OKT-3 to activate T cells. CD86 and CD137L are ligands of CD28 and 4-1BB (CD137) on T cells, which help to activate as secondary signals and help maintain the function and survival of T cells. Membrane-bound IL-21 was also introduced into K562-based aAPC to support the proliferation and expansion of T cells, especially CD8 ⁺ T cells (17). In addition, when CD19 antigen is introduced into aAPC based on B2MKO K562, aAPC can be used to efficiently enrich and expand anti-CD19 CAR-T cells, and the expanded CAR-T cells show potent antigen-specific cytotoxicity.
references:

1. DM Barrett, SA Grupp, CH June, Chimeric Antigen Receptor- and TCR-Modified T Cells Enter Main Street and Wall Street. Journal of immunology 195 , 755 (Aug 1, 2015).

2. MV Maus, SA Grupp, DL Porter, CH June, Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood 123 , 2625 (Apr 24, 2014).

3. MH Kershaw, JA Westwood, CY Slaney, PK Darcy, Clinical application of genetically modified T cells in cancer therapy. Clinical & translational immunology 3 , e16 (May, 2014).

4. CM Paulos et al. , Adoptive immunotherapy: good habits instilled at youth have long-term benefits. Immunologic research 42 , 182 (2008).

5. JV Kim, JB Latouche, I. Riviere, M. Sadelain, The ABCs of artificial antigen presentation. Nature biotechnology 22 , 403 (Apr, 2004).

6. M. Wolfl, PD Greenberg, Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8 + T cells. Nature protocols 9 , 950 (Apr, 2014).

7. CJ Turtle, SR Riddell, Artificial antigen-presenting cells for use in adoptive immunotherapy. Cancer journal 16 , 374 (Jul-Aug, 2010).

8. MV Maus et al. , Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nature biotechnology 20 , 143 (Feb, 2002).

9. MO Butler et al. , Long-lived antitumor CD8 + lymphocytes for adoptive therapy generated using an artificial antigen-presenting cell. Clinical cancer research: an official journal of the American Association for Cancer Research 13 , 1857 (Mar 15, 2007).

10. MM Suhoski et al. , Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules. Molecular therapy: the journal of the American Society of Gene Therapy 15 , 981 (May, 2007).

11. W. Gong et al. , Establishment and characterization of a cell based artificial antigen-presenting cell for expansion and activation of CD8 + T cells ex vivo. Cellular & molecular immunology 5 , 47 (Feb, 2008).

12. MO Butler, N. Hirano, Human cell-based artificial antigen-presenting cells for cancer immunotherapy. Immunol Rev 257 , 191 (Jan, 2014).

13. MC Ngo et al. , Complementation of antigen-presenting cells to generate T lymphocytes with broad target specificity. J Immunother 37 , 193 (May, 2014).

14. K. Tanimoto et al . , Genetically engineered fixed K562 cells: potent "off-the-shelf" antigen-presenting cells for generating virus-specific T cells. Cytotherapy 16 , 135 (Jan, 2014).

15. J. Yuan et al. , In vitro expansion of Ag-specific T cells by HLA-A * 0201-transfected K562 cells for immune monitoring. Cytotherapy 8 , 498 (2006).

16. T. Numbenjapon et al. , Antigen-independent and antigen-dependent methods to numerically expand CD19-specific CD8 + T cells. Experimental hematology 35 , 1083 (Jul, 2007).

17. H. Singh et al. , Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies. Cancer research 71 , 3516 (May 15, 2011).

18. H. Singh et al. , Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer research 68 , 2961 (Apr 15, 2008).

19. D. Rushworth et al. , Universal artificial antigen presenting cells to polar propagate T cells expressing chimeric antigen receptor independent of specificity. J Immunother 37 , 204 (May, 2014).

20. I. Caruana et al. , K562-Derived Whole-Cell Vaccine Enhances Antitumor Responses of CAR-Redirected Virus-Specific Cytotoxic T Lymphocytes In Vivo. Clinical cancer research: an official journal of the American Association for Cancer Research 21 , 2952 ( Jul 1, 2015).

21. ZL Chang, PA Silver, YY Chen, Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors. Journal of translational medicine 13 , 161 (2015).

22. E. Klein et al. , Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia. International journal of cancer. Journal international du cancer 18 , 421 (Oct 15, 1976).

23. T. Inoue, A. Swain, Y. Nakanishi, D. Sugiyama, Multicolor Analysis of Cell Surface Marker of Human Leukemia Cell Lines Using Flow Cytometry. Anticancer Research 34 , 4539 (Aug, 2014).

24. SI Drew et al. , Group-specific human granulocyte antigens on a chronic myelogenous leukemia cell line with a Philadelphia chromosome marker. Blood 49 , 715 (May, 1977).

25. EJ Benz, Jr. et al. , Embryonic-fetal erythroid characteristics of a human leukemic cell line. Proceedings of the National Academy of Sciences of the United States of America 77 , 3509 (Jun, 1980).

26. N. Lapteva et al. , Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications. Cytotherapy 14 , 1131 (Oct, 2012).

27. M. Zijlstra et al. , Beta 2-microglobulin deficient mice lack CD4-8 + cytolytic T cells. Nature 344 , 742 (Apr 19, 1990).

28. BH Koller, P. Marrack, JW Kappler, O. Smithies, Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8 + T cells. Science 248 , 1227 (Jun 8, 1990).

29. L. Riolobos et al. , HLA engineering of human pluripotent stem cells. Molecular therapy: the journal of the American Society of Gene Therapy 21 , 1232 (Jun, 2013).

30. P. Lu et al. , Generating hypoimmunogenic human embryonic stem cells by the disruption of beta 2-microglobulin. Stem cell reviews 9 , 806 (Dec, 2013).

31. FD Urnov, EJ Rebar, MC Holmes, HS Zhang, PD Gregory, Genome editing with engineered zinc finger nucleases. Nature reviews. Genetics 11 , 636 (Sep, 2010).

32. L. Cong et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819 (Feb 15, 2013).

33. P. Mali et al. , RNA-guided human genome engineering via Cas9. Science 339 , 823 (Feb 15, 2013).

34. PK Mandal et al. , Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR / Cas9. Cell stem cell 15 , 643 (Nov 6, 2014).

35. HJ Kim, HJ Lee, H. Kim, SW Cho, JS Kim, Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome research 19 , 1279 (Jul, 2009).

36. RC DeKelver et al. , Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome research 20 , 1133 (Aug, 2010).

37. FC Tay et al. , Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector-mediated zinc finger nuclease expression in human-induced pluripotent stem cells. The journal of gene medicine 15 , 384 (Oct, 2013).

38. H. Zhu et al. , Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre / LoxP cassette exchange in human-induced pluripotent stem cells. Nucleic acids research 41 , e180 (Oct, 2013).

39. J. Zeng, J. Du, Y. Zhao, N. Palanisamy, S. Wang, Baculoviral vector-mediated transient and stable transgene expression in human embryonic stem cells. Stem Cells 25 , 1055 (Apr, 2007).

40. KJ Airenne et al. , Baculovirus: an insect-derived vector for diverse gene transfer applications. Molecular therapy: the journal of the American Society of Gene Therapy 21 , 739 (Apr, 2013).

41. JD Sander et al. , ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic acids research 38 , W462 (Jul, 2010).

42. J. Sutherland et al. , Induction of the expression of HLA class I antigens on K562 by interferons and sodium butyrate. Human immunology 12 , 65 (Feb, 1985).

43. P. Krief et al. , Modulation of expression of class II histocompatibility antigens by secretion of a cellular inhibitor in K562 leukemic cells. Eur J Immunol 17 , 1021 (Jul, 1987).

44. A. Liu et al. , Regulation of the expression of MHC class I and II by class II transactivator (CIITA) in hematopoietic cells. Hematol Oncol 17 , 149 (Dec, 1999).

45. J. Zeuthen, U. Friedrich, A. Rosén, E. Klein, Structural abnormalities in chromosome 15 in cell lines with reduced expression of beta-2 microglobulin. Immunogenetics 4 , 567 (1977).

46. MG Morvan, LL Lanier, NK cells and cancer: you can teach innate cells new tricks. Nature reviews. Cancer 16 , 7 (Jan, 2016).

47. A. Ziegler et al. , K562 cells express human major histocompatibility antigens. Immunogenetics 13 , 359 (1981).

48. DR Johnson, Differential expression of human major histocompatibility class I loci: HLA-A, -B, and -C. Human immunology 61 , 389 (Apr, 2000).

49. S. Boegel, M. Lower, T. Bukur, U. Sahin, JC Castle, A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines. Oncoimmunology 3 , e954893 (2014).

50. K. Malinowski, C. Pullis, AP Raisbeck, FT Rapaport, Modulation of human lymphocyte marker expression by gamma irradiation and mitomycin C. Cellular immunology 143 , 368 (Sep, 1992).

51. DJ Hanlon, CL Berger, RL Edelson, Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes. International journal of cancer. Journal international du cancer 78 , 70 (Sep 25, 1998).

52. DB Kazansky, MHC restriction and allogeneic immune responses. Journal of immunotoxicology 5 , 369 (Oct, 2008).

53. F. Bettens, S. Buhler, JM Tiercy, Allorecognition of HLA-C Mismatches by CD8 + T Cells in Hematopoietic Stem Cell Transplantation Is a Complex Interplay between Mismatched Peptide-Binding Region Residues, HLA-C Expression, and HLA-DPB1 Disparities Frontiers in immunology 7 , 584 (2016).

54. H. Torikai et al. , Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 122 , 1341 (Aug 22, 2013).

55. H. Torikai et al. , Genetic editing of HLA expression in hematopoietic stem cells to broaden their human application. Scientific reports 6 , 21757 (2016).

56. FA Ran et al. , Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154 , 1380 (Sep 12, 2013).

57. B. Shen et al. , Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods 11 , 399 (Apr, 2014).

58. B. Zetsche et al. , Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163 , 759 (Oct 22, 2015).

59. MF Bolukbasi et al. , DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nature methods 12 , 1150 (Dec, 2015).

60. JP Guilinger, DB Thompson, DR Liu, Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature biotechnology 32 , 577 (Jun, 2014).

61. SQ Tsai et al. , Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nature biotechnology 32 , 569 (Jun, 2014).

Examples and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying drawings.

FIGS. 1A and 1B. Graphs and bar graphs showing the performance of B2M and MHC class I molecules on K562 cells after stimulation with IFN-γ or a cell culture supernatant collected from a T cell / K562 cell co-culture. ( 1A ) Representative flow cytometry histogram of surface B2M and HLA-A, B, C performance. Non-activated, mitomycin C-treated or γ-irradiated K562 cells were treated with IFN-γ (500 IU / ml, light gray) or from T cell / K562 cell co-cultures (1: 1 ratio for 48 hours, (Black line) The collected supernatant layer was stimulated for 48 hours. Unstimulated K562 cells were included as controls (grey). ( 1B ) Bar graph showing the mean ± SD of three independent experiments. Stimulation was performed with IFN-γ or culture supernatants produced from three separate donors. ***: p <0.001.

Figures 2A and 2B. Graphs showing the performance of isotype controls and MHC class II molecules on K562 cells. ( 2A ) Flow cytometry histogram of an isotype control labeled on the surface of K562 cells. ( 2B ) Representative flow cytometry histogram of HLA-DR performance. K562 cells were stimulated with IFN-γ (500 IU / ml) or T cell / K562 cell co-cultures (1: 1 ratio for 48 hours) for 48 hours. Unstimulated K562 cells were included as controls. Percent gated positive performance based on isotype controls.

Figures 3A to 3D. Schematic and bar graphs related to the generation of B2M gene knockouts in K562 cells using CRISPR / Cas9 technology. ( 3A ) Schematic of the CRISPR / Cas9 system targeting B2M exon 1 (EX1). The system is used for electroporation-based genetic modification in K562 cells. (3 B) a schematic B2M EX1 and exon 2 (EX2) baculovirus CRISPR / Cas9 targeting system. For genetic modification, K562 cells were transduced with the baculovirus system. Arrows show the binding sites of PCR primers for genotyping. ( 3C ) and ( 3D ) are bar graphs showing the B2M performance of K562 single cell pure lines after B2M gene knockout. The K562 single-cell pure lines tested were stimulated with 500 IU / mL IFN-γ and subsequently analyzed by flow cytometry to detect B2M performance.

Figures 4A to 4E. Images, diagrams and graphics related to the characterization of B2M gene knockout K562 pure lines. ( 4A ) Genotyping of B2M knockout single cell pure lines by PCR. Two pure lines were from K562 cells transfected with electroporation, and five pure lines were from K562 cells transduced with baculovirus. As a control, wild-type K562 was included. The figure above shows the specific amplification of the selectable marker integrated in the B2M site. (+) Positive control for B2M site-specific integration. The figure below shows the specific amplification of the wild-type B2M EX1 allele. ( 4B ) Schematic representation of the sequencing results of the targeting regions in EX1 and EX2 from five B2M knockout single cell pure lines. The three alleles of the B2M gene are aligned with the wild type. CRISPR / Cas9 targets are shown in light gray. The start codon for B2M is dark gray "ATG". Deletions are shown by dashed lines and the inserted "T" nucleotides are underlined. ( 4C ) Representative Sanger sequencing traces from mutant B2M alleles. The electropherogram displayed with the wild-type B2M sequence shows the sites of deletions and insertions. ( 4D ) Western blots on B2M expression in B2M gene knockout single cell pure lines. Wild-type K562 was included as a control and β-actin was detected for housekeeping performance. ( 4E ) Representative flow cytometry histogram of B2M gene knockout K562 single cell pure line upper surface B2M and HLA-A, B, C performance. Prior to analysis, B2M gene knockout single cell pure lines were stimulated with IFN-γ or T cell / K562 cell co-culture supernatants. Percent gated positive performance based on isotype controls.

Figures 5A to 5C. Bar graphs and graphs showing the analysis results of MHC class I performance and function of B2M knockout K562 cells. After stimulation with IFN-γ or T cell / K562 cell co-culture supernatants, B2M ( 5A ) and MHC class I molecules ( 5B ) were examined on wild-type (WT) K562 and B2M gene knockout K562 single cell pure lines Surface performance. The bar graph shows the mean ± SD of three independent experiments. ( 5C ) Figure showing B2M knockout K562 cells with reduced MHC class I expression on the upper surface leading to increased susceptibility to NK cell lysis, such as by using DELFIA EuTDA cytotoxicity analysis (2-hour Eu-ligand release) using K562 B2MKO Pure line EX1EX2 # 5 was determined by analysis of target cells. Three independent analyses were performed with NK cells from three different donors. The percentage of lysis of K562 cells at different effect (E): target (T) cell ratios (mean ± SD of triplicate samples) is shown. *: P <0.05; ***: p <0.001.

6A to 6E. Schematic diagrams and graphs related to the generation of aAPC based on B2M gene knockout K562. ( 6A ) Schematic of an electroporated ZFN construct targeting the AAVS1 site for integration of a co-stimulatory molecule. Arrows indicate the binding sites of PCR primers for genotyping. ( 6B ) Performance of costimulatory molecules on K562A and K562B cells. aAPC was stained with antibodies and analyzed by flow cytometry analysis. A representative FACS plot is shown and the percentage of gated positive performance relative to the isotype control. ( 6C ) AAVS1 locus genotyping was performed on K562B by PCR. Includes unmodified B2M gene knockout K562 pure line EX1EX2 # 5 as a control. The left panel shows the specific amplification of the donor cassette integrated at the AAVS1 site. The right panel shows the specific amplification of the wild-type AAVS1 allele. ( 6D and 6E ) K562B cells are characterized by red blood cell and bone marrow-specific cell markers. As a control, wild-type K562 cells were included. Cells were stained with antibodies and analyzed by flow cytometry analysis. A representative figure is shown in 6E . The percentage of positive cells was determined by gating relative to the isotype control. The bar graph shows the mean ± SD of three independent experiments.

7A to 7D. Schematic diagrams and graphs related to allogeneic reactivity analysis of T cells expanded with aAPC based on B2M gene knockout K562 cells. ( 7A ) Schematic of an in vitro T cell expansion protocol. T cells were stimulated with K562A (aAPC based on WT K562) or K562B (aAPC based on B2M gene knockout) every seven days before being used for cytotoxicity analysis for two weeks. ( 7B to 7D ) CD8 + T cells expanded in the presence of K562A or K562B cells against wild-type K562 cells and express the designated HLA-C allele (HLA-C * 03 or HLA-C * 05) expressed by K562 cells Target cells or target cells that do not exhibit HLA-C alleles HLA-C * 03 or HLA-C * 05 allogeneic reactivity. Three independent analyses were performed using T cells from three different donors ( 7B , 7C, and 7D , respectively). Shows the percentage of target cell lysis at different E: T ratios (mean ± SD of triplicate samples). *: P &lt;0.05; ***: p &lt; 0.001.

8A to 8D. Images and graphs related to allogeneic reactivity analysis of T cells expanded with aAPC based on B2M gene knockout K562 cells. ( 8A and 8B ) HLA-C genotyping of K562 cells, tumor cell lines used as targets, and PBMCs used as effectors. PBMCs are grouped according to whether they show allo-reactivity. ( 8A ) Specific amplification of the HLA-C * 03 allele. ( 8B ) Specific amplification of the HLA-C * 05 allele. ( 8C ) Multiple growth of T cells from three different allo-reactive donors co-cultured with the designated K562 cells. K562 cells were deactivated with mitomycin C, and a T cell / K562 cell ratio of 1: 1 was used for co-cultures. ( 8D ) Immunophenotyping of expanded cells by flow cytometry. Representative FACS plots of three alloactive donors are shown, and the percentage of positive cells is determined by gating relative to the isotype control.

9A to 9H. Schematic diagrams, bar graphs, and tables showing the expansion of antigen-independent T cells using aAPC based on B2M gene knockout K562. ( 9A ) Schematic of in vitro antigen-independent T cell expansion protocol. Prior to harvest, T cells were stimulated with K562A or K562B at a T cell / K562 cell ratio of 1:50 for two weeks. OCT-3 is included to promote T cell expansion. ( 9B to 9D ) T-fold growth of T cells co-cultured with WT K562 and B2M k / o K562 cells. K562 cells were deactivated with mitomycin C. A T cell / K562 cell ratio of 1:50 was used and OCT-3 was included to facilitate T cell expansion. Three independent analyses were performed using T cells from three different donors ( 9B , 9C, and 9D , respectively). ( 9E to 9H ) Immunophenotyping of expanded cells by flow cytometry. ( 9E and 9F ) Bar graphs showing the percentage of major cell types and CD8 + T cell subpopulations expanded in the experiments shown in 9B . ( 9G and 9H ) A table showing the percentage of major cell types and CD8 + T cell subsets expanded in the experiments shown in 9G and 9H , respectively.

10A to 10I. Patterns and schematic diagrams related to the expansion of anti-CD19 CAR T cells using aAPC based on B2M gene knockout K562 expressing CD19 antigen. ( 10A ) Analysis of CD19 and CAR expression based on aAPC of B2MKO K562 and T cells expressing anti-CD19 CAR. Cells were stained with antibodies and analyzed by flow cytometry. A representative FACS plot is shown and positive performance is determined by gating relative to an isotype control. ( 10B ) Schematic of an in vitro expansion protocol for anti-CD19 CAR T cells. Prior to harvest, T cells were stimulated with K562B + CD19 or K562B at a T cell / K562 cell ratio of 1: 1 every 7 days for four weeks. ( 10C and 10D ) Multiple growth of anti-CD19-CAR-T cells derived from two different donors co-cultured with K562 cells. K562 cells were deactivated with mitomycin C, and a T cell / K562 cell ratio of 1: 1 was used for co-cultures. ( 10E and 10F ) shows a scatter plot of the number of anti-CD19 CAR-positive T cells after co-culture of anti-CD19-CAR-T cells from two different donors with K562B or K562B + CD19 cells for a specified number of days. Cells were collected and stained with anti-mouse IgG Fab and anti-human CD3 antibodies, and analyzed by flow cytometry. ( 10G ) Immunophenotyping of expanded cells by flow cytometry. The representative graph is displayed. ( 10H and 10I ) Analysis of in vitro cytolysis of CD19-positive tumor cell lines (Daudi and Raji) or CD19-negative cell line (MCF-7), in which anti-CD19 CAR-expressing T cells were derived from two different donors, such as Determined by DELFIA EuTDA cytotoxicity analysis (2-hour Eu-ligand release). Three or more independent experiments were performed on PBMCs from individual donors (mean ± SD of triplicate samples). *: P <0.05.

Sequence Listing

<110> 1. Singapore Commercial Tessa Therapy Pte Ltd
2.National University of Singapore


<120> Modified K562 cells

<150> US 62/588661
<151> 2017-11-20

<160> 36

<170> PatentIn version 3.5

<210> 1
<211> 6673
<212> DNA
<213> Homo sapiens B2M gene sequence (NCBI reference sequence: NG_012920.1)

<400> 1
aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60

atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 120

atccagcgtg agtctctcct accctcccgc tctggtcctt cctctcccgc tctgcaccct 180

ctgtggccct cgctgtgctc tctcgctccg tgacttccct tctccaagtt ctccttggtg 240

gcccgccgtg gggctagtcc agggctggat ctcggggaag cggcggggtg gcctgggagt 300

ggggaagggg gtgcgcaccc gggacgcgcg ctacttgccc ctttcggcgg ggagcagggg 360

agacctttgg cctacggcga cgggagggtc gggacaaagt ttagggcgtc gataagcgtc 420

agagcgccga ggttggggga gggtttctct tccgctcttt cgcggggcct ctggctcccc 480

cagcgcagct ggagtggggg acgggtaggc tcgtcccaaa ggcgcggcgc tgaggtttgt 540

gaacgcgtgg aggggcgctt ggggtctggg ggaggcgtcg cccgggtaag cctgtctgct 600

gcggctctgc ttcccttaga ctggagagct gtggacttcg tctaggcgcc cgctaagttc 660

gcatgtccta gcacctctgg gtctatgtgg ggccacaccg tggggaggaa acagcacgcg 720

acgtttgtag aatgcttggc tgtgatacaa agcggtttcg aataattaac ttatttgttc 780

ccatcacatg tcacttttaa aaaattataa gaactacccg ttattgacat ctttctgtgt 840

gccaaggact ttatgtgctt tgcgtcattt aattttgaaa acagttatct tccgccatag 900

ataactacta tggttatctt ctgcctctca cagatgaaga aactaaggca ccgagatttt 960

aagaaactta attacacagg ggataaatgg cagcaatcga gattgaagtc aagcctaacc 1020

agggcttttg cgggagcgca tgccttttgg ctgtaattcg tgcatttttt tttaagaaaa 1080

acgcctgcct tctgcgtgag attctccaga gcaaactggg cggcatgggc cctgtggtct 1140

tttcgtacag agggcttcct ctttggctct ttgcctggtt gtttccaaga tgtactgtgc 1200

ctcttacttt cggttttgaa aacatgaggg ggttgggcgt ggtagcttac gcctgtaatc 1260

ccagcactta gggaggccga ggcgggagga tggcttgagg tccgtagttg agaccagcct 1320

ggccaacatg gtgaagcctg gtctctacaa aaaataataa caaaaattag ccgggtgtgg 1380

tggctcgtgc ctgtggtccc agctgctccg gtggctgagg cgggaggatc tcttgagctt 1440

aggcttttga gctatcatgg cgccagtgca ctccagcgtg ggcaacagag cgagaccctg 1500

tctctcaaaa aagaaaaaaa aaaaaaaaga aagagaaaag aaaagaaaga aagaagtgaa 1560

ggtttgtcag tcaggggagc tgtaaaacca ttaataaaga taatccaaga tggttaccaa 1620

gactgttgag gacgccagag atcttgagca ctttctaagt acctggcaat acactaagcg 1680

cgctcacctt ttcctctggc aaaacatgat cgaaagcaga atgttttgat catgagaaaa 1740

ttgcatttaa tttgaataca atttatttac aacataaagg ataatgtata tatcaccacc 1800

attactggta tttgctggtt atgttagatg tcattttaaa aaataacaat ctgatattta 1860

aaaaaaaatc ttattttgaa aatttccaaa gtaatacatg ccatgcatag accatttctg 1920

gaagatacca caagaaacat gtaatgatga ttgcctctga aggtctattt tcctcctctg 1980

acctgtgtgt gggttttgtt tttgttttac tgtgggcata aattaatttt tcagttaagt 2040

tttggaagct taaataactc tccaaaagtc ataaagccag taactggttg agcccaaatt 2100

caaacccagc ctgtctgata cttgtcctct tcttagaaaa gattacagtg atgctctcac 2160

aaaatcttgc cgccttccct caaacagaga gttccaggca ggatgaatct gtgctctgat 2220

ccctgaggca tttaatatgt tcttattatt agaagctcag atgcaaagag ctctcttagc 2280

ttttaatgtt atgaaaaaaa tcaggtcttc attagattcc ccaatccacc tcttgatggg 2340

gctagtagcc tttccttaat gatagggtgt ttctagagag atatatctgg tcaaggtggc 2400

ctggtactcc tccttctccc cacagcctcc cagacaagga ggagtagctg ccttttagtg 2460

atcatgtacc ctgaatataa gtgtatttaa aagaatttta tacacatata tttagtgtca 2520

atctgtatat ttagtagcac taacacttct cttcattttc aatgaaaaat atagagttta 2580

taatattttc ttcccacttc cccatggatg gtctagtcat gcctctcatt ttggaaagta 2640

ctgtttctga aacattaggc aatatattcc caacctggct agtttacagc aatcacctgt 2700

ggatgctaat taaaacgcaa atcccactgt cacatgcatt actccatttg atcataatgg 2760

aaagtatgtt ctgtcccatt tgccatagtc ctcacctatc cctgttgtat tttatcgggt 2820

ccaactcaac catttaaggt atttgccagc tcttgtatgc atttaggttt tgtttctttg 2880

ttttttagct catgaaatta ggtacaaagt cagagagggg tctggcatat aaaacctcag 2940

cagaaataaa gaggttttgt tgtttggtaa gaacatacct tgggttggtt gggcacggtg 3000

gctcgtgcct gtaatcccaa cactttggga ggccaaggca ggctgatcac ttgaagttgg 3060

gagttcaaga ccagcctggc caacatggtg aaatcccgtc tctactgaaa atacaaaaat 3120

taaccaggca tggtggtgtg tgcctgtagt cccaggaatc acttgaaccc aggaggcgga 3180

ggttgcagtg agctgagatc tcaccactgc acactgcact ccagcctggg caatggaatg 3240

agattccatc ccaaaaaata aaaaaataaa aaaataaaga acataccttg ggttgatcca 3300

cttaggaacc tcagataata acatctgcca cgtatagagc aattgctatg tcccaggcac 3360

tctactagac acttcataca gtttagaaaa tcagatgggt gtagatcaag gcaggagcag 3420

gaaccaaaaa gaaaggcata aacataagaa aaaaaatgga aggggtggaa acagagtaca 3480

ataacatgag taatttgatg ggggctatta tgaactgaga aatgaacttt gaaaagtatc 3540

ttggggccaa atcatgtaga ctcttgagtg atgtgttaag gaatgctatg agtgctgaga 3600

gggcatcaga agtccttgag agcctccaga gaaaggctct taaaaatgca gcgcaatctc 3660

cagtgacaga agatactgct agaaatctgc tagaaaaaaa acaaaaaagg catgtataga 3720

ggaattatga gggaaagata ccaagtcacg gtttattctt caaaatggag gtggcttgtt 3780

gggaaggtgg aagctcattt ggccagagtg gaaatggaat tgggagaaat cgatgaccaa 3840

atgtaaacac ttggtgcctg atatagcttg acaccaagtt agccccaagt gaaataccct 3900

ggcaatatta atgtgtcttt tcccgatatt cctcaggtac tccaaagatt caggtttact 3960

cacgtcatcc agcagagaat ggaaagtcaa atttcctgaa ttgctatgtg tctgggtttc 4020

atccatccga cattgaagtt gacttactga agaatggaga gagaattgaa aaagtggagc 4080

attcagactt gtctttcagc aaggactggt ctttctatct cttgtactac actgaattca 4140

cccccactga aaaagatgag tatgcctgcc gtgtgaacca tgtgactttg tcacagccca 4200

agatagttaa gtggggtaag tcttacattc ttttgtaagc tgctgaaagt tgtgtatgag 4260

tagtcatatc ataaagctgc tttgatataa aaaaggtcta tggccatact accctgaatg 4320

agtcccatcc catctgatat aaacaatctg catattggga ttgtcaggga atgttcttaa 4380

agatcagatt agtggcacct gctgagatac tgatgcacag catggtttct gaaccagtag 4440

tttccctgca gttgagcagg gagcagcagc agcacttgca caaatacata tacactctta 4500

acacttctta cctactggct tcctctagct tttgtggcag cttcaggtat atttagcact 4560

gaacgaacat ctcaagaagg tataggcctt tgtttgtaag tcctgctgtc ctagcatcct 4620

ataatcctgg acttctccag tactttctgg ctggattggt atctgaggct agtaggaagg 4680

gcttgttcct gctgggtagc tctaaacaat gtattcatgg gtaggaacag cagcctattc 4740

tgccagcctt atttctaacc attttagaca tttgttagta catggtattt taaaagtaaa 4800

acttaatgtc ttcctttttt ttctccactg tctttttcat agatcgagac atgtaagcag 4860

catcatggag gtaagttttt gaccttgaga aaatgttttt gtttcactgt cctgaggact 4920

atttatagac agctctaaca tgataaccct cactatgtgg agaacattga cagagtaaca 4980

ttttagcagg gaaagaagaa tcctacaggg tcatgttccc ttctcctgtg gagtggcatg 5040

aagaaggtgt atggccccag gtatggccat attactgacc ctctacagag agggcaaagg 5100

aactgccagt atggtattgc aggataaagg caggtggtta cccacattac ctgcaaggct 5160

ttgatctttc ttctgccatt tccacattgg acatctctgc tgaggagaga aaatgaacca 5220

ctcttttcct ttgtataatg ttgttttatt cttcagacag aagagaggag ttatacagct 5280

ctgcagacat cccattcctg tatggggact gtgtttgcct cttagaggtt cccaggccac 5340

tagaggagat aaagggaaac agattgttat aacttgatat aatgatacta taatagatgt 5400

aactacaagg agctccagaa gcaagagaga gggaggaact tggacttctc tgcatcttta 5460

gttggagtcc aaaggctttt caatgaaatt ctactgccca gggtacattg atgctgaaac 5520

cccattcaaa tctcctgtta tattctagaa cagggaattg atttgggaga gcatcaggaa 5580

ggtggatgat ctgcccagtc acactgttag taaattgtag agccaggacc tgaactctaa 5640

tatagtcatg tgttacttaa tgacggggac atgttctgag aaatgcttac acaaacctag 5700

gtgttgtagc ctactacacg cataggctac atggtatagc ctattgctcc tagactacaa 5760

acctgtacag cctgttactg tactgaatac tgtgggcagt tgtaacacaa tggtaagtat 5820

ttgtgtatct aaacatagaa gttgcagtaa aaatatgcta ttttaatctt atgagaccac 5880

tgtcatatat acagtccatc attgaccaaa acatcatatc agcatttttt cttctaagat 5940

tttgggagca ccaaagggat acactaacag gatatactct ttataatggg tttggagaac 6000

tgtctgcagc tacttctttt aaaaaggtga tctacacagt agaaattaga caagtttggt 6060

aatgagatct gcaatccaaa taaaataaat tcattgctaa cctttttctt ttcttttcag 6120

gtttgaagat gccgcatttg gattggatga attccaaatt ctgcttgctt gctttttaat 6180

attgatatgc ttatacactt acactttatg cacaaaatgt agggttataa taatgttaac 6240

atggacatga tcttctttat aattctactt tgagtgctgt ctccatgttt gatgtatctg 6300

agcaggttgc tccacaggta gctctaggag ggctggcaac ttagaggtgg ggagcagaga 6360

attctcttat ccaacatcaa catcttggtc agatttgaac tcttcaatct cttgcactca 6420

aagcttgtta agatagttaa gcgtgcataa gttaacttcc aatttacata ctctgcttag 6480

aatttggggg aaaatttaga aatataattg acaggattat tggaaatttg ttataatgaa 6540

tgaaacattt tgtcatataa gattcatatt tacttcttat acatttgata aagtaaggca 6600

tggttgtggt taatctggtt tatttttgtt ccacaagtta aataaatcat aaaacttgat 6660

gtgttatctc tta 6673


<210> 2
<211> 987
<212> DNA
<213> Homo sapiens B2M mRNA (NCBI reference sequence: NM_004048.2)

<400> 2
aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 60

atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 120

atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 180

aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 240

aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 300

tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 360

cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgtaa 420

gcagcatcat ggaggtttga agatgccgca tttggattgg atgaattcca aattctgctt 480

gcttgctttt taatattgat atgcttatac acttacactt tatgcacaaa atgtagggtt 540

ataataatgt taacatggac atgatcttct ttataattct actttgagtg ctgtctccat 600

gtttgatgta tctgagcagg ttgctccaca ggtagctcta ggagggctgg caacttagag 660

gtggggagca gagaattctc ttatccaaca tcaacatctt ggtcagattt gaactcttca 720

atctcttgca ctcaaagctt gttaagatag ttaagcgtgc ataagttaac ttccaattta 780

catactctgc ttagaatttg ggggaaaatt tagaaatata attgacagga ttattggaaa 840

tttgttataa tgaatgaaac attttgtcat ataagattca tatttacttc ttatacattt 900

gataaagtaa ggcatggttg tggttaatct ggtttatttt tgttccacaa gttaaataaa 960

tcataaaact tgatgtgtta tctctta 987


<210> 3
<211> 360
<212> DNA
<213> Homo sapiens B2M protein coding sequence

<400> 3
atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60

atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 120

aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 180

aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 240

tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 300

cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgtaa 360


<210> 4
<211> 119
<212> PRT
<213> Homo sapiens B2M (UniProt: P61769-1, v1)

<400> 4

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser
1 5 10 15


Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg
20 25 30


His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser
35 40 45


Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu
50 55 60


Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp
65 70 75 80


Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp
85 90 95


Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile
100 105 110


Val Lys Trp Asp Arg Asp Met
115


<210> 5
<211> 99
<212> PRT
<213> Homo sapiens mature B2M (UniProt: P61769-1, residues 21-199 of v1)

<400> 5

Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu
1 5 10 15


Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro
20 25 30


Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys
35 40 45


Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu
50 55 60


Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys
65 70 75 80


Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp
85 90 95


Arg Asp Met



<210> 6
<211> 26
<212> DNA
<213> Artificial sequence

<220>
<223> Forward B2M EX1 Target

<400> 6
aaacggccga gatgtctcgc tccggt 26


<210> 7
<211> 26
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse B2M EX1 Target

<400> 7
taaaaccgga gcgagacatc tcggcc 26


<210> 8
<211> 26
<212> DNA
<213> Artificial sequence

<220>
<223> Forward B2M EX2 Target

<400> 8
aaacgaagtt gacttactga agaagt 26


<210> 9
<211> 26
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse B2M EX2 Target

<400> 9
taaaacttct tcagtaagtc aacttc 26


<210> 10
<211> 24
<212> DNA
<213> Artificial sequence

<220>
<223> Forward B2M EX1 WT

<400> 10
tctcgaatga aaaatgcagg tccg 24


<210> 11
<211> 25
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse B2M EX1 WT

<400> 11
tgacgcttat cgacgcccta aactt 25


<210> 12
<211> 22
<212> DNA
<213> Artificial sequence

<220>
<223> Forward B2M EX1 HDR

<400> 12
gactccacca ccacgaaatg gc 22


<210> 13
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse B2M EX1 HDR

<400> 13
ccccatcaag ctgatccgga 20


<210> 14
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Forward B2M EX2 WT

<400> 14
gcttgacacc aagttagccc 20


<210> 15
<211> 21
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse B2M EX2 WT

<400> 15
tggatgggac tcattcaggg t 21


<210> 16
<211> 23
<212> DNA
<213> Artificial sequence

<220>
<223> Forward AAVS1 WT

<400> 16
cactcgctgg gttccctttt cct 23


<210> 17
<211> 25
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse AAVS1 WT

<400> 17
ggctggctac tggccttatc tcaca 25


<210> 18
<211> 24
<212> DNA
<213> Artificial sequence

<220>
<223> Forward AAVS1 CD64 HDR

<400> 18
ctttggcagc ctgtgctgac ccat 24


<210> 19
<211> 24
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse AAVS1 CD64 HDR

<400> 19
tcgaggtctg agtggctgtg ccat 24


<210> 20
<211> 24
<212> DNA
<213> Artificial sequence

<220>
<223> Forward AAVS1 EpCAM HDR

<400> 20
ctttggcagc ctgtgctgac ccat 24


<210> 21
<211> 24
<212> DNA
<213> Artificial sequence

<220>
<223> Inverse AAVS1 EpCAM HDR

<400> 21
aagagcccgc tctcatcgca gtca 24


<210> 22
<211> 19
<212> DNA
<213> Artificial sequence

<220>
<223> Forward HLA-C * 03 typing

<400> 22
cacagactga ccgagtgag 19


<210> 23
<211> 20
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse HLA-C * 03 typing

<400> 23
agcgtctcct tcccattctt 20


<210> 24
<211> 19
<212> DNA
<213> Artificial sequence

<220>
<223> Forward HLA-C * 05 classification

<400> 24
ccgagtgaac ctgcggaaa 19


<210> 25
<211> 18
<212> DNA
<213> Artificial sequence

<220>
<223> Reverse HLA-C * 05 typing

<400> 25
cgcgcgctgc agcgtctt 18


<210> 26
<211> 42
<212> DNA
<213> Artificial sequence

<220>
<223> crRNA targeting B2M EX1

<400> 26
ggccgagatg tctcgctccg gttttagagc tatgctgttt tg 42


<210> 27
<211> 42
<212> DNA
<213> Artificial sequence

<220>
<223> crRNA targeting B2M EX2

<400> 27
gaagttgact tactgaagaa gttttagagc tatgctgttt tg 42


<210> 28
<211> 84
<212> DNA
<213> Artificial sequence

<220>
<223> tracrRNA

<400> 28
ggaaccattc aaaacagcat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 60

agtggcaccg agtcggtgct tttt 84


<210> 29
<211> 46
<212> DNA
<213> Artificial sequence

<220>
<223> EX1 WT allele

<400> 29
tgacagcatt cgggccgaga tgtctcgctc cgtggcctta gctgtg 46


<210> 30
<211> 47
<212> DNA
<213> Artificial sequence

<220>
<223> B2M-EX1-G # 7 allele 2

<400> 30
tgacagcatt cgggccgaga tgtctcgctt ccgtggcctt agctgtg 47


<210> 31
<211> 44
<212> DNA
<213> Artificial sequence

<220>
<223> B2M-EX1-G # 7 allele 3

<400> 31
tgacagcatt cgggccgaga tgtctcgccg tggccttagc tgtg 44


<210> 32
<211> 27
<212> DNA
<213> Artificial sequence

<220>
<223> B2M-EX1EX2-G # 5/6/7 allele 2

<400> 32
tgacagcatt ccgtggcctt agctgtg 27


<210> 33
<211> 47
<212> DNA
<213> Artificial sequence

<220>
<223> B2M-EX1EX2-G # 2/5/6/7 allele 3

<400> 33
tgacagcatt cgggccgaga tgtctcgctt ccgtggcctt agctgtg 47


<210> 34
<211> 43
<212> DNA
<213> Artificial sequence

<220>
<223> EX2 WT allele

<400> 34
catccgacat tgaagttgac ttactgaaga atggagagag aat 43


<210> 35
<211> 43
<212> DNA
<213> Artificial sequence

<220>
<223> B2M-EX1EX2-G # 2/5/6/7 WT allele 2

<400> 35
catccgacat tgaagttgac ttactgaaga atggagagag aat 43


<210> 36
<211> 43
<212> DNA
<213> Artificial sequence

<220>
<223> B2M-EX1EX2-G # 2/5/6/7 WT allele 3

<400> 36
catccgacat tgaagttgac ttactgaaga atggagagag aat 43

Claims (42)

A modified K562 cell that has reduced MHC class I performance compared to a wild-type K562 cell. The modified K562 cell of claim 1, comprising a modification of a gene encoding a MHC class I polypeptide relative to a wild-type K562 cell. The modified K562 cell of claim 2, wherein the modification reduces or prevents the performance of a polypeptide encoded by the gene encoding a MHC class I polypeptide. For example, the modified K562 cell of claim 2 or claim 3, wherein the gene encoding a MHC class I polypeptide is B2M . The modified K562 cell of any one of claims 1 to 4, further comprising a modification to increase the performance of one or more factors capable of increasing the activation or proliferation of immune cells. The modified K562 cell of claim 5, comprising a nucleic acid encoding the one or more factors capable of increasing the activation / proliferation of immune cells. For example, the modified K562 cells of claim 5 or claim 6, wherein the one or more factors capable of increasing the activation / proliferation of immune cells are selected from the group consisting of a costimulatory molecule, a cytokine, or an antigen. The modified K562 cell of claim 7, wherein the costimulatory molecule is selected from the group consisting of CD40L, CD86, CD137L, CD80, or CD83. The modified K562 cell according to claim 7 or claim 8, wherein the interleukin is selected from the group consisting of IL-21, IL-15, membrane-bound IL-21 and membrane-bound IL-15. The modified K562 cell of any one of claims 1 to 9, further comprising a modification to increase the performance of one or more Fc receptors. A modified K562 cell comprising a modification to reduce or prevent the performance of a polypeptide encoded by B2M . The modified K562 cells of claim 11, further comprising modifications to increase the performance of one or more of the following: CD64, CD86, CD137L, and membrane-bound IL-21. The modified K562 cells as claimed in claim 11 or claim 12, further comprising modifications to increase the expression of an antigen. The modified K562 cell of any one of claims 11 to 13, wherein the modified K562 cell comprises a modification to increase the performance of CD19. A method for generating a modified K562 cell with reduced MHC class I performance compared to a wild-type K562 cell, comprising modifying a K562 cell to reduce or prevent MHC class I performance. The method of claim 15, wherein the modification reduces or prevents the performance of a polypeptide encoded by a gene encoding a MHC class I polypeptide. The method of claim 15 or 16, wherein the gene encoding a MHC class I polypeptide is B2M . The method of any one of claims 15 to 17, further comprising modifying the K562 cells to increase the performance of one or more factors capable of increasing the activation or proliferation of immune cells. The method of claim 18, comprising introducing into the K562 cells a nucleic acid encoding the one or more factors capable of increasing the activation / proliferation of immune cells. The method according to claim 18 or claim 19, wherein the one or more factors capable of increasing the activation / proliferation of immune cells are selected from the group consisting of a costimulatory molecule, a cytokine, or an antigen. The method of claim 20, wherein the costimulatory molecule is selected from the group consisting of CD40L, CD86, CD137L, CD80, or CD83. The method of claim 20 or claim 21, wherein the interleukin is selected from the group consisting of IL-21, IL-15, membrane-bound IL-21, and membrane-bound IL-15. The method of any one of claims 20 to 22, comprising introducing into the K562 cells a nucleic acid encoding one or more Fc receptors. The method of any one of claims 20 to 23, further comprising modifying the K562 cells to increase the expression of an antigen. A modified K562 cell obtained or obtained by a method according to any one of claims 15 to 24. A method for generating or expanding a population of immune cells, comprising contacting immune cells in vitro, in vivo, or ex vivo with modified K562 cells as in any one of claims 1 to 14 or 25 . The method of claim 26, which is a method for generating or expanding an antigen-specific immune cell population, wherein the method comprises a method of any one of claims 1 to 14 or 25 comprising or expressing the antigen. Immune cells are cultured in the presence of modified K562 cells. The method of claim 27, wherein the antigen-specific immune cell is a CAR-modified immune cell, and wherein the modified K562 cell comprises or expresses the CAR-specific antigen. An immune cell population produced or expanded by a method according to any one of claims 26 to 28. The immune cell population of claim 29, which is used in a method of medical treatment or prevention of a disease or condition. An immune cell population as claimed in claim 29 for use in the manufacture of a medicament for use in a method of medical treatment or prevention of a disease or condition. A method of treating or preventing a disease or condition in an individual, comprising administering to a subject an immune cell population as claimed in claim 29. A method of treating or preventing a disease or condition in a subject, comprising: (a) isolating immune cells from a body; (b) generating or expanding an immune cell population by culturing the immune cells isolated in step (a) in the presence of modified K562 cells as in any of claims 1 to 14 or claim 25; and (c) administering to a body the population of immune cells produced or expanded in step (b). Immune cell population for use as in claim 30, as in claim 31 or as in a method as in any of claim 32 or claim 33, wherein the disease or condition is a T cell dysfunction disorder, a cancer or An infectious disease. The method of claim 34, wherein the cancer is selected from the group consisting of colon cancer, colon cancer, colorectal cancer, nasopharyngeal cancer, cervical cancer, oropharyngeal cancer, gastric cancer, Hepatocellular carcinoma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), oral cancer, laryngeal cancer, prostate cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, urethral epithelial cancer, melanoma, advanced melanoma Tumor, renal cell carcinoma, ovarian cancer, or mesothelioma. A nucleic acid or a plurality of nucleic acids encoding a site-specific nuclease (SSN) system targeting B2M . The nucleic acid or nucleic acids of claim 36, wherein the nucleic acid or nucleic acids encode a CRISPR / Cas9 system. The nucleic acid or nucleic acids of claim 37, wherein the nucleic acid or nucleic acids encode a CRISPR RNA (crRNA) that targets one of the exons of B2M . For example, the nucleic acid or multiple nucleic acids of claim 37 or claim 38, wherein the nucleic acid or multiple nucleic acids encode a crRNA targeting B2M exon 1 and / or a crRNA targeting B2M exon 2. A vector or a plurality of vectors encoding a nucleic acid or a plurality of nucleic acids as claimed in any one of claims 36 to 39. A method for producing a modified cell having reduced MHC Class I performance compared to a comparable unmodified cell, comprising introducing a modification into a cell for use in any of claims 36 to 39 A nucleic acid of the item or a plurality of nucleic acids or a vector or a plurality of vectors as claimed in the item 40. The method of claim 41, wherein the modified cell is a modified K562 cell.