WO2019097083A1

WO2019097083A1 - Modified k562 cell

Info

Publication number: WO2019097083A1
Application number: PCT/EP2018/081928
Authority: WO
Inventors: Wang Shu; Shijun ZHA; Zhendong Li; Can Chen
Original assignee: Tessa Therapeutics Pte. Ltd.; National University Of Singapore; CLEGG, Richard Ian
Priority date: 2017-11-20
Filing date: 2018-11-20
Publication date: 2019-05-23
Also published as: TW201923073A

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 Cell

Field of the Invention

The present invention relates to the fields of cellular biology, genetic engineering, and methods of medical treatment and prophylaxis.

Background to the Invention

Adoptive cell-based cancer immunotherapy uses autologous or allogeneic immune cells to treat malignancies. Cytotoxic T lymphocytes engineered with antigen-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) have been studied and have shown promising therapeutic effects in a variety of cancers (1-3). Many methods have been investigated 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, co-stimulatory molecules, CD40 ligand, B7-2 (CD86) or 4-1 BB ligand (CD137L), and membrane- bound interleukins have been shown to dramatically promote the proliferation and maturation of cytotoxic T lymphocytes (8-11 ). Feeder cells can also be used as artificial antigen presenting cells (aAPCs) through genetic modification to express molecules which stimulate antigen-specific T cells (12). K562 cells have been used in many studies as aAPCs to facilitate the expansion of cytotoxic T lymphocytes (13-15), and CAR-T cells (16-21 ).

The K562 cell line is a human myelogenous leukemia cell line which was derived from a patient in blastic crisis (22). This cell line is a mixture of colony forming unit-erythroid cells equivalent to erythroblasts with a near-triploid karyotype (22, 23). K562 cells outperform other cell types as feeder cells because they are to culture and propagate in serum-free medium, are amenable to transfection and are readily modifiable to be used as artificial antigen presenting cells. K562 cells also expresses molecules like ICAM (CD54) and LFA-3 (CD58) through which they can interact with T cells (8, 9). After being engineered to express co-stimulatory molecules and tumor-associated antigens, K562 cell-based aAPCs can support both antigen-independent and antigen-dependent T cell expansion, and expansion of specific CAR-T cells.

K562 cells have been widely reported to lack expression of major histocompatibility complex (MHC) molecules (24, 25), which support the use of K562 cells as feeder cells in methods for expanding T cells in vitro. However, a recent study has shown that K562 cells can up-regulate MHC class I molecule expression during co-culture with immune cells, and the authors also detected the presence of alloreactive cytotoxic T cells capable of recognising and killing K562 feeder cells in immune cells populations expanded from HLA class I mismatched donors (26).

Summary of the Invention

The present invention relates to modified K562 cells, methods for producing the same, articles used to the produce the modified K562 cells and uses of the K562 cells. In particular the modified K562 cells are useful for generating/expanding populations of immune cells displaying reduced alloreactivity as compared to populations of immune cells generated/expanded using wildtype K562 cells. This is achieved through inhibition of MHC class I expression by the modified K562 cells. In one aspect the present invention provides a modified K562 cell having reduced expression of MHC class I as compared to a wildtype K562 cell. In some embodiments the modified K562 cell comprises modification to a gene encoding an MHC class I polypeptide relative to a wildtype K562 cell.

In some embodiments the modification reduces or prevents expression of a polypeptide encoded by the gene encoding an MHC class I polypeptide. In some embodiments the gene encoding an MHC class I polypeptide is B2M.

In some embodiments the modified K562 cell comprises modification to increase expression of one or more factors capable of increasing immune cell activation or proliferation. In some embodiments the modified K562 cell comprises nucleic acid encoding the one or more factors capable of increasing immune cell activation/proliferation. In some embodiments the one or more factors capable of increasing immune cell activation/proliferation are selected from: a costimulatory molecule, a cytokine or an antigen. In some embodiments the costimulatory molecule is selected from CD40L, CD86, CD137L, CD80 or CD83. In some embodiments the cytokine is selected from IL-21 , IL-15, membrane-bound IL-21 and membrane-bound IL-15.

In some embodiments the modified K562 cell comprises modification to increase expression of one or more Fc receptors.

In another aspect the present invention provides a modified K562 cell comprising modification to reduce or prevent expression of a polypeptide encoded by B2M.

In some embodiments the modified K562 cell comprises modification to increase expression of one or more of: CD64, CD86, CD137L and membrane-bound IL-21. In some embodiments the modified K562 cell comprises modification to increase expression of an antigen. In some embodiments the modified K562 cell comprises modification to increase expression of CD19.

In another aspect the present invention provides a method for producing a modified K562 cell having reduced expression of MHC class I as compared to a wildtype K562 cell, comprising modifying a K562 cell to reduce or prevent expression of MHC class I.

In some embodiments the modification reduces or prevents expression of a polypeptide encoded by a gene encoding an MHC class I polypeptide. In some embodiments the gene encoding an MHC class I polypeptide is B2M.

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

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

In some embodiments the method comprises modifying the K562 cell to increase expression an antigen.

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

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

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

embodiments the antigen-specific immune cells are CAR-modified immune cells, and wherein the modified K562 cell comprises or expresses the antigen for which the CAR is specific.

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

In another aspect the present invention provides the population of immune cells according to the present invention is provided for use in a method of medical treatment or prophylaxis of a disease or condition.

In another aspect the present invention provides the use of the population of immune cells according to the present invention in the manufacture of a medicament for use in a method of medical treatment or prophylaxis of a disease or condition.

In another aspect the present invention provides a method of treating or preventing a disease or condition in a subject, comprising administering a population of immune cells according to the present invention to a subject.

In another aspect the present invention provides a method of treating or preventing a disease or condition in a subject, comprising:

(a) isolating immune cells from a subject;

(b) generating or expanding a population of immune cells by culturing the immune cells isolated at step (a) in the presence of a modified K562 cell according to the present invention; and (c) administering the population of immune cells generated or expanded at step (b) to a subject.

In some embodiments the disease or condition is a T cell dysfunctional disorder, a cancer or an infectious disease. In some embodiments the cancer is selected from the group consisting of: colon cancer, colon carcinoma, colorectal cancer, nasopharyngeal carcinoma, cervical carcinoma, oropharyngeal carcinoma, gastric carcinoma, 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, urothelial carcinoma, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or mesothelioma.

In another aspect the present invention provides a nucleic acid, or a plurality of nucleic acids, encoding a site-specific nuclease (SSN) system targeting B2M.

In some embodiments the nucleic acid or plurality of nucleic acids encodes a CRISPR/Cas9 system. In some embodiments the nucleic acid or plurality of nucleic acids encodes a CRISPR RNA (crRNA) targeting an exon of B2M. In some embodiments the nucleic acid or plurality of nucleic acids encodes a crRNA targeting exon 1 and/or a crRNA targeting exon 2 of B2M.

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

In another aspect the present invention provides a method for producing a modified cell having reduced expression of MHC class I as compared to a comparable non-modified cell, comprising introducing into a cell modifying the nucleic acid, plurality of nucleic acids, vector or plurality of vectors according to the present invention. In some embodiments the modified cell is a modified K562 cell.

Description

The invention relates to a modified K562 cell useful in methods for generating/expanding populations of immune cells for use e.g. in adoptive cell transfer, in which the generated/expanded population comprises fewer immune cells specific for MHC class I.

K562 cells

The K562 cell line is a human myelogenous leukemia cell line which was derived from pleural effusion of 53 year old female with chronic myelogenous leukaemia in terminal blast crisis, and is described in Klein et al., International Journal of Cancer (1976) 18, 421. K562 cells are available from various commercial and non-commercial sources, e.g. ATCC (American Type Culture Collection) and Sigma (Sigma Catalog No. 89121407). The K562 cell line was obtained by a method which does no not involve destruction of a human embryo.

In some embodiments in accordance with various aspects of the present invention, a K562 cell (also referred to herein as a“wildtype” or“WT” K562 cell, or an“unmodified” K562 cell) is a cell of the cell line deposited under ATCC accession number CCL-243. In some embodiments, a K562 cell is a human myelogenous leukemia cell comprising the following DNA profile: STR-PCR Data: Amelogenin: X;

CSF1 PO: 9,10; D13S317: 8; D16S539: 1 1 ,12; D5S818: 11 ,12; D7S820: 9,1 1 ; TH01 : 9.3; TPOX: 8,9; vWA: 16. In some embodiments, a K562 cell is a human myelogenous leukemia cell comprising the genome of a cell of the cell line deposited under ATCC accession number CCL-243.

Aspects of the present invention relate to modified K562 cells. Herein, a“modified” K562 cell refers to a K562 cell which has been altered in some way, such that the modified K562 cell is different to a wildtype K562 cell. The alteration may change one or more structural and/or functional properties of the K562 cell.

Reduced expression of MHC class I

In some embodiments, a modified K562 cell according to the present invention has reduced expression of MHC class I as compared to a wildtype K562 cell. Expression may refer to gene expression and/or protein expression.

MHC class I molecules are heterodimers of an alpha (a) chain and a beta ( )2-microglobulin (B2M). The a-chain has three domains designated cd , a2 and a3. The a1 and a2 domains together form the groove to which the peptide presented by the MHC class I molecule binds, to form the peptide-MHC complex. MHC class I a-chains are polymorphic, and different a-chains are capable of binding and presenting different peptides. In humans MHC class I a-chains are encoded by human leukocyte antigen (HLA) genes. B2M is required for the cell surface expression of MHC class I molecules; its deficiency can disrupt the functional structure of MHC class I complex and reduce surface expression of MHC class I molecules (27, 28). Since the B2M gene in human genome is extremely conservative, disruption of B2M can generate hypoimmunogenic cells which are devoid of MHC class I molecule expression.

Reduced expression of MHC class I may refer to reduced expression of one or more polypeptides of an MHC class I molecule. In some embodiments the modified K562 cell has reduced expression of b 2 microglobulin (B2M) polypeptide as compared to a wildtype K562 cell. In some embodiments the modified K562 cell has reduced expression of an MHC class I a chain polypeptide as compared to a wildtype K562 cell. In some embodiments the modified K562 cell has reduced expression of MHC class I complex as compared to a wildtype K562 cell.

Expression of a polypeptide or a polypeptide complex by a cell can be determined by analysis according to a variety of methods which are well known to the skilled person. Such methods include analysis using an antigen-binding molecule (e.g. an antibody or aptamer) specific for the polypeptide/polypeptide complex of interest, e.g. western blot, immunohistochemistry, immunocytochemistry, flow cytometry or ELISA.

In some embodiments the modified K562 cell has reduced surface expression of one or more

polypeptides of MHC class I complex as compared to a wildtype K562 cell, e.g. reduced surface expression of B2M, an MHC class I a chain polypeptide and/or a MHC class I complex. Surface expression refers to expression of the relevant polypeptide/polypeptide complex which is detectable at the cell surface (i.e. in or at the cell membrane). Surface expression of a given polypeptide or polypeptide complex can be analysed e.g. on intact cells using an antigen-binding molecule specific for a region of the polypeptide/polypeptide complex which is extracellular to the cell when the polypeptide/polypeptide complex is expressed at the cell surface.

In some embodiments the modified K562 cell has reduced gene expression of one or more polypeptides of an MHC class I molecule as compared to a wildtype K562 cell. Gene expression by a cell can be determined e.g. quantifying of mRNA encoding the polypeptide/polypeptides, for example by quantitative real-time PCR (qRT-PCR), or by reporter-based methods. For example, reduced gene expression of one or more polypeptides of an MHC class I molecule may be determined by detection of a reduced level of nucleic acid encoding B2M polypeptide (e.g. wildtype B2M polypeptide), and/or a reduced level of nucleic acid encoding an MHC class I a chain polypeptide.

As used herein“an MHC class I polypeptide” refers to a constituent polypeptide of an MHC class I molecule (i.e. a polypeptide complex of an MHC class I a chain polypeptide and a B2M polypeptide).

In some embodiments expression of MHC class I by the cell is analysed under certain environmental conditions, e.g. in response to treatment of the cell with an agent capable of upregulating gene or protein expression of MHC class I. In some embodiments the modified K562 cell of the present invention displays reduced gene or protein expression of one or more polypeptides of an MHC class I molecule (e.g. B2M or an MHC class I a chain) following stimulation with IFNy as compared to the level of gene/protein expression by a wildtype K562 cell in response to similar stimulation. In some embodiments the modified K562 cell displays reduced gene or protein expression of one or more polypeptides of an MHC class I molecule (e.g. B2M or an MHC class I a chain) following stimulation with cell culture supernatant of a coculture of K562 cells with immune cells (e.g. PBMCs or T cells) as compared to the level of gene/protein expression by a wildtype K562 cell in response to similar stimulation.

A“reduced” level of gene or protein expression of a given factor relative to the level of expression by a wildtype K562 cell may be determined by measuring the level of expression of the factor in the modified K562 cell and measuring the level of expression of the factor in a wildtype K562 cell, and by comparing the values to determine whether the level of expression is reduced in the modified K562 cell relative to the level of expression in the wildtype K562 cell. In some embodiments the reduced level of expression may be a level of expression which is less than 1 times, e.g. 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 expression by a wildtype K562 cell under the same conditions.

In some embodiments the modified K562 cell according to the present invention has substantially no gene/protein expression of MHC class I, B2M or MHC class I a chain. In some embodiments the modified K562 cell has an undetectable level of gene/protein expression of MHC class I, B2M or MHC class I a chain (e.g. as determined by a standard method for detecting gene and/or protein expression).

In some embodiments the modified K562 cell displays substantially no surface expression of MHC class I, e.g. as determined by analysis by flow cytometry using an antibody capable of binding to MHC class I. In some embodiments the modified K562 cell displays substantially no surface expression of B2M, e.g. as determined by analysis by flow cytometry using an antibody capable of binding to B2M. In such assays, the level of staining of the modified K562 cells by the relevant antibody may not be significantly different from the level of staining of the cells by an appropriate negative control antibody of the same isotype.

In some embodiments, the modified K562 cell may be referred to as a MHC class l-negative or MHC class l-knockout K562 cell. In some embodiments, the modified K562 cell may be referred to as a B2M- negative or B2M-knockout K562 cell.

The modified K562 cell of the present invention may have reduced expression of MHC class I e.g. as a consequence of treatment with an agent for reducing gene and/or protein expression of one or more polypeptides of an MHC class I molecule (e.g. B2M polypeptide or an MHC class I a chain polypeptide). An agent capable of preventing or reducing of the expression of one or more polypeptides of an MHC class I molecule may do so e.g. through inhibiting transcription of a gene encoding B2M polypeptide or an MHC class I a chain polypeptide, inhibiting post-transcriptional processing of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, reducing the stability of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, inhibiting translation of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, promoting degradation of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, inhibiting post-translational processing of B2M polypeptide or an MHC class I a chain polypeptide, inhibiting association of B2M polypeptide and MHC class I a chain polypeptide, inhibiting formation of an MHC class I polypeptide complex, reducing the stability of B2M polypeptide, an MHC class I a chain polypeptide or an MHC class I polypeptide complex, or promoting degradation of B2M polypeptide, an MHC class I a chain polypeptide or an MHC class I polypeptide complex. In some embodiments the agent may inhibit gene or protein expression of MHC class I through RNA interference (RNAi). In some embodiments the agent may be, or may encode, shRNA or siRNA targeting nucleic acid encoding B2M or an MHC class I a chain.

In some preferred embodiments the modified K562 cell of the present invention comprises a modification to nucleic acid encoding an MHC class I polypeptide. The modification causes the cell to have a reduced level of gene and/or protein expression of one or more polypeptides of an MHC class I molecule (e.g.

B2M or an MHC class I a chain) as compared to a wildtype K562 cell.

In some embodiments the modified K562 cell comprises a modification to a gene encoding an MHC class I polypeptide. In some embodiments the modified K562 cell comprises a modification to a gene encoding B2M polypeptide relative to a wildtype K562 cell. In some embodiments the modified K562 cell comprises a modification to a gene encoding an MHC class I a chain relative to a wildtype K562 cell. 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 the protein-coding sequence thereof is shown in SEQ ID NO:3 .

In some embodiments the modified K562 cell comprises a non-wildtype B2M allele. That is, in some embodiments the modified K562 cell comprises a B2M allele which comprises a modification relative to a B2M allele possessed by a wildtype K562 cell.

The K562 cell genome contains three alleles of the B2M gene (45). For this reason, it is challenging to produce a B2M knockout K562 cell, as the K562 cell must be modified in such a way that all three copies of B2M are disrupted.

In some embodiments the modified K562 cell comprises more than one non-wildtype B2M allele. In some embodiments the modified K562 cell comprises a modification to each B2M allele. In some embodiments the modified K562 cell lacks a B2M allele possessed by a wildtype K562 cell.

Human B2M polypeptide is translated as a 119 amino acid polypeptide having the amino acid sequence shown in SEQ ID NO:4 (UniProt: P61769-1 , v1 ). After processing to remove the 20 amino acid signal peptide, mature B2M has the amino acid sequence shown in SEQ ID NO:5.

In some embodiments the modification to a B2M allele comprises an insertion, substitution or deletion in the nucleic acid sequence encoding B2M polypeptide. In some embodiments the modification reduces or prevents the expression of a polypeptide according to SEQ ID NO:4 or SEQ ID NO:5 from the modified nucleic acid sequence. In some embodiments the modified K562 cell comprises 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 cell lacks 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 the sequence transcribed from the non-wildtype B2M allele. In some embodiments the non-wildtype B2M allele encodes a truncated and/or non-functional B2M polypeptide. In some embodiments the non-wildtype B2M allele encodes a B2M polypeptide which is misfolded and/or degraded. In some embodiments the non-wildtype B2M allele encodes a B2M polypeptide which is incapable of participating in a functional MHC class I polypeptide complex. In some embodiments the non-wildtype B2M allele encodes a B2M polypeptide which is incapable of associating with an MHC class I a chain.

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

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

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

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

The skilled person is able to determine whether a given cell comprises a wildtype B2M allele or a non- wildtype B2M allele, and is also able to determine the nucleotide sequence(s) of B2M allele(s), by methods well known to the skilled person, including sequencing by the classic chain termination method, or by next generation sequencing (NGS), reviewed e.g. by Metzker, M.L., Nat Rev Genet 2010 Jan;11 (1 ): 31-46 (hereby incorporated by reference).

Increased expression of factors capable of increasing immune cell activation/proliferation and/or Fc receptors

In some embodiments, the modified K562 cell according to the present invention has increased expression of one or more factors capable of increasing immune cell activation/proliferation as compared to a wildtype K562 cell. In some embodiments, the modified K562 cell according to the present invention has increased expression of one or more Fc receptors as compared to a wildtype K562 cell.

Expression may be gene and/or protein expression. Gene expression can be measured by various means known to those skilled in the art, e.g. by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or by reporter-based methods. Protein expression can be measured by various methods well known in the art, e.g. by antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

An increased level of gene or protein expression of a given factor relative to the level of expression by a wildtype K562 cell may be determined by measuring the level of expression of the factor in the modified K562 cell and measuring the level of expression of the factor in a wildtype K562 cell, and by comparing the values to determine whether the level of expression is increased in the modified K562 cell relative to the level of expression in the wildtype K562 cell. In some embodiments the increased level of expression may be a level of expression which is more than 1 times, e.g. more 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 the level of expression by a wildtype K562 cell under the same conditions.

A factor capable of increasing immune cell activation can be identified by contacting an immune cell or a population of immune cells with the factor, and subsequently analysing the immune cell for one or more markers of activation of the immune cell. A factor capable of increasing immune cell activation may be identified by the detection of an increase in the level of expression of one or more markers of activation of the immune cell and/or an increase in the proportion of cells expressing one or more markers of activation of the immune cell following treatment with the relevant factor (as compared to an appropriate control condition). Markers of T cell activation include e.g. CD69 and CD45R0. Markers of NK cell activation include e.g. 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 analysing proliferation by the immune cell(s). A factor capable of increasing immune cell proliferation may be identified by the detection of an increase in the level of proliferation by the immune cell(s) following treatment with the relevant factor (as compared to an appropriate control condition). The level of cell proliferation can be determined by analysing cell division over a period of time. Cell division can be analysed, for example, by in vitro analysis of incorporation of 3H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.

In some embodiments the factor capable of increasing immune cell activation/proliferation is selected from a costimulatory molecule, a cytokine or an antigen.

K562 cells are commonly engineered to increase expression of costimulatory molecules; see e.g. 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 hereby incorporated by reference in their entirety. K562 cells having increased expression of costimulatory molecules promote activation and proliferation of immune cells expressing the ligands for the costimulatory molecules.

In some embodiments the modified K562 cell according to the present invention has increased expression of one or more of the following costimulatory molecules as compared to a wildtype K562 cell: CD70, CD40, LFA3, ICAM1 , CD80, CD86, CD137L, OX40L, ICOSL, LIGHT, LTb and GITRL. In some embodiments the modified K562 cell has increased expression of one or more of the following costimulatory molecules as compared to a wildtype K562 cell: CD40L, CD70, CD80, CD83, CD86,

ICOSL, GITRL, CD137L and OX40L. In some embodiments the modified K562 cell has increased expression of one or more of the following costimulatory molecules as compared to a wildtype K562 cell: CD40L, CD86 and CD137L.

K562 cells have also been engineered to increase expression of other molecules capable of stimulating immune cell activation/proliferation , such as cytokines. For example. Wang et al., Clin Exp Immunol. (2013) 172(1 ): 104-12 (hereby incorporated by reference in its entirety) describes the use of K562 cells engineered to express membrane-bound IL-21 (mblL-21 ) on the cell surface (also modified for cell surface expression of CD137L) to expand NK cells from within a population of PBMCs, and Denman et al., PLoS One (2012) 7(1 ):e30264 describes the use of K562 cells engineered to express membrane- bound membrane bound IL-15 (mblL-15) to expand NK cells.

In some embodiments the modified K562 cell according to the present invention has increased expression of one or more of the following molecules as compared to a wildtype K562 cell: IL-21 , membrane-bound IL-21 , IL-15 and membrane-bound IL-15. In some embodiments the modified K562 cell has increased expression of membrane-bound IL-21 and/or membrane-bound IL-15 as compared to a wildtype K562 cell.

In some embodiments the modified K562 cell according to the present invention has increased expression of one or more antigens as compared to a wildtype K562 cell. As used herein, an“antigen” refers to a molecule capable of activating an immune cell expressing a receptor specific for the antigen.

In some embodiments the antigen is a cancer cell antigen. A cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell. A cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell. In some embodiments, the antigen is expressed at the cell surface of the cancer cell (i.e. the cancer cell antigen is a cancer cell surface antigen). In some embodiments the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer. The cancer- associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g. as compared to the level of expression of by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type). In some embodiments, the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein. In some embodiments the antigen is CD19. Disease association of CD19 is reviewed e.g. in Wang et al., Exp Hematol Oncol. (2012) 1 :36. CD19 expression is highly conserved on most B cell tumors, and is expressed in most acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL) and B cell lymphomas (Cooper et al. Blood Cells Mol Dis. (2004) 33(1 ):83-9). The majority of B cell malignancies express CD19 at normal to high levels (80% of ALL, 88% of B cell lymphomas and 100% of B cell leukemias). CD19 has also been observed in cases of myeloid malignancies, including in 2% of AML cases.

In some embodiments the modified K562 cell has increased expression of one or more Fc receptors as compared to a wildtype K562 cell. Feeder cells and artificial antigen presenting cells are often engineered for increased expression of Fc receptors such as CD64, CD32 and CD16 for improved presentation of e.g. agonist anti-CD3 antibody (e.g. clone OKT3) and/or agonist anti-CD28 antibody for the activation of CD3-expressing immune cells - see e.g. Turtle and Riddell Cancer J. (2010) 16(4): 374-381 , incorporated by reference herein. Cells expressing increased levels of Fc receptors are particularly useful in methods for generating/expanding populations of immune cells employing agonist anti-CD3 and/or ant- CD28 antibody for antigen-independent T cell activation.

Accordingly in some embodiments the modified K562 cell according to the present invention has increased expression of one or more Fc receptors as compared to a wildtype K562 cell. In some embodiments the Fc receptor is a receptor for Fc gamma. In some embodiments the Fc receptor is selected from CD64, CD32 and CD 16.

In some embodiments the modified K562 cell according to the present invention has increased expression of one or more of the following factors as compared to a wildtype K562 cell: CD19, CD40L, CD86, CD137L, mblL-21 and CD64.

The modified K562 cell according to the present invention may have increased expression of one or more factors capable of increasing immune cell activation/proliferation and/or increased expression of one or more Fc receptors as compared to a wildtype K562 cell as a consequence of having been engineered to have increased expression of the one or more factors.

In some embodiments the modified K562 cell comprises exogenous nucleic acid encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors. As used herein,“exogenous” nucleic acid refers to nucleic acid which is non-endogenous to a wildtype K562 cell; i.e. nucleic acid which is not comprised in the genome of a wildtype K562 cell.

In some embodiments a the modified K562 cell comprises exogenous nucleic acid encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors as a consequence of having had nucleic acid introduced into the cell or a precursor thereof, e.g. by transfection, electroporation or transduction. In some embodiments the modified K562 cell stably expresses the exogenous nucleic acid encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors. In some embodiments the exogenous nucleic acid may be integrated into the genome of the modified K562 cell.

In some embodiments the exogenous nucleic acid may be integrated into the genome of the modified K562 cell at a particular locus. In some embodiments, the exogenous nucleic acid may be integrated into the genome of the modified K562 cell at a genomic safe harbour (GSH). A GSH is a site which supports stable integration and expression of exogenous nucleic acid while minimising the risk of unwanted interactions with the host cell genome (see e.g. Sadelain et al., Nat Rev Cancer. (201 1 ) 12(1 ):51 -8).

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

In some embodiments the modified K562 cells according to the present invention may have been treated to inhibit/prevent cell proliferation (i.e. the cells may be“inactivated”). The inactivated cells may lack the capacity to undergo cell division (and may therefore lack the capacity to proliferate). In some embodiments the K562 cells may have been inactivated by treatment with mitomycin C or cyclosporin A, or exposure to ionising radiation (e.g. gamma irradiation, X-rays or UV light). Suitable conditions for such treatment/exposure are known to the skilled person, and can be determined e.g. by reference to Roy et al., J Hematother Stem Cell Res (2001 ) 10(6):873-80.

In some embodiments the modified K562 cell or a population of modified K562 cells according to the present invention is provided in isolated form or substantially purified form. The modified K562 cells may be isolated/purified e.g. from one or more other cell types, e.g. wildtype K562 cells.

In particular embodiments, the modified K562 cell of the present invention may comprise one of the following phenotypes (e.g. as determined by flow cytometry using antibodies specific for the relevant factor(s)): MHC class l-negative/B2M-negative; MHC class l-negative/B2M-negative, CD40L-positive, CD86-positive, CD137L-positive and mblL-2-positive; MHC class l-negative/B2M-negative, CD40L- positive, CD86-positive, CD137L-positive, mblL-2-positive and CD64-positive; MHC class I- negative/B2M-negative, CD40L-positive, CD86-positive, CD137L-positive, mblL-2-positive and CD19- positive; MHC class l-negative/B2M-negative, CD40L-positive, CD86-positive, CD137L-positive, mblL-2- positive, CD64-positive and CD 19-positive.

Functional properties of the modified K562 cells

The modified K562 cells of the present invention may be characterised by reference to one or more functional properties. In some embodiments modified K562 cells according to the present invention possess one of more of the following properties:

Reduced surface expression of B2M and/or MHC class I as compared to wildtype K562 cells following stimulation with an agent for upregulating surface expression of B2M/MHC class I by wildtype K562 cells;

Increased lysis by NK cells as compared to wildtype K562 cells;

Expand T cells displaying reduced cytotoxicity to cells expressing HLA-C*03 or HLA-C*05 as compared to T cells expanded using wildtype K562 cells;

Expand fewer T cells specific for cells expressing HLA-C*03 or HLA-C*05 as compared to T cells expanded using wildtype K562 cells;

In some embodiments the modified K562 cells have reduced surface expression of B2M and/or MHC class I as compared to wildtype K562 cells following stimulation with an agent for upregulating surface expression of B2M/MHC class I by wildtype K562 cells. In some embodiments the agent is IFNy. In some embodiments the agent is a co-culture supernatant of T cells and wildtype K562 cells. Surface expression of B2M/MHC class I may be determined e.g. in vitro by flow cytometry using an antibody specific for B2M/MHC Class I. In some embodiments the modified K562 cells can be analysed for surface expression of B2M and/or MHC class I as compared to wildtype K562 cells following stimulation with an agent for upregulating surface expression of B2M/MHC class I as described in the Examples of the present disclosure. In some embodiments the modified K562 cells display less than 1 times, e.g. 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 surface expression of B2M and/or MHC class I as compared to wildtype K562 cells following stimulation with an agent for upregulating surface expression of B2M/MHC class I by wildtype K562 cells, in a comparable assay.

In some embodiments the modified K562 cells are more susceptible to lysis by NK cells as compared to wildtype K562 cells. Susceptibility to lysis by NK cells can be determined e.g. in vitro using an assay of NK cell cytotoxicity. Cytotoxicity assays are known to the skilled person are reviewed e.g. in Zaritskaya et al., Expert Rev Vaccines (201 1 ), 9(6):601-616, hereby incorporated by reference in its entirety. In some embodiments lysis by NK cells is analysed using the DELFIA EuTDA cytotoxicity assay as described in the Examples of the present disclosure. In some embodiments the level of cytotoxicity (e.g. determined as the level of cell lysis) displayed by NK cells to the modified K562 cells is more than 1 times, e.g. more 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 or more than 10 times the level of cytotoxicity (e.g. determined as the percentage of cell lysis) displayed by NK cells to wildtype K562 cells, in a comparable assay. It will be appreciated that the modified K562 cells of the present invention are useful for

generating/expanding populations of immune cells having reduced alloreactivity as compared to populations of immune cells generated/expanded using wildtype K562 cells. In particular, the modified K562 cells of the present invention are useful for generating/expanding populations of immune cells having a reduced number of cells specific for cells expressing HLA-C*03 or HLA-C*05.

In some embodiments the modified K562 cells expand T cells displaying reduced cytotoxicity to cells expressing HLA-C*03 or HLA-C*05 as compared to T cells expanded using wildtype K562 cells. The cytotoxicity of expanded T cell population to cells expressing HLA-C*03 or HLA-C*05 can be analysed e.g. in vitro using an assay of cytotoxicity, e.g. an assay described in Zaritskaya et al., Expert Rev Vaccines (201 1 ), 9(6):601-616. In some embodiments the cytotoxicity of T cells to cells expressing HLA- C*03 or HLA-C*05 is analysed using the DELFIA EuTDA cytotoxicity assay as described in the Examples of the present disclosure. In some embodiments T cells expanded by culture in the presence of the modified K562 cells display a level of cytotoxicity to cells expressing HLA-C*03 or HLA-C*05 which is less than 1 times, e.g. 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 displayed by T cells expanded by culture in the presence of wildtype K562 cells, in a comparable assay.

In some embodiments the modified K562 cells expand fewer T cells specific for (i.e. reactive to, e.g. displaying cytotoxicity towards) cells expressing HLA-C*03 or HLA-C*05 as compared to T cells expanded using wildtype K562 cells. In some embodiments the modified K562 cells expand fewer than 1 times, e.g. fewer 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 fewer than 0.1 times the number of T cells specific for cells expressing HLA-C*03 or HLA-C*05 expanded by wildtype K562 cells, in a comparable assay.

Methods for producing modified K562 cells

The present invention also provides methods for producing a modified K562 cell according to the invention.

In some embodiments the method comprises modifying a K562 cell (e.g. a wildtype K562 cell) to reduce or prevent expression of MHC class I. In some embodiments the method comprises modifying a K562 cell to reduce or prevent expression of MHC class I at the cell surface. In some embodiments the method comprises modifying a K562 cell to reduce or prevent expression of B2M and/or an MHC class I a chain polypeptide.

In some embodiments the method comprises modifying a K562 cell to reduce or prevent expression of a polypeptide encoded by a gene encoding an MHC class I polypeptide. In some embodiments the method comprises modifying a K562 cell to reduce or prevent expression of a polypeptide encoded by B2M. In some embodiments the method comprises modifying a K562 cell to reduce or prevent expression of a polypeptide encoded by a gene encoding an MHC class I a chain polypeptide (e.g. an HLA gene).

In some embodiments the modification comprises treating a K562 cell with an agent capable of reducing gene and/or protein expression of one or more polypeptides of an MHC class I molecule (e.g. B2M polypeptide or an MHC class I a chain polypeptide). In some embodiments the agent may be capable of inhibiting transcription of a gene encoding B2M polypeptide or an MHC class I a chain polypeptide, inhibiting post-transcriptional processing of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, reducing the stability of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, inhibiting translation of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, promoting degradation of RNA encoding B2M polypeptide or an MHC class I a chain polypeptide, inhibiting post-translational processing of B2M polypeptide or an MHC class I a chain polypeptide, inhibiting association of B2M polypeptide and MHC class I a chain polypeptide, inhibiting formation of an MHC class I polypeptide complex, reducing the stability of B2M polypeptide, an MHC class I a chain polypeptide or an MHC class I polypeptide complex, or promoting degradation of B2M polypeptide, an MHC class I a chain polypeptide or an MHC class I polypeptide complex. In some embodiments the agent may inhibit gene or protein expression of MHC class I through RNA interference (RNAi). In some embodiments the agent may be, or may encode, shRNA or siRNA targeting nucleic acid encoding B2M or an MHC class I a chain.

In some embodiments the method comprises modifying a nucleic acid encoding an MHC class I polypeptide. The modification causes the cell to have a reduced level of gene and/or protein expression of one or more polypeptides of an MHC class I molecule (e.g. B2M polypeptide or an MHC class I a chain polypeptide) as compared to a wildtype K562 cell.

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

In some embodiments the method comprises modifying one or more alleles of the 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 B2M polypeptide.

In some embodiments the method comprises introducing a modification which reduces or prevents the expression of a polypeptide according to SEQ ID NO:4 or SEQ ID NO:5 from the modified nucleic acid sequence. In some embodiments the method comprises modifying the K562 cell to 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 method comprises modifying the K562 cell to lack nucleic acid encoding a polypeptide according to SEQ ID NO:4 or SEQ ID NO:5. In some embodiments the method comprise modifying a B2M allele to introduce a premature stop codon in the sequence transcribed from the B2M allele. In some embodiments the method comprise 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 B2M polypeptide which is misfolded and/or degraded. In some embodiments the method comprises modifying a B2M allele to encode a B2M polypeptide which is incapable of participating in a functional MHC class I polypeptide complex. In some the method comprises modifying a B2M allele to encode a B2M polypeptide which is incapable of associating with an MHC class I a chain.

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

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

In some embodiments the method comprises inserting a nucleotide (e.g. thymidine (T)) between positions corresponding to 70 and 71 of SEQ ID NO:1. In some embodiments method deleting positions corresponding to 51 to 69 of SEQ ID NO:1.

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

Modification of a nucleic acid encoding an MHC class I polypeptide (e.g. B2M polypeptide or an MHC class I a chain polypeptide) in accordance with the methods of the present invention can be achieved in a variety of ways known to the skilled person, including modification of the target nucleic acid by homologous recombination, and target nucleic acid editing using site-specific nucleases (SSNs).

Researches have previously achieved disruption of B2M using adeno-associated virus (AAV) vector- mediated traditional gene targeting, or using Transcription activator-like effector nucleases (TALENs) on human embryonic stem cells (29, 30).

In some embodiments the methods employ targeting by homologous recombination, which is reviewed, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4:Unit 4.29 and Vasquez et al., PNAS 2001 , 98(15): 8403-8410 both of which are hereby incorporated by reference in their entirety. Targeting by homologous recombination involves the exchange of nucleic acid sequence through crossover events guided by homologous sequences.

In some embodiments the methods employ target nucleic acid editing using SSNs. Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequence(s) of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively DSBs may be repaired by highly homology-directed repair (HDR), in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB.

SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) systems.

ZFN systems are reviewed e.g. in Umov et al., Nat Rev Genet. (2010) 11(9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zince Finger array capable of binding to the target nucleic acid sequence.

TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA-cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: “HD” binds to C,“Nl” 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 e.g. CRISPR/Cpf1 , CRISPR/C2c1 , CRISPR/C2c2 and CRISPR/C2c3 are reviewed e.g. in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the singleguide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

In some particular embodiments modifying a nucleic acid encoding an MHC class I polypeptide (e.g. B2M polypeptide or an MHC class I a chain polypeptide) in accordance with the methods of the present invention comprises modification using a CRISPR/Cas9 system. In some embodiments the methods comprise introducing into a K562 cell a nucleic acid or plurality of nucleic acids encoding: a CRISPR RNA (crRNA) targeting a nucleic acid encoding an MHC class I polypeptide (e.g. a nucleic acid encoding B2M polypeptide or an MHC class I a chain polypeptide), and a Cas9 endonuclease. In some embodiments the crRNA targets a nucleic acid encoding B2M polypeptide. In some embodiments the crRNA targets B2M. In some embodiments the crRNA targets an exon of B2M. In some embodiments the crRNA targets exon 1 of B2M. In some embodiments the crRNA targets exon 2 of B2M. The nucleic acid or plurality of nucleic acids may be comprised in one or more vectors.

Nucleic acid(s)/vector(s) may be introduced into a K562 cell by any suitable means, e.g. by

transformation, transfection, electroporation or transduction. In some embodiments, the methods comprise introducing nucleic acid(s)/vector(s) into a K562 cell by electroporation, e.g. as described in Delgado-Cahedo et al., Cytotechnology. (2006) 51(3):141-8 (hereby incorporated by reference in its entirety), or as described in Example 1 of the present disclosure. In some embodiments, the methods comprise introducing nucleic acid(s)/vector(s) into a K562 cell by transduction, e.g. as described in Example 1 of the present disclosure.

In some embodiments the methods of the present invention comprise modifying a K562 cell to increase expression of one or more factors capable of increasing immune cell activation/proliferation (e.g. a costimulatory molecule, a cytokine or an antigen). In some embodiments, the methods comprise modifying a K562 cell to increase expression of one or more Fc receptors.

In some embodiments the methods of the present invention comprise modifying a K562 cell to increase expression 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 a K562 cell to increase expression of one or more of: CD40L, CD70, CD80, CD83, CD86, ICOSL, GITRL, CD137L and OX40L.

In some embodiments the methods comprise modifying a K562 cell to increase expression of one or more of: CD40L, CD86 and CD137L. In some embodiments the methods comprise modifying a K562 cell to increase expression of CD86 and/or CD137L.

In some embodiments the methods of the present invention comprise modifying a K562 cell to increase expression 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 a K562 cell to increase expression of membrane-bound IL-21 and/or membrane-bound IL-15.

In some embodiments the methods of the present invention comprise modifying a K562 cell to increase expression of one or more antigens. In some embodiments the methods comprise modifying a K562 cell to increase expression of an immune cell of interest comprises a specific receptor (e.g. a TCR or CAR). In some embodiments the methods comprise modifying a K562 cell to increase expression of a cancer cell antigen, e.g. a cancer cell antigen as described herein. In some embodiments the methods comprise modifying a K562 cell to increase expression of CD 19. In some embodiments the methods of the present invention comprise modifying a K562 cell to increase expression of one or more Fc receptors. In some embodiments the methods comprise modifying a K562 cell to increase expression of a receptor for Fc gamma. In some embodiments the methods comprise modifying a K562 cell to increase expression of an Fc receptor selected from CD64, CD32 and CD16.

In some embodiments the methods of the present invention comprise modifying a K562 cell to increase expression of one or more of: CD19, CD40L, CD86, CD137L, mblL-21 and CD64.

In some embodiments modifying a K562 cell to increase expression 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 encoding the one or more factors capable of increasing immune cell

activation/proliferation and/or one or more Fc receptors into the K562 cell.

In some embodiments modifying a K562 cell to increase expression 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 the relevant proteins into the cell. Nucleic acid(s)/vector(s) may be introduced into a K562 cell by any suitable means, e.g. by transformation, transfection, electroporation or transduction.

In some embodiments the one or more nucleic acids encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors are integrated into the genome of the K562 cell. In some embodiments the one or more nucleic acids encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors are integrated into the genome of the K562 cell at a genomic safe harbour (GSH), e.g. a GSH described herein. In some embodiments the GSH is AAVS1. In some embodiments method comprises introducing nucleic acid(s) encoding one or more SSNs targeting AAVS1 into a K562 cell. In some embodiments method comprises introducing nucleic acid(s) encoding a ZFN targeting AAVS1 into a K562 cell.

In some embodiments, the one or more nucleic acid(s)/vector(s) encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors additionally comprises sequence(s) homologous to sequence(s) of AAVS1 for site-specific integration of the nucleic acid(s) encoding the one or more factors capable of increasing immune cell activation/proliferation and/or one or more Fc receptors, e.g. following cleavage of AAVS1 with an SSN targeting AAVS1 (e.g. a ZFN targeting AAVS1).

Modified K562 cells comprising the desired modification(s) may be cultured/expanded, e.g. from a single cell clone. Such methods may comprise culture in the presence of a selection agent corresponding to a selectable marker for successful introduction of a nucleic of interest into a cell.

In some embodiments the methods comprise treating the modified K562 cell to inhibit/prevent cell proliferation (i.e.“inactivating” the modified K562 cell). In some embodiments the methods comprise treating the cells with mitomycin C or cyclosporin A, or exposing the cells to ionising radiation (e.g.

gamma irradiation, X-rays or UV light).

In some embodiments the methods comprise isolating/separating the modified K562 cell or population of modified K562 cells, e.g. from one or more other cell types, e.g. wildtype K562 cells.

Nucleic acids/vectors

The present invention also provides a nucleic acid, or a plurality of nucleic acids, for producing a modified K562 cell according to the present invention. The nucleic acid(s) encoding a site-specific nuclease (SSN) system targeting B2M. In some embodiments the SSN system is a ZFN system, a TALEN system, 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(s) encode a CRISPR/Cas9 system. In some embodiments the nucleic acid(s) encode a CRISPR RNA (crRNA) targeting an exon of B2M. In some embodiments the nucleic acid(s) encode a crRNA targeting an exon 1 of B2M. In some embodiments the nucleic acid(s) encode a crRNA targeting an exon 2 of B2M. In some embodiments the nucleic acid(s) encode crRNAs targeting exon 1 and exon 2 of B2M. The CRISPR/Cas9 system also comprises a trans-activating crRNA (tracrRNA) for processing the crRNA to its mature form. Accordingly, in some embodiments the nucleic acid(s) encode a tracrRNA for the crRNA.

In some embodiments the crRNA targeting exon 1 of B2M comprises or consists of the nucleic acid of SEQ ID NO:26. In some embodiments the crRNA targeting exon 2 of B2M 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 plasmid 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 plasmid pX260 (Addgene #42229; pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS- H1-shorttracr-PGK-puro).

The nucleic acid(s) may be provided in one or more vectors. The present invention also provides a vector or a plurality of vectors comprising a nucleic acid or plurality of nucleic acids according to the present invention. A“vector” as used herein is nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell. The vector may be a vector for expression of the nucleic acid in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. A vector may also include a termination codon and expression enhancers.

In some embodiments the nucleic acid(s) encoding the crRNA(s) are operably linked to the ubiquitin 6 (U6) promoter. In some embodiments the nucleic acid(s) encoding Cas9 are operably linked to the Chicken b-Actin Promoter (CBh) promoter. In some embodiments the nucleic acid(s) encoding tracRNA are operably linked to H1 promoter.

Any suitable vectors may be used, including e.g. the pX260 plasmid containing a CRISPR/Cas9 system described in Cong et al., Science 339, 819 and pFastBac plasmids described in Zeng et al., Stem Cells (2007) 25, 1055. Baculoviral vectors derived from the insect Autographa californica multiple

nucleopolyhedrovirus (AcMNPV) have previously been used to transduce human pluripotent stem cells and other cell lines with high efficiency (37-40). This vector is reported to be able to carry large and multiple DNA cassettes, with low cytotoxicity and is very safe as it is non-integrating.

Other suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 or Morgan and Boyerinas, Biomedicines 2016 4, 9, which are both hereby incorporated by reference in its entirety.

Methods using the modified K562 cells

The modified K562 cells of the present invention are useful in methods for generating/expanding populations of immune cells. In particular, the modified K562 cells are useful for generating/expanding populations of immune cells having reduced alloreactivity as compared to populations of immune cells generated/expanded using wildtype K562 cells. In some embodiments the modified K562 cells are useful for generating/expanding populations of immune cells having reduced reactivity for a HLA molecule expressed by a wildtype K562 cell (e.g. HLA-C*03 and/or HLA-C*05).

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

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

Culture of cells in accordance with the methods of the invention is performed using suitable cell culture medium and under suitable environmental conditions (e.g. temperature, pH, humidity, atmospheric conditions, agitation etc.) for the in vitro culture of immune cells, which are well known to the person skilled in the art of cell culture.

Conveniently, cultures of cells may be maintained at 37°C in a humidified atmosphere containing 5%

CO2. Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc. The cell cultures can be established and/or maintained at any suitable density, as can readily be determined by the skilled person. For example, cultures may be established at an initial density of ~0.5 x 10^® to -5 x 10^® cells/ml of the culture (e.g. -1 x 10^® cells/ml). Cells may be cultured in any suitable cell culture vessel. In some embodiments cells are cultured in a bioreactor. In some embodiments, cells are cultured in a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, which is hereby incorporated by reference in its entirety. In some embodiments cells are cultured in a GRex cell culture vessel, e.g. a GRex flask or a GRex 100 bioreactor.

Suitable conditions for the coculture of the modified K562 cells with immune cells can be determined with reference to Example 1 of the present disclosure. Suitable conditions for the use of the modified K562 cells in methods for generating/expanding populations of immune cells (e.g. periods of cell culture, ratios of the modified K562 cells to immune cells cell, culture medium etc.) can also be readily determined by the skilled person, e.g. with reference to the present Examples.

In some embodiments the modified K562 cells are employed as feeder cells to support the growth and/or survival of cells of the population of immune cell in culture, e.g. the immune cells being

generated/expanded. In some embodiments the modified K562 cells are employed as factors for increasing activation/proliferation of the population of immune cells being generated/expanded.

In some embodiments the population of immune cells generated/expanded according to the present invention is generated/expanded from within a population of immune cells, e.g. a population of peripheral blood mononuclear cells (PBMCs) or Peripheral Blood Lymphocytes (PBLs). The immune cells to be generated/expanded may be present within the starting population of immune cells (e.g. PBMCs or PBLs) at low frequency, and culture of the starting population of immune cells in accordance with the invention preferably causes an increase the number of the immune cells to be generated/expanded, and/or results in an increased proportion of such cells in the cell population at the end of the culture.

By way of example, where the method is for generating/expanding a population of T cells, the population of T cells may be generated/expanded from within a population of PBMCs, and the methods may increase the number of T cells and/or result in an increased proportion of T cells in the cell population at the end of the culture.

The immune cells (e.g. PBMCs, PBLs) from which the populations of immune cells are

generated/expanded according to the methods of the present invention may be freshly obtained, or may be thawed from a sample of immune cells which has previously been obtained and frozen.

In embodiments of the methods disclosed herein, generation/expansion of a population of immune cells may involve culture of a population of PBMCs. In some embodiments, a population of immune cells may be generated/expanded from within a population of T cells (e.g. a population of T cells of heterogeneous type and/or specificity), which may have been obtained from a blood sample or a population of PBMCs. The immune cell population which is generated/expanded according to the methods of the present invention may be any desired population of immune cells. In some embodiments the immune cell population expanded/generated according to the method of the present 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, innate lymphoid cells (ILC), antigen-specific immune cells (i.e. cells expressing a receptor specific for an antigen; e.g. antigen-specific T cells and/or antigen-specific NK cells), TCR-expressing cells, CAR-expressing cells (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 the immune cell population expanded/generated according to the method of the present invention is a population of T cells (e.g. CD4+ T cells, CD8+ T cells, CD8+ cytotoxic T cells or antigen-specific T cells). In some embodiments the immune cell population expanded/generated according to the method of the present invention is a population of NK cells (e.g. antigen-specific NK cells).

In some embodiments the method is for the generation/expansion of immune cells (e.g. T cells, NK cells) in an antigen-independent manner. In some embodiments T cells may be activated in an antigen- independent fashion, e.g. stimulation through CD3, optionally in combination with stimulation through CD28. In some embodiments T cells may be activated by stimulation through treatment with agonist anti- CD3 antibody (e.g. clone OKT3), optionally in combination with treatment with agonist anti-CD28 antibody. In embodiments employing anti-CD3 and/or anti-CD28 for antigen-independent activation of T cells, the modified K562 cells may be modified to increase expression of one or more Fc receptors (e.g. one or more Fc gamma receptors, e.g. one or more of CD64, CD32 and/or CD16). The Fc receptors facilitate presentation of the antibodies and thus facilitate T cell activation.

In some embodiments the methods comprise culturing a population of immune cells (e.g. PBMCs or PBLs) in the presence of modified K562 cells modified to increase expression of one or more Fc receptors (e.g. one or more Fc gamma receptors, e.g. one or more of CD64, CD32 and/or CD16) and in the presence of agonist anti-CD3 antibody (e.g. clone OKT3), optionally in the presence of agonist anti- CD28 antibody.

In some embodiments the immune cell population expanded/generated according to the methods is a population of cells expressing a receptor specific for an antigen (e.g. TCR-expressing cells or CAR- expressing cells). In some embodiments binding of the antigen or a fragment thereof to an immune cell expressing a receptor specific for the antigen causes phosphorylation of one or more immunoreceptor tyrosine-based activation motifs (ITAMs) in an immune cell expressing a receptor specific for the antigen. In some embodiments the ITAMs are ITAMs of a CD3 polypeptide.

In some embodiments the immune cell population expanded/generated according to the methods is a population of TCR-expressing T cells or CAR-expressing T cells. In some embodiments the receptor specific for an antigen is a chimeric antigen receptor (CAR). CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), hereby incorporated by reference in its entirety. CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signaling region. An optional hinge region may provide separation between the antigenbinding region and cell membrane anchor region, and may act as a flexible linker. The signalling region of a CAR allows for activation of the T cell. The CAR signalling regions may comprise the amino acid sequence of the intracellular domain of Oϋ3-z, which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing cell. Signalling regions comprising sequences of other ITAM-containing proteins such as FcyRI have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1 ): 182-187). Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein. Suitable co-stimulatory molecules include CD28, 0X40, 4-1 BB, ICOS and CD27.

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

In some embodiments the receptor specific for an antigen specific for the antigen is a T cell receptor (TCR). TCRs are heterodimeric, antigen-binding molecules typically comprising an a-chain and a b-chain. In nature, a-chain and a b-chains are expressed at the cell surface of T cells (ab T cells) as a complex with invariant CD3 polypeptides. An alternative TCR comprising y and d chains is expressed on a subset of T cells (gd T cells).

In some embodiments the modified K562 cells may be employed as antigen presenting cells (APCs) in methods for antigen-dependent expansion of immune cells. The modified K562 cells may be employed as APCs presenting antigen for the generation/expansion of immune cells specific for an antigen. The modified K562 cell may be comprise or express an antigen.

In embodiments where the modified K562 cells are employed in methods for generating/expanding immune cells expressing a receptor specific for an antigen, the modified K562 cell may be modified to increase expression by the K562 cell of the antigen for which the receptor is specific. For example, where the modified K562 cells are employed in methods for generating/expanding immune cells expressing a receptor specific for a cancer cell antigen the modified K562 cell may be modified to increase expression by the K562 cell of the cancer cell antigen.

By way of illustration, the Examples of the present disclosure describe expansion of a population of T cells 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 (e.g. PBMCs or PBLs) in the presence of modified K562 cells modified to increase expression of an antigen. In some embodiments the methods comprise culturing a population of immune cells (e.g. T cells) modified to express a receptor specific for an antigen (e.g. a CAR) in the presence of modified K562 cells modified to increase expression of the antigen.

In some embodiments the modified K562 cells may be employed as artificial costimulatory factors, e.g. providing costimulatory signals to immune cells to be generated/expanded. In some embodiments the methods comprise culturing a population of immune cells (e.g. PBMCs or PBLs) in the presence of modified K562 cells modified to increase expression of one or more factors capable of increasing immune cell activation/proliferation.

Uses of populations of immune cells qenerated/expanded by culture in the presence of the modified K562 cells

Populations of immune cells expanded by culture in the presence of modified K562 cells according to the present invention find use e.g. in therapy or prophylaxis of a disease/condition. The disease/condition may be any disease/condition which would derive therapeutic or prophylactic benefit from an increase in the number of the immune cells generated/expanded according to the methods described herein. In some embodiment the disease/condition is a T cell dysfunctional disorder, an infectious disease or a cancer.

A T cell dysfunctional disorder may be a disease/condition in which normal T cell function is impaired causing downregulation of the subject’s immune response to pathogenic antigens, e.g. generated by infection by exogenous agents such as microorganisms, bacteria and viruses, or generated by the host in some disease states such as in some forms of cancer (e.g. in the form of tumor-associated antigens).

The T cell dysfunctional disorder may comprise T cell exhaustion or T cell anergy. T cell exhaustion comprises a state in which CD8+ T cells fail to proliferate or exert T cell effector functions such as cytotoxicity and cytokine (e.g. IFNy) secretion in response to antigen stimulation. Exhausted T cells may also be characterised by sustained expression of one or more markers of T cell exhaustion, e.g. PD-1 , CTLA-4, LAG-3, TIM-3. The T cell dysfunctional disorder may be manifest as an infection, or inability to mount an effective immune response against an infection. The infection may be chronic, persistent, latent or slow, and may be the result of bacterial, viral, fungal or parasitic infection. As such, treatment may be provided to patients having a bacterial, viral or fungal infection. Examples of bacterial infections include infection with Helicobacter pylori. Examples of viral infections include infection with HIV, hepatitis B or hepatitis C. The T-cell dysfunctional disorder may be associated with a cancer, such as tumor immune escape. Many human tumors express tumor-associated antigens recognised by T cells and capable of inducing an immune response.

An infectious disease may be e.g. bacterial, viral, fungal, or parasitic infection. In some embodiments it may be particularly desirable to treat chronic/persistent infections, e.g. where such infections are associated with T cell dysfunction or T cell exhaustion. It is well established that T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections (including viral, bacterial and parasitic), as well as in cancer (Wherry Nature Immunology Vol.12, No.6, p492-499, June 2011 ).

Examples of bacterial infections that may be treated include infection by Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) and Pseudomonas aeruginosa. For example, the bacterial infection may be sepsis or tuberculosis. Examples of viral infections that may be treated include infection by influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplex virus and human papilloma virus (HPV). Examples of fungal infections that may be treated include infection by Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungal infection may be fungal sepsis or histoplasmosis. Examples of parasitic infections that may be treated include infection by Plasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli, Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi). The parasitic infection may be a disease such as malaria, leishmaniasis and toxoplasmosis.

In particular embodiments, the disease/condition to be treated/prevented is a cancer. The cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumor. The cancer may be benign or malignant and may be primary or secondary (metastatic). A neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g. renal epithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells. In some embodiments, the cancer to be treated may be a cancer of a tissue selected from the group consisting of colon, rectum, nasopharynx, cervix, oropharynx, stomach, liver, head and neck, oral cavity, oesophagus, lip, mouth, tongue, tonsil, nose, throat, salivary gland, sinus, pharynx, larynx, prostate, lung, bladder, skin, kidney, ovary or mesothelium.

Tumors to be treated may be nervous or non-nervous system tumors. Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma. Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancer and sarcoma. In some embodiments the cancer is selected from the group consisting of: colon cancer, colon carcinoma, colorectal cancer, nasopharyngeal carcinoma, cervical carcinoma, oropharyngeal carcinoma, gastric carcinoma, 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, urothelial carcinoma, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or mesothelioma.

In some embodiments the cancer to be treated/prevented is a virus-associated cancer, e.g. an EBV- associated cancer or a HPV-associated cancer.“EBV associated” and“HPV associated” cancers may be a cancers which are caused or exacerbated by infection with the respective viruses, cancers for which infection is a risk factor and/or cancers for which infection is positively associated with onset,

development, progression, severity or metastasis. EBV-associated cancers which may be treated with cells produced by methods of the disclosure include nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). HPV-associated medical conditions that may be treated with cells produced by methods of the disclosure include at least dysplasias of the genital area(s), cervical intraepithelial neoplasia, vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, anal intraepithelial neoplasia, cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, genital cancers, oral papillomas, oropharyngeal cancer. In some embodiments, the cancer to be treated in accordance with various aspects of the present disclosure is one or more of nasopharyngeal carcinoma (NPC; e.g. Epstein-Barr Virus (EBV)-positive NPC), cervical carcinoma (CC; e.g. human papillomavirus (HPV)-positive CC), oropharyngeal carcinoma (OPC; e.g. HPV-positive OPC), gastric carcinoma (GC; e.g. EBV-positive GC), hepatocellular carcinoma (HCC; e.g. Hepatitis B Virus (HBV)-positive HCC), lung cancer (e.g. non-small cell lung cancer (NSCLC)) and head and neck cancer (e.g. cancer originating from tissues of the lip, mouth, nose, sinuses, pharynx or larynx, e.g. head and neck squamous cell carcinoma (HNSCC)).

Populations of immune cells generated/expanded according to the methods described herein are also useful in connection with methods comprising adoptive cell transfer (ACT). In particular the modified K562 cells may be used to generate/expand populations of immune cells which may then be administered to a subject in order to treat/prevent a disease/condition.

The present invention provides a method of treatment or prophylaxis comprising adoptive transfer of immune cells (e.g. T cells, effector T cells, antigen-specific T cells, NK cells) produced (i.e. generated or expanded) according to the methods of the present invention. Adoptive cell transfer generally refers to a process by which immune cells are obtained from a subject, typically by drawing a blood sample from which the immune cells are isolated. The immune cells are then typically treated or altered in some way, optionally expanded, and then administered either to the same subject or to a different subject. The treatment is typically aimed at providing an immune cell population with certain desired characteristics to a subject, or increasing the frequency of immune cells with such characteristics in that subject. The adoptively transferred cells may be e.g. 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 (i.e. CTLs) NK cells or antigen-specific NK cells.

In some cases, the immune cells are derived from the patient that they are introduced to (autologous cell therapy). That is, cells may have been obtained from the patient, generated according to methods described herein, and then returned to the same patient.

Methods disclosed herein may also be used in allogeneic cell therapy, in which cells obtained from a different individual are introduced into the patient. Populations of immune cells generated/expanded by methods comprising culture in the presence of the modified K562 cells of the present invention are particularly well suited to use in allogeneic adoptive cell therapy because they display reduced alloreactivity as compared to methods immune cells generated/expanded by methods comprising culture in the presence of wildtype K562 cells.

Adoptive T cell transfer is described, for example, in 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 are hereby incorporated by reference in their entirety.

In the present invention, adoptive transfer is performed with the aim of introducing, or increasing the frequency of, immune cells in a subject.

Accordingly, the present invention provides a method of treating or preventing a disease or condition in a subject, comprising:

(a) generating or expanding a population of immune cells by culturing a population of immune cells (e.g. PBMCs or PBLs) in the presence of a modified K562 cell according to the present invention; and

(b) administering the population of immune cells generated or expanded at step (a) to a subject.

The present invention also provides a method of treating or preventing a disease or condition in a subject, comprising:

(a) isolating immune cells (e.g. PBMCs or PBLs) from a subject;

(b) generating or expanding a population of immune cells by culturing the immune cells (e.g. PBMCs) in the presence of a modified K562 cell according to the present invention, and;

(c) administering the generated/expanded population of immune cells to a subject.

In some embodiments, the subject from which the immune cells (e.g. PBMCs or PBLs) are isolated at step (a) is the subject administered with the generated/expanded population of immune cells at step (c) (i.e., adoptive transfer is of autologous cells). In some embodiments, the subject from which the immune cells (e.g. PBMCs or PBLs) at step (a) is a different subject to the subject to which the generated/expanded population of immune cells are administered to at step (c) (i.e., adoptive transfer is of allogeneic cells).

In some embodiments the method may comprise one or more of the following steps: taking a blood 5 sample from a subject; isolating PBMCs or PBLs from the blood sample; generating or expanding a population of immune cells by culture in the presence of a modified K562 cell according to the present invention; collecting the generated or expanded population of immune cells; mixing the generated or expanded population of immune cells with an adjuvant, diluent, or carrier; administering the generated or expanded population of immune cells or composition to a subject.

10

The skilled person is able to determine appropriate reagents and procedures for adoptive transfer of immune cells generated or expanded according to the methods of the present invention for example by reference 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.

15

Sequence identity

As used herein,“sequence identity” refers to the percent of nucleotides/amino acid residues in a subject sequence that are identical to nucleotides/amino acid residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum percent sequence identity 20 between the sequences. Pairwise and multiple sequence alignment for the purposes of determining percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved in various ways known to a person of skill in the art, for instance, using publicly available computer software such as ClustalOmega (Soding, 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)) 25 and MAFFT (Katoh and Standley 2013, Molecular Biology and Evolution, 30(4) 772-780 software. When using such software, the default parameters, e.g. for gap penalty and extension penalty, are preferably used.

Sequences

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

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

Aspects and embodiments of the present invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word“comprise,” and variations such as“comprises” and“comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from“about” one particular value, and/or to“about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent“about,” it will be understood that the particular value forms another embodiment.

Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

Methods described herein may preferably performed in vitro. The term“in vitro” is intended to encompass experiments with cells in culture whereas the term“in vivo" is intended to encompass experiments with intact multi-cellular organisms.

Brief Description of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures. Figures 1A and 1B. Graphs and bar charts showing expression of B2M and MHC Class I molecules on K562 cells after stimulation with IFN-g or cell culture supernatant collected from T cell/K562 cell cocultures. (1A) Representative flow cytometry histograms of surface B2M and HLA-A, B, C expression. Non-inactivated, mitomycin C-treated, or g-irradiated K562 cells were stimulated with either IFN-g (500 lU/ml, light gray) or the supernatants collected from T cell/K562 cell co-cultures (1 :1 ratio for 48 hours, black lines) for 48 hours. K562 cells without stimulation were included as controls (gray). (1B) Bar charts showing mean ± SD of three independent experiments. Stimulation was performed with IFN-g or the culture supernatants generated from three individual donors. ***: p < 0.001.

Figures 2A and 2B. Graphs showing isotype controls and the expression of MHC class II molecules on K562 cells. (2A) Flow cytometry histograms of isotype controls for surface markers on K562 cells. (2B) Representative flow cytometry histograms for HLA-DR expression. K562 cells were stimulated with either IFN-g (500 lU/ml) or T cell/K562 cell co-culture (1 :1 ratio for 48 hours) supernatants for 48 hours. K562 cells without stimulation were included as a control. The percentages of positive expression were gated according to the isotype control.

Figures 3A to 3D. Schematics and bar charts relating to generation of B2M knockout in K562 cells with the CRISPR/Cas9 technology. (3A) Schematic of a CRISPR/Cas9 system targeting B2M exon 1 (EX1 ). The system was used for electroporation-based genetic modification in K562 cells. (3B) Schematic of a baculoviral CRISPR/Cas9 system targeting B2M EX1 and exon 2 (EX2). For genetic modification, K562 cells were transduced with the baculoviral system. Arrows show the binding sites of PCR primers for genotyping. (3C) and (3D) Bar charts showing B2M expression by K562 single cell clones after B2M knockout. The tested K562 single cell clones were stimulated with 500 lU/mL IFN-g before being analysed by flow cytometry to detect B2M expression.

Figures 4A to 4E. Images, schematics and graphs relating to characterisation of B2M knockout K562 clones. (4A) Genotyping of B2M knockout single cell clones by PCR. Two clones were from electroporation-transfected K562 cells and five clones were from baculovirus-transduced K562 cells. Wild type K562 was included a control. Upper panel shows the specific amplification of integrated selection marker in B2M site. (+) was a positive control of B2M site-specific integration. Lower panel shows the specific amplification of wild type B2M EX1 allele. (4B) Schematic representation of sequencing results of targeting regions in EX1 and EX2 from five B2M knockout single cell clones. Three alleles of B2M gene are aligned with the wild type. CRISPR/Cas9 targets are shown in light grey. Start codon of B2M is darker grey“ATG”. Deletion is shown by dashed line, and inserted“T” nucleotides are underlined. (4C)

Representative Sanger sequencing traces from mutant B2M alleles. The electropherograms shown with wild type B2M sequence to show the sites of deletion and insertion. (4D) Western blots for B2M expression in B2M knockout single cell clones. Wild type K562 was included a control and b-actin was detected for housekeeping expression. (4E) Representative flow cytometry histograms of surface B2M and HLA-A, B, C expression on B2M knockout K562 single cell clones. B2M knockout single cell clones were stimulated with IFN-g or T cell/K562 cell co-culture supernatants before prior to analysis.

Percentages of positive expression were gated according to isotype controls.

Figures 5A to 5C. Bar charts and graph showing results of analysis of MHC class I expression and function of B2M knockout K562 cells. Surface expression of B2M (5A) and MHC class I molecules (5B) on wild type (WT) K562 cells and B2M knockout K562 single cell clones was examined after stimulation with IFN-g or T cell/K562 cell co-culture supernatants. Bars show the mean ± SD of three independent experiments. (5C) Graphs showing that the decrease in surface MHC class I expression on B2M knockout K562 cells resulted in increased susceptibility to lysis by NK cells as determined by analysis using the DELFIA EuTDA cytotoxicity assay (2 hours Eu-ligand release), using K562 B2MKO clone EX1 EX2#5 as target cells. Three independent assays were performed with NK cells from three different donors. Percentage lysis of K562 cells at varying effector (E):target (T) cell ratios (Mean ± SD of triplicate samples) is shown. *: p < 0.05; ***: p < 0.001.

Figures 6A to 6E. Schematic and graphs relating to generation of B2M knockout K562-based aAPCs. (6A) Schematics of eletroporated ZFN constructs targeting AAVS1 site for integration of costimulatory molecules. Arrows represent the binding sites of PCR primers for genotyping. (6B) Expression of co-stimulatory molecules on K562A and K562B cells. aAPCs were stained with antibodies and analysed by flow cytometry assay. Representative FACS plots are shown and the percentages of positive expression were gated relative to isotype controls. (6C) Genotyping of K562B for AAVS1 site by PCR. Unmodified B2M knockout K562 clone EX1 EX2#5 was included as a control. Left panel shows the specific amplification of integrated donor cassette at the AAVS1 site. Right panel shows the specific amplification of wild type AAVS1 allele. (6D and 6E) Characterization of expression of erythroid and myeloid-specific cell markers by K562B cells. Wild type K562 cells were included as a control. Cells were stained by antibodies and analysed with flow cytometry assay. Representative plots are shown in 6E. Percentages of positive cells were determined by gating relative to isotype controls. Bars show the mean ± SD of three independent experiments.

Figures 7A to 7D. Schematic and graphs relating to analysis of alloreactivity of T cells expanded with B2M knockout K562 cell-based aAPCs. (7A) Schematic of in vitro T cell expansion protocol. T cells were stimulated with K562A (WT K562-based aAPC) or K562B (B2M knockout K562-based aAPC) every seven days for two weeks before being used in cytotoxicity assays. (7B to 7D) Alloreactivity of CD8+ T cells expanded in the presence of K562A or K562B cells against wildtype K562 cells, target cells expressing the indicated HLA-C alleles which are expressed by K562 cells (HLA-C*03 or HLA-C*05), or target cells that do not express HLA-C alleles HLA-C*03 or HLA-C*05. Three independent assays were performed using T cells from three different donors (7B, 7C and 7D, respectively). Percentage lysis of target cells at varying E:T ratios is shown (Mean ± SD of triplicate samples). *: p <0.05; ***: p < 0.001.

Figures 8A to 8D. Images and graphs relating to analysis of alloreactivity of T cells expanded with B2M knockout K562 cell-based aAPCs. (8A and 8B) HLA-C genotyping for K562 cells, tumor cell lines used as targets and PBMCs used as effectors. PBMCs are grouped according to whether or not they showed alloreactivity. (8A) Specific amplification of HLA-C*03 allele. (8B) Specific amplification of HLA- C*05 allele. (8C) Fold growth of T cells from three different alloreactive donors co-cultured with the indicated K562 cells. K562 cells were inactivated with mitomycin C and a T cell/K562 cell ratio of 1 :1 was used for the co-cultures. (8D) Immunophenotyping of the expanded cells by flow cytometry.

Representative FACS plots of three alloreactive donors are shown and the percentages of positive cells were determined by gating relative to isotype controls.

Figures 9A to 9H. Schematic, graphs bar charts and tables showing antigen-independent T cell expansion with B2M knockout K562-based aAPCs. (9A) Schematic representation of the antigen- independent T cell expansion protocol in vitro. T cells were stimulated with K562A or K562B at a T cell/K562 cell ratio of 1 :50 for two weeks before being harvested. OCT-3 was included to promote T cell expansion. (9B to 9D) Fold growth of T cells in co-culture with WT K562 and B2M k/o K562 cells. K562 cells were inactivated with mitomycin C. A T cell/K562 cell ratio of 1 :50 was used and OCT-3 was included to promote T cell expansion. Three independent assays were performed using T cells from three different donors (9B, 9C and 9D, respectively). (9E to 9H) Immunophenotyping of the expanded cells by flow cytometry. (9E and 9F) Bar charts showing percentages of major cell types and CD8+ T cell subsets expanded in the experiment shown in 9B. (9G and 9H) Tables showing percentages of major cell types and CD8+ T cell subsets expanded in the experiments shown in 9G and 9H, respectively.

Figures 10A to 101. Graphs and schematics relating to expansion of anti-CD19 CAR-T cells using B2M knockout K562-based aAPCs expressing CD19 antigen. (10A) Analysis of CD19 and CAR expression by B2MKO K562-based aAPCs and T cells expressing anti-CD19 CAR. Cells were stained with antibodies and analysed by flow cytometry. Representative FACS plots are shown and the positive expression was determined by gating relative to isotype controls. (10B) Schematic of protocol for in vitro expansion of anti-CD19 CAR-T cells. T cells were stimulated with K562B+CD19 or K562B every 7 days at a T cell/K562 cell ratio of 1 : 1 for four weeks before being harvested. (10C and 10D) Fold growth of anti- CD19-CAR-T cells derived from two different donors in co-culture with K562 cells. K562 cells were inactivated with mitomycin C and a T cell/K562 cell ratio of 1 : 1 was used for the co-cultures. (10E and 10F) Scatterplots showing numbers of anti-CD19 CAR-positive T cells after the indicated number of days of co-culture of anti-CD 19-CAR-T cells derived from two different donors with K562B or K562B+CD19 cells. Cells were collected and stained with anti-mouse IgG Fab and anti-human CD3 antibodies, and analysed by flow cytometry. (10G) Immunophenotyping of the expanded cells by flow cytometry.

Representative plots are shown. (10H and 101) Results of analysis of in vitro cell lysis of CD19-positive tumor cell lines (Daudi and Raji) or a CD19-negative cell line (MCF-7) with anti-CD19 CAR-expressing T cells derived from two different donors, as determined by DELFIA EuTDA cytotoxicity assay (2 hours Eu- ligand release). More than three independent experiments with PBMCs from individual donors were performed (Mean ± SD of triplicate samples). *: p < 0.05. Examples

In the following Examples, the inventors describe the use of a CRISPR/Cas9-based system to knockout the B2M gene in K562 cells, thereby generating MHC class I molecule-deficient K562 cells. The inventors demonstrate that the B2M knockout K562 cells do not express MHC class I molecules at the cell surface even after stimulation with wildtype K562 cell/T cell co-culture supernatants or interferon-g. The inventors show that the B2M/MHC class I molecule-deficient K562 cells can be used as a‘scaffold’ cells for the generation of aAPCs, which are capable of supporting robust antigen independent T cell expansion in vitro, as well as in vitro expansion of antigen-specific anti-CD19 CAR-T cells. Importantly, T cells expanded with the B2M knockout K562 cell aAPC are demonstrated to display attenuated alloreactivity as compared to T cells expanded with comparable K562 cells not having had the B2M gene knocked out.

Example 1 : Materials and methods

1.1 Cell culture

Human myelogenous leukemia cell line K562 cells (ATCC, Manassas, VA, USA) were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Lonza, Basel, Switzerland) with 10% fetal bovine serum (FBS, Hyclone™ GE Healthcare, Little Chalfont, UK). When used as aAPC co-culture feeder cells, K562 cells were inactivated by 100Gy (10,000 rads) g-irradiation or treated with 20 pg/mL Mitomycin C (Roche Diagnostics, Basel, Switzerland) for one hour, and then washed three times with phosphate-buffered saline (PBS, Lonza) and transferred into co-culture medium. For the induction of B2M and human leukocyte antigen (HLA), K562 cells were treated with 500 lU/mL IFN-g (PeproTech, Rocky Hill, NJ, USA) or supernatants collected from K562 and T cell co-cultures (1 :1 ratio for 48 hours) for 48 hours.

Fresh peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor's buffy coat with Ficoll®-Paque PREMIUM 1.084 (GE Healthcare) by density gradient centrifugation. PBMCs were cocultured with Dynabeads® Human T-Activator CD3/CD28 (Gibco, Thermo Fisher Scientific, Waltham,

MA, USA) in AIM V® Medium (Invitrogen, Thermo Fisher Scientific) with 5% human AB serum (Valley Biomedical, Winchester, VA, USA) for T cell activation. Primary activated CD3+ T cells were collected and the microbeads were depleted on day 7. For in vitro antigen-independent T cell expansion, T cells and B2M-knockout K562-based aAPCs were co-cultured at 1 :50 ratio in AIM V® Medium with 5% human AB serum and 300 lU/mL IL-2 (PeproTech). Anti-CD3 (OKT-3) antibody (60 ng/mL, eBioscience, San Diego, CA, USA) was added in the first week and fresh medium was changed or topped up during coculture accordingly. For in vitro antigen-dependent T cell expansion, activated T cells were transduced with lentiviral vectors encoding anti-CD19 CAR and stimulated with aAPCs expressing CD19 at 1 :1 ratio every week in AIM V® Medium with 5% human AB serum, 300 lU/mL IL-2, 5 ng/mL IL-7 (PeproTech) and 5 ng/mL IL-15 (PeproTech). CD8+ T cells were isolated by magnetic beads with CD8+ T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Magnetic sorting was performed according to the manufacturer's instructions.

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 pg/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. Cells were passaged every alternative days accordingly.

1.2 Plasmids and baculoviral vector construction

The pX260 plasmid (Addgene, Cambridge, MA, USA) containing a 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. crRNA sequences targeting B2M gene exon 1 (EX1 ) and exon 2 (EX2) (Table 1 , Example 1.8) were designed and selected using ZiFiT Targeter online software (41 ), and cloned into pX260. The donor plasmid for B2M EX1 homology-direct integration was constructed, containing the EF1 a (eukaryotic translation elongation factor 1a) promoter driving the expression of an EGFP gene and the PGK (Mouse phosphoglycerate kinase 1 ) promoter driving the expression of the Neo gene (neomycin resistance gene), 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). The donor plasmid for B2M EX1-EX2 homology-direct integration was constructed containing the same cassette as the donor for B2M EX1 homology-direct integration, flanked by homologous DNA sequences from 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). The vector containing ZFNs is described in Tay et al., Journal of gene medicine 15, 384 (37). The donor plasmids for AAVS1 site homology-direct integration were constructed, containing co-stimulatory molecule-expression cassettes flanked by homologous DNA sequences from the AAVS1 locus (chromosome 19: nucleotides

55,116,611-55,115,767 and nucleotides 55,1 15,765-55,114,929, GRCh38.p2 Primary Assembly). The coding sequences for CD64 (FcyRI, GenBank accession no. BC032634), CD86 (B7-2, GenBank accession no. NM_175862), 4-1 BBL (CD137L, GenBank accession no. NM_003811 ), and membrane- bound IL-21 (mblL21 , GenBank accession no. NM_021803.3) were PCR-amplified from a human PBMC cDNA library and cloned into a pFastBac™1 (Invitrogen) vector as expression cassettes. The CD64-2A- CD137L-2A-CD86 expression cassette under the control of the CMV (cytomegalovirus) promoter was subcloned into one donor plasmid and the EpCAM-2A-mblL21 expression cassette under the control of a CMV promoter was subcloned into another donor plasmid. The donor plasmid for CD19 expression was constructed with a CMV promoter to drive the expression of a CD19-IRES-Puro (puromycin resistant gene) expression cassette. The plasmid for anti-CD19 CAR expression was constructed with the CMV promoter to drive the expression of an scFv (anti-CD19) -CD8TM (CD8 transmembrane domain) -CD28- ΰϋ3z expression cassette. The anti-CD 19 CAR expression cassette was subcloned into a lentiviral vector for lentivirus production.

For baculoviral vectors construction, the pFastBac™1 (Invitrogen) plasmid was described previously (39). crRNAs targeting both EX1 and EX2, tracRNA with Cas9, and the donor for B2M EX1-EX2 homology- direct integration were subcloned into three pFastBac™1 plasmids respectively. Three recombinant bacmids were generated according to the protocol of Bac-to-Bac® Baculovirus Expression System (Invitrogen). Insect Sf9 cells (ATCC) were cultured in Sf-900™ II SFM (Gibco) medium and transfected with those recombinant bacmids 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 Generation of B2M knockout K562 clones

To generate B2M knockout cells by electroporation, 5 x 10^® K562 cells were electroporated with pX260- B2M-EX1 and a donor vector in Opti-MEM® I Reduced Serum Medium (Gibco) at 2 pg DNA per vector via electroporator (Nepa Gene, Chiba, Japan). Cells were subsequently transferred into culture medium. Four days after electroporation, cells were selected by culture in the presence of 500 pg/nnL Geneticin® (G418 Sulfate, Gibco) for 2 weeks, before single cell seeding. To generate B2M knockout clones with baculovirus transduction, 5 x 10^® K562 cells were plated per well of 12-well plate and were transduced with BVs at a multiplicity of infection (MOI) of 500 pfu of each BV per cell in Opti-MEM® overnight. Cells were then transferred into culture medium. Two weeks after transduction, cells were selected by culture in the presence of 500 pg/nnL Geneticin®. Two weeks after selection, fluorescence-activated cell sorting (FACS) was conducted with BD FACSAria™ I Flow Cytometer (BD Biosciences, Franklin Lakes, NJ,

USA) for single cell seeding. EGFP positive cells were gated and seeded at one cell per well in a 96-well plate. Single cell clones were then cultured and expanded. Once reaching 80 - 90% confluence, K562 single cell clones in 96-well plate were duplicated for genotyping and subculturing. One plate was used for genomic DNA extraction, followed by genotyping by PCR. For subsequent sequencing, PCR products were purified and subcloned into pGEM®-T Easy Vector Systems (Promega, Fitchburg, Wl, USA).

Monoclonal vectors were collected for sequencing. The identified clones were then expanded for further studies.

1.4 Generation of K562 cell-based aAPCs

To generate K562-based aAPCs for use in methods of T cell expansion, WT K562 cells or B2M knockout clone EX1 EX2#5 K562 cells were electroporated with ZFNs and donor plasmids and selected by culture in the presence of 1 pg/ml puromycin (Gibco). Cells were subsequently stained with anti-CD64 APC (Miltenyi Biotec, Bergisch Gladbach, Germany) antibody and seeded as one cell per well in 96-well plate with culture medium by FACS for APC positive population. Single-cell cloning was performed and two clones with the highest expression of CD64, CD86, 4-1 BBL, and mblL21 , K562A from WT K562 cells and K562B from EX1 EX2#5, were selected. For the expansion of anti-CD19 CAR expressing T cells, the CD19 gene was introduced into K562B cells through transfection. After puromycin selection for two weeks, anti-CD19 APC (eBioscience) antibody was used to stain cells and APC positive cells were gated and seeded as one cell per well in 96-well plate with culture medium. Single cell clones with high and stable CD19 expression were selected and expanded for further analysis.

1.6 Cell cytotoxicity assays

Cell cytotoxicity was analysed in a standard 2 hour Europium-release assay with DELFIA® EuTDA Cytotoxicity Reagents (PerkinElmer, Waltham, MA, USA). Assays were performed according to the manufacturer's instructions. Target cells were first labeled with BATDA Reagent at 37°C for 5 to 15 minutes and then washed three times with PBS. Effector cells and labeled target cells were mixed in triplicate at different effector to target (E:T) ratios in AIM V® Medium with 5% human AB serum. Mixed cells were incubated at 37°C in humid incubator for 2 hours. Spontaneous and maximum release were determined by incubating target cells without effector cells and with lysis buffer respectively. After incubation, supernatants were transferred to mix with the Europium solution and analysed using VICTOR™ Time-resolved fluorometer (PerkinElmer). The percentage of specific lysis was calculated as: % lysis = [(experimental release - spontaneous release) / (maximum release - spontaneous release)] x

100.

1.7 Statistical analysis

Data were collected as described above and analysed using Prism Version 7 software (GraphPad). Data were presented as mean (± SD) and analysed by Two-way Anova, Tukey's multiple comparisons test and Independent Samples Student's t-test. Representative histograms and graphs were chosen from independent repetitions on the basis of the average values.

1.8 Oligos and primers

Table 1 :

1.9 Antibodies

Table 2:

1.5 Western blotting, flow cytometry analysis, and intracellular staining

For Western blot analysis, sample proteins were extracted by lysing cells with Radioimmunoprecipitation assay (RIPA) buffer (Nacalai Tesque, Kyoto, Japan), analysed in SDS-PAGE gel under reducing conditions and then electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Rabbit Anti-B2M antibody clone EP2978Y (1 :5000 dilution, Abeam, Cambridge, UK) and mouse Anti- -actin antibody clone GT5512 (1 :1000 dilution, Abeam) were used as primary antibodies. Goat antirabbit 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 visualized for chemiluminescence using an MYECL™ Imager (Thermo Fisher Scientific).

For flow cytometry analysis, cells were stained with antibodies in autoMACS® Running Buffer (Miltenyi Biotec) and analysed by BD Accuri™ C6 Flow Cytometer (BD Biosciences). Data were analysed using CFlow® Sampler software (BD Biosciences). Multicolour staining was analysed using BD™ LSR II Flow Cytometer (BD Biosciences) and data were analysed using FlowJo software (TreeStar, Ashland, OR, USA). For anti-F(ab')2 staining, T cells were first stained with Biotin-conjugated AffiniPure F(ab')2 Fragment (goat anti-mouse IgG, F(ab')2 Fragment Specific, Jackson ImmunoResearch Laboratories,

West Grove, PA, USA) for 1 hour and then sub-stained with Streptevdin APC (BD Biosciences). For intracellular staining, K562 cells were first fixed with 0.01 % paraformaldehyde (PFA, Thermo Fisher Scientific) and permeabilized with 0.5% Tween-20 (Bio-Rad Laboratories), and then analysed by flow cytometry.

Example 2: Expression of B2M and MHC class I molecules on K562 cells can be induced by factors in T cell co-culture conditioned medium

The inventors first investigated whether soluble factors produced by co-cultures of T cells and K562 cells were able to stimulate upregulation of MHC class I molecules on wildtype K562 cells.

Wildtype 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 in culture in another cell culture plate. In another condition K562 cells were stimulated with IFNy, and in another condition K562 cells were not stimulated.

To mimic the situation where inactivated K562 cells are used as aAPC feeder cells for immune cell expansion cultures K562 cells were inactivated by treatment with mitomycin C or by gamma irradiation. In another condition K562 cells were not inactivated.

Expression of MHC class I molecules was analysed by flow cytometry. A moderate increase in the expression levels of B2M and HLA-A, B, C on K562 cells was observed after mitomycin C treatment or gamma irradiation as compared with their expression levels on non-inactivated K562 cells (Figures 1A and 1 B). With the stimulation using supernatants collected from the K562/T cell co-cultures, the expression levels reached up to 90% of cells, and the levels were comparable to that induced by treatment with IFN-g treatment (Figures 1 A and B). Non-inactivated and inactivated K562 cells showed similar levels of up-regulation, suggesting that inhibition of K562 cell proliferation does not disrupt the mechanism underlying the up-regulation of B2M and MHC class I molecules. Expression of the MHC class II molecule HLA-DR was also analysed, but it was not detectable under any condition (Figure 2B), consistent with previous studies (43, 44). Example 3: B2M knockout K562 cells display a MHC class l-neqative phenotype

The inventors designed two different clustered regulatory interspaced short palindromic repeat

(CRISPR)/Cas9 systems to knockout the B2M gene in order to generate MHC class I expression-deficient K562 cells.

For the first system, K562 cells were co-electroporated with a CRISPR/Cas9 construct targeting the exon 1 (EX1 ) of the B2M gene and a donor cassette comprising EGFP and Neomycin resistance genes flanked by homologous sequences, in order to integrate the sequence of the donor cassette in place of EX1 (Figure 3A).

Since K562 cells contain three alleles of B2M gene (45), it is challenging to knockout all of the alleles simultaneously in a single K562 cells. To address the issue, the inventors further designed a second CRISPR/Cas9 system targeting both B2M EX1 and exon 2 (EX2), with the aim of achieving complete disruption of the B2M gene (Figure 3B). Baculoviral vectors, which have excellent transduction efficiency in K562 cells, were used to deliver the second CRISPR/Cas9 system.

K562 cells electroporated with the EX1-targeting system and K562 cells transduced with the EX1 EX2- targeting system were then selected by culture in the presence of geneticin for 2 weeks to enrich stable EGFP-positive cells. Single cells were then seeded into individual wells of 96 well plates based on EGFP- positive cell sorting by FACS. Single cell clones - 78 from the electroporated cells and 60 from the transduced cells - were collected, and subjected to PCR genotyping to select the clones bearing the B2M site-specific integration of the selection marker (Figure 4A). Ten representative positive clones were collected from each method and expanded for further analysis. To detect the deficiency in B2M expression, the single cell clones were stimulated by IFN-g for 48 hours and then analysed by flow cytometry. Deficient B2M expression was observed in one of the clones derived from the EX1 system electroporated K562 cells (EX1#7) and four clones derived from the EX1 EX2 system transduced K562 cells (EX1 EX2#2, #5, #6, #7) (Figure 3C and 3D). B2M knockout was also confirmed in the five clones was by Western blot analysis (Figure 4D). Through DNA sequencing, B2M site-specific integration of the selection marker into one allele and mutation or early stop codons in other two alleles for the B2M gene was detected in the five B2M-deficient clones (Figure 4B and 4C).

MHC class I molecule expression was further examined in the five B2M knockout (B2MKO) K562 clones. K562 cells inactivated by treatment with mitomycin C or gamma irradiation, or non-inactivated K562 cells were stimulated by treatment with IFN-g or cell culture supernatants collected from the K562/T cell cocultures. By contrast to the upregulation of B2M and HLA-A, B, C molecule expression observed in wildtype K562 cells, expression of these molecules in the B2MKO K562 cells was barely detectable by flow cytometry (Figures 5A, 5B and 4E).

Since the cells without MHC class I expression would be very sensitive to lysis by NK cells (46), the inventors performed a functional assay based on NK cell-mediated lysis to further characterise MHC class I deficiency using the EX1 EX2#5 clone. WT and B2MKO K562 cells were stimulated by IFN-g and then co-cultured with human primary NK cells at varying effector (E):target (T) cell ratios. As expected, the percentages of NK-specific lysis of B2MKO K562 cells were much higher as compared to the lysis of WT K562 cells (Figure 5C). These findings confirmed that the B2MKO K562 cells displayed a MHC class l-deficient phenotype.

Example 4: T cells co-cultured with B2M-knockout K562 cell-based aAPCs show attenuated alloreactivitv

The K562 B2MKO clone EX1 EX2#5 was used to generate aAPCs. Genes encoding Fc receptor CD64, the essential co-stimulatory molecules CD86 and CD137L, and membrane-bound IL-21 (mblL21 ) were stably introduced into EX1 EX2#5 cells by ZFNs-mediated AA\/S1 site-specific integration (Figure 6A).

Modified cells underwent single cell cloning and a clone with high level expression of the four molecules was selected and designated“K562B” (Figures 6B and 6C). MHC class I phenotype deficiency and the expression of myelogenous lineage markers in K562B was examined and confirmed (Figure 6D and 6E).

Wildtype K562 cells (i.e. not B2M knockout) were also modified to express CD64, CD86, CD137L and mblL21. This cell line was designated“K562A” (Figure 6B), and was used as a MHC l-expressing control aAPC in the following experiments.

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

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

Cytotoxicity assays were then performed by mixing the CD8⁺ T cells generated by expansion with K562A or K562B cells, after the target cells had been stimulated with IFN-g to up-regulate MHC class I expression (Figures 7B to 7D). CD8⁺ T cells isolated immediately after the PBMC priming (i.e. without expansion by culture in the presence of K562A or K562B cells) were able to effectively kill the IFN-y stimulated wildtype K562 cells (Figure 7B, top left panel, triangles). After the expansion of CD8⁺ T cells with K562A cells, an increase in cytotoxicity against the IFN-g stimulated WT K562 cells was observed (Figure 7B, top left panel, diamonds). By contrast, CD8⁺ T cells expanded with K562B cells, did not have significantly increased cytotoxicity against WT K562 cells as compared to the cytotoxicity displayed by CD8⁺ T cells isolated immediately after the PBMC priming (Figure 7B, top left panel, squares).

Since K562 cells can only up-regulate expression of HLA-C molecules amongst the MHC class I molecules they encode (A* 1 1 ,31 ; B*18,40; C*03,05) (26), the inventors investigated alloreactivity of the expanded T cells against tumor cells expressing an HLA-C allele expressed by K562 (HLA-C*03 or HLA- C*05), or tumor cells that do not express HLA-C alleles HLA-C*03 or HLA-C*05 (Figures 8A and 8B).

CD8⁺ T cells expanded by culture in the presence of K562A cells displayed much higher cytotoxicity against tumor cells expressing an HLA-C allele expressed by K562 cells than the CD8⁺ T cells were expanded by culture in the presence of K562B (Figures 7B to 7D). Irrespective of whether the T cells were expanded by culture in the presence of K562A or K562B cells, no cytotoxicity observed when tumor cells not expressing an HLA-C allele expressed by K562 cells were used as target cells (Figures 7B to 7D).

These results indicate that while alloreactive cytotoxic T lymphocytes (CTLs) can be expanded and probably enriched when wildtype K562 cells are used as feeder cells or artificial APCs, B2MKO K562 cells do not stimulate alloreactive CTLs. T cells expanded by culture in the presence of B2M knockout K562 cells will comprise fewer alloreactive T cells, and a greater proportion of T cells having desired specificity.

Example 5: B2M knockout does not affect the ability of K562 cells to be used as aAPCs for antigen- independent and antigen-dependent T cell expansion

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

The CD8⁺ ab-T cell population constituted the major portion (60% to 75%), followed by the CD4⁺ ab-T cell population (20% to 40%). CD3 CD56⁺ NK cells were 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 obvious expansion of the gd-T cell population (1.5% to 3%). Among the expanded CD8⁺ ab-T cells, over 90% were effector memory T cells (CCR7 CD45RA ) and less than 3% expressed the T-cell exhaustion marker PD-1 (i.e. PD1 ⁺CD8⁺). The results obtained with K562B and K562A cells were comparable among different donors.

The results demonstrated that B2M knockout K562 cells are able to be used as aAPCs for antigen- independent expansion of T cells. To investigate the use of B2MKO K562-based aAPCs for expansion of antigen-dependent T cells, CD19 antigen was introduced into K562B cells and a CD 19-expressing single cell clone K562B+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-CD 19-CAR-T cells (Figure 10A).

The anti-CD19 CAR-T cells were cultured in the presence of inactivated K562B+CD19 cells or K562B cells at a 1 :1 ratio and re-stimulated every week for T cell expansion (Figure 10B). Robust T cell expansion when the CAR-T cells were co-cultured with K562B+CD19 cells, but not with K562B cells (Figures 10C and 10D).

When the CAR-T cells were stimulated with K562B+CD19 cells, the stable expression of anti-CD19 CAR increased from 9% of T cells at day 4 to 93% of the expanded T cells by day 25, indicating a significant enrichment of CAR-T cells after expansion (Figures 10E and 10F). When the T cells were stimulated with K562B cells, anti-CD19 CAR-positive T cells slightly decreased from the original 9% to 6% by day 25 (Figures 10E and 10F).

The CAR-T cells expanded by co-culture with K562B+CD19 cells were phenotyped and were found mostly to be CD8⁺ effector memory T cells (CCR7 CD45RA ) (Figure 10G).

To evaluate the cytotoxicity of the expanded CAR-T cells, in vitro cytotoxicity assays were performed using the DELFIA cytotoxicity kit. Dose-dependent cell lysis of CD19-positive tumor target cells was observed with anti-CD 19-CAR-T cells expanded with K562B+CD19 cells, but no cytotoxicity against CD19-negative tumor cells was displayed by these T cells (Figures 10H and 101, circles), which demonstrated that CD19 antigen-specific tumor cell killing was mediated by the expanded CAR-T cells.

The results demonstrated that B2M knockout in K562 cells are able to be used as aAPCs for expansion of antigen-specific T cells.

Example 6: Conclusions

The K562 leukemia cell line is broadly used to construct aAPCs for immune cell expansion in vitro due to its property of null MHC class I expression. However, a recent study reported that its MHC class I molecules, at least HLA-C, could be up-regulated during the co-culture with PBMCs for NK cell expansion (26).

In the present study the inventors confirmed the up-regulation of B2M and MHC class I molecules on K562 cells in the condition of K562/T cell co-cultures. The up-regulated MHC class I molecules on K562- based aAPCs can be alloantigens for MHC mismatched donors, activating and triggering specific expansion of alloreactive T cells.

To address this issue, the inventors disrupted the B2M gene in K562 with a baculoviral CRISPR/Cas9 system. A total of five single cell-derived B2M knockout K562 clones were selected and characterized, and were confirmed not to express MHC class I expression even after stimulation with IFN-g or T cell/K562 co-culture supernatants.

A K562-based aAPC was generated from one of the B2M knockout clones by introduction of essential costimulatory molecules with ZFNs mediated AA\/S1 site-specific integration. T cells co-cultured with this B2M knockout K562-based aAPC showed attenuated alloreactivity against K562 cells and target cells expressing an HLA-C allele expressed by K562. The B2MKO K562-based aAPC was also shown to support robust antigen-independent T cell expansion and antigen-specific anti-CD19 CAR-T cell expansion.

K562 cells were first reported to be devoid of MHC class I molecule expression and to bear only traces of endogenous B2M expression (22, 47, 48). Based on these properties and other advantageous properties as feeder cells, K562 cells are widely used as feeder cells for immune cell expansion in vitro.

Many studies have claimed that K562 cells are HLA-negative. However, reduced B2M expression is associated with a t(15; 18) chromosome translocation near B2M gene (45) but the cell line still bears wildtype human B2M alleles, which means it could express or up-regulate B2M in certain conditions.

Previous studies have shown that IFN-g can up-regulate MHC class I molecule expression on K562 cells, mostly HLA-C (42, 48, 49). Lapteva et al. also reported the up-regulation of MHC class I molecules on K562 in co-culture with NK cells (26). The present inventors also observed up-regulation of both B2M and MHC class I molecules on K562 cells in co-culture with T cells. Such up-regulation could be induced by cytokines such as IFN-g, which are secreted by T cells or other immune cells in the co-culture environment. The inventors also showed that K562 inactivated by treatment with mitomycin C or g- irradiation can still upregulate B2M and MHC class I expression in response to this stimulus (irrespective of inhibition of proliferation).

Mitomycin C treatment and g-irradiation were also found to induce moderate upregulation of the expression of B2M and MHC class I on K562 cells, in agreement with the findings of previous studies (50, 51 ). This could be a consequence of accumulated DNA and cytoplasmic protein damage within K562 cells after mitomycin C treatment or g-irradiation. Intracellular damage may trigger up-regulation of MHC complex to present abnormal peptides to circulating immune cells.

Since K562 cells can upregulate MHC class I molecule expression in co-culture conditions, K562-based aAPCs may induce the expansion of allospecific T cells during T cell expansion (specific for the MHC class I molecule(s) expressed by the K562 cells). The alloresponses of T cells against K562 could occur either in a direct or an indirect manner (52). As a direct allorecognition, alloreactive T cells could be activated when their TCRs recognise an allo-peptide-MHC complex on K562 cells directly. As an indirect allorecognition, dendritic cells and other monocytes within PBMCs would first take up K562 cell alloantigens and then help present K562 alloantigens to activate T cells. The alloreactive T cells will subsequently be highly expanded due to coexistence of allo-antigens and co-stimulatory molecules on K562-based aAPCs in a T cell expansion scenario. Lapteva et al. detected alloreactive CD8⁺ T cells triggered and expanded by K562-based aAPC after K562/PBMC co-cultures and even confirmed their alloresponses against K562’s HLA-C*05 (26). Allorecognition of HLA-C is less common among MHC molecule mismatches, but it still happens frequently (53).

The inventors also detected such alloreactivity against K562's HLA-C after K562/PBMC co-cultures from at least three donors whose HLA-C is mismatched with K562 HLA-C (Figures 8A and 8B). The inventors expanded alloreactive T cell populations with wildtype (K562A) and B2MKO (K562B) K562-based aAPCs respectively. Their studies demonstrate that B2M knockout K562-based aAPC can attenuate the alloreactivity during expansion. The inventors observed the preferable expansion of alloreactive T cells with wildtype K562-based aAPC but not with B2M knockout aAPC. The expanded alloreactive T cells were terminally differentiated and activated effector memory T cells with high expression of CD8, CD86 and HLA-DR. The cytotoxicity of the expanded T cells was at least restricted to K562's HLA-C alleles HLA-C*03 and HLA-C*05. The alloreactive T cells were found not to target cell lines expressing e.g. HLA- C*07, HLA-C*12 or HLA-C*16. The alloreactive T cells may display some cross-reactivity against HLA- C*02 or C*04 because they exhibit a moderate cytolytic effect on MDA-MB-468 (HLA-C*02 *04) (Data not shown). Therefore, B2M knockout is necessary for K562 cells to generate aAPCs with minimal immunogenicity and ensure the specificity of T cell expansion.

Many approaches have been investigated to reduce the immunogenicity of specific cells. HLA disruption is a direct method and it was achieved on T cells, human embryonic stem cells (hESCs) (54) and hematopoietic stem cells (55). However, this strategy is not universal due to the high level of

polymorphism in MHC class I molecules. B2M knockout is an alternative choice since this microglobulin is conserved and indispensable for the formation of MHC class I molecules. Reduction of the expression of MHC class I molecules and B2M have been achieved in hESCs after B2M knockout and no abnormality was reported on those B2M knockout cells (29, 30).

In the present Examples the inventors generated B2M knockout K562 cells at single cell level. Apart from the site-specific integration to disrupt B2M gene, deletions and insertions were also introduced into B2M EX1 coding sequence by CRISPR/Cas9, which would lead to a reading frame shift and an early stop codon in the new reading frame. As a result, expression of B2M and MHC class I was fully depleted in B2M knockout K562 clones. It is reported that K562 cell line has two normal copies of human chromosome 15 and a t(15; 18) chromosome translocation (45), which suggests three copies of B2M gene in K562 cells. Within each of the knockout single cell clones, besides the integration of selection marker, at most two types of B2M mutant alleles were detected. This finding is consistent with there being three copies of B2M gene in K562 cells, and that all the three copies were disrupted in the B2M deficient clones. In addition, the B2M knockout clones were cultured for more than sixty passages without abnormality compared to wildtype K562 cells, suggesting that the B2M knockout is a safe and efficacious manner by which to generate stable hypoimmunogenic cells. B2M knockout could make cells more sensitive to NK cell lysis due to lack of inhibitory ligands against NK cells (46), but this is not an important consideration for cells to be used as feeder cells in methods for T cell expansion expansion because new aAPCs can be added as necessary. Also, NK cells would be more easily activated by B2M knockout K562 cells, which means that B2M knockout K562-based aAPCs may benefit NK cell expansion.

In the present Examples two different strategies were used to knockout B2M gene in K562. One strategy used a CRSIPR/Cas9 system which targeted B2M EX1 directly introduced into the K562 cells by electroporation, and the other strategy used a CRSIPR/Cas9 system to target B2M EX1 and EX2 simultaneously, which was introduced into K562 by baculoviral transduction. Mutations were only observed within EX1 region in disrupted B2M alleles (Figure 4B), which indicates the disruption efficacy of EX1 target was higher than the EX2 target. Consistently, this EX1 target sequence was also reported with high efficacy by Mandal et al. (34). The CRISPR/Nickase system (56, 57), CRISPR/Cpfl (58) or fusion with additional DNA-binding-domain (59) or Fokl nuclease (60, 61 ), could be used to reduce the random genome damage and facilitate the precise gene knockout.

For genome modification, some safe gene harbors have been identified and used for exogenous gene expression. The AA\/S1 locus has been investigated, and stable AA\/S1 site-specific integration has demonstrated in previous studies (36-38). In the present examples the AA\/S1 site was used for stable expression of integrated co-stimulatory molecules and this precise engineering did not interfere with the normal function of K562 cells. In addition, the lineage-specific markers CD71 and CD235a were confirmed to be highly expressed on the B2M knockout K562-based aAPCs after genome modification, despite variation in the expression of some markers (Figure 6D). As it is reported that K562 cell line is a mixture of colony-forming erythroid progenitors and erythroblasts (23), the B2M knockout K562-based aAPC could be derived from a more mature erythroblast of the K562 cell line due to two rounds of single cell selection. This single cell selection may contribute to the changes of some of the cell markers' expression patterns and narrow their spectrums down to a certain level.

K562 cells have been used extensively as aAPCs for T cell expansion in vitro (8-11 ). In the present examples the inventors demonstrate that B2MKO K562-based aAPC can support robust T cell expansion as good as the wild type K562-based aAPCs in an either antigen-independent or -dependent manner. According to the methods described herein thousands fold of T cells can be expanded in vitro within three weeks from PBMCs in an antigen-independent scenario. Expanded T cells are activated by anti-CD3 antibody, OKT-3, and co-stimulatory molecules on B2M knockout K562-based aAPCs. CD64 as an Fc receptor helps the binding of OKT-3 to activate T cells. CD86 and CD137L are the ligands of CD28 and 4- 1 BB (CD137) on T cells to help activation as secondary signals and help to maintain function and survival of T cells. Membrane-bound IL-21 was also introduced into the K562-based aAPCs to support the proliferation and expansion of T cells, in particular CD8⁺ T cells (17). In addition, when CD19 antigen was introduced into B2MKO K562-based aAPCs, the aAPCs were useful for enriching and expanding anti- CD19 CAR-T cells efficiently, and the expanded CAR-T cells displayed potent antigen-specific cytotoxicity. References:

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Claims

Claims:

1. A modified K562 cell having reduced expression of MHC class I as compared to a wildtype K562 cell.

2. The modified K562 cell according to claim 1 , comprising modification to a gene encoding an MHC class I polypeptide relative to a wildtype K562 cell.

3. The modified K562 cell according to claim 2, wherein the modification reduces or prevents expression of a polypeptide encoded by the gene encoding an MHC class I polypeptide.

4. The modified K562 cell according to claim 2 or claim 3, wherein the gene encoding an MHC class I polypeptide is B2M.

5. The modified K562 cell according to any one of claims 1 to 4, additionally comprising modification to increase expression of one or more factors capable of increasing immune cell activation or proliferation.

6. The modified K562 cell according to claim 5, which comprises nucleic acid encoding the one or more factors capable of increasing immune cell activation/proliferation.

7. The modified K562 cell according to claim 5 or claim 6, wherein the one or more factors capable of increasing immune cell activation/proliferation are selected from: a costimulatory molecule, a cytokine or an antigen.

8. The modified K562 cell according to claim 7, wherein the costimulatory molecule is selected from CD40L, CD86, CD137L, CD80 or CD83.

9. The modified K562 cell according to claim 7 or claim 8, wherein the cytokine is selected from IL-21 , IL- 15, membrane-bound IL-21 and membrane-bound 11-15.

10. The modified K562 cell according to any one of claims 1 to 9, additionally comprising modification to increase expression of one or more Fc receptors.

1 1. A modified K562 cell comprising modification to reduce or prevent expression of a polypeptide encoded by B2M.

12. The modified K562 cell according to claim 1 1 , additionally comprising modification to increase expression of one or more of: CD64, CD86, CD137L and membrane-bound IL-21.

13. The modified K562 cell according to claim 1 1 or claim 12, additionally comprising modification to increase expression of an antigen.

14. The modified K562 cell according to any one of claims 1 1 to 13, wherein the modified K562 cell comprises modification to increase expression of CD 19.

15. A method for producing a modified K562 cell having reduced expression of MHC class I as compared to a wildtype K562 cell, comprising modifying a K562 cell to reduce or prevent expression of MHC class I.

16. The method according to claim 15, wherein the modification reduces or prevents expression of a polypeptide encoded by a gene encoding an MHC class I polypeptide.

17. The method according to claim 15 or 16, wherein the gene encoding an MHC class I polypeptide is B2M.

18. The method according to any one of claims 15 to 17, additionally comprising modifying the K562 cell to increase expression of one or more factors capable of increasing immune cell activation or proliferation.

19. The method according to claim 18, comprising introducing into the K562 cell nucleic acid encoding the one or more factors capable of increasing immune cell activation/proliferation.

20. The method according to claim 18 or claim 19, wherein the one or more factors capable of increasing immune cell activation/proliferation are selected from: a costimulatory molecule, a cytokine or an antigen.

21. The method according to claim 20, wherein the costimulatory molecule is selected from CD40L,

CD86, CD137L, CD80 or CD83.

22. The method according to claim 20 or claim 21 , wherein the cytokine is selected from IL-21 , IL-15, membrane-bound IL-21 and membrane-bound IL-15.

23. The method according to any one of claims 20 to 22, comprising introducing into the K562 cell nucleic acid encoding one or more Fc receptors.

24. The method according to any one of claims 20 to 23, additionally comprising modifying the K562 cell to increase expression of an antigen.

25. A modified K562 cell obtained or obtainable by the method according to any one of claims 15 to 24.

26. A method for generating or expanding a population of immune cells, comprising contacting immune cells in vitro, in vivo or ex vivo with a modified K562 cell according to any one of claims 1 to 14 or claim 25.

27. The method according to claim 26, which is a method for generating or expanding a population of antigen-specific immune cells, wherein the method comprises culturing immune cells in the presence of a modified K562 cell according to any one of claims 1 to 14 or claim 25 comprising or expressing the antigen.

28. The method according to claim 27, wherein the antigen-specific immune cells are CAR-modified immune cells, and wherein the modified K562 cell comprises or expresses the antigen for which the CAR is specific.

29. A population of immune cells generated or expanded by the method according to any one of claims 26 to 28.

30. The population of immune cells according to claim 29 for use in a method of medical treatment or prophylaxis of a disease or condition.

31. Use of the population of immune cells according to claim 29 in the manufacture of a medicament for use in a method of medical treatment or prophylaxis of a disease or condition.

32. A method of treating or preventing a disease or condition in a subject, comprising administering a population of immune cells according to claim 29 to a subject.

33. A method of treating or preventing a disease or condition in a subject, comprising:

(a) isolating immune cells from a subject;

(b) generating or expanding a population of immune cells by culturing the immune cells isolated at step (a) in the presence of a modified K562 cell according to any one of claims 1 to 14 or claim 25; and

(c) administering the population of immune cells generated or expanded at step (b) to a subject.

34. The population of immune cells for use according to claim 30, the use according to claim 31 , or the method according to any claim 32 or claim 33, wherein the disease or condition is a T cell dysfunctional disorder, a cancer or an infectious disease.

35. The method according to claim 34, wherein the cancer is selected from the group consisting of: colon cancer, colon carcinoma, colorectal cancer, nasopharyngeal carcinoma, cervical carcinoma, oropharyngeal carcinoma, gastric carcinoma, 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, urothelial carcinoma, melanoma, advanced melanoma, renal cell carcinoma, ovarian cancer or mesothelioma.

36. A nucleic acid, or a plurality of nucleic acids, encoding a site-specific nuclease (SSN) system targeting B2M.

37. The nucleic acid or plurality of nucleic acids according to claim 36, wherein the nucleic acid or plurality of nucleic acids encodes a CRISPR/Cas9 system.

38. The nucleic acid or plurality of nucleic acids according to claim 37, wherein the nucleic acid or plurality of nucleic acids encodes a CRISPR RNA (crRNA) targeting an exon of B2M.

39. The nucleic acid or plurality of nucleic acids according to claim 37 or claim 38, wherein the nucleic acid or plurality of nucleic acids encodes a crRNA targeting exon 1 and/or a crRNA targeting exon 2 of B2M.

40. A vector, or a plurality of vectors, encoding the nucleic acid or plurality of nucleic acids according to any one of claims 36 to 39.

41. A method for producing a modified cell having reduced expression of MHC class I as compared to a comparable non-modified cell, comprising introducing into a cell modifying the nucleic acid or plurality of nucleic acids according to any one of claims 36 to 39, or a vector or a plurality of vectors according to claim 40.

42. The method of claim 41 , wherein the modified cell is a modified K562 cell.