Immunologically distinguishable cell surface variants for use in cell therapy

Abstract

To provide a mammalian cell, particularly a human cell, expressing a first isoform of a surface protein, for use in treatment of a patient having cells expressing a second isoform of the surface protein.SOLUTION: The first isoform and the second isoform are functionally indistinguishable but immunologically distinguishable. The invention further provides an agent for use in a method of treating a medical condition, where the agent is selected from (1) and (2) in the following: (1) a compound comprising or consisting of an antibody or antibody-like molecule; and (2) an immune effector cell bearing an antibody-like molecule or an immune effector cell bearing a chimeric antigen receptor. The agent is specifically reactive to either a first or a second isoform of a surface protein. The agent is administered to ablate a cell bearing the isoform that the agent is reactive to.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the use of cells bearing mutant but functional cell surface proteins in medical applications where selective depletion or enrichment of cell populations is desired. The mutant but functional cell surface proteins can be introduced into cells by gene editing methods such as homologous recombination repair of DNA double strand breaks (in particular CRISPR / Cas gene editing) or by using base editors. The present invention further relates to agents and methods for selectively depleting edited cells in vivo. [Background technology]

[0002] Cell therapy is a very powerful treatment option, but is often associated with severe and unwanted side effects. Transgenes and / or genetic manipulation of the cells may lead to malignant transformation. The introduction of CAR-T cells may result in severe on-target and off-target effects (cytokine release syndrome). The introduction of allogeneic T cells may also lead to graft-versus-host disease (GvHD). The success of cell therapy in oncology is likely to promote cell therapy for other indications, including non-malignant diseases. To increase the safety of cell therapy, especially when used to treat non-lethal diseases, it is important to ensure that the transplanted cells are safe for several years after transplantation. The safety of cell therapy would be significantly increased if there was a possibility to actively and selectively deplete the transplanted cells by a "safety switch" or "kill switch" in the event of severe unwanted side effects.

[0003] The underlying problem of the present invention is to provide a system for permanently labeling and tracking cells, which allows for selective depletion of labeled or unlabeled cells in vitro or in vivo. These problems are solved by the features of the independent claims. Summary of the Invention

[0004] A first aspect of the invention relates to a mammalian cell expressing a first isoform of a surface protein, the first isoform of the surface protein is functionally indistinguishable from a second isoform of the surface protein, but is immunologically distinguishable; The mammalian cell is for use in treating a patient whose cells express the second form of the surface protein.

[0005] The phrase "immunologically distinct" refers to a first and a second isoform of a surface protein that can be distinguished by a ligand that specifically binds to either the first or the second isoform.

[0006] In the context of this specification, the expression "specific binding" refers to a dissociation constant: K D <= 10 -7 represents a bond where

[0007] In the present context, the expression "ligand" relates to an antibody or an antibody-like molecule. The antibody or antibody-like molecule may be linked to another molecule (e.g., to an immunotoxin). Alternatively, the antibody or antibody-like molecule may be present on the surface of a cell, in particular an immune cell.

[0008] In the context of this specification, the term "antibody" is used in the sense known in the art of cell biology and immunology. This term refers to (i) full-length antibodies, including but not limited to immunoglobulin types G (IgG), A (IgA), D (IgD), E (IgE) or M (IgM), (ii) any antigen-binding fragments or single chains thereof, and (iii) constructs related thereto or derived therefrom. A full-length antibody is a glycoprotein that contains two or more heavy chains (H chains) and two or more light chains (L chains), which are interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain constant region is composed of one domain (CL). The variable regions of the heavy and light chains have a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues (or host factors). Such host tissues or host factors include various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

[0009] In the context of this specification, the term "antibody-like molecule" refers to a molecule that is capable of specifically binding to another molecule or target with high affinity (K D <= 10 -7 mol / L, especially K D <= 10 -8mol / L). Antibody-like molecules bind to targets in a manner similar to the specific binding of antibodies. The term "antibody-like molecules" includes repeat-containing proteins (such as engineered ankyrin repeat-containing proteins (Molecular Partners, Zuerich)), polypeptides derived from armadillo repeat-containing proteins, polypeptides derived from leucine-rich repeat-containing proteins, Affimers, antibody-derived molecules (such as chimeric antigen receptors (CARs)), and polypeptides derived from tetratricopeptide repeat-containing proteins.

[0010] The term "antibody-like molecule" further includes polypeptides derived from protein A domains, polypeptides derived from domain FN3 of fibronectin, polypeptides derived from consensus fibronectin domains, polypeptides derived from lipocalins, polypeptides derived from zinc fingers, polypeptides derived from Src homology domain 2 (SH2), polypeptides derived from Src homology domain 3 (SH3), polypeptides derived from PDZ domains, polypeptides derived from gamma crystallins, polypeptides derived from ubiquitin, polypeptides derived from cystine-knot polypeptides, and polypeptides derived from knottins.

[0011] Ideally, the first and second isoforms of a surface protein can be distinguished by two ligands. One ligand can specifically recognize the first isoform. The other ligand can specifically recognize the second isoform. That is, each ligand can specifically bind to one isoform but not the other. In other words, the ligand can distinguish between the two isoforms by specifically binding to only one isoform and not the other.

[0012] The term "functionally indistinguishable" refers to the first and second isoforms being capable of performing the same function in a cell in the same manner, without significant functional impairment. That is, the first and second isoforms are functionally largely indistinguishable. In certain embodiments, minor functional impairment may be tolerated.

[0013] In the context of this specification, the expression "a first and / or a second isoform of a cell surface protein" refers to a first allele and a second allele of said cell surface protein.

[0014] In certain embodiments, the mammalian cell is a human cell.

[0015] In certain embodiments, the second isoform of the surface protein relates to the wild-type form of the protein (i.e., the form that normally occurs in nature), whereas the first isoform relates to an isoform obtained by introducing a mutation into the nucleic acid sequence encoding the second isoform.

[0016] In certain embodiments, the second isoform of the surface protein is a naturally occurring isoform, while the first isoform is an engineered isoform that is derived from the naturally occurring isoform.

[0017] In the context of this specification, the expression "native protein" refers to a protein that is encoded by a nucleic acid sequence in the genome of a cell. This nucleic acid sequence has not been inserted or mutated by genetic engineering. In other words, a native protein is a protein that is not a recombinant or engineered protein.

[0018] In certain embodiments, the surface protein has an extracellular polypeptide sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 amino acid insertions, deletions, and / or substitutions when comparing the first isoform to the second isoform.

[0019] In certain embodiments, the surface protein comprises an extracellular polypeptide sequence in which between 1 and 20 (particularly between 1 and 5, more particularly between 1 and 3) amino acids are inserted, deleted and / or substituted when comparing the first isoform to the second isoform.

[0020] In certain embodiments, the surface protein has an extracellular polypeptide sequence, where the first isoform is compared to the second isoform and has a single amino acid insertion, deletion and / or substitution.

[0021] In certain embodiments, the insertions, deletions, and / or substitutions are located at sites that are not conserved among different mammalian species.

[0022] In certain embodiments, the insertions, deletions and / or substitutions do not alter the secondary structure of the surface protein.

[0023] In certain embodiments, the insertions, deletions and / or substitutions are located at potential ligand binding sites identified by crystallography or computer-aided structure prediction.

[0024] In certain embodiments, the insertions, deletions and / or substitutions are located at sites that have a unique topology compared to other mammalian proteins, as identified by crystallography or computer-aided structure prediction.

[0025] In the context of this specification, the expression "unique topology" refers to a topology that is present only in one surface protein (which is modified by insertion, deletion and / or substitution of 1 to 20 amino acids) and is not present in other mammalian proteins (in particular, not present in other human surface proteins). The presence of an identical or very similar topology in other proteins would prevent the production of specific antibodies that recognize the epitope located at said site.

[0026] In certain embodiments, the insertions, deletions, and / or substitutions are not located at sites of the surface protein involved in predicted (or experimentally established or confirmed) protein-protein interactions.

[0027] In certain embodiments, the insertion, deletion and / or substitution does not eliminate or introduce disulfide bonds, intermolecular or intramolecular interactions, hydrophobic stacking. When the insertion, deletion and / or substitution eliminates salt-bridge intermolecular or intramolecular interactions, it must be confirmed that the elimination of the salt-bridge interaction is compensated for by new interactions in the protein. If not, the elimination of the salt-bridge interaction should be avoided.

[0028] In certain embodiments, the insertions, deletions and / or substitutions do not remove or introduce sites of post-translational modification of the protein that are important for protein folding, in particular glycosylation sites.

[0029] In certain embodiments, the insertions, deletions and / or substitutions remove or introduce sites of post-translational modification of the protein that are not involved in protein folding, particularly glycosylation sites, thereby creating novel epitopes.

[0030] In certain embodiments, the first and second isoforms are distinguishable by the binding of an antibody-like molecule or by the binding of an antibody.

[0031] In certain embodiments, the first and second isoforms can be distinguished by the response of immune effector cells bearing antibodies. In certain embodiments, the first and second isoforms can be distinguished by the response of immune effector cells bearing antibody-like molecules. In certain embodiments, the first and second isoforms can be distinguished by the response of immune effector cells bearing antibodies. In certain embodiments, the first and second isoforms can be distinguished by the response of immune effector cells bearing antibody-like molecules. In certain embodiments, the first and second isoforms can be distinguished by the response of T cells (particularly activated T cells) bearing a chimeric antigen receptor (CAR).

[0032] In the context of this specification, chimeric antigen receptor (CAR) refers to an engineered receptor that transfers binding specificity (particularly that of a monoclonal antibody) to a T cell. A common form of CAR includes (i) an extracellular domain derived from a monoclonal antibody having the desired binding specificity, (ii) a transmembrane domain, and (iii) an intracellular domain (Gill and June, Imm Rev, 2014). The extracellular domain includes a single chain variable fragment that includes the variable regions of the heavy and light chains of an immunoglobulin. The nucleotide sequence encoding the extracellular domain of the CAR may be derived from a hybridoma cell that produces an antibody having the desired binding specificity (Gill and June, Imm Rev, 2014; Fields, Nat Prot, 2013).

[0033] In certain embodiments, the surface protein is selected from: CD1a、CD1b、CD1c、CD1e、CD1e、CD2、CD3、CD3d、CD3e、CD3g、CD4、CD5、CD6、CD7、CD8a、CD8b、CD9、CD10、CD11a、CD11b、CD11c、CD11d、CDwl2、CD13、CD14、CD15、CD15u、CD15s、CD15su、CD16、CD16b、CD17、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD31、CD32、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、CD42b、CD42c、CD42d、CD43、CD44、CD45、CD45RA、CD45RB、CD45RC、CD45RO、CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、CD49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55、CD56、CD57、CD58、CD59、CD60a、CD60b、CD60c、CD61、CD62E、CD62L、CD62P、CD63、CD64、CD65、CD65s、CD66a、CD66b、CD66c、CD66d、CD66e、CD66f、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85a、CD85d、CD85j、CD85k、CD86、CD87、CD88、CD89、CD90、CD91、CD92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD99R、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD107b、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158e、CD158i、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210b、CD212、CD213a1、CD213a2、CD215、CD217a、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240DCE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD266、CD267、CD268、CD269(BCMA)、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD286、CD289、CD290、CD292、CDw293、CD294、CD295、CD296、CD297、CD298、CD299、CD300a、CD300c、CD300e、CD301、CD302、CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD31 7, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD 336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, immunoglobulin light chains (lambda or kappa), HLA proteins, and β2-microglobulin.

[0034] In the context of this specification, HLA stands for "human leukocyte antigens." HLA includes HLA-A, HLA.B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR.

[0035] In certain embodiments, the surface protein is selected from: CD2, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD23, CD33, CD34, CD90, CD45, CD123, CD269 (BCMA), immunoglobulin light chains (lambda or kappa), HLA proteins and β2-microglobulin.

[0036] In certain embodiments, the surface proteins are selected from CD45, CD3, CD4, CD8a, CD8b, and CD279.

[0037] In certain embodiments, the surface protein is selected from CD45, CD45RA and CD45RO.

[0038] In certain embodiments, the surface proteins are selected from CD45, CD34, CD38, CD59, CD90, and CD117.

[0039] In certain embodiments, the surface protein is selected from CD45, CD19, CD20, CD22, CD22, CD23, CD38, CD138, CD268, CD269 (BCMA), and CD319.

[0040] In certain embodiments, the surface proteins are selected from CD5, CD19, CD20, CD33, CD123, CD38 and CD269.

[0041] In certain embodiments, the surface proteins are selected from CD45, CD19, CD4 and CD8.

[0042] In a specific embodiment, the surface protein is CD45.

[0043] In a specific embodiment, the surface protein is Thy1 (CD90).

[0044] In a specific embodiment, the surface protein is CD19.

[0045] In the context of this specification, "Thy1" stands for "thymus cell antigen 1, theta" (also known as CD90; UniProt ID P04216: (human)).

[0046] In the context of this specification, "CD45" stands for "protein tyrosine phosphatase, receptor type, C (Ptprc)" (UniProt ID: P08575 (human)).

[0047] CD45 and CD90 in the animal experiments in the Examples represent mouse homologs of human genes and human proteins. In claims 30 to 31 and 34 to 36, the mouse homologs "mouse CD45" and "mouse CD90 / Thy1" are particularly represented.

[0048] In certain embodiments, the surface protein is CD4. In certain embodiments, the surface protein is CD2. In certain embodiments, the surface protein is CD8. In certain embodiments, the surface protein is an HLA protein.

[0049] In certain embodiments, the first isoform of the surface protein is not encoded in the patient's original genomic DNA.

[0050] In certain embodiments, the cells are allogeneic cells. Allogeneic cells refer to cells derived from a donor that is genetically similar to the recipient of the cells. The donor may or may not be related.

[0051] In certain embodiments, the cells are autologous cells. Allogeneic cells refer to cells that are derived from the same individual as the recipient of the cells.

[0052] In a particular embodiment, the first isoform was obtained by altering the sequence encoding the surface protein in the patient's original genomic DNA, inducing amino acid insertions, deletions and / or substitutions.

[0053] In a particular embodiment, the first isoform was obtained by altering the mRNA encoding the surface protein using RNA editing techniques (Zhang, 2017), which does not alter the genomic DNA but inserts, deletes and / or substitutes amino acids in the amino acid sequence of the surface protein.

[0054] In certain embodiments, the first isoform is obtained by inducing an insertion, deletion and / or substitution of 1, 2, 3, 4 or 5 (or 6, 7, 8, 9, 10, 11, 12, 15 or 20) amino acids in the amino acid sequence of the second isoform of the surface protein.

[0055] In a particular embodiment, the first isoform is obtained by inducing an insertion, deletion and / or substitution of 1 to 20 (particularly 1 to 5, more particularly 1 to 3) amino acids in the amino acid sequence of the second isoform of the surface protein.

[0056] In a particular embodiment, the first isoform was obtained by introducing an insertion, deletion and / or substitution of a single amino acid in the amino acid sequence of the second isoform of the surface protein.

[0057] Amino acid insertions, deletions and / or substitutions can be achieved by altering the sequence that codes for the surface protein in the patient's original genomic DNA (gene editing) or by altering the mRNA that codes for the surface protein (RNA editing). Both forms of editing result in changes to the amino acid sequence of the protein.

[0058] In certain embodiments, the insertion, deletion, and / or substitution is performed by the following steps: (a) comparing (aligning) one or several homologous sequences of different mammalian species (in particular mouse, rat, primate and human homologs) and locating insertions, deletions and / or substitutions at non-conserved sites; (b) selecting insertions, deletions and / or substitutions that are predicted by crystal structure analysis or computer-aided structure prediction to not alter the secondary structure of the region to be inserted, deleted and / or substituted; (c) selecting insertions, deletions and / or substitutions located at potential ligand binding sites according to crystallography computer-aided structure prediction; (d) selecting insertions, deletions and / or substitutions in the sequence that are not involved in predicted or experimentally established protein-protein interactions of said surface proteins; (e) selecting insertions, deletions and / or substitutions that do not eliminate or introduce disulfide bonds, intermolecular or intramolecular interactions, hydrophobic stacking; or (f) selecting insertions, deletions and / or substitutions that do not remove or introduce sites of post-translational modification of the protein that are important for protein folding, in particular glycosylation sites.

[0059] In certain embodiments, the insertion, deletion, and / or substitution is performed by the following steps: (a) comparing (aligning) one or several homologous sequences of different mammalian species (in particular mouse, rat, primate and human homologs) and locating insertions, deletions and / or substitutions at non-conserved sites; (b) selecting insertions, deletions and / or substitutions that are predicted by crystal structure analysis or computer-aided structure prediction to not alter the secondary structure of the region to be inserted, deleted and / or substituted; (c) selecting insertions, deletions and / or substitutions located at potential ligand binding sites according to crystallography computer-aided structure prediction; (d) selecting insertions, deletions and / or substitutions in the sequence that are not involved in predicted or experimentally established protein-protein interactions of said surface proteins; (e) selecting insertions, deletions and / or substitutions that do not eliminate or introduce disulfide bonds, intermolecular or intramolecular interactions, hydrophobic stacking; and (f) selecting insertions, deletions and / or substitutions that do not remove or introduce sites of post-translational modification of the protein that are important for protein folding, in particular glycosylation sites.

[0060] In certain embodiments, the insertions, deletions and / or substitutions are selected to be located at sites that have a unique topology compared to other mammalian proteins according to crystal structure analysis or computer-aided structure prediction.

[0061] In the context of this specification, non-conserved sites refer to sites that have frequently undergone mutations during evolution (as deduced from multiple sequence alignments (MSA) of a large number of homologous sequences). Site-specific conservation indicates the presence of functional or structural constraints acting at a particular site, which allows assessment of their importance in preserving the structure or function of the protein. The degree of solvent accessibility at each site is predicted (or observed) from the available experimentally determined structures. This degree of solvent accessibility allows distinction between structurally important sites (highly conserved and often internal) and functionally important sites (involved in ligand binding, substrate binding or protein-protein interactions, also highly conserved and exposed). Thus, in the context of this specification, non-conserved sites refer to sites that are less evolutionarily conserved and solvent accessible.

[0062] Methods for searching similar sequences are well established and routinely used to estimate homology, including the widely used BLAST as well as more sensitive profile-based and hidden Markov model-based methods (PSI-BLAST, HMMER, HHblits, etc.).

[0063] By effectively combining local and global alignment information, multiple sequence alignments of homologous sequences can be obtained (as T-COFFEE does), or, where possible, by incorporating structural information to build the MSA (MAFFT, PROMALS3D, 3D Coffee).

[0064] Effective methods to estimate site-specific conservation from MSA are based on Shannon entropy measures, similarity-based matrices (i.e., BLOSUM), or stochastic evolution models, such as maximum likelihood and empirical Bayes paradigms (Rate4Site).

[0065] Effective methods for predicting the three-dimensional structure of proteins include, but are not limited to, comparison-based methods (such as SWISS-MODEL, MODELLER, Raptorx, and IntFOLD).

[0066] Methods for predicting B cell epitopes utilize the physicochemical properties of amino acids (i.e., hydrophobicity, flexibility, polarity, and exposed surface) to provide a probability, on a residue-by-residue basis, of being part of either a discontinuous or linear epitope. Such tools include, but are not limited to, Ellipro, SEPPA, BepiPred, ABCpred, DiscoTope, and EpiSearch.

[0067] In certain embodiments, the insertions, deletions and / or substitutions are located in extracellular sites of the first surface protein, particularly in extracellular loops. Mutations in the extracellular loops are unlikely to affect the function of the protein.

[0068] If an antibody or antibody-like molecule reactive with a given surface protein already exists, knowledge of the epitope of the given surface protein recognized by this antibody can be used to select sites for insertion, deletion and / or substitution. The isoform of the given protein comprising the epitope bound by the existing antibody corresponds to a second isoform. Also, the new modified isoform of the given protein comprising the modified epitope corresponds to a second isoform.

[0069] CDR modeling is described in [Messih, Bioinformatics, 2014].

[0070] In certain embodiments, the cells are administered prior to, simultaneously with, or after specific removal of cells expressing the second isoform of the surface protein. In certain embodiments, cells expressing the second isoform of the surface protein are removed by administering to the patient an agent selected from an antibody-like molecule, an antibody, an immune effector cell bearing an antibody or antibody-like molecule, and an immune effector cell (particularly a T cell) bearing a chimeric antigen receptor, wherein the agent specifically reacts with the second isoform of the cell surface protein but not with the first isoform.

[0071] In certain embodiments, the cells express an antibody or antibody-like molecule reactive against the second isoform of the surface protein.

[0072] In certain embodiments, the cells express a chimeric antigen receptor reactive to a second isoform of the surface protein.

[0073] In a particular embodiment, the cell expresses an antibody or antibody-like molecule (particularly a chimeric antigen receptor) reactive against a second isoform of a surface protein, said surface protein being selected from CD45, CD19, CD8 and CD4. In particular, the surface protein is CD45.

[0074] In certain embodiments, the cells express: (a) a first isoform of a first surface protein, the first isoform of the first surface protein being functionally indistinguishable from a second isoform of the first surface protein, but immunologically distinguishable from the second isoform of the first surface protein; and (b) a first isoform of a second surface protein, which first isoform of the second surface protein is functionally indistinguishable from a second isoform of the second surface protein, but is immunologically distinguishable from the second isoform of the second surface protein.

[0075] In a specific embodiment, the first surface protein is CD19 and the second surface protein is CD45.

[0076] In certain embodiments, the mammalian cell is selected from the group comprising hematopoietic stem cells (hemoblasts), CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells (Treg), natural killer cells (NK), innate lymphoid cells (ILCs), dendritic cells (DCs), B lymphocytes, mucosal-associated invariant T cells (MAITs), and gamma delta T cells (γδTs).

[0077] In certain embodiments, the mammalian cell is a hematopoietic cell. In certain embodiments, the mammalian cell is a hematopoietic stem cell. In certain embodiments, the mammalian cell is an immune cell. In certain embodiments, the mammalian cell is a T cell. In certain embodiments, the mammalian cell is a B cell.

[0078] In certain embodiments, the cells are genetically modified to correct or counteract a disease-associated genetic abnormality present in the patient. The term "genetic modification to correct a disease-associated genetic abnormality" also includes the insertion of a transgene, the genetic correction of a disease-associated mutation, the deletion of a gene (particularly the deletion of a gene having a disease-associated mutation), the change of an epigenetic modification important for the expression of a gene, or a combination thereof. The term "deletion of a gene" also includes the deletion of the function of a gene (i.e., inhibiting the expression of a gene), for example, by inserting a stop codon in the middle or by inserting a regulatory repressor sequence.

[0079] In certain embodiments, the cell contains a transgene. In certain embodiments, the transgene is a nucleic acid sequence encoding a functional isoform of a protein affected by a disease-associated genetic abnormality. The transgene does not have the disease-associated genetic abnormality.

[0080] In certain embodiments, the cells contain a genetic correction of a disease-associated mutation. When a cell contains a genetic correction of a disease-associated genetic abnormality, a disease-causing mutation in a native gene is corrected using gene editing techniques.

[0081] In the context of this specification, gene editing or genetic engineering refers to the technique of inserting, deleting or replacing nucleic acid sequences in the genome of an organism. Gene editing involves site-specific double-strand breaks in genomic DNA. Alternatively, gene editing involves site-specific single-strand breaks (nicks) in genomic DNA. As a non-limiting example, gene editing can be achieved by (i) HDR after CRISPR / Cas-mediated site-specific double-strand breaks, or (ii) by using site-specific base editors.

[0082] In certain embodiments, the disease-associated genetic abnormality is a mutation in a gene selected from the Foxp3 gene, the CD25 gene, the Stat5b gene, the Stat1 gene, and the Itch gene.

[0083] In certain embodiments, the disease-associated genetic abnormality is a mutation in the Foxp3 gene.

[0084] Certain embodiments are relevant when graft-versus-host disease (GvHD) develops after administration of mammalian cells to a patient. In these cases, the cells are specifically removed by administering to the patient an antibody or antibody-like molecule that specifically binds to a first isoform of a cell surface protein but not to a second isoform. Alternatively, the cells are specifically removed by administering to the patient immune effector cells (particularly CAR-T cells) that specifically react with a first isoform of a cell surface protein but not to a second isoform.

[0085] In certain embodiments, the treatment includes hematopoietic stem cell transplantation.

[0086] In certain embodiments, the treatment includes transplantation of T cells (i.e., T cell therapy).

[0087] In certain embodiments, the treatment includes organ transplantation.

[0088] In certain embodiments, the treatment relates to the treatment of an inherited hematopoietic disorder. Non-limiting examples of inherited hematopoietic disorders include thalassemia.

[0089] In certain embodiments, the treatment relates to the treatment of a T cell mediated disease, particularly an inherited T cell mediated disease, in particular IPEX-like syndrome, CTLA-4 associated immunoregulatory disorder, hemophagocytic syndrome, ALPS syndrome, or syndrome resulting from heterozygous germline PTEN mutations.

[0090] In certain embodiments, the T cell mediated disease is dysimmunoregulation with endocrinopathy-enteropathy (X-linked) syndrome (IPEX, OMIM: http: / / www.omim.org / entry / 304790) or IPEX-like syndrome. In this case, the genomic locus is a mutation in a gene selected from Foxp3, CD25, Stat5b, Stat1 and Itch genes (Verbsky and Chatila, Curr Opin Pediatr. 2013 Dec;25(6):708-14). Mutations in these genes prevent expression or normal function of the gene product. Editing these genomic loci to eliminate the mutation restores gene and protein expression.

[0091] In certain embodiments, the treatment relates to the treatment of an immune deficiency, in particular severe combined immune deficiency syndrome (SCID).

[0092] In a particular embodiment, the disease-associated genetic abnormality is a mutation in the Foxp3 gene and the disease is dysimmunodeficiency (X-linked) syndrome with endocrinopathy-enteropathy (IPEX). In a particular embodiment, the disease-associated genetic abnormality is a mutation in Foxp3 (particularly Foxp3 K276X This mutation prevents the normal function of the gene product. Editing this genomic location reverts the mutant DNA sequence to the wild-type DNA sequence, thus restoring expression of the Foxp3 gene and Foxp3 protein (Figure 20).

[0093] Although the mechanism is different, IPEX can be considered a malignancy comparable to tumors / cancers due to its aggressive nature, and elimination of pathogenic hematopoietic / immune cells harboring disease-associated genetic abnormalities is justified, at least temporarily.

[0094] In the present specification, the term "Foxp3 gene" refers to "human forkhead box P3, NCBI GENE ID: 50943". In the animal experiments described in the Examples of the present application, mouse Foxp3 k276X The mutation was repaired by gene editing and recapitulates clinically relevant human Foxp3 mutations (Ramsdell et al., Nature reviews. Immunology 14, 343-349 (2014); Lin et al., The Journal of allergy and clinical immunology 116, 1106-1115 (2005)).

[0095] The inventors used gene editing to inhibit Foxp3 in mouse T cells. k276XWe have shown that the mutation can be corrected (Figs. 20, 21, 22, 26). We also demonstrated that the repaired T cells are functional and can suppress other immune cells (Figs. 24, 25). These T cells can be (i) directly repaired, (ii) derived from repaired HSCs, or (iii) iPS-derived T cells. We further demonstrated that adoptively transferred T cells expand in Foxp3-deficient hosts. Up to half of the T cells become Tregs and suppress the disease (Fig. 29). This indicates the feasibility of T cell therapy for scurfy / IPEX syndrome. We have shown that the disease can be prevented by depleting CD4 (Figs. 30-32). Therefore, CD4+ cells are pathogenic cells. Thus, the therapeutic approach would in principle be as follows: First, deplete CD4+ T cells. Foxp3-repaired CD4+ T cells are then transplanted to restore immunological balance. However, both gene-repaired and pathogenic Foxp3-deficient T cells express CD4. Therefore, continuous depletion of CD4+ T cells will kill not only pathogenic cells but also adoptively transferred gene-repaired cells. A possible solution to this would be allelic modification in conjunction with Foxp3 gene correction. We demonstrate the feasibility of modifying the Foxp3 gene to convert CD45.2 to CD45.1 (Figure 20). As a result, antibodies against the native CD45 epitope (e.g., CD45.2) can be used to deplete pathogenic Foxp3-deficient cells. If Foxp3-repaired cells have been converted to modified CD45 alleles (e.g., CD45.1), the transplanted gene-repaired cells are no longer depleted. This allows for simultaneous depletion of pathogenic cells and replenishment of functional repair cells until immune balance is restored, at which point selective elimination of pathogenic host cells can be halted. Indeed, we have shown that CD45 depletion substantially extends survival of Foxp3-deficient mice.

[0096] Furthermore, Foxp3 gene corrected cells not only re-express Foxp3 protein but also up-regulate CD25 (Figures 20, 21, 22, 26). CD25 is a high affinity receptor for interleukin-2 (IL-2) in vitro and in vivo. Physiologically, Foxp3 positively regulates CD25 expression in wild-type T cells, and the IL-2 / CD25 / Stat5 axis is required for regulatory T cell survival. We therefore hypothesized that gene corrected T cells re-expressing Foxp3 could be expanded in vivo by therapeutic administration of IL-2 (Figure 23). It has been well established that T cells can be expanded in vivo by IL-2 / anti-IL-2 mAb complexes, low-dose IL-2 therapy, (modified) IL-2 mutants, and other Treg expansion protocols. Figure 27 shows that the repaired T cells can be expanded in vivo by utilizing IL-2 / anti-IL-2 mAb complexes.

[0097] In a particular embodiment, the disease-associated genetic abnormality is a mutation in the CTLA-4 gene, and the disease is human immune dysregulation syndrome associated with CTLA-4 mutations (Schubert et al., Science Translational Medicine 5, 215ra174-215ra174 (2013); Kuehn et al., Science (New York, NY) 345, 1623-1627 (2014)).

[0098] A second aspect of the invention provides an agent selected from the following for use in the treatment of a medical condition: (a) a compound comprising an antibody or antibody-like molecule, or a compound consisting of an antibody or antibody-like molecule; and (b) An immune effector cell having an antibody-like molecule or an immune effector cell having a chimeric antigen receptor.

[0099] The agent specifically reacts with either a first isoform of a surface protein or a second isoform of the surface protein, the first isoform of the surface protein being functionally indistinguishable but immunologically distinguishable from the second isoform of the surface protein, and administration of the agent results in the elimination of cells carrying the isoform to which the agent reacts.

[0100] In certain embodiments, the medical condition is a hematopoietic disorder.

[0101] In certain embodiments, the medical condition is a hematopoietic malignancy.

[0102] In certain embodiments, the medical condition is a malignant hematopoietic disease that is refractive to treatment with anti-CD19 CAR-T cells (CAR19 cells).

[0103] In certain embodiments, the medical condition is a non-malignant hematopoietic disease.

[0104] In certain embodiments, the medical condition is an autoimmune disease.

[0105] In certain embodiments, the medical condition is graft-versus-host disease (GvHD).

[0106] In the context of this specification, graft-versus-host disease refers to a medical complication following receipt of transplanted tissue from a genetically different individual: immune cells in the donor tissue (the graft) recognize the recipient (the host) as foreign.

[0107] In certain embodiments, the medical condition is graft-versus-host disease resulting from hematopoietic stem cell transplantation.

[0108] In certain embodiments, the medical condition is graft-versus-host disease resulting from adoptive transfer. In the context of this specification, adoptive transfer or adoptive cell therapy refers to the transplantation of human cells (usually immune cells) to a patient. The cells can be autologous or allogeneic. Targeted modifications achieved by gene editing technology allow the transplanted cell product to be remodeled to (i) repair genetic defects, (ii) increase the efficacy of transplanted cells, or (iii) incorporate desired additional features (such as induction molecules or safety switches) into the cells.

[0109] In certain embodiments, the medical condition is graft-versus-host disease resulting from organ transplantation.

[0110] In certain embodiments, the antibody or antibody-like molecule is linked to a toxin, thereby forming an immunotoxin, hi certain embodiments, the antibody or antibody-like molecule is linked to saporin.

[0111] In certain embodiments, the agent is a bispecific antibody or a bispecific antibody-like molecule, that is, the agent is an antibody or antibody-like molecule that can simultaneously bind to two different types of antigens.

[0112] In certain embodiments, the agent is an immune effector cell carrying a bispecific antibody or a bispecific antibody-like molecule.

[0113] In certain embodiments, the agent is an immune effector cell comprising: (a) a first antibody or a first antibody-like molecule that specifically reacts with either a first isoform or a second isoform of a first surface protein, a first antibody or a first antibody-like molecule, wherein the first and second isoforms of the first surface protein are functionally indistinguishable but immunologically distinct; and (b) a second antibody or a second antibody-like molecule that specifically reacts with either the first isoform or the second isoform of the second surface protein, A second antibody or second antibody-like molecule, wherein the first and second isoforms of the second surface protein are functionally indistinguishable but immunologically distinct.

[0114] In a specific embodiment, the first surface protein is CD19.

[0115] In a specific embodiment, the second surface protein is CD45 or CD34.

[0116] In a specific embodiment, the first surface protein is CD19 and the second surface protein is CD45.

[0117] In a particular embodiment, the agent is an immune effector cell bearing an antibody or antibody-like molecule, or an immune effector cell bearing a chimeric antigen receptor (in particular a T cell), for use in a method for the treatment of a hematopoietic malignancy.

[0118] In a particular embodiment, the agent is a T cell bearing a chimeric antigen receptor for CD45 for use in a method for the treatment of a hematopoietic disorder, particularly a malignant hematopoietic disorder.

[0119] CD45 is a particularly suitable target for therapeutic applications in hematopoietic disorders because it is expressed on all hematopoietic cells, including malignant cells, and is important for cell survival, making it very difficult for cells to develop resistance.

[0120] Another aspect of the present invention provides a pharmaceutical combination comprising: (a) a first agent according to the second aspect, which is reactive against a first surface protein; and (b) a second agent according to the second embodiment, which is reactive against a second surface protein.

[0121] In certain embodiments, a combination agent is provided for the treatment of a hematopoietic disorder.

[0122] In certain embodiments, a combination agent is provided for the treatment of a hematopoietic malignancy.

[0123] In certain embodiments, a combination agent is provided for the treatment of a non-malignant hematopoietic disorder.

[0124] In a specific embodiment, the first and second agents are T cells bearing a chimeric antigen receptor.

[0125] In a specific embodiment, the first surface protein is CD19 and the second surface protein is CD45.

[0126] In a specific embodiment, the first agent is an anti-CD19 CAR-T cell and the second agent is an anti-CD45 CAR-T cell.

[0127] In certain embodiments, the hematopoietic malignancy is refractive to treatment with anti-CD19 CAR-T cells (CAR19 cells) alone.

[0128] Another aspect of the invention is to culture a cell expressing the first isoform of the surface protein in A method of tracking in vivo is provided, wherein the first isoform of the surface protein is functionally indistinguishable but immunologically distinguishable from a second isoform of the surface protein, the method comprising administering to the patient a ligand that specifically reacts with the first isoform.

[0129] Alternatively, the above aspect of the invention provides a method of tracking cells expressing a first isoform of a surface protein in tissue derived from a patient, wherein the first isoform of the surface protein is functionally indistinguishable but immunologically distinguishable from a second isoform of the surface protein, the method comprising administering to the tissue derived from the patient a ligand that specifically reacts with the first isoform.

[0130] In a particular embodiment of the above aspect of the present invention, the tissue from the patient is related to a blood sample. Such blood sample may be analyzed by FACS. In a particular embodiment of the above aspect of the present invention, the tissue from the patient is related to an organ tissue sample (e.g., a biopsied liver sample, etc.). Such tissue sample may be analyzed using histological methods.

[0131] Yet another aspect of the present invention provides a method for selectively depleting or enriching cells in vivo, comprising the steps of: (a) providing a cell, the cell expresses a first isoform of the surface protein; the first isoform differs from the second isoform of the surface protein with respect to an amino acid marker; The first isoform comprises an amino acid marker A encoded by a nucleic acid sequence A; the second isoform comprises an amino acid marker B encoded by a nucleic acid sequence B; (b) inducing a mutation from nucleic acid sequence A to nucleic acid sequence B in the genomic DNA of the cell; and (c) selectively enriching / depleting said cells based on expression of said first isoform or said second isoform of said surface protein.

[0132] In the context of this specification, the term "selective depletion of cells" relates to selectively reducing the total number or concentration of cells expressing a certain marker / allele / isoform.

[0133] One of skill in the art will understand that when cells expressing a first isoform and cells expressing a second isoform are contained in a given volume, selective depletion of cells expressing the first isoform corresponds to enrichment of cells expressing the second isoform.

[0134] By way of non-limiting example, selective depletion can be achieved by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-drug conjugates (ADC). Alternatively, selective depletion can be achieved by the reaction of cells (particularly immune receptor cells) bearing natural antigen receptors or chimeric antigen receptors (CARs). Selective depletion can also be achieved by administration of antibodies or antibody-like molecules that are not linked to effector compounds (such as drugs or toxins).

[0135] The inventors have demonstrated that selective depletion in vivo is possible using antibodies against CD45.2 or CD45.1 (Figure 12). Using an antibody against an epitope common to two cell populations (e.g., CD45.2+T cells and CD45.1+T cells) results in non-selective depletion of both cell populations (e.g., depletion against CD4). In contrast, depletion using an antibody that selectively binds to one allele (e.g., CD45.2) but not to the other allele (e.g., CD45.1) of a commonly expressed surface protein (e.g., CD45) depletes only cells expressing the allele that a particular mAb binds. For example, depletion of CD45.2+ cells leaves behind CD45.1+ cells, which are therefore relatively enriched. More efficient depletion can be achieved when a toxin is linked to the mAb.

[0136] The advantage of using antibodies or antibody-like molecules that are not linked to toxins is that treatments using immunotoxins result in the depletion of HSCs, whereas treatments using antibodies or antibody-like molecules that are not linked to toxins leave HSCs, which may be desirable since it results in partial preservation of the hematopoietic system.

[0137] The inventors have demonstrated that single amino acid differences can be introduced into cells and can be distinguished by two different ligands (natural / engineered) that specifically bind to the two isoforms / alleles (see EP16196860.7, EP16196858.1, PCT / EP2017 / 059799). Specifically designed artificial mutations or rare but naturally occurring mutations (e.g., single nucleotide polymorphisms (SNPs)) are introduced into endogenous surface-expressed genes to alter the antigen. As will be appreciated by those skilled in the art, this mutation can be introduced by any method known in the art (such as HDR and base editors). The modified epitope is then utilized to selectively deplete the edited cells with a ligand that specifically and selectively recognizes the artificial epitope. Alternatively, the edited cells are resistant to depletion with a ligand that recognizes the natural epitope but not the modified epitope, and thus the transplanted cells remain (while the host cells can be depleted).

[0138] When the "edited / modified cells (cells in which the first isoform of a cell surface protein has been changed to the second isoform)" are then used for transplantation (especially adoptive transfer), the two different isoforms can be utilized to distinguish between transplanted and host cells. This allows the transplanted cells to be tracked, since they are permanently labeled. Tracking can be achieved either in vivo or ex vivo using labeled ligands (e.g., by flow cytometry or by histochemistry on cells or tissues). When a ligand specific for either the transplanted or host cells is applied in vivo, either the transplanted or host cells can be selectively depleted using a ligand that only binds to the transplanted (modified) or host cells. Selective cell depletion can also be achieved by cells bearing natural or chimeric antigen receptors (CARs) that recognize either the transplanted or host cells.

[0139] The selective depletion of modified cells constitutes an important safety feature by providing a "safety or killing switch". The basic concept of safety switches and suicide genes is explained in [Jones et al., Front Pharmacol.; 5:254. doi: 10.3389]. Our approach is simpler, safer and more versatile. In principle, any adoptively transferred cell can be modified to have a modified allele / epitope and an in vitro or in vivo selection switch, a tracking switch, a safety switch and / or a selective removal switch. Non-limiting examples include (i) cells that only have a modified allele and are otherwise not genetically engineered, and (ii) cells that have additional modified features, such as CAR cells. For example, allogeneic transplanted cells utilized for the graft-versus-leukemia effect may give rise to graft-versus-host disease (GvHD). If the modified allele is incorporated prior to transplantation, the transplanted cells can be purged with the modified allele to alleviate / treat GvHD (Figure 2). Similarly, transplanted autologous tumor-infiltrating lymphocytes (TILs) or pathogen-specific lymphocytes can be modified to carry the modified allele. This allows the transplanted cells to be purged if they cause undesirable side effects due to off-target effects or excessive on-target effects (Figure 2). CARs may be derived from mAbs, combining known mAb specificities with the characteristics of cells (e.g., killer T cells). In principle, the principle of redirecting the cytotoxic (or suppressive) activity of a given cell (T cell) to a specific target antigen by introducing a CAR can be applied to a wide range of diseases (besides malignancies, autoimmune diseases, transplantation or other hematopoietic diseases, etc.). The success of CAR19 T cells shows the potential of CAR cells as therapeutic agents. Importantly, however, although CAR19 T cells are highly effective in curing some types of CD19+ tumors, some types of CAR-T cells that react against a variety of different target molecules can sometimes cause severe and even fatal side effects.Such side effects include cytokine release syndrome and / or neurotoxicity, which have been demonstrated for several CAR constructs targeting CD19, as well as other targets (such as but not limited to CD123). Thus, the ability to control / remove CAR-T cells after transplantation is important (Figures 2, 10, 19). In the case of CAR cells, the modified allele can act as a safety switch. The modified cells can also be eliminated if (possibly after several years) the transplanted modified cells become malignant or cause any kind of unwanted on-target or off-target damage. Alternatively, disease-causing host cells can be selectively eliminated while leaving the autologous modified cells. In contrast to our method, existing technologies are limited to the removal of transplanted cells, and host cells cannot be easily removed. The modified isoform allows the transplantation of autologous cells (e.g., gene-repaired or otherwise modified autologous cells) while removing host cells. Without the isoform switch introduced by the method of the present invention, the removal of host cells would need to be stopped when transplanting healthy cells. In this case, the newly transplanted repaired cells will grow, but the host cells will also grow and can no longer be eliminated, and there is a risk that the disease-causing host cells will eliminate the repaired cells. Therefore, the method of the present invention will make the modified cells resistant to depletion, which is a very meaningful therapeutic approach. For example, the CD19 epitope recognized by anti-CD19-CAR cells could be mutated in autologous hematopoietic cells so that the depleting anti-CD19 mAb or anti-CD19-CAR cells cannot bind to and destroy the modified cells while maintaining the functionality of CD19. This would eliminate the major complications of currently effective anti-CD19-CAR cells. Anti-CD19-CARs have a very high success rate in eliminating malignant hematopoietic tumors expressing CD19, but at the same time, they also lead to the elimination of healthy host cells expressing CD19. This leads to hypogammaglobulinemia and therefore increased risk of infection.Mutant CD19 allows reconstitution of the host immune system with healthy autologous hematopoietic stem cells (HSCs). This generates B cells that are resistant to anti-CD19-CAR cells. Thus, the resistant edited cells provide natural protection against infection, while CAR19 T cells can prevent relapse over time. Therefore, patients would no longer be dependent on IVIG infusions. With non-genotoxic preconditioning (e.g., with antibodies), HSC transplantation could potentially be achieved as partial chimerism (Nat Biotech,. 2016). Alternatively, anti-CD45-CAR cells that recognize epitopes (e.g., CD45.2 analogs) found in the general human population can be used to eliminate all hematopoietic host cells (e.g., hematopoietic cells causing malignant or other diseases). CD45 is a good target because it is expressed on all hematopoietic cells (e.g., most malignant cells). Also, CD45 is important for lymphocyte survival. Therefore, when CD45 is targeted by CAR-T cells, the cells are less likely to downregulate CD45 or mutate CD45 to escape targeting by CAR-T cells. This would reduce the risk of relapse. Transplantation of healthy autologous hematopoietic stem cells (HSCs) or other hematopoietic cells carrying modified CD45 epitopes (e.g., CD45.1) can reconstitute a healthy hematopoietic system in the host that is no longer depleted by anti-CD45-CAR cells (see CD45.2 to CD45.1 switching experiments). A major advantage is the ability to target all malignancies expressing CD45 (including but not limited to T-cell and myeloid malignancies) without the need for tumor- or cell-type-specific antigens. Thus, the present invention provides a universally applicable system for treating malignant hematopoietic tumors and other non-malignant hematopoietic diseases. This overcomes a major obstacle for CAR-T therapy: identifying the appropriate target antigen (Klebanoff, Nat Med 2016). Thus, the application of CAR-T therapy to diseases that respond to CAR-T therapy can be substantially expanded. Although CAR-T therapy has been highly successful in treating CD19+ tumors, it remains a major challenge for other malignant hematopoietic tumors. As an example, the treatment of multiple myeloma remains a major challenge, due in part to the lack of suitable target antigens (Mikkilineni, Blood, 2017; Sadelain, Nature 2017). Also, hematopoietic tumors could be treated without the need for allogeneic cells, thus eliminating GvHD as a major complication, and reconstitution could be initiated during depletion, shortening the time to recovery.Importantly, the mutations utilized to render transplanted cells resistant to depletion can also be utilized to deplete the transplanted cells again if necessary later. CAR cell-dependent depletion of HSCs could be used as an alternative method to achieve gentle (i.e., non-genotoxic) preconditioning. CAR45 cells also eliminate HSCs. The "hit and replace" strategy currently employed for "bone marrow" or HSC transplantation could be achieved using CAR45. Instead of toxic chemotherapy or radiation, patients could be preconditioned with CAR45. The advantage of depleting HSCs and hematopoietic system with CAR45 cells and replacing them with allelically modified resistant HSCs is that the dangerous time after transplantation could be eliminated, since hematopoietic reconstitution could begin during the elimination of unwanted cells. Thus, patients would have a functional immune system at all times, without long-term bone marrow depletion or immune suppression. Thus, our strategy eliminates infection, a major complication of HSC transplantation today. CAR cells directed against an antigen (or combination of antigens) to specifically restrict target cells to HSCs can be used to deplete endogenous HSCs. In a synthetic biology approach (e.g., using an AND gate) to specifically and exclusively direct CAR cells to HSCs, the combination of antigens can be, for example, anti-CD45 or anti-CD34 and a second antigen.

[0140] CAR45 therapy combined with replacement of the hematopoietic system with allelically modified HSCs may be an alternative to CAR19 therapy (since all CD19+ cells also express CD45) and may be used as a combination therapy of CAR19 and CAR45. CAR45 therapy can also be applied to recurrent CD19-negative or CD19-mutated malignancies after CAR19 therapy treatment.

[0141] This aspect of the invention represents a universal strategy for replacing cells. The cells may be autologous or allogeneic hematopoietic cells. When the replacing cells are HSCs, the above method can be used to treat any malignant hematopoietic tumor or other hematopoietic disorder.

[0142] Other advantages of our approach compared to existing "safety switch" approaches include: Our approach uses an endogenous protein and does not require the introduction of a transgene or tag into the cells. The two epitopes are functionally identical but can be distinguished by the ligand that specifically binds them. This approach can deplete either the transplanted or the host cells, depending on which ligand is used. Because the designed mutation is introduced into the genome, the safety feature remains permanently in the cell and cannot be silenced (as would happen with a genetically engineered safety switch introduced by a virus). The modified epitope is also less antigenic than large artificial safety switch / suicide gene constructs. Therefore, the modified epitope is less likely to be rejected by the host cell. Furthermore, the use of modified isoforms relies on targeted mutations. Therefore, it is likely to be safer than other safety switch / suicide genes (which are usually delivered by viruses and randomly integrated into the genome, which can lead to insertional mutagenesis) (Cornu, Nat. Med, 2017).

[0143] Those skilled in the art understand that other methods than HDR can be applied to change cell surface protein from a first isoform to a second isoform.Non-limiting examples include isoform switching using base editors (see Komor et al., Nature 533, 420-424, doi:10.1038 / nature17946).This approach allows editing of desired amino acids without the need for dsDNA cleavage, so safety can be further increased.Base editors or related technologies can be delivered as plasmids or minicircles (dsDNA), mRNA, or RNP.

[0144] When combining the switching of a cell surface protein from a first isoform to a second isoform with the repair of a disease-causing gene (e.g., Foxp3 gene), the depletion of cells expressing the first isoform can result in the depletion of non-repaired cells in vivo (i.e., after introduction into a host). The inventors have demonstrated that in examples where both isoform switching and repair of gene defects are achieved by HDR, the probability of successful gene repair in cells where isoform switching has occurred is increased (Figure 20). By combining isoform switching in a first gene with genetic modification in a second gene, a safety feature can be introduced into the genetically engineered cells.

[0145] The isoform switch can also be used as a marker to track the transplanted edited cells within the host.

[0146] According to another aspect, there is provided a method of selectively depleting or enriching cells in a composition of unedited and edited cells, comprising the steps of: (a) providing a cell, the cell expresses a first isoform of the surface protein; the first isoform differs from the second isoform of the surface protein with respect to an amino acid marker; The first isoform comprises an amino acid marker A encoded by a nucleic acid sequence A; the second isoform comprises an amino acid marker B encoded by a nucleic acid sequence B; (b) inducing in the cell a site-specific genetic exchange of nucleic acid sequence A with nucleic acid sequence B, thereby changing expression of the first isoform to expression of the second isoform in the cell; and (c) selectively enriching / depleting said cells based on expression of said first isoform or said second isoform of said surface protein.

[0147] In certain embodiments, genetic engineering resulting in the insertion, deletion and / or substitution of amino acids is achieved by homology directed repair following a double-stranded break induced by a CRISPR-associated endonuclease (Cas9) and a guide RNA, where the guide RNA can anneal to the first genomic location.

[0148] In the present specification, "CRISPR-associated endonuclease" refers to the Cas9 endonuclease known in the art, which promotes the DNA strand cleavage induced by CRISPR-like sequence.Non-limiting examples of CRISPR-associated endonuclease include Streptococcus pyogenes Cas9 endonuclease (SpyCas9); Francisella Cpf1 endonuclease (FnCpf1), Acidaminococcus Cpf1 endonuclease (AsCpf1) and Lachnospiraceae bacterium Cpf1 endonuclease (LbCpf1); any ortholog of SpyCas9, FnCpf1, AsCpf1 or LbCpf1; or any modified protein of SpyCas9, FnCpf1, AsCpf1 or LbCpf1 or its ortholog; As will be appreciated by those of skill in the art, the present invention also includes newly discovered or engineered CRISPR / Cas mutants.

[0149] In the context of this specification, the term "ortholog" refers to a gene and corresponding polypeptide that have evolved by vertical inheritance from a single ancestral gene. That is, orthologous genes / orthologous polypeptides have a common ancestor and differentiated when a species diverged into two different species. The copies of a single gene in the two species are called orthologs. To confirm that two genes are orthologous, a person skilled in the art can perform phylogenetic analysis of gene lineage by comparing the aligned nucleotide sequences of the genes or the aligned amino acid sequences of the polypeptides.

[0150] In the context of this specification, the term "guide RNA" refers to a synthetic RNA that can guide a CRISPR-associated endonuclease to a predetermined genomic location where the endonuclease cleaves a phosphodiester bond in genomic DNA. As understood by those skilled in the art, when using Cas9 endonuclease, the "guide RNA" expressed can be (i) a single guide RNA (sgRNA) that has both the sequence required for Cas9 binding and a user-defined "target sequence", or (ii) a combination of two RNA molecules, one of which has the sequence required for Cas9 binding (tracrRNA) and the other has a user-defined "target sequence" (crRNA). When using Cpf1 endonuclease, the "guide RNA" expressed can be (i) a single RNA molecule that has both the sequence required for Cpf1 binding and a user-defined "target sequence", or (ii) several guide RNAs that are introduced as a single crRNA array (Zetsche, Nat Biotech, 2016). A "target sequence" is capable of annealing to a given genomic location, thereby defining the genomic target to be modified (usually around 20 nucleotides).

[0151] DNA cleavage by Cas9 depends on the presence of a short protospacer adjacent motif (PAM) in the target DNA, and the selection of targetable sequences is limited. For example, CAS9 from Streptococcus pyogenes (SpyCas9) corresponds to the PAM sequence: 5'-NGG-3'. In certain embodiments, the DNA repair construct has a mutated PAM sequence. This mutation eliminates the function of the PAM sequence, but does not affect the expression, stability or function of the protein. The use of a DNA repair construct with a mutated PAM sequence increases HDR efficiency.

[0152] As those skilled in the art understand, there are alternative means for site-specific DNA editing other than CRISPR system.That is, zinc finger endonucleases, transcription activator-like effector nucleases (TALEN), meganucleases, or Argonaute-based systems (Nat Biotechnol. 2016 Jul;34(7):768-73), or base editors (Komor et al., Nature 533, 420-424, doi:10.1038 / nature17946) can be used.The present invention also includes the use of these alternative means for site-specific DNA editing.

[0153] In certain embodiments, the first DNA repair construct is not the substrate of the CRISPR system used in the first step of the above method (the step of cutting genomic DNA strand).This is because the first DNA repair construct does not have a PAM sequence.Therefore, after the insertion by the second endonuclease event, the insertion sequence cannot be cut.

[0154] In certain embodiments, genetic engineering resulting in amino acid insertion, deletion and / or substitution is achieved by transfecting / electroporating cells with a base editor (see Komor et al., Nature 533, 420-424, doi:10.1038 / nature17946; Gaudelli, Nature 2017) and a guide RNA. The base editor can convert a nucleic acid sequence A encoding an amino acid marker A to a nucleic acid sequence B encoding an amino acid marker B. The guide RNA can also guide the base editor to the nucleic acid sequence A encoding the amino acid marker A. As described below in the Examples, a base editor can be designed to convert mouse CD45.1 to mouse 45.2 (Figure 9). This shows the feasibility of allelic modification using a base editor as an alternative to the proven HDR approach. However, due to constraints on whether nucleotides in the genome are amenable to base editing, we could not design a base editor that converts CD90.2 to CD90.1 or CD90.1 to CD90.2. Furthermore, based on base editors designed in the context of NGG PAM (see Komor et al., Nature 2016), we could not design a base editor that converts CD45.1 to CD45.2 or CD45.2 to CD45.1. In contrast, by creating modified Cas9 variants that recognize modified PAM site specificity, the number of nucleotides that can be targeted by the base editor was increased (Kim, Nat Biotech 2017). We identified a PAM site in Staphylococcus aureus (SaKKH-BE3) that is near a G present in the CD45.1 sequence (which, when mutated to A, converts to a CD45.2 allele). Therefore, we designed an sgRNA to replace the G at position 5 of the editing window with an A because SaKKH-BE3 most efficiently edits the G at position 5 compared to other positions in the editing window.Next, we designed a guide RNA / base editor pair to convert CD45.2 to CD45.1 (Figure 9b). The newly designed A / T adenine base editor (ABE) can convert G / C. In principle, this new ABE can be used to convert the essential A in the CD45.2 allele to G in the CD45.1 allele. However, the ABE available now requires a NGG PAM in the vicinity of the desired mutation, and no such PAM is available to precisely position the ABE. We therefore designed a putative ABE based on the rules that apply to cytidine deaminase base editors. Since the known cytidine deaminase base editors and the new ABE edit within similar editing windows, it is reasonable to assume that this strategy will work. We found two options. One was putatively based on the Cas9 SaKH-ABE fusion and the other was putatively based on the Cas9 VQR-ABE fusion (Figure 9b). It cannot be excluded that the flanking A was also converted, which would change the epitope. Therefore, when using a base editor to convert CD45.2 to CD45.1, a highly specific base editor with a narrow editing window is required. A recently reported base editor is suitable because it has an editing window of 1-2 nucleotides (Kim, Nat Biotech, 2017). In summary, the above examples illustrate that the principle of converting bases for allele editing is platform independent and may be achieved by HDR-mediated approaches or alternative means (e.g. base editing). Depending on the cell type and the genomic context of the desired mutation, one can choose one or the other.

[0155] In the context of this specification, "DNA repair construct" refers to a DNA construct that is used as a template for repairing DNA strand damage (especially double-strand breaks (DSBs)) in genomic DNA by HDR. The DNA repair construct has homology arms and a predetermined recombination sequence. The homology arms are homologous to the genomic DNA sequences at 5' and 3' of the DSB. The predetermined recombination sequence is located between the homology arms. During genomic DNA repair by HDR, the predetermined recombination sequence is inserted into genomic DNA. As understood by those skilled in the art, the DNA repair construct can be linear (single-stranded or double-stranded) or circular (e.g., plasmid, minicircle plasmid).

[0156] As understood by those skilled in the art, the expression "guide RNA can anneal to a predetermined genomic position" refers to a part of guide RNA (user-defined "target sequence") that can anneal to a predetermined genomic position under high stringency conditions. Guide RNA has another part that cannot anneal to the predetermined genomic position. By (partially) annealing to a predetermined genomic position, guide RNA guides CRISPR-associated endonuclease to the predetermined genomic position. This causes DSB to occur at the predetermined genomic position.

[0157] In certain embodiments, HDR enhancement reagents (particularly vanillin or rucaparib) are used during gene editing protocol.In the context of this specification, vanillin refers to 4-hydroxy-3-methoxybenzaldehyde (CAS number: 121-33-5).In the context of this specification, rucaparib refers to 8-fluoro-2-{4-[(methylamino)methyl]phenyl}-1,3,4,5-tetrahydro-6H-azepino[5,4,3-cd]indol-6-one (CAS number: 283173-50-2).

[0158] Whenever alternatives of a single separable feature are presented as "embodiments" in this specification, it should be understood that the alternatives can be freely combined to form further embodiments of the invention disclosed in this specification.

[0159] Applications EP16196860.7, EP16196858.1, PCT / EP2017 / 059799, and EP17197820.8 are incorporated herein by reference.

[0160] The present invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived, and which are intended to illustrate the invention without limiting its scope. [Brief description of the drawings]

[0161] [Figure 1] The principle of modification of immunologically distinguishable mutants of cell surface proteins is shown. Isoform A and isoform B can perform the same function (are functionally equivalent). In this example, isoform A represents the naturally occurring surface protein and is modified to become mutant isoform B. [Diagram 2] We show how immunologically distinguishable cell surface variants could improve the safety of human cell therapies. [Figure 3A] The two isoforms of the surface protein can be distinguished by monoclonal antibodies (mAbs). Top sequence: SEQ ID NO:020, bottom sequence: SEQ ID NO:021. [Figure 3B] See Figure 3A. [Figure 4]Proof-of-concept experiment: conversion of CD90.2 (allele A) to CD90.1 (allele B) in primary cells. The flow cytometry panel on the left shows a pure starting population of CD90.2+ primary T cells. The cells are then modified in vitro to convert from CD90.2 to CD90.1. After editing, four different populations can be distinguished: (i) CD90.2+ homozygous cells (unedited starting population; top left), (ii) CD90.1+ / CD90.2+ heterozygous cells (top right), (iii) CD90.1+ homozygous cells (bottom right), and (iv) CD90 deleted cells (bottom left). The allelically modified cells, represented by the bottom right box, no longer express the starting (original) endogenous allele (CD90.2). The pure population of CD90.1+CD90.2- cells can be used to isolate correctly edited cells in vitro prior to transplantation into the patient. [Figure 5A] Proof of concept experiment (part 2): Conversion of CD45.2 (allele A) to CD45.1 (allele B) in primary cells. Top sequence: SEQ ID NO: 022, bottom sequence: SEQ ID NO: 023. As shown for the allele conversion from CD90.2 to CD90.1 (Figure 4), the allele-modified cell in the bottom right box no longer expresses the starting (original) endogenous allele (CD45.2). This cell can be used to isolate a pure population of correctly edited cells prior to transplantation into the patient. [Figure 5B] See Figure 5A. [Figure 6] We show gene editing in EL4 cells using Cas9 ribonucleoprotein particles (RNPs). Similar to the plasmid-based approach, crRNA:tracrRNA / Cas9 complex and + / -HDR 2kb template were transfected into EL4 cells, except for the electroporation conditions (described in the Methods section). This result demonstrates that allele editing can be achieved by different approaches (e.g., plasmid or RNP). That is, allele editing is platform independent. [Figure 7]We show gene editing in primary mouse T cells using Cas9 ribonucleoprotein particles (RNPs). Similar to the plasmid-based approach, crRNA:tracrRNA / Cas9 complexes and + / -HDR 2kb templates were transfected into primary mouse T cells. Similar to Figure 6, the results demonstrate that allele editing to convert allele A to allele B is platform independent. [Figure 8] Highly pure ex vivo selection of allele-modified cells is shown. Left panel: After modification of CD45.2 cells to CD45.1 cells, four populations result (similar to Fig. 4). All four populations were then purified to very high purity by flow cytometry. Right panel shows the post-purification purity for each of the four populations. Conclusion: All four possible cell populations can be purified to high purity ex vivo based on the modified allele. Correct gene editing was confirmed by Sanger DNA sequencing (not shown). [Figure 9A]Design of (A) base editors converting CD45.1 to CD45.2 and (B) base editors converting CD45.2 to CD45.1. (A) Top sequence: SEQ ID NO: 024, bottom sequence: SEQ ID NO: 025. (B) Top sequence for option 1 and option 2: SEQ ID NO: 026, bottom sequence for option 1 and option 2: SEQ ID NO: 027. (A) Guide RNA was designed and used in conjunction with SaKKH-BE3 base editor restricted by PAM (NNRRT). The selected PAM sequence is represented by a black dotted line (ATTTGT). The guide RNA is represented by a grey line, showing the base editing window (nucleotides 4-7 of the guide RNA). This base editor converts the G at position 5 to A. Thus, the CD45.1 allele is converted to the CD45.2 allele. (B) The modified adenine base editor can convert A to G. Two options were discovered to convert CD45.2 to CD45.1. Option 1: sgRNA (gray line) was designed to be used in conjunction with Cas9 SaKKH (PAM is black dotted line), which converts an A at position 5 of the base editing window to a G. Option 2: sgRNA (gray line) to be used in conjunction with Cas9 VQR, which has a PAM represented by a black dashed line, which converts an A at position 6 of the base editing window to a G. Option 1 and Option 2 convert a CD45.2 allele to a CD45.1 allele. [Figure 9B] See Figure 9A. [Figure 10] The concept behind optional removal of transplanted cells is presented. Cells are harvested from the patient and then edited ex vivo to convert one allele to another. Successfully edited cells are selected and a pure population of cells carrying the epitopes required for selective depletion is injected. Thus, allelic modification adds the option to remove transplanted cells if desired. [Figure 11A] The application of selectively depleting transplanted or host cells in vivo is demonstrated. [Figure 11B] See Figure 11A. [Figure 12]Proof-of-concept experiments for selective in vivo depletion are shown. Model for in vivo depletion. (1) Reconstitution of immunodeficient mice lacking all T cells (Rag KO) with a 1:1 mixture of purified CD45.2+ and CD45.1+ T cells. The T cells used are congenic, but not edited. (2) Antibody depletion. Comparison of a non-selective antibody (anti-CD4) with a selective antibody (anti-CD45.2). The anti-CD45.2 antibody is either unlinked to a toxin or linked to a toxin (saporin). (3) Analysis after 1 week. [Figure 13] Selective depletion of CD45.2+ cells in vivo: Quantification of T cells in blood. [Figure 14] Selective depletion of CD45.2+ cells in vivo: Quantification of relative numbers of T cells in lymphoid organs. Same procedure as in Figure 12, except lymph nodes (LN) and mesenteric lymph nodes (mesLN) were analyzed. [Figure 15] Selective depletion of CD45.2+ cells in vivo: quantification of relative numbers of T cells in lymphoid organs. Same procedure as in FIG. 12, except spleen (SP) was analyzed. [Figure 16] Selective depletion of CD45.2+ cells in vivo: Quantification of absolute numbers of T cells in lymphoid organs. Same procedure as in Figure 12, except lymph nodes (LN) and mesenteric lymph nodes (mesLN) were analyzed. [Figure 17] Selective depletion of CD45.2+ cells in vivo: Quantification of absolute numbers of T cells in lymphoid organs. Same procedure as in FIG. 12, except spleen (SP) was analyzed. [Figure 18]We present a proof-of-concept experiment (part 2) for selective removal of allele-edited cells in vivo. Experimental method: (1) CD45.2 T cells were electroporated ex vivo and modified to CD45.1. (2) Adoptive transfer into T cell-deficient mice (Rag KO). No selection for purity was performed before transfer. (3) After several weeks, quality control demonstrated allele editing. (4) CD45.2 cells were depleted with antibodies, leaving only the edited cells. Conclusion: The PoC demonstrates that CD45.2+ host cells and unedited cells were depleted in vivo, while allele-modified CD45.1+ cells remained. [Figure 19] The application of the present invention is shown. Allelic modification improves safety (e.g., of CAR-T cell therapy). The modified cells express a first isoform of a cell surface protein (e.g., CD45) and a predefined target (e.g., CD19 (CAR-19)) that is reactive to a chimeric antigen receptor. CAR19 is incorporated into T cells and the second allele of CD45 is converted to the first allele of CD45. This allows the transplanted CAR-T cells to be distinguished from unmodified host cells. In the event of treatment-related toxicity, the pathogenic CAR-T cells are selectively eliminated to stop the toxicity associated with CAR-T. [Figure 20A] Gene correction of scurfy cells and cells carrying the human Foxp3K276X mutation, and the increased relative frequency of gene-repaired cells when gated on surrogate surface markers with switched isoforms. (A) Alignment of wild-type Foxp3 (C57BL / 6) genomic DNA sequence (SEQ ID NO: 028), the Foxp3 locus containing the targeted mutation Foxp3K276X (SEQ ID NO: 029; a premature stop codon has been introduced), and the Foxp3 locus of scurfy mice (B6.Cg-Foxp3sf / J) (SEQ ID NO: 030; a spontaneous 2 bp insertion resulting in a frameshift). sgRNA binding site (green line) and PAM sequence (black line). [Figure 20B](B) Protocol for gene editing of all CD4+ T cells from Foxp3K276X C57BL / 6 mice. (Step 1) Activate in vitro. Electroporate with a plasmid encoding an sgRNA targeting the Foxp3K276X mutation and a circular plasmid carrying a 1 kb wild-type (wt) Foxp3 repair template. (Step 2) Isolate successfully transfected cells based on GFP expression. (Step 3) Propagate cells in vitro for gene editing in the presence of rhIL-2, TGF-β (or a combination of TGF-β and retinoic acid (RA)), and cytokine-neutralizing antibodies (anti-IL-4 and anti-IFNγ) for 7 days. [Figure 20C] (C) The same experimental procedure as in B was used with total CD4+ T cells from control mice (WT) or total CD4+ T cells from Foxp3K276X mice. CD25 and Foxp3 expression was analyzed by flow cytometry (gating on viable CD4+ T cells). Wild-type cells electroporated with empty px458 plasmid differentiated into CD4+Foxp3+CD25+ T cells (left panel). Foxp3K276X cells electroporated with sgRNA containing Foxp3K276X alone did not differentiate into Foxp3 (middle panel). Foxp3K276X cells electroporated with sgRNA containing Foxp3K276X and 1kb Foxp3 dsDNA repair template restored Foxp3 protein expression (right panel). Top: Foxp3 induction by TGF-β alone. Bottom: Foxp3 induction by combining TGF-β and RA. Compared to TGF-β alone, the combination of TGF-β and RA results in a higher frequency of cells expressing Foxp3 in cells with an intact Foxp3 locus (i.e., in wild-type and repaired cells). Representative data from two experiments with Foxp3K276X cells and one experiment with Foxp3sf / J cells. [Figure 20D](D) Enrichment of gene-repaired cells expressing Foxp3 using multiple CD45 isoform switching as a surrogate marker. The experimental procedure was the same as in B, but with electroporation of plasmids encoding two sgRNAs (sgRNA Foxp3K276X and sgRNACD45.2_R1) and two 1 kb dsDNA templates (Foxp3 wild type and CD45.1). After 7 days, CD45.2, CD45.1, CD25 and Foxp3 were analyzed by flow cytometry (gated on viable CD4+ cells). Upper panel: pre-gating on CD45.1- cells (green line) and CD45.1+ cells (red line). Lower panel: enrichment of CD25+Foxp3+ cells in isoform-switched CD45.1+ cells. Representative data from two experiments with Foxp3K276X cells and one experiment with Foxp3sf / J cells. [Figure 21A] Restoration of the Foxp3 gene by plasmid-based and RNP-based approaches. (A) CD4 T cells from Foxp3 KO mice were transfected with sgRNA plasmid alone (or sgRNA plasmid together with Foxp3 wild-type HDR template). 24 hours after transfection (plasmid transfection), GFP+ and GFP- cells were sorted. Immediately after cell sorting, cells were expanded in the presence of Foxp3 differentiation cocktail until the end of the experiment. [Figure 21B] (B) CD4 T cells from Foxp3 KO mice were transfected with crRNA:tracrRNA / Cas9 RNP complexes alone (or together with + / - HDR templates (180bp ssDNA or 2kb plasmids)). The entire population of RNP-transfected cells was expanded in the presence of Foxp3 differentiation cocktail until the end of the experiment. [Figure 22]We successfully repaired the Foxp3 gene in T cells in vitro. Top: Alignment of the genomic DNA sequence of wild-type Foxp3 (C57BL / 6) (SEQ ID NO: 031) and the genomic DNA sequence of the Foxp3 locus containing the targeted mutation Foxp3K276X (SEQ ID NO: 032; a stop codon has been introduced prematurely). Binding site of sgRNA (green line) and PAM sequence (black line). Total CD4+ T cells from wt control or total CD4+ T cells from Foxp3K276X mice were transfected. Expression of CD25 and Foxp3 was analyzed by flow cytometry (gating on viable CD4+ T cells). Wt cells electroporated with empty px458 plasmid differentiated into CD4+Foxp3+CD25+ T cells (upper panel). Foxp3K276X cells electroporated with sgRNA containing only Foxp3K276X did not differentiate into Foxp3 (middle panel). Foxp3K276X cells electroporated with sgRNA containing Foxp3K276X and the 1kb Foxp3 dsDNA repair template restored Foxp3 protein expression (lower panel). Foxp3 was induced with a combination of TGFβ and RA. [Figure 23] We present a protocol for gene editing of all CD4+ T cells derived from Foxp3K276X C57BL / 6 mice. Cells are activated in vitro. They are electroporated with a circular plasmid containing a plasmid encoding an sgRNA targeting the Foxp3K276X mutation and a 1 kb wt Foxp3 repair template. Successfully transfected cells are isolated based on GFP expression. Cells are transplanted in vivo for Foxp3 differentiation and then subjected to an IL-2 regimen. [Figure 24] Clinical phenotype of mice 14 weeks after adoptive transfer of cells. WT and repair mice were free of disease. Transplantation of KO cells caused skin inflammation and alopecia. Experimental procedure as in FIG. 23. [Diagram 25]Correlation of the clinical phenotype (T cell infiltration) of mice transplanted with KO cells with infiltration of CD4 / CD3+ T cells in the ear and tail skin. No or very few CD4 / CD3+ T cells were found in the ear and tail skin of mice transplanted with wild-type or repair T cells. Data are presented as % and absolute numbers. [Figure 26] Shows the presence of CD25 / Foxp3+ Treg cells in mice transplanted with WT and repaired cells. No CD25 / Foxp3+ T cells were found in mice transplanted with KO cells. Data are shown as percentage and MFI of Foxp3. Experimental procedure as in Figure 23. [Figure 27] We show that repaired Treg cells respond to IL-2. Additional IL-2 treatment in the final week of the experiment increased CD25 / Foxp3+ Treg cells in mice transplanted with WT and repaired cells. No CD25 / Foxp3+ T cells were found in mice transplanted with KO cells. These data demonstrate that WT and repaired Treg cells respond to IL-2 to a similar extent. [Figure 28] We show that WT and restored CD25 / Foxp3 Treg cells express similar levels of various cell surface markers (GITR, ICOS, TIGIT, PD1 and KLRG1). A combination of CD25 and additional markers (e.g., GITR) can be used to enrich for these populations. T cells lacking Foxp3 have a different phenotype, e.g., they do not express GITR and KLRG1. [Figure 29]We show that adoptive transfer of CD45.1+ WT cells rescued Foxp3-deficient mice and extended their life span to 8 weeks (without signs of disease at the end of the study). WT Treg cells, found in high proportions (50%) in Foxp3 KO mice, suppress host T cell activation (at a lower percentage (%) than CD44High cells), demonstrating that WT T cells can fully restore immune regulation and control scurfy disease. Furthermore, the Foxp3-deficient microenvironment significantly expands Foxp3-bearing T cells. [Diagram 30] CD4 depletion experiments in Foxp3 KO mice (lymph node (LN)) are shown. Combination of T cell gene repair and selective antibody depletion: pathogenic cells are depleted followed by transplantation of gene corrected T cells. CD4 depletion prevented scurfy disease to a large extent and significantly increased life expectancy. FACS data showing low or absent CD4 T cells in KO depleted mice compared to untreated KO or WT mice support this observation. [Diagram 31] CD4 depletion experiments in Foxp3 KO mice are shown (spleen (SP)). Combining T cell gene repair and selective antibody depletion: pathogenic cells are depleted followed by transplantation of gene corrected T cells. CD4 depletion greatly suppressed scurfy disease and significantly increased life expectancy. FACS data showing low or absent CD4 T cells in KO depleted mice compared to untreated KO or WT mice support this observation. [Diagram 32] (A) shows that CD4 depletion suppresses the development of scurfy disease in Foxp3-deficient mice, and (B) shows that CD4 depletion greatly suppresses the development of dermatitis in the tail skin of Foxp3-deficient mice. EXAMPLES

[0162] For a description of efficient plasmid-based gene ablation in primary T cells, introduction of targeted point mutations in primary T cells, enrichment of HDR edited cells by monitoring isoform switching of surrogate cell surface markers, and gene correction of mouse scurfy cells, see EP16196860.7, EP16196858.1 and PCT / EP2017 / 059799.

[0163] General considerations for designing engineered mutations (allele modifications) Modifying gene alleles by introducing targeted small mutations (which can be single nucleotide or single amino acid mutations) to alter the specific binding of a ligand and allow the binding of a second specific ligand may be useful as a general principle in therapeutic applications. The mutations are designed to preserve the function of the modified protein as much as possible. The immunogenicity must be altered to give rise to specifically binding ligands (such as monoclonal antibodies (mAbs)) or antibody-like molecules (such as Affimers, DARPINS, nanobodies). Such ligands must be screened for specific binding. Also, the characteristics of the immunogen must be carefully designed. At the same time, the mutations are unlikely to cause a strong immune response in vivo, since they only give a small change to the antigen. Congenic markers in mice fulfill these criteria exactly. Both the difference between CD90.1 and CD90.2 and the difference between CD45.1 and CD45.2 are single nucleotides, resulting in a single amino acid difference. In both cases, the difference can be detected by a specific mAb. So there is a pair of mAbs for each gene. These differences can be used to generate mAbs, but when the congenic cells are transplanted into the corresponding congenic host (i.e., CD90.1+ cells are transplanted into a CD90.2+ host mouse (or vice versa); or CD45.1+ cells are transplanted into a CD45.2+ host), the cells are not immunologically rejected. This means that such small antigenic differences are tolerated by the immune system in vivo. In summary, these properties have led to the creation of congenitally labeled cells, which have been used for decades as a very useful tool for immunological research. Immunologically, these cells are used as surrogate cells for autologous transplantation, because they are genetically identical except for the one mutation mentioned above. However, the small congenic differences allow the transplanted cells to be distinguished from the host cells.

[0164] [Selection of protein to manipulate] To design a similar system for therapy in humans, several points must be considered. In principle, mutations can be introduced into any gene encoding a protein. The expression pattern of the given protein will also be relevant (i.e., it may be ubiquitously expressed or, if desired, cell or tissue specific). Proteins expressed on the surface can be most directly targeted. They are therefore the proteins of choice in most cases. An example includes, but is not limited to, proteins characterized by the cluster of differentiation CD1-CD371 (Engel, J Immunol November 15, 2015, 195 (10) 4555-4563; http: / / www.hcdm.org / ). Other given proteins may be ubiquitously expressed proteins. Such proteins include β-2-microglobulin, constant sites of HLA class I, etc., which are expressed in all cells (including non-immune cells). Alternatively, the given protein may be a protein expressed in a particular cell type. Examples include human CD45 on hematopoietic cells, human CD3 on T cells, constant regions of human T cell receptor components, constant regions of B cell immunoglobulins (such as the kappa and lambda light chains or the heavy chain constant regions), human CD4 coreceptor or human CD8 coreceptor, B cell markers (such as CD19, CD20, CD21, CD22, CD23), costimulatory molecules (such as CD28 or CD40), CD34 on hematopoietic stem cells, and specific isoforms expressed on subsets of cells (such as CD45RA or CD45RO). In the last case, mutations are designed within the variable regions (within alternatively spliced ​​exons) that differ between CD43RA and CD45RO. Introducing mutations into ubiquitously expressed molecules (such as beta-2-microglobulin or HLA-I) may serve as a unique system that can be used in virtually any mammalian cell (more specifically, any human cell). This modification can be used to track and remove the modified cells while leaving the unmodified host cells. Such features might be useful, for example, as kill switches in cell therapy.The cells include cells derived from various types of stem cells (e.g., muscle stem cells), hepatocytes, or induced pluripotent stem cells (iPS cells). Introducing mutations into cell type-specific proteins (e.g., CD45) may be useful to target all cells derived from a particular tissue. In the case of the hematopoietic system, modification of human CD45 may be useful to mark hematopoietic stem cells (HSCs) that are used to replace the endogenous hematopoietic system. All progeny of this HSC carry the same mutation introduced into the genome of the original HSC. If the original host hematopoietic system is ablated by any means (e.g., irradiation, chemotherapy, depleting ligands (e.g., mAbs or antibody-like molecules), mAbs linked to toxins, cellular ablation (e.g., CAR-T cell ablation), etc.), the replaced hematopoietic system, even if autologous, can be identified by the introduced mutation. Alternatively, the host cells can be ablated while the modified cells remain.

[0165] [Design Considerations] ◆ Protein expression pattern (ubiquitous or cell or tissue specific) Extracellular proteins: Most often, mutations are introduced at the extracellular site of a given protein that is accessible to a specific ligand. If subset-specific expression is desired, mutations can be introduced within exons that are always expressed or within alternatively spliced ​​exons. ◆ Conserved among species: Conserved amino acids (aa) or structures are more likely to be functionally relevant, and therefore should not be touched. Rather, mutations should be introduced into non-conserved regions. Chemical properties of amino acids (e.g., polarity, charge, hydrophobicity) Amino acid similarity: To preserve function, mutations should convert a given amino acid into a related one, but still be a change that is detectable by a specific ligand. Naturally occurring mutations: Mutations that correspond to amino acid mutations observed at a given position in multiple sequence alignments of members of a protein family (homologous sequences in different organisms) are unlikely to affect the function and structure of the protein, and therefore may be selected for design mutations. Structural considerations: Structural data can aid in the rational design of possible mutations. The amino acids to be modified must be accessible for ligand binding. Mutations in loops are functionally more tolerated and therefore may be the preferred sites. ◆Avoid known disease-causing mutations. Consider known human genetic variation: Functionally permissive single nucleotide polymorphisms (SNPs) are good candidates for mutations. ◆Sites important for secondary modifications (such as glycosylation) should not be mutated. * Sites important for disulfide bonding should not be mutated. Sites important for published interactions (e.g., hydrogen bonds, salt bridges, hydrophobic stacking interactions), including those occurring within the protein as well as those involved in protein-protein interactions for multiple protein complexes or receptor-ligand interactions, should not be mutated. ◆Consider systems that have been successful in the past (e.g., CD90.1 / CD90.2(Q→R)). Consider the binding site of a known binding ligand (such as a known mAb) and computational modeling of the complementarity determining regions (CDRs). ◆ Avoid known "active sites" (e.g., the catalytic site of an enzyme). * Avoid structurally very similar domains present in other human proteins, as this will likely increase the risk of cross-specific ligands. ◆ Consider immunogenicity: Select a site likely to give rise to a suitable peptide immunogen.

[0166] Allelic modification as a generally applicable, platform-independent principle In EP16196860.7, EP16196858.1, and PCT / EP2017 / 059799, the inventors have demonstrated and characterized a method for introducing targeted point mutations for allele modification and subsequent selective removal using the CRISPR / Cas platform. The inventors have demonstrated that two different point mutations can be introduced into two different genes, and the introduced mutations can be used to track and / or selectively remove modified cells in vivo. The inventors have relied on the CRISPR / Cas9 system and dsDNA templates to achieve homology directed repair (HDR). However, the example used is only one way to introduce mutations. Alternatives include other types of nucleases. Examples include zinc finger proteins, TALENs, other natural or modified CRISPR / Cas systems (Cas9, cpf-1, etc.), high fidelity nucleases, or nucleases with modified PAM dependency (Komor, Cell, 2017). Furthermore, the above principle does not depend on the delivery form of the nuclease. The nuclease can be delivered as a plasmid, mRNA, recombinant protein, recombinant protein complexed with guide RNA (i.e., ribonucleoprotein complex, RNP), split recombinase, or integrating or non-integrating virus (e.g., retrovirus, lentivirus, baculovirus, or other viral delivery platform). Methods of delivery include electroporation or other forms (e.g., lipofection, nanoparticle delivery, cell squeeze or physical perforation). The HDR template can also be ssDNA or dsDNA. These forms can be short ssDNA, long ssDNA, or circular or linear minicircle DNA, plasmid DNA, or viral DNA templates (e.g., adeno-associated virus (AAV)). Depending on the target cell, specific AAV serotypes are used to deliver the contents to the cell. For human T cells and human hematopoietic stem cells, HDR templates are often provided as AAV6.However, endonuclease RNP and short ssDNA HDR templates can also be successfully used, thus allowing flexibility in the method of introducing mutations.

[0167] Allele modification using other approaches (e.g., base converters or base editors) We have demonstrated how targeted dsDNA breaks following HDR can be used to introduce small targeted mutations (i.e., precise mutations) for allele modification. There are also other approaches on how to introduce engineered point mutations into the genome of living cells. For example, newly designed chimeric fusion proteins can directly convert bases of target DNA to other bases (Komor, Nature 2016; Nishida, Science 2016; Yang, Nat Comm, 2016; Ma, Nat Meth 2016). Enzymes such as deaminases can be fused with DNA binding modules (such as, but not limited to, zinc finger proteins, TALENs or CRISPR / Cas systems). Naturally occurring cytidine deaminases (APOBEC1, APOBEC3F, APOBEC3G) and activation-induced deaminase (AID) or the AID orthologue PmcdA1 (from sea lamprey) can convert cytidine (C) in DNA to uracil (U). The DNA replication machinery treats U as T if DNA replication occurs before repairing U. This leads to conversion of C:G base pairs to T:A base pairs (Yang, Nat Comm, 2016; Komor, Nature 2016). Therefore, several groups have developed engineered chimeric proteins that have deaminases fused to DNA-binding modules. These engineered chimeric proteins are used to deliver the deaminase to specific genomic loci. For example, the CRISPR / Cas system can be used as a delivery system when using a catalytically deficient mutant of Cas9 nuclease (dCas9). This approach has been successfully used to target fused effector molecules to specific genomic loci, including fluorescent proteins to specific loci and transactivators or repressors to specific genomic loci to control the expression of specific genes (Wang, Ann Rev Biochem, 2016).Fusing cytidine deaminase or AID to the nickase Cas9 and further modifying it to improve the efficiency of base editing allows for directly targeted base conversion (Komor, Nature 2016; Nishida, Science 2016). In certain circumstances, this approach may have advantages over HDR approaches, since the base editors do not induce dsDNA breaks and do not require delivery of a DNA HDR template. Thus, this alternative approach can reduce indels more. Therefore, it may be a safer (or safer) alternative to HDR-based genome modification. Furthermore, delivery of the base editor as mRNA or RNP is quite feasible, which may reduce toxicity for certain cell types. For human T cells, Cas9 RNP provides a successful genome editing approach (Schumann, PNAS, 2015). It can therefore be expected that base converters in the form of RNPs would be well suited for hematopoietic cells (such as hematopoietic stem cells (HSCs) and T cells). In principle, however, base conversion can be a suitable approach for allele modification that can be applied to any cell (such as mammalian cells). After introducing the designed point mutations, they can be used for downstream applications (such as cell labeling, cell tracking, and selective removal). Base editors delivered as RNPs have successfully edited targeted nucleotides in mammalian cells, mouse and zebrafish embryos, and the inner ear of live mice (Rees, Nat Comm, 2017; Kim, Nat Biotech, 2017 doi:10.1038 / nbt.3816). Thus, the above studies show the feasibility of base editing without specific DNA. However, the number of nucleotides in the (human) genome that are amenable to base conversion is more limited than in HDR approaches. This is because base conversion depends on the PAM sequence and has additional constraints (in the case of CRISPR / Cas-based base converters). Cytidine deaminase base editors can only convert C to T (or G to A).However, the newly engineered adenine base editor can convert A to G (or T to C) (Gaudelli, Nature 2017). However, in addition to the PAM constraint, base conversion can only occur within a specific window defined by the specific design and / or modification of the fusion protein. Thus, with base editors, the number of nucleotides that can be edited is much more limited compared to point mutations introduced by HDR (Komor, Nature 2016). Moreover, the window of base conversion contains several nucleotides. For example, for the so-called base editor 3 (BE3), which uses Cas9 from S. pyogenes (SpBE3; see Komor et al., Nature 2016), this window contains about 5 nucleotides. Thus, it is not possible to edit a C to a T in a sequence flanked by several C nucleotides. This would result in additional undesired mutations that may change the amino acid of the resulting protein. A similar editing window applies to adenine base editors. Also, a different BE3 (SaBE3, based on Cas9 from S. aureus) targets Cs that are outside the canonical BE3 editing window, giving detectable base editing. To address these limitations, novel base converter mutants have been engineered to have narrow editing windows and PAM specificities different from NGG (e.g., NGA, NGAG, NGCG, NNGRRT and NNNRRT). However, designing a specific base editor to convert a given single nucleotide remains challenging (Kim, Nat Biotech 2017, doi:10.1038 / nbt.3803). Although novel base converters expand the range of targetable nucleotides in mammalian genomes, they do not freely edit all nucleotides.But still, to take advantage of some of the advantages of base converters, we tried to design base editors for CD90.1 to CD90.2 allele conversion, CD90.2 to CD90.1 allele conversion, CD45.1 to CD45.2 allele conversion, or CD45.2 to CD45.1 allele conversion. However, none of these conversions can be achieved with the available base editors, which are conventional NGG-restricted base editors. This is despite the fact that for both genes (CD90 and CD45), the allelic difference is encoded by a G to A substitution, which is in principle amenable to conversion by deaminases. The only solution was to utilize a SaCas9-based mutant of BE3, which has three mutations that relax the PAM constraint to NNRRT (Kleinstiver, Nature 2015; Kim, Nat Biotech, 2016 (doi:10.1038 / nbt.3803)). By selecting this base editor, we were able to design a base editor that converts CD45.1 alleles to CD45.2 alleles, and the corresponding sgRNA (Figure 9A). However, this base editor only converts CD45.1 to CD45.2, but does not convert CD45.2 to CD45.1. Therefore, we designed a putative adenine base editor (adenine base editor fused with Cas9 SaKKH or Cas9 VQR; Figure 9B). On the other hand, even mutants of the existing base editors cannot convert CD90 alleles.

[0168] 〔method〕 For detailed methods regarding gene editing of primary mouse CD4+ T cells, gene editing of EL-4 cells, and Foxp3 restoration protocols, see EP16196860.7, EP16196858.1 and PCT / EP2017 / 059799.

[0169] [Isolation of human T cells and antibodies] Primary human T cells were isolated from buffy coats (Blutspendezentrum, Basel) of healthy donors using a density gradient of Lymphoprep (Stemcell Technologies; trademark). Easysep Human naive CD4 + Naive CD4+ T cells were pre-enriched using a T-cell enrichment kit (Stemcell Technologies) according to the manufacturer's protocol. Alternatively, umbilical cord blood was used as the source of PMBCs without the naive T cell isolation step (due to the high frequency of naive T cells). Before and after enrichment, CD4+ T cell samples were stained with the following antibodies to assess purity: αCD4-FITC (OKT-4), αCD25-APC (BC96), αCD45RA-BV711 (HI100), αCD45RO-BV450 (UCHL1), αCD62L-BV605 (DREG-56), αCD3-PerCP (HIT3a), and Zombie-UV viability dye (all purchased from Biolegend).

[0170] The procedure is summarized as follows: For one sample of buffy coat (50 mL), prepare 2 x 50 mL filtered Falcon tubes, fill each tube with 16 mL Lymphoprep and centrifuge at 300 g for 1 min. Dispense an equal aliquot of blood into each filtered tube (50 mL) and fill to 50 mL with PBS. Centrifuge at 2000 rpm for 15 min (acceleration time: 4, deceleration time 1). Remove and discard a portion of the serum. Carefully pool the buffy coat of leukocytes in a new 50 mL Falcon tube. Add sterile PBS to the enriched PBMC fraction to approximately 50 mL and centrifuge at 300 g for 5 min. Discard the supernatant and resuspend the pellet in 10 mL PBS, fill to 50 mL and centrifuge at 300 g for 5 min. If necessary, lyse red blood cells with red blood cell lysis buffer prior to the purification step.

[0171] [Protocol for transfecting human T cells] Naive CD4 derived from blood or umbilical cord blood+ T cells or whole PBMCs were used for transfection. To activate T cells, 2 × 10 6 Cells were seeded in 24-well plates (Corning). The plates were coated with monoclonal antibodies (mAbs) a-CD3 (hybridoma clone OKT3) and a-CD28 (hybridoma clone CD28) (both from Biolegend). The coating concentrations were 5 μg / mL (high), 2.5 μg / mL (medium), or 1 μg / mL (low) for a-CD3 and 2.5 μg / mL (high), 1 μg / mL (medium), or 0.5 μg / mL (low) for a-CD28. The plates were incubated for 24 hours at 37°C in the presence of 50 IU / mL recombinant human interleukin-2 (rhIL-2; RD systems) and 5% CO2. After 24 hours, T cells were harvested and washed with PBS. 2 × 10 activated mAbs were added to the plates. 6T cells were electroporated. Amaxa Transfection System, T-020 program (for plasmid) or Neon® Transfection System (ThermoFisher) was used for electroporation. Electroporation conditions were as follows: voltage (1600V), pulse width (10ms), pulse (3 times), 100μL tip, buffer R (for RNP). Cells were transfected with empty plasmid px458 (Addgene plasmid number: 48138) or crRNA:tracerRNA-Atto550 (IDT)-Cas9 (Berkeley) complex. After electroporation, cells were seeded in 24-well plates. The plates were filled with 650μL of complete medium supplemented with 50IU / mL rhIL-2 and inoculated with mAb. The concentration of the mAb was half that used for the initial activation. That is, anti-CD3 was 2.5 μg / mL, 1.25 μg / mL, or 0.5 μg / mL, and anti-CD28 was 1.25 μg / mL, 0.5 μg / mL, or 0.25 μg / mL. After 24 hours, GFP was detected using a Fortessa analyzer (BD Biosciences). + Cells or Atto550 + The cells were evaluated for expression.

[0172] [CD45.2 depletion experiment] From C57BL6 mice (CD45.2) and C57BL6 congenic mice (CD45.1), EasySep Mouse CD4 + CD4+ T cells were isolated using a T Cell Isolation Kit (Stem cell Technologies). RAG KO mice were reconstituted with donor CD4+ T cells. The CD4+ T cells had a 1:1 ratio of CD45.2 to CD45.1 (10 × 10 6Mice were injected intraperitoneally with PBS (untreated group) or anti-CD4 depleting antibody (clone GK1.5, 250 μg) on ​​three consecutive days starting from the same day as T cell transfer. CD45.2-ZAP immunotoxin was made by combining a biotinylated CD45.2 antibody (molecular weight: 160 kDa, Biolegend) with a streptavidin-SAP complex (2.8 saporin molecules per streptavidin, molecular weight: 135 kDa, Advanced Targeting Systems). The combination was in a 1:1 molar ratio and then diluted in PBS immediately before use (as described in the original publication, https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5179034 / ). Immunotoxin or a control with unconjugated CD45.2 antibody was administered in vivo by intravenous injection. One week later, blood, peripheral lymph nodes (LN), mesenteric lymph nodes (mesLN) and spleens (SP) were collected and cells were stained with fluorochrome-conjugated mAbs (all Biolegend): anti-CD45.2 (104), anti-CD45.1 (A20), anti-CD4 (RM4-5) and anti-CD3 (145-2C11). Samples were run on a BD Fortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star).

[0173] 〔item〕 <Item 1> A method for determining a first homology-directed repair (HDR) event at a first genomic location in a eukaryotic cell, comprising: the cell expresses a first isoform of a first surface protein, the first isoform differing from a second isoform of the first surface protein with respect to an amino acid marker, the first isoform comprising amino acid marker A encoded by nucleic acid sequence A and the second isoform comprising amino acid marker B encoded by nucleic acid sequence B; The first genomic location has the nucleic acid sequence A, comprising the steps of: (a) inducing a first DNA double-stranded break at the first genomic location; (b) providing a first DNA repair construct having said nucleic acid sequence B and first homology arms that are homologous to DNA sequences 5' and 3' of said first genomic location; (c) determining the expression of the first and / or the second isoform of the first surface protein in the cell, and optionally purifying the cell based on expression of the first and / or the second isoform of the surface protein; (d) determining the occurrence of the first HDR event, wherein expression of the second isoform of the first surface protein in the cell is synonymous with the occurrence of the first HDR event.

[0174] <Item 2> The occurrence of the first HDR event is determined under two or more experimental conditions, and when the expression ratio of the second isoform relative to the first isoform is increased under the first experimental condition compared to the second experimental condition, it is determined that the HDR efficiency under the first experimental condition is increased.

[0175] <Item 3> The method according to Item 1 or 2, wherein steps (a) and (b) are carried out in a cell culture medium containing vanillin and / or rucaparib, particularly wherein the medium contains 50 μM to 500 μM vanillin and / or 0.5 μM to 2.5 μM rucaparib, and more particularly wherein the medium contains about 300 μM vanillin and / or about 1 μM rucaparib.

[0176] <Item 4> The method according to any one of Items 1 to 3, wherein the first isoform and the second isoform of the first surface protein can be distinguished from each other by a ligand (particularly, an antibody), and the ligand can distinguishably either (i) bind to the amino acid marker A and cannot bind to the amino acid marker B, or (ii) bind to the amino acid marker B and cannot bind to the amino acid marker A.

[0177] <Item 5> The method according to any one of Items 1 to 4, wherein the first surface protein is a natural protein.

[0178] <Item 6> The method according to any one of Items 1 to 5, wherein the first surface protein is a recombinant protein.

[0179] <Item 7> The method according to any one of Items 1 to 6, wherein the purification is achieved by fluorescence-activated cell sorting (FACS).

[0180] <Item 8> The method according to any one of Items 1 to 6, wherein the purification comprises a step of concentrating cells expressing the first isoform or the second isoform of the first surface protein using magnetic beads.

[0181] <Item 9> The method according to any one of Items 1 to 8, wherein the first surface protein is Thy1 or CD45.

[0182] <Item 10> The method according to any one of Items 1 to 9, wherein the first double-stranded break at the first genomic location is induced by transfecting the cell with a DNA expression construct encoding a CRISPR-associated endonuclease (Cas9) and a guide RNA, and the guide RNA can anneal to the first genomic location.

[0183] <Item 11> The method according to any one of Items 1 to 10, wherein each of the homology arms has about 2000 base pairs (bp).

[0184] <Item 12> A method for selectively depleting or enriching edited cells from a composition of unedited cells and edited cells, comprising: (a) the unedited cell expresses a first isoform of a surface protein and the edited cell has been edited to express a second isoform of the surface protein by the method of any one of items 1 to 11, the second isoform differing from the first isoform with respect to an amino acid marker, the first isoform comprising amino acid marker A encoded by nucleic acid sequence A and the second isoform comprising amino acid marker B encoded by nucleic acid sequence B; and (b) selectively enriching or depleting the edited cells based on expression of the first or second isoform of the surface protein; method.

[0185] <Item 13> A method for selectively depleting or enriching cells in a cell composition, comprising the steps of: (c) providing a cell, the cell expressing a first isoform of a surface protein, the first isoform differing from a second isoform of the surface protein in terms of an amino acid marker, the first isoform comprising amino acid marker A encoded by nucleic acid sequence A and the second isoform comprising amino acid marker B encoded by nucleic acid sequence B; (d) inducing a DNA double-strand break at the genomic location having nucleic acid sequence A; (e) providing a DNA repair construct having nucleic acid sequence B and homology arms that are homologous to DNA sequences 5' and 3' of the genomic location; (f) selectively enriching / depleting said cells based on expression of said first or second isoform of said surface protein.

[0186] [Item 2] <1> A mammalian cell (particularly a human cell) expressing a first isoform of a surface protein, the first isoform of the surface protein is functionally indistinguishable from a second isoform of the surface protein, but is immunologically distinguishable; A mammalian cell for use in treating a patient having cells expressing said second form of said surface protein. <2> the surface protein having an extracellular polypeptide sequence, The first isoform has one, two, three, four or five amino acid insertions, deletions and / or substitutions compared to the second isoform. <1> 2. A mammalian cell for use in a treatment as described in claim 1. <3> The first and second isoforms can be distinguished by (i) antibody-like molecule binding, (ii) antibody binding, or (iii) response of immune effector cells bearing antibodies or antibody-like molecules, or (iv) response of immune effector cells (particularly T cells) bearing chimeric antigen receptors (CARs). <1> or <2> 2. A mammalian cell for use in a treatment as described in claim 1. <4> The surface protein is selected from: CD1a、CD1b、CD1c、CD1e、CD1e、CD2、CD3、CD3d、CD3e、CD3g、CD4、CD5、CD6、CD7、CD8a、CD8b、CD9、CD10、CD11a、CD11b、CD11c、CD11d、CDwl2、CD13、CD14、CD15、CD15u、CD15s、CD15su、CD16、CD16b、CD17、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD31、CD32、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、CD42b、CD42c、CD42d、CD43、CD44、CD45、CD45RA、CD45RB、CD45RC、CD45RO、CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、CD49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55、CD56、CD57、CD58、CD59、CD60a、CD60b、CD60c、CD61、CD62E、CD62L、CD62P、CD63、CD64、CD65、CD65s、CD66a、CD66b、CD66c、CD66d、CD66e、CD66f、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85a、CD85d、CD85j、CD85k、CD86、CD87、CD88、CD89、CD90、CD91、CD92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD99R、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD107b、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158e、CD158i、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210b、CD212、CD213a1、CD213a2、CD215、CD217a、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240DCE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD266、CD267、CD268、CD269、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD286、CD289、CD290、CD292、CDw293、CD294、CD295、CD296、CD297、CD298、CD299、CD300a、CD300c、CD300e、CD301、CD302、CD303、CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD318 , CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD3 37, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, BCMA, immunoglobulin light chain (lambda or kappa), HLA proteins, and β2-microglobulin; In particular, said surface proteins are selected from: CD2, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD23, CD33, CD34, CD90, CD45, CD123, BCMA, immunoglobulin light chains (lambda or kappa), HLA proteins and β2-microglobulin; <1> ~ <3> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <5> The first isoform is not encoded in the original genomic DNA of the patient. <1> ~ <4> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <6> The first isoform is obtained by altering a sequence encoding the gene for the surface protein in the original genomic DNA of the patient. <1> ~ <5> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <7> the first isoform is obtained by inserting, deleting and / or substituting 1, 2, 3, 4 or 5 amino acids (or 6, 7, 8, 9, 10, 11, 12, 15 or 20 amino acids) in the amino acid sequence of the second isoform of the surface protein, <1> ~ <6> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <8> The above insertions, deletions and / or substitutions are achieved by the following steps: <1> ~ <7> 2. A mammalian cell for use in a treatment according to any one of the preceding claims: (a) comparing one or several homologous sequences of different mammalian species (in particular mouse, rat, primate and human homologs) and locating insertions, deletions and / or substitutions at non-conserved sites; (b) selecting insertions, deletions and / or substitutions that are predicted by computer-aided structural prediction to not alter the secondary structure of the region to be inserted, deleted and / or substituted; (c) selecting insertions, deletions and / or substitutions that are located in sites capable of ligand binding according to computer-aided structural prediction; (d) selecting insertions, deletions and / or substitutions in the sequence that are not involved in predicted or experimentally established protein-protein interactions of said surface proteins; (e) selecting insertions, deletions and / or substitutions that do not eliminate or introduce disulfide bonds, intermolecular or intramolecular interactions, hydrophobic stacking; and (f) selecting insertions, deletions and / or substitutions that do not remove or introduce post-translational modification sites of the protein that are important for protein folding. <9> The insertion, deletion and / or substitution is located in an extracellular site of the first surface protein (particularly an extracellular loop); <1> ~ <8> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <10> the cells are administered before, simultaneously with, or after specific removal of cells expressing the second isoform of the surface protein; In particular, the removal of cells expressing said second isoform of said surface protein is achieved by administering to said patient an agent selected from: (i) an antibody-like molecule, (ii) an antibody, (iii) an immune effector cell carrying an antibody or an antibody-like molecule, and (iv) an immune effector cell (in particular a T cell) carrying a chimeric antigen receptor, the agent specifically reacts with the second isoform of the cell surface protein but not with the first isoform; <1> ~ <9> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <11> the cell expresses an antibody or antibody-like molecule (particularly a chimeric antigen receptor) reactive to the second isoform of the surface protein; <1> ~ <10> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <12> the surface protein is selected from CD45, CD19, CD8 and CD4; In particular, the surface protein is CD45. <11> 2. A mammalian cell for use in a treatment as described in claim 1. <13> The mammalian cell is a hematopoietic cell (particularly a hematopoietic stem cell or a T cell), <1> ~ <12> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <14> The cells have a genetic modification that corrects a disease-associated genetic abnormality present in the patient. <1> ~ <13> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <15> The above disease-related genetic abnormality is a mutation in Foxp3. <14> 2. A mammalian cell for use in a treatment as described in claim 1. <16> The treatment is for a T cell mediated disease, in particular an IPEX-like syndrome, a CTLA-4 associated immunoregulatory disorder, a hemophagocytic syndrome, an ALPS syndrome, or a syndrome resulting from a heterozygous germline PTEN mutation. <1> ~ <15> 2. A mammalian cell for use in a treatment according to any one of claims 1 to 11. <17> 1. For use in the treatment of a medical condition, an agent selected from (a) a compound comprising an antibody or antibody-like molecule, or a compound consisting of an antibody or antibody-like molecule; and (b) an immune effector cell bearing an antibody or antibody-like molecule, or an immune effector cell bearing a chimeric antigen receptor; the agent reacts specifically with either the first or second isoform of the surface protein; the first isoform of the surface protein is functionally indistinguishable but immunologically distinguishable from the second isoform of the surface protein; By administering the drug, cells having an isoform to which the drug reacts are eliminated. Drugs. <18> The antibody or antibody-like molecule is linked to a toxin, in particular saporin. <17> The drug described in. <19> The agent is a bispecific antibody or bispecific antibody-like molecule. <17> or <18> The drug described in. <20> The agent is an immune effector cell carrying a bispecific antibody or bispecific antibody-like molecule. <17> The drug described in. <21> The agent is an immune effector cell comprising: <17> Medications listed in: (a) a first antibody or a first antibody-like molecule that specifically reacts with either a first isoform or a second isoform of a first surface protein, a first antibody or a first antibody-like molecule, wherein the first and second isoforms of the first surface protein are functionally indistinguishable but immunologically distinct; and (b) a second antibody or a second antibody-like molecule that specifically reacts with either the first isoform or the second isoform of the second surface protein, A second antibody or second antibody-like molecule, wherein the first and second isoforms of the second surface protein are functionally indistinguishable but immunologically distinct. <22> The agent is an antibody-like molecule (particularly, a T cell) having an antibody-like molecule (particularly, a chimeric antigen receptor), The medical condition is a hematopoietic disease (particularly a malignant hematopoietic disease); <17> , <20> or <21> 2. The drug according to claim 1 . <23> For use in the treatment of graft-versus-host disease, <17> ~ <22> 2. The drug according to claim 1 . <24> 1. A combination drug comprising: (a) reactive to a first surface protein; <17> ~ <23> and (b) reactive to a second surface protein; <17> ~ <23> a second agent according to any one of the preceding claims; In particular, combination drugs for the treatment of hematopoietic disorders (especially combination drugs for the treatment of malignant hematopoietic disorders). <25> the first agent and the second agent are T cells bearing a chimeric antigen receptor; <24> The combination drug according to claim 1. <26> The first surface protein is CD19 and the second surface protein is CD45. <24> or <25> The combination drug according to claim 1. <27> 1. A method for tracking cells expressing a first isoform of a surface protein in a patient in vivo, comprising: said first isoform of said surface protein being functionally indistinguishable from said second isoform of said surface protein but being immunologically distinguishable; Administering to the patient a ligand that specifically reacts with the first isoform. method. <28> 1. A method for selectively depleting or enriching cells in vivo, comprising the steps of: (a) providing a cell, the cell expresses a first isoform of the surface protein; the first isoform differs from the second isoform of the surface protein with respect to an amino acid marker; The first isoform comprises an amino acid marker A encoded by a nucleic acid sequence A; the second isoform comprises an amino acid marker B encoded by a nucleic acid sequence B; (b) inducing a mutation from nucleic acid sequence A to nucleic acid sequence B in the genomic DNA of the cell; and (c) selectively enriching / depleting said cells based on expression of said first isoform or said second isoform of said surface protein. <29> A kit comprising the following components: (a) a base editor guide RNA that targets a genomic location of a gene encoding a cell surface protein, (i) the gene has two isoforms that differ with respect to a nucleic acid marker sequence, isoform 1 having a first marker sequence and isoform 2 having a second marker sequence; (ii) a base editor guide RNA, wherein the genomic location comprises a base editor PAM sequence and the first marker sequence or the second marker sequence; and (b) optionally, a first antibody and a second antibody that specifically bind to the gene products of isoform 1 and isoform 2, respectively. <30> The cell surface protein is mouse Thy1 or mouse CD45. <29> The kit according to claim 1, <31> the cell surface protein is mouse CD45; The base editor guide RNA has a nucleic acid sequence selected from SEQ ID NO: 004, SEQ ID NO: 005, and SEQ ID NO: 006. <30> The kit according to claim 1, <32> A kit containing the following components: (a) a guide RNA that targets a genomic location of a gene encoding a cell surface protein, (i) the gene has two isoforms that differ with respect to a nucleic acid marker sequence, isoform 1 having a first marker sequence and isoform 2 having a second marker sequence; (ii) a guide RNA, wherein the genomic location has a PAM sequence and the first marker sequence or the second marker sequence; (b) A DNA construct having the following (i) to (iii): (i) the first marker sequence or the second marker sequence (ii) the PAM sequence (particularly, the PAM sequence that is mutated and non-functional); (iii) a pair of homology arms that are homologous to genomic DNA sequences 5' and 3' to the genomic location of the gene encoding the cell surface protein. (c) optionally, a first antibody and a second antibody that specifically bind to the gene products of isoform 1 and isoform 2, respectively. <33> The homology arms each contain at least 85 base pairs (bp), particularly at least 450 bp, and more particularly about 2000 bp. <32> The kit according to claim 1, <34> The cell surface protein is mouse Thy1 or mouse CD45. <32> or <33> The kit according to claim 1, <35> the cell surface protein is mouse Thy1, Satisfying either (a) or (b) below, <32> The kit includes: (a) the guide RNA is SEQ ID NO: 001; The DNA construct is selected from SEQ ID NO:007 (no mut), SEQ ID NO:008 (mut), SEQ ID NO:009 (4x mut), SEQ ID NO:010 (2kb), SEQ ID NO:011 (4kb), SEQ ID NO:012 (1kb), and SEQ ID NO:013 (160bp); or (b) the guide RNA is SEQ ID NO: 002; Said DNA construct is selected from SEQ ID NO: 014 (120 bp) and SEQ ID NO: 015 (180 bp). <36> the cell surface protein is mouse CD45; The guide RNA is SEQ ID NO: 003, The DNA construct is selected from SEQ ID NO: 016, SEQ ID NO: 017 (1 kb), SEQ ID NO: 018 (2 kb) and SEQ ID NO: 019 (4 kb), <32> The kit according to claim 1, <37> containing mouse T cells genetically engineered for stable Cas9 expression, <32> ~ <36> 2. The kit according to claim 1 ,

Claims

1. 1. A pharmaceutical composition for use in treating a human patient having cells expressing a second isoform of a surface protein, comprising: a human cell expressing the first isoform of said surface protein, the first isoform of the surface protein and the second isoform of the surface protein are functionally indistinguishable but immunologically distinguishable; the cells are administered prior to, concurrently with, or following specific removal of cells expressing the second isoform of the surface protein; removing cells expressing the second isoform of the surface protein by administering to the patient an antibody-like molecule or immune effector cells bearing an antibody-like molecule; the antibody-like molecule or an immune effector cell bearing the antibody-like molecule specifically reacts with the second isoform of the surface protein of the cell; the first isoform comprises one, two, three, four or five amino acid insertions, deletions and / or substitutions relative to the amino acid sequence of the second isoform of the surface protein, The first isoform is obtained by the following pharmaceutical composition (i) or (ii): (i) changing the sequence encoding the gene for the surface protein contained in the patient's original genomic DNA by gene editing; (ii) The mRNA encoding the surface protein is altered by RNA editing.

2. 2. The pharmaceutical composition of claim 1, wherein the surface protein is selected from: CD1a、CD1b、CD1c、CD1d、CD1e、CD2、CD3、CD3d、CD3e、CD3g、CD4、CD5、CD6、CD7、CD8a、CD8b、CD9、CD10、CD11a、CD11b、CD11c、CD11d、CDwl2、CD13、CD14、CD15、CD15u、CD15s、CD15su、CD16、CD16b、CD17、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD31、CD32、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、CD42b、CD42c、CD42d、CD43、CD44、CD45、CD45RA、CD45RB、CD45RC、CD45RO、CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、CD49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55、CD56、CD57、CD58、CD59、CD60a、CD60b、CD60c、CD61、CD62E、CD62L、CD62P、CD63、CD64、CD65、CD65s、CD66a、CD66b、CD66c、CD66d、CD66e、CD66f、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85a、CD85d、CD85j、CD85k、CD86、CD87、CD88、CD89、CD90、CD91、CD92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD99R、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD107b、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158e、CD158i、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210b、CD212、CD213a1、CD213a2、CD215、CD217a、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240DCE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD266、CD267、CD268、CD269、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD286、CD289、CD290、CD292、CDw293、CD294、CD295、CD296、CD297、CD298、CD299、CD300a、CD300c、CD300e、CD301、CD302、CD303、CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD31 8, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD 337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, BCMA, immunoglobulin light chains (lambda or kappa), HLA proteins and β2-microglobulin.

3. The first isoform is not encoded in the patient's original genomic DNA; 3. The pharmaceutical composition according to claim 1 or 2.

4. The sequence changes are accomplished through the use of a base editor. The pharmaceutical composition according to any one of claims 1 to 3.

5. The base editor is a deaminase fused to a DNA binding module. The pharmaceutical composition of claim 4.

6. The base editor is a cytidine deaminase or an adenine deaminase. The pharmaceutical composition of claim 5.

7. The deaminase is fused to a nickase Cas9. The pharmaceutical composition of claim 5.

8. the insertion, deletion and / or substitution is located in an extracellular site of the surface protein; A pharmaceutical composition according to any one of claims 1 to 7.

9. The base editor is Cas9 SaKKH-BE3, Cas9 SaKKH, or Cas9 VQR. The pharmaceutical composition of claim 4.

10. The human cells are human hematopoietic cells. The pharmaceutical composition according to any one of claims 1 to 9.

11. The pharmaceutical composition of claim 10, wherein the human hematopoietic cells are selected from the group consisting of: Hematopoietic stem cells, CD4+ T cells, CD8+ T cells, memory T cells, regulatory T cells (Treg), natural killer cells (NK), innate lymphoid cells (ILCs), dendritic cells (DCs), B lymphocytes, mucosal-associated invariant T cells (MAITs) and gamma delta T cells (γδTs).

12. The above treatment includes hematopoietic stem cell transplantation. A pharmaceutical composition according to any one of claims 1 to 11.

13. The antibody-like molecule is a bispecific antibody or an antibody linked to a toxin. A pharmaceutical composition according to any one of claims 1 to 12.

14. The immune effector cell having the antibody-like molecule is a T cell having a chimeric antigen receptor (CAR). A pharmaceutical composition according to any one of claims 1 to 13.

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