IL5RA cell surface marker

Abstract

The invention relates to an IL5RA cell surface marker. A cell tag comprising an extracellular region, a transmembrane region, and optionally an intracellular region is provided. The extracellular region comprises an IL5 receptor alpha (IL5Ra) sequence linked to a transmembrane domain, and the recombinant polypeptide cannot function in signal transduction. The cell tag can be operably linked to a transgene. Expression of the cell tag allows for the identification, detection, selection, and ablation of cells expressing the transgene and the cell tag. In some embodiments, the genetically modified host cell comprises a transgene comprising a polynucleotide encoding a chimeric antigen receptor comprising a ligand binding domain and a polynucleotide encoding a cell tag. Pharmaceutical formulations produced by the methods and methods of using the pharmaceutical formulations are also described.

Description

This invention application is a divisional application based on an invention patent application with an application date of September 22, 2022, application number 202280074739.X (international application number PCT / US2022 / 076862) and name “IL5RA Cell Surface Marker”. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 247,239, filed on September 22, 2021, which is hereby incorporated by reference in its entirety. Reference to an electronic sequence listing

[0002] The contents of the electronic Sequence Listing (237752000640SEQLIST.xml; size: 314,569 bytes; and creation date: September 21, 2022) are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates to cell tags comprising an extracellular region and a transmembrane region. The cell tags allow identification, detection, selection and ablation of cells modified to express the cell tags. In some embodiments, the present disclosure provides cell tags that are incapable of signal transduction, which can be expressed on the surface of cells modified to express chimeric antigen receptors. Background Art

[0004] CD19 CAR T-cell therapy (marketed by Novartis Pharmaceuticals Corp. Marketed by Kite Pharma, Inc. and marketed by Juno Therapeutics, Inc. ) has changed the way patients with B cell malignancies are treated. In fact, this success is changing the way doctors and scientists seek to treat cancer and other diseases via cell therapy. Scientists use different synthetic biology methods to transduce cells with transgenes, which give cells new or enhanced abilities such as fighting cancer. In order for these therapies to be effective, scientists must be able to understand the percentage of cells expressing the transgene (if the percentage is low, enrich it), know the transport location of the transgenic cells in the body, and ablate the transgenic cells if the therapy has side effects. Cell surface markers have been used to effectively meet all or some of these criteria. Cell surface markers are usually designed by membrane proteins, which are then truncated to make them relatively inert on the cell surface. Truncated proteins are cell surface tags and usually have the ability to be bound by small molecules or antibodies. CD19, CD20, CD34, CTLA-4, EGFR and HER2 are some surface proteins that produce cell surface tags. Only tags from EGFR, HER2, CD19 and hybrid tags of CD20 and CD34 have all the properties listed above and have entered human clinical trials.

[0005] While these tags exist, there is a clear need to have more cell tags. Ideally, the tag would be orthogonal to the cell type on which it is used to serve as a unique identifier. As cell therapy expands to use cells other than effector T cells, this need becomes more pressing. Furthermore, as the use of multiplexing in cell therapy increases, so does the need to have multiple tags.

[0006] The most well-known tag is EGFRt. The coding region of the tag is roughly 1100 base pairs long, which covers a large percentage of the transgenic payload that can be effectively transduced. Therefore, the ability to reduce the size of the tag coding region is important for the effective delivery of transgenic payloads. Finally, more tags are needed that can promote ex vivo purification of transgenic cells, monitoring the in vivo transport of transgenic cells, and ablation of transgenic cells in vivo via antibodies. The present invention addresses these needs. Summary of the invention

[0007] The present disclosure provides a recombinant polypeptide comprising a cell surface tag. The tag comprises an extracellular region, a transmembrane region and optionally an intracellular region, wherein the extracellular region comprises an IL5 receptor alpha (IL5Ra) sequence linked to the transmembrane domain, wherein the recombinant polypeptide cannot function in signal transduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate exemplary embodiments and, together with the description, further serve to enable those skilled in the relevant art to make and use these embodiments and other embodiments that will be apparent to those skilled in the art. The present invention will be described in more detail in conjunction with the following drawings.

[0009] Figure 1 A diagram showing some possible uses of cell labels, including in vitro selection, in vivo detection, and ablation of cells in a subject. The reference for flow and IHC images is Wang et al., Blood 118, 1255-1263, 2011.

[0010] Figure 2 Depicted are flow cytometric graphs showing the expression of surface proteins (CD25, VEGFR2, and IL5Ra) in primary Treg cells from two human donors.

[0011] Figure 3A An expression cassette for expressing the IL5Rat tag (pSB_0166) in K562 cells is depicted. The promoter is the human elongation factor-1 alpha promoter (EF1a). The coding region of the chimeric antigen receptor (CAR) is separated from the coding region of the IL5Ra tag by the coding region of the P2A ribosomal skipping peptide. A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is included to increase the stability of the mRNA and the expression of the protein. Figure 3B-Figure 3E Shown are flow cytometry plots of transiently transfected and untransfected K652 cells stained with: CV-biotin conjugated to streptavidin-FITC for CAR detection, and commercial anti-IL5Ra-PE or benralizumab (Creative Biolab) followed by secondary anti-human Fab-PE. Figure 3B Shown are images of non-transfected K562 control cells stained with the full antibody cocktail. Figure 3C Shown are images of transfected K562 cells stained with anti-human IgG Fab secondary antibody only. Figure 3D Shown are images of transfected K562 cells stained with: CV-biotin conjugated to streptavidin-FITC for CAR detection, and a commercial anti-IL5Ra-PE antibody. Figure 3E Shown are images of transfected K562 cells stained with: CV-biotin conjugated to streptavidin-FITC for CAR detection, and benralizumab (CreativeBiolab) followed by a secondary anti-human Fab-PE antibody for IL5Rat tag detection.

[0012] Figure 4AThe expression cassettes used to express the IL5Rat tag and CONIM EGFRt tag in Jurkat cells are depicted. Figure 4B is a flow cytometry graph showing the expression of anti-CV CAR and EGFRt in transduced Jurkat cells. Figure 4C is a flow cytometry graph showing the expression of anti-CV CAR and IL5Rat tag (pSB_0166) in transduced Jurkat cells.

[0013] Figure 5 Shown is the expression of the IL5Rat tag on transduced Jurkat cells (pSB_0166) that underwent positive selection via the IL5Rat tag to generate a pure population of transduced cells.

[0014] Figure 6 is a graph of an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay showing selective ablation of transduced cells (pSB_0166) expressing the L5Rat tag.

[0015] Fig. 7A The structures of three different IL5Rat tags are shown. Figure 7B Included are flow cytometry graphs showing expression of the IL5Rat tag on transduced Jurkat cells (pSB195, pSB196, or pSB198). Figure 7C is a graph showing the expression level of IL5Rat tag on transduced Jurkat cells. Fig.7D is a graph of an ADCC reporter assay showing selective ablation of transduced cells expressing the L5Rat tag.

[0016] Fig. 8A The structures of three different IL5Rat tags are shown. Figure 8B is a flow cytometry graph showing the expression of the IL5Rat tag on transduced Jurkat cells (pSB198, pSB323 or pSB326). Figure 8C is a graph showing the expression level of IL5Rat tag on transduced Jurkat cells. Fig.8D is a graph of an ADCC reporter assay showing ablation of transduced cells expressing the IL5Rat tag.

[0017] Fig.9A is a graph showing the percentage of transduced Jurkat cells (pSB198 or pSB590) expressing the IL5Rat tag. Fig. 9Bis a graph showing the expression level of IL5Rat tag on transduced Jurkat cells. Fig. 9C is a graph of an ADCC reporter assay showing ablation of transduced cells expressing the IL5Rat tag.

[0018] Fig.10 Is a graph showing the binding of recombinant IL-5 to transduced Jurkat cells expressing an IL5Rat tag. Relative to transduced cells expressing an IL-5Rat tag with a wild-type extracellular domain (pSB511 or pSB198), transduced cells expressing an IL-5Rat tag with a mutant extracellular domain (pSB540, pSB541, pSB546, or pSB552) exhibited reduced IL-5 binding.

[0019] Fig.11A Included are flow cytometry graphs showing expression of the IL5Rat tag and CAR on transduced Jurkat cells. Fig. 11B is a graph showing the expression level of IL5Rat tag on transduced Jurkat cells. Fig. 11C is a graph showing the percentage of transduced Jurkat cells expressing CAR and IL5Rat tags.

[0020] Fig.12 is a graph of an ADCC reporter assay showing ablation of transduced cells expressing the IL5Rat tag.

[0021] Fig.13A Included are flow cytometry graphs showing expression of the IL5Rat tag on transduced Jurkat cells, and graphs showing expression levels of the IL5Rat tag on transduced Jurkat cells. Fig. 13B is a graph showing the binding of recombinant IL-5 to transduced Jurkat cells expressing the IL5Rat tag. Fig. 13C is a graph of an ADCC reporter assay showing ablation of transduced cells expressing the IL5Rat tag.

[0022] Fig.14A is a graph showing the percentage of transduced human Treg cells expressing CAR and cell surface tags after the cells were subjected to a 14-day expansion protocol. Fig. 14B is showing the expression level of IL5Rat tag on transduced human Treg cells after the cells were subjected to a 14-day expansion protocol. DETAILED DESCRIPTION

[0023] The present disclosure provides novel cell surface markers that can be used for detection, selection and enrichment of engineered cells and for in vivo cell ablation. One aspect of the present disclosure provides gene tags for transgene expression that provide stable expression of transgenes in cells. Figure 1 Some possible uses of gene tags in cell therapy are shown. In some embodiments, the gene tags provide for selection of transduced cells that express a transgene. In some embodiments, the gene tags are expressed on the cell surface, have reduced immunogenicity, do not substantially increase the gene payload in the vector, and / or provide transgene expression in a variety of cells.

[0024] In some embodiments, the gene signature is a fragment of the IL-5 receptor alpha designated as IL5Rat, which comprises at least an epitope recognized by an anti-IL5Ra antibody. In some embodiments, the antibody specifically binds to domain I of IL5Ra. In some embodiments, the anti-IL5Ra antibody is an antibody that can be used therapeutically to treat a disease or disorder (e.g., cancer). In some embodiments, the epitope is recognized by benralizumab. definition

[0025] As used herein, unless otherwise indicated, the following meanings apply. The word "may" is used in a permissible sense (i.e., meaning that it is possible) rather than a mandatory sense (i.e., meaning that it must). The singular forms "one / a kind (a)", "one / a kind (an)" and "the" include plural indicators. Therefore, for example, although other terms and phrases (such as "one or more") are used for one or more elements, the reference to "one / a kind of element" includes a combination of two or more elements. The phrase "at least one / a kind" includes "one / a kind", "one / a kind or more / multiple" and "multiple / multiple". Unless otherwise indicated, the term "or" is non-exclusive, i.e., covers both "and" and "or". The term "any" between a modifier and a sequence means each member of the modifier modification sequence. Therefore, for example, the phrase "at least 1, 2 or 3 any one" means "at least 1, at least 2 or at least 3".

[0026] A composition or method that "comprising" or "includes" one or more listed elements may include other elements that are not specifically listed (e.g., an open term that means including but not limited to). For example, a composition that "comprising" or "includes" Treg cells may contain a single Treg cell or a combination with other ingredients (such as excipients, culture medium, etc.). In contrast, the phrase "consisting of..." is closed, indicating that such embodiments do not include additional elements. The term "consisting essentially of..." refers to including the listed elements and other elements (e.g., partially closed terms) that do not substantially affect the basic and novel features of the claimed combination. It should be understood that the aspects and embodiments described herein as "comprising" include "consisting of..." and "consisting essentially of..." embodiments.

[0027] As used herein, the terms "antigen", "immunogen" and "antibody target" refer to a molecule, compound or complex that is recognized by an antibody (i.e., can be bound by an antibody). The term can refer to any molecule that can be recognized by an antibody, such as a polypeptide, a polynucleotide, a carbohydrate, a lipid, a chemical moiety or a combination thereof (e.g., a phosphorylated or glycosylated polypeptide, etc.). The skilled artisan will understand that the term does not indicate that a molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody.

[0028] As used herein, the term "epitope" refers to a local site on an antigen that is recognized and bound by an antibody. An epitope can include multiple parts of several amino acids or several amino acids, such as 5 or 6 or more (e.g., 20 or more) amino acids or multiple parts of those amino acids. In some cases, the epitope includes non-protein components, such as components from carbohydrates, nucleic acids, or lipids. In some cases, the epitope is a three-dimensional portion. Therefore, for example, in the case where the target is a protein, the epitope can be composed of continuous amino acids or amino acids from different parts of the protein approached by protein folding (e.g., discontinuous epitopes).

[0029] As used herein, the term "antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene that specifically binds and recognizes an antigen. Typically, the "variable region" contains the antigen binding region of an antibody (or its functional equivalent) and is the most critical in terms of specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kD) and a "heavy" chain (about 50-70 kD).

[0030] Antibodies can be (i) any of the five major classes of immunoglobulins based on the identity of their heavy chain constant domains - α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), or (ii) a subclass (isotype) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The light chain can be λ or κ.

[0031] As used herein, the term "about" refers to a value, encompassing 90% to 110% of that value (eg, about 50 amino acids refers to 45 to 55 amino acids).

[0032] As used herein, an amino acid sequence "consists" only of the amino acids in that sequence.

[0033] As used herein, a first amino acid sequence "consists essentially of" a second amino acid sequence if the first amino acid sequence (1) comprises the second amino acid sequence and (2) is no more than 1, no more than 2, or no more than 3 amino acids longer than the second amino acid sequence.

[0034] As used herein, if the second amino acid sequence comprises the first amino acid sequence, the first amino acid sequence is a "fragment" of the second amino acid sequence. In certain embodiments, the first amino acid sequence, which is a fragment of the second amino acid sequence, may have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or any one of 10 amino acids less than the second amino acid sequence.

[0035] As used herein, a "functional equivalent" of a reference amino acid sequence is a sequence that is not identical to the reference sequence but contains minor changes (e.g., insertion, deletion, or substitution of one or several amino acids). A functionally equivalent sequence retains the function (e.g., immunogenicity) of the reference sequence to which it is equivalent. If a functionally equivalent amino acid sequence contains one or more amino acid substitutions relative to the reference sequence, these are typically conservative amino acid substitutions.

[0036] As used herein, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue without eliminating the desired properties of the protein. Suitable conservative amino acid substitutions can be made by replacing one amino acid with another having similar hydrophobicity, polarity, and R chain length. Examples of conservative amino acid substitutions include the following (note that certain categories are not mutually exclusive):

[0037] As used herein, the term "substantially identical" refers to the identity between a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are i) identical to the aligned amino acid residues in the second amino acid sequence or ii) conservatively substituted for the aligned amino acid residues in the second amino acid sequence, such that the first and second amino acid sequences have a common domain and / or a common functional activity and / or a common immunogenicity. For example, amino acid sequences containing a common domain or antigenic domain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are referred to as being sufficiently or substantially identical. In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence, such that the first nucleotide sequence and the second nucleotide sequence encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode a polypeptide having the same immunogenic properties.

[0038] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. In order to determine the percentage of identity of two amino acid sequences or two nucleic acid sequences, the sequences are compared for the purpose of optimal comparison (for example, a vacancy can be introduced in the sequence of the first amino acid or nucleic acid sequence to achieve the best comparison with the second amino acid or nucleic acid sequence). Then compare the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (that is, the number of identical overlapping positions / total number of positions multiplied by 100%). In one embodiment, the two sequences have the same length. The determination of the percentage of identity between the two sequences can also be completed using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set (e.g., for score = 100, wordlength = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameter set (e.g., score -50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules described herein. In order to obtain gapped alignments for comparison purposes, gapped BLAST (Gapped BLAST) can be used as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used for iterative searches, which detects long-range relationships between molecules (ibid.). When using BLAST, gapped BLAST and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, e.g., NCBI website). Another preferred non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is a part of the GCG sequence alignment software package.When utilizing the ALIGN program to compare amino acid sequences, the PAM120 weight residue table can be used, a gap length penalty of 12, and a gap penalty of 4. Techniques similar to those described above can be used to determine the percent identity between the two sequences with or without allowing gaps. When calculating percent identity, typically only exact matches are counted.

[0039] The amino acid sequence identity percentage can be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program can be obtained from the National Institutes of Health (Bethesda, Maryland). NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmasked = yes, chain = all, expected number of occurrences = 10, minimum low complexity length = 15 / 5, multi-pass e value = 0.01, multi-pass constant = 25, decay of final gapped alignment = 25, and scoring matrix = BLOSUM62.

[0040] In the case where NCBI-BLAST2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: Multiply the fraction X / Y by 100 Where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in the program's alignment of A and B, and Where Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0041] As used herein, the terms "nucleic acid sequence" and "nucleotide sequence" refer to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages and include cDNA. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequence of the present application may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may comprise naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine, and uracil; as well as xanthine and hypoxanthine. It should be understood that polynucleotides comprising non-transcribable nucleotide bases may be used as probes in, for example, hybridization assays. Nucleic acids may be double-stranded or single-stranded, and represent sense or antisense strands. In addition, the term "nucleic acid" includes complementary nucleic acid sequences and optimized codons or synonymous codon equivalents.

[0042] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of the sequences that naturally flank the nucleic acid from which it is derived (i.e., the sequences located at the 5' and 3' ends of the nucleic acid).

[0043] As used herein, the term "expression construct" refers to a polynucleotide comprising an expression control sequence operably linked to a heterologous nucleotide sequence (the heterologous nucleotide sequence, i.e., a sequence to which the expression control sequence is not usually linked in nature), which is the object of expression. As used herein, the term "expression vector" refers to a polynucleotide comprising an expression construct and a sequence sufficient to replicate in a host cell or insert into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term "expression control sequence" refers to a nucleotide sequence that regulates the transcription and / or translation of a nucleotide sequence operably linked thereto. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome binding sites (translation regulatory sequences).

[0044] As used herein, a nucleotide sequence is "operably linked" to an expression control sequence when the expression control sequence functions in a cell to regulate the transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through interaction between a polymerase and a promoter.

[0045] As used herein, the term "vector" includes any intermediate vehicle for a nucleic acid molecule that enables, for example, the nucleic acid molecule to be introduced into a prokaryotic and / or eukaryotic cell and / or integrated into the genome, and includes plasmids, phagemids, phages, or viral vectors (e.g., retroviral-based vectors, lentiviral vectors, adeno-associated viral vectors, etc.). As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material (usually a circular DNA duplex) that can replicate independently of chromosomal DNA.

[0046] "Transfection" refers to the introduction of new genetic material into cells. It includes transformation (direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (introduction of foreign DNA into host cells by bacteriophage viruses) and conjugation.

[0047] As used herein, "host cell" refers to a recombinant cell that contains an expression construct.

[0048] As used herein, the term "biological sample" refers to a sample containing cells (eg, peripheral blood mononuclear cells) or biomolecules derived from cells.

[0049] As used herein, the terms "therapy", "treatment", "therapeutic intervention" and "improvement" refer to any activity that causes a decrease in the severity of symptoms. The terms "treatment" and "prevention" are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, improvement of symptoms, improvement in patient survival, increase in survival time or survival rate, etc. Treatment and prevention can be complete or partial. The therapeutic effect can be compared with an individual or individual set that has not received treatment, or with the same patient at different times before or during treatment. In some aspects, for example, compared with an individual before administration or a control individual that has not undergone treatment, the severity of the disease is reduced by at least 10%. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80% or 90%, or in some cases, it is no longer detectable using standard diagnostic techniques. "Treatment" and "treatment" can also mean to extend the survival period compared with the expected survival period when not receiving treatment. As used herein, "treatment" and "treatment" also include preventive treatment.

[0050] A composition or method that "comprising" or "includes" one or more listed elements may include other elements not specifically listed (e.g., an open-ended term intended to include, but not limited to). For example, a composition that "comprising" or "includes" an antibody may contain a single antibody or a combination of the antibody and other ingredients. In contrast, the phrase "consisting of" is closed, indicating that such embodiments do not include additional elements. The term "consisting essentially of" means including the listed elements and other elements that do not substantially affect the basic and novel features of the claimed combination (e.g., partially closed terms). It should be understood that aspects and embodiments described herein as "comprising" include "consisting of" embodiments and "consisting essentially of" embodiments. IL-5 receptor alpha

[0051] The interleukin-5 receptor is a type I cytokine receptor. It is a heterodimer of the interleukin 5 receptor alpha subunit (IL5Ra) and CSF2RB. The IL5 receptor (IL5R) belongs to the type I cytokine receptor family and is a heterodimer composed of two polypeptide chains, an alpha subunit (which binds IL5 and confers cytokine specificity to the receptor) and a beta subunit (which contains the signal transduction domain).

[0052] The IL5Rα chain is expressed by eosinophils, some basophils and mouse B1 cells or B cell precursors. Like many other cytokine receptors, the alternative splicing of the α chain gene leads to the expression of the bα chain in a membrane-bound or soluble form. The soluble form does not lead to signal transduction and therefore has an antagonistic effect on IL5 signal transduction. Both monomeric forms of IL5Rα are low-affinity receptors, while dimerization with the β chain produces high-affinity receptors. In either case, the α chain only binds to IL5, and the intracellular part of IL5Rα associates with Janus kinase (JAK) 2 (protein tyrosine kinase essential in IL5 signal transduction).

[0053] The present disclosure provides novel IL5Ra-derived cell surface tags. In some embodiments, these tags are truncated (i.e., non-full-length) IL5Ra surface proteins that have been truncated to remove part or all of the intracellular signaling domain, making the protein relatively inert. In some embodiments, these proteins lack the ligand binding function and / or signal transduction function of wild-type IL5Ra, but can still be recognized by common anti-IL5Ra antibodies. In some embodiments, the extracellular domain of the IL5Rat tag can no longer bind IL5, allowing the cell surface tag to be even more inert on the surface. However, in some variants, the IL5Rat tag still has the ability to bind IL5 and is still suitable for clinical use.

[0054] In some embodiments, the length of the IL5Rat tag is about 250 to 450 amino acids. In some embodiments, the length of the IL5Rat tag is greater than (lower limit) about 250, 275, 300, 325, 350, 375, 400, or 425 amino acids. In some embodiments, the length of the IL5Rat tag is less than (upper limit) about 450, 425, 400, 375, 350, 325, 300, or 275 amino acids. That is, the length is in the range of about 250 to 450, wherein the lower limit is less than the upper limit. For example, in some embodiments, the length of the IL5Rat tag is about 325 to 425 amino acids. Unless otherwise indicated, the length range refers to the IL5Ra tag comprising the signal peptide, as opposed to the mature form of the IL5Ra tag in which the signal peptide has been removed.

[0055] In some embodiments, the IL5Rat cell tag of the present disclosure is expressed on the cell surface and does not substantially increase the gene payload in the vector, and / or promotes transgene expression in a variety of cells.

[0056] In some embodiments, the IL5Rat tag of the present invention can be expressed at high levels on the cell surface and can therefore be used as a safety switch for cell ablation in cell therapy. When the engineered cells in therapy are no longer needed in the body, a pharmaceutical grade anti-IL5Ra antibody (such as benralizumab) can be administered to the patient to remove the engineered cells by antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP). The benefit of the use of benralizumab for in vivo cell ablation is that the side effects of benralizumab are very mild for the patient.

[0057] The IL5Rat tag of the present disclosure can be used on all different cell types. In some embodiments, the IL5Rat tag of the present disclosure is used for Treg cells.

[0058] Unless otherwise indicated, IL5Ra as used herein refers to human IL5Ra. The human IL5Ra polypeptide sequence can be found in the Uniprot database (identifier number Q01344) and may have the following sequence:

[0059] In the above sequence, the various IL5RA domains are described as follows. The signal peptide spans amino acids 1-20 (*…*). The extracellular region (SEQ ID NO:59) spans amino acids 21-342 (#…#), wherein domain I (SEQ ID NO:60), domain II (SEQ ID NO:61), and domain III (SEQ ID NO:62) span amino acids 32-123 (single underline), 124-242 (double underline), and 243-334 (single underline), respectively. The transmembrane domain (SEQ ID NO:12) spans amino acids 343-362 (&…&). The intracellular domain (SEQ ID NO:13) spans amino acids 363-420 ($..$). IL-5 receptor α-derived cell labeling

[0060] The cell tags of the present disclosure are derived from IL5Ra comprising at least a portion of the extracellular sequence of IL5Ra. They do not comprise the entire sequence of IL5Ra, for example, they may comprise a truncated sequence of IL5Ra, for example, wherein the intracellular domain is truncated.

[0061] The cell tags of the present disclosure are not formulated to play a role in signal transduction. This can be accomplished by truncating the intracellular domain of IL5Ra so that it cannot perform signal transduction activity. It can also be accomplished by truncating the extracellular sequence of IL5Ra so that the sequence cannot bind to its natural target as required for signal transduction. a. Extracellular domain

[0062] The extracellular region of the IL5Ra-derived cell tag of the present invention comprises an epitope bound by an anti-IL5Ra antibody. In some embodiments, the antibody is benralizumab. For example, the region may comprise domain I of IL5Ra, such as the following domain I sequence or a functional variant thereof:

[0063] In some embodiments, the fragment of IL5Ra comprises, consists essentially of, or consists of amino acids 32-123 (domain I) or 32-242 (domains I and II) or 32-334 (domains I, II, and III) or 1-334 (domains I, II, and III). An amino acid sequence "consists essentially of" a second amino acid sequence if the amino acid sequence comprises the second amino acid sequence and any of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acids.

[0064] In some embodiments, domain I of IL5Ra comprises any one of the amino acid sequences listed below: and

[0065] In some embodiments, domain II of IL5Ra comprises any one of the amino acid sequences listed below: and

[0066] In some embodiments, domain III of IL5Ra comprises any one of the amino acid sequences listed below: and

[0067] In some embodiments, the IL5Ra-derived cell tag comprises a variant of an IL5Ra fragment having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the extracellular domain sequence of SEQ ID NO: 1, or a percentage of sequence identity between the ranges defined by any two of the above percentages. In some embodiments, the variant fragment has at least any of 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (preferably conservative amino acid substitutions).

[0068] In some embodiments, the IL5Ra-derived cell tag comprises a variant of domain I of IL5Ra that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the extracellular domain sequence of SEQ ID NO: 2. In some embodiments, the extracellular region may further comprise additional IL5Ra sequences, such as sequences derived from domains II and / or III. In other embodiments, the extracellular region excludes a portion or all of the sequence of domains II and / or III.

[0069] In some embodiments, the IL5Ra-derived cell tag comprises a variant of domain II of IL5Ra that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the extracellular domain sequence of SEQ ID NO: 61. In some embodiments, the extracellular region may further comprise additional IL5Ra sequences, such as sequences derived from domains I and / or III. In other embodiments, the extracellular region excludes a portion or all of the sequence of domains I and / or III.

[0070] In some embodiments, the IL5Ra-derived cell tag comprises a variant of domain III of IL5Ra that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the extracellular domain sequence of SEQ ID NO: 73. In some embodiments, the extracellular region may further comprise additional IL5Ra sequences, such as sequences derived from domains I and / or II. In other embodiments, the extracellular region excludes a portion or all of the sequence of domains I and / or II.

[0071] In some embodiments, the IL5Ra-derived cell tag comprises a variant of an IL5Ra fragment that has reduced binding to IL-5.

[0072] In some embodiments, the IL5Ra-derived cell tag comprises a variant of domain I of IL5Ra that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the extracellular domain of the sequence of SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the extracellular region may further comprise additional IL5Ra sequences, such as sequences derived from domains II and / or III. In other embodiments, the extracellular region excludes a portion or all of the sequence of domains II and / or III.

[0073] In some embodiments, the IL5Ra-derived cell tag comprises a variant of domain II of IL5Ra that has at least any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the extracellular domain of the sequence of SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In some embodiments, the extracellular region may further comprise additional IL5Ra sequences, such as sequences derived from domains I and / or III. In other embodiments, the extracellular region excludes a portion or all of the sequence of domains I and / or III.

[0074] In some embodiments, the IL5Ra-derived cell tag comprises a variant of domain III of IL5Ra that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the extracellular domain of the sequence of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77. In some embodiments, the extracellular region may further comprise additional IL5Ra sequences, such as sequences derived from domains I and / or II. In other embodiments, the extracellular region excludes a portion or all of the sequence of domains I and / or II.

[0075] In some embodiments, the gene tag comprises an amino acid sequence that is heterologous to IL5Ra, ie, a sequence that is not native to the IL5Ra protein. An example of a heterologous sequence is a sequence from the transmembrane region of a gene other than IL5Ra. b. Transmembrane domain

[0076] The transmembrane region of the polypeptide of the present invention contains a hydrophobic sequence. This region may contain an artificial sequence or may be derived from any transmembrane protein. When the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane region includes, for example, T cell receptors, CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, endothelial growth factor receptor family members (EGRF / ErbB1 / HER1; ErbB2 / HER2 / neu ErbB3 / HER3; ErbB4 / HER4), hepatocyte growth factor receptor (HGFR / c-MET), insulin-like growth factor receptor-1 (IGF-1R), EpCAM, VEGFR, integrin, TNF receptor superfamily (e.g., TRAILR1, TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3 or other differentiation One or more transmembrane regions of the α, β or ζ chain of a group (e.g., CD2, CD5, CD11, CD11a / LFA-1, CD15, CD18 / ITGB2, CD19, CD20, CD23 / IgE receptor, CD30, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD125, CD147 / basic immunoglobulin (basigin), CD152 / CTLA-4, CD195 / CCR5, CD319 / SLAMF7). In a specific alternative, the transmembrane domain comprises the amino acid sequence of the IL5Ra transmembrane domain having the sequence of amino acids 343-362 of SEQ ID NO: 1.

[0077] In a specific alternative, the transmembrane domain can be derived from any transmembrane protein, which can be, for example, CD28, EGFR, Her2, SlamF7, VEGFR2, CD34, PDGFRa, CD8 or CD4. In some embodiments, the transmembrane domain comprises any one of the amino acid sequences listed below: FWVLVVVGGVLACYSLLVTVAFIIFWV(SEQ ID NO:3); IATGMVGALLLLLVVALGIGLFM(SEQ ID NO:4); SIISAVVGILLVVVLGVVFGILI(SEQ ID NO:5); VLLCLLLVPLLLSLFVLGLFL(SEQ ID NO:6); IIILVGTAVIAMFFWLLLVII (SEQ ID NO:7); LIALVTSGALLAVLGITGYFL(SEQ ID NO:8); AAVLVLLLVIVIISLIVLVVIW(SEQ ID NO:9); IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 10); and MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 11).

[0078] In some embodiments, the transmembrane domain comprises any one of the amino acid sequences listed below: FVIVIMATICFILLILSLIC(SEQ ID NO:12); IIILVGTAVIAMFFWLLLVII (SEQ ID NO:7); SIISAVVGILLVVVLGVVFGILI(SEQ ID NO:5); AAVLVLLLVIVIISLIVLVVIW(SEQ ID NO:9); VSAVTLAYLIFCLCSLVGILHL(SEQ ID NO:78); VLLCLLLVPLLLSLFVLGLFL (SEQ ID NO: 6); and IVAVIFGLLLGAALLLGILVF (SEQ ID NO:79).

[0079] In some embodiments, synthetic membrane spaning domain or variant membrane spaning domain mainly comprise hydrophobic residues, such as leucine and valine.In some embodiments, membrane spaning domain can have at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with membrane spaning domain FVIVIMATICFILLILSLIC (SEQ ID NO:12) any one or the sequence identity percentage between the scope defined by any two of the above percentages.Variant membrane spaning domain preferably has the hydrophobicity score of at least 50 as calculated by Kyte Doolittle.

[0080] In some embodiments, the fragment of IL5Ra comprises the transmembrane domain described above and an extracellular domain comprising amino acids 32-123 (domain I) or 32-242 (domains I and II) or 32-334 (domains I, II, and III). c. Intracellular region

[0081] In some embodiments, the polypeptide of the present invention contains an intracellular region. In some embodiments, the intracellular region may have at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with the intracellular region KICHLWIKLFPPIPAPKSNIKDLFVTTNYEKAGSSETEIEVICYIEKPGVETLED SVF (SEQ ID NO: 13) or a sequence identity percentage between the ranges defined by any two of the above percentages. The length of the intracellular region of the cell tag described herein may be 1 to 9 (e.g., 2-9, 3-9, 4-9, 5-9, 1-4, 1-5, 1-6 or 5-8) amino acids. They may also be more than 9 amino acids in length.

[0082] In some embodiments, polypeptides of the invention contain an intracellular region. In some embodiments, the intracellular region may have at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with the intracellular region KICHLWIK (SEQ ID NO: 14) or a sequence identity percentage between the ranges defined by any two of the above percentages.

[0083] In some embodiments, the cytoplasmic domain comprises any one of the amino acid sequences listed below: KICHLWIK (SEQ ID NO: 14); LRTVKRANGG (SEQ ID NO: 80); KRRQQKIRK (SEQ ID NO: 81); KQKPRYEIRWRVIESIS(SEQ ID NO:82); QRALVLRRKRKR (SEQ ID NO: 83); WFLKRERQ (SEQ ID NO:84); and RSRRAQRQRQQRQRDR (SEQ ID NO:85).

[0084] In some embodiments, the fragment of IL5Ra comprises the intracellular domain described above and an extracellular domain comprising amino acids 32-123 (domain I) or 32-242 (domains I and II) or 32-334 (domains I, II, and III). In some embodiments, the fragment of IL5Ra comprises, consists essentially of, or consists of amino acids 32-370 (domains I, II, and III, a transmembrane domain, and a fragment of the intracellular domain). d. Signal peptide

[0085] In some embodiments, cell labels as described herein include peptides for enhancing the surface expression of cell labels.Signal peptides (also referred to herein as signal sequences) can be derived from those of any cell surface protein or secretory protein.Such polypeptides include, for example, including granulocyte macrophage stimulating factor signal sequence, endogenous HER2 leader peptide (aa 1-22), type I signal peptide, IgGK signal peptide, GM-CSFRa signal sequence and / or CD8 leader sequence.In some embodiments, the signal peptide has the following sequence: MIIVAHVLLILLGATEILQA (SEQ ID NO: 58) or MLLLVTSLL LCELPHPAFLLIP (SEQ ID NO: 15).In some embodiments, the signal peptide includes any one of the following listed amino acid sequences: MIIVAHVLLILLGATEILQA(SEQ ID NO:58); MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:15); MQSKVLLAVALWLCVETRA(SEQ ID NO:63); MELAALCRWGLLLALLPPGAAS(SEQ ID NO:64); MGTSHPAFLVLGCLLTGLLSLILC (SEQ ID NO:65); or MAGSPTCLTLIYILWQLTGSAA (SEQ ID NO: 66).

[0086] The various domains described above for the extracellular region, transmembrane region and intracellular region of the polypeptide of the present invention may be connected directly or via a peptide linker. e. Linker sequence

[0087] Optionally, the linker sequence can precede the cell tag sequence and / or separate one or more functional domains of the cell tag (e.g., peptides, gene tags, transmembrane domains that enhance surface expression). The linker sequence is optionally cleavable (e.g., T2A sequence or IRES sequence). Typically, the cleavable linker sequence is placed before the cell tag sequence in the nucleic acid construct. Other linker sequences are typically short peptides of about 2 to 15 amino acids and are located between the functional domains of the cell tag (including peptides, cell tags, and transmembrane domains that enhance surface expression). In some embodiments, the linker is between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids and is located between the functional domains of the cell tag (including peptides, cell tags, and transmembrane domains that enhance surface expression). In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a cleavable T2A sequence. In some embodiments, the linker comprises an IRES sequence.

[0088] In some embodiments, the linker comprises any of the following sequences: IgG4 hinge: ESKYGPPCPPCP (SEQ ID NO: 16); or IgG4 hinge to CH3: or IgG4 to CH2 (with optional mutations L235D, N297Q) to CH3: or CD28 hinge: or CD8:

[0089] In some embodiments, the linker comprises any of the following glycine-rich sequences: GGGGS repeats (1-6 repeats or more): GGGGS (SEQ ID NO: 21); or GGGSGGG Connector: GGGSGGG (SEQ ID NO: 22). f. Example of IL5Rat cell labeling

[0090] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain I, domain II, domain III, a transmembrane domain, and with or without a signal peptide (e.g., an endogenous signal sequence) (shown in the sequence):

[0091] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain I, domain II, domain III, a transmembrane domain, and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0092] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of a fragment of IL5Ra domain I, domain II, domain III, transmembrane domain, intracellular domain, and with or without a signal peptide (shown in the sequence):

[0093] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of a fragment of IL5Ra domain I, domain II, domain III, transmembrane domain, intracellular domain (with a C to G mutation), and with or without a signal peptide (e.g., GM-CSFRα signal sequence) (shown in the sequence):

[0094] In some embodiments, polypeptides of the invention comprise, consist of, or consist essentially of a fragment of IL5Ra domain I, domain II, domain III, transmembrane domain, intracellular domain (with an additional four amino acids at the termini), and with or without a signal peptide (e.g., GM-CSFRα signal sequence) (shown in the sequence):

[0095] In some embodiments, the polypeptides of the invention comprise IL5Ra domains I, G 4 SG 3 Linker, transmembrane domain, consisting of, or consisting essentially of, and with or without a signal peptide (e.g., GM-CSFRa signal sequence) (shown in the sequence):

[0096] In some embodiments, the polypeptides of the invention comprise IL5Ra domain I, (G 3 S) 3Linker, transmembrane domain, consisting of or consisting essentially of, and with or without a signal peptide (e.g., GM-CSFRa signal sequence) (shown in the sequence):

[0097] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of an IL5Ra domain I, an IgG4 hinge linker, a transmembrane domain, and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0098] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of an IL5Ra domain I, an IgG4 hinge linker, a transmembrane domain, and with or without a signal peptide (e.g., an endogenous signal sequence) (shown in the sequence):

[0099] In some embodiments, the polypeptides of the invention comprise IL5Ra domain I, (G 3 SG 3 ) linker, a transmembrane domain (with an additional four amino acids at the terminus), and with or without a signal peptide (e.g., GM-CSFRα signal sequence) (shown in the sequence):

[0100] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain 1, a (G3S)3 linker, a transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0101] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain I, an IgG4 hinge linker, a transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0102] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain I, an IgG4 hinge linker, a transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., an endogenous signal sequence) (shown in the sequence):

[0103] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain I, an IgG4-CH3 hinge linker, a transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0104] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of an IL5Ra domain I, an IgG4-CH2 hinge (L235D)-CH3 linker, a transmembrane domain, and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0105] In some embodiments, a polypeptide of the invention comprises, consists of, or consists essentially of IL5Ra domain I, a (G3S)3_D1_IgG4) linker and hinge, a transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0106] In some embodiments, the polypeptides of the invention comprise IL5Ra domain I, (G 4 S)3_IgG4) linker and hinge, transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., GM-CSFRα signal sequence) (shown in the sequence):

[0107] In some embodiments, the polypeptides of the invention comprise IL5Ra domain I, ((G 4 S) 3 _D1_G3SG3) linker and hinge, transmembrane domain (with an additional four amino acids), and with or without a signal peptide (e.g., GM-CSFRα signal sequence) (shown in the sequence):

[0108] In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of the amino acid sequence of the IL5Ra tag [IL5Rat(K186A)EC_Her2(TMIC)(S1)] of pSB_0693, with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0109] In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of the amino acid sequence of the IL5Ra tag [IL5Rat(K186A)] of pSB_0540, with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence):

[0110] In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of the amino acid sequence of the IL5Ra tag [IL5RatEC_Her2(TMIC)(S1))] of pSB_0590, with or without a signal peptide (e.g., a GM-CSFRa signal sequence) (shown in the sequence):

[0111] In some embodiments, the recombinant polypeptide comprises, consists of, or consists essentially of the amino acid sequence of the IL5Ra tag [IL5Rat(S3)] of pSB_0198, with or without a signal peptide (e.g., a GM-CSFRα signal sequence) (shown in the sequence): Nucleic acids and vectors.

[0112] Another aspect of the present disclosure includes nucleic acid constructs encoding a cell tag as described herein, and variants thereof.

[0113] In some embodiments, the nucleic acid encodes an amino acid sequence of a fragment IL5Ra or a variant thereof. In some embodiments, the cell tag sequence is an IL5 receptor alpha subunit fragment as described herein. Exemplary polynucleotides encoding truncated IL5Rat tags are shown in SEQ ID NOs: 41-57 and 86-147. The nucleic acid includes codon-optimized nucleic acid sequences, degenerate sequences, and / or variant sequences for expression in humans. carrier.

[0114] In some embodiments, the vector comprises a nucleic acid encoding a cell tag. The nucleic acid encoding the cell tag can be packaged in the vector as a separate construct or connected to the nucleic acid encoding the transgene. In some embodiments, the nucleic acid encoding the cell tag can be packaged in the vector as a separate construct or connected to the nucleic acid encoding the transgene.

[0115] A variety of vector combinations can be constructed to provide efficiency of transduction and transgenic expression. In some embodiments, the vector is a dual packaging or single (integrated) viral vector. In other embodiments, the vector can include a combination of a viral vector and a plasmid vector. Other viral vectors include foamy viruses, adenoviral vectors, retroviral vectors, and lentiviral vectors. In some embodiments, the vector is a lentiviral vector.

[0116] In some embodiments, the plasmid vector or viral vector comprises a nucleic acid comprising a polynucleotide encoding a cell tag. In some embodiments, the cell tag comprises a polynucleotide encoding IL5Rat, and further comprises a promoter, a polynucleotide encoding a peptide that enhances surface expression, and / or a polynucleotide encoding a transmembrane domain. In a specific alternative, the first nucleic acid encodes a polypeptide having a sequence of SEQ ID NO: 2, SEQ ID NO: 23-40, or a variant thereof having at least 80%, 85%, 90%, 95% or 100% sequence identity with the polypeptide, and is operably connected to a promoter.

[0117] In some embodiments, the plasmid or viral vector comprises a promoter operably connected to a polynucleotide encoding a chimeric antigen receptor, which is operably connected to a polynucleotide encoding a cell tag. In some embodiments, the polynucleotide encoding CAR is operably connected to a cell tag with a self-cleavable linker.

[0118] Each element of the nucleic acid can be separated from each other by a linker sequence (eg, a self-cleaving linker, such as a T2A self-cleaving sequence).

[0119] In some embodiments, IRES can be used. IRES sequences are commonly used in molecular biology to co-express several genes under the control of the same promoter, thereby mimicking polycistronic mRNA. In some embodiments, several genes can be placed on a plasmid with a promoter and terminator. The advantage of this technology is that it improves molecular processing.

[0120] In other embodiments, the heterogeneous (heterogeneous to the vector (e.g., lentiviral vector)) nucleic acid sequence is limited by the amount of additional genetic components that can be packaged in the vector. In some embodiments, the construct contains at least two genes that are heterogeneous to the viral vector. In some embodiments, the construct contains at least 4 genes that are heterogeneous to the viral vector. The number of genes that are heterogeneous to the viral vector that can be packaged in the vector can be determined by detecting the expression of one or more transgenes and selecting a vector construct that provides transduction of at least 10% of the cells and / or a level of transgene expression that is detectable in at least 10% of the cells.

[0121] In some embodiments, the lentivirus is a double packaged virus. The double packaged virus contains at least one nucleic acid, and the at least one nucleic acid includes a polynucleotide encoding a chimeric antigen receptor and a first cell label. Optionally, the nucleic acid further includes a polynucleotide encoding a cytokine and / or a chemokine receptor. The double packaged virus contains at least one nucleic acid, and the at least one nucleic acid includes a polynucleotide encoding a chimeric antigen receptor and a second cell label. Optionally, the nucleic acid further includes a polynucleotide encoding a cytokine and / or a chemokine receptor. In some embodiments of a system with two constructs, each construct can be packaged in a separate viral vector, and the viral vector can be mixed together for transduction in a cell population. In some embodiments, the first cell label and the second cell label are different from each other. In some embodiments, the double packaged virus provides expression of at least two different transgenics (e.g., CAR constructs) in a single cell type. The use of different cell labels provides the selection of dual transduced cells.

[0122] In some embodiments, the vector is a minicircle. The minicircle is an episomal DNA vector that is produced as a circular expression cassette lacking any bacterial plasmid DNA backbone. Compared with standard plasmid vectors that only work for a few days, their smaller molecular size enables more efficient transfection and provides sustained expression over a period of several weeks. In some embodiments, the minicircle comprises a promoter connected to a polynucleotide encoding a chimeric antigen receptor operably connected to a cell tag. One or more minicircles can be used. In some embodiments, the minicircle comprises a promoter connected to a polynucleotide encoding a chimeric antigen receptor and a first cell tag, and another minicircle comprises a promoter connected to a polynucleotide encoding a chimeric antigen receptor and a second and different cell tag. In some embodiments, each element of the construct is separated by a nucleic acid (such as a nucleic acid encoding a self-cleaving T2A sequence). In some embodiments, each minicircle is different from each other in terms of chimeric antigen receptors, including but not limited to spacer length and sequence, intracellular signaling domain and / or cell tag sequence.

[0123] In some embodiments, the vector is a PiggyBac transposon. PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between a vector and a chromosome via a "cut and paste" mechanism. During transposition, the PB transposase recognizes the transposon-specific reverse terminal repeats (ITRs) at both ends of the transposon vector and efficiently moves the contents from the original site and efficiently integrates them into the TTAA chromosome site. The powerful activity of the PiggyBac transposon system enables the target gene between the two ITRs in the PB vector to be easily mobilized into the target genome.

[0124] In some embodiments, PB contains a promoter connected to a polynucleotide encoding a chimeric antigen receptor operably connected to a gene tag. One or more PB transposons can be used. In some embodiments, PB includes a promoter connected to a polynucleotide encoding a chimeric antigen receptor and a first gene tag, and another PB includes a promoter connected to a polynucleotide encoding a chimeric antigen receptor and a second and different cell tag. Each element of the construct is separated by a nucleic acid (such as a nucleic acid encoding a self-cleaving T2A sequence). In some embodiments, each PB is different from each other in terms of chimeric antigen receptor, including but not limited to spacer length and sequence, intracellular signaling domain and / or cell tag sequence.

[0125] In some embodiments, the first nucleic acid comprises a first promoter operably linked to: a chimeric antigen receptor comprising a ligand binding domain, wherein the ligand binding domain binds to a ligand, wherein the ligand is a disease-specific molecule, a viral molecule, or any other molecule expressed on a target cell population suitable for mediating recognition by lymphocytes; a polynucleotide encoding a polypeptide spacer, wherein the spacer provides increased T cell proliferation and / or cytokine production in response to the ligand compared to a reference chimeric receptor; a polynucleotide encoding a transmembrane domain; and d) a polynucleotide encoding an intracellular signaling domain. In some embodiments, the first nucleic acid further comprises a cell tag.

[0126] In some embodiments, the second nucleic acid comprises a polynucleotide encoding a second and different chimeric antigen receptor. The first chimeric antigen receptor and the second chimeric antigen receptor may differ from each other in terms of the ligand binding domain, the target antigen, the epitope of the target antigen, the length and sequence (short, medium or long) of the spacer domain, and the intracellular signaling domain. In some embodiments, the second nucleic acid further comprises a second and different cell tag from the first nucleic acid.

[0127] In some embodiments, in a single lentiviral construct, the first nucleic acid and the second nucleic acid can be separated by a genomic insulator nucleic acid, such as a sea urchin insulator chromatin domain.

[0128] In some embodiments, the promoter used herein can be an inducible or constitutive promoter. Inducible promoters include tamoxifen inducible promoters, tetracycline inducible promoters, and doxycycline inducible promoters. Constitutive promoters include SV40, CMV, UBC, EF1α, PGK, and CAGG.

[0129] One or more of these vectors can be combined with each other to transduce target cells and provide expression of the chimeric antigen receptor. genetically modified

[0130] Several transgenes are also aspects of the present invention. Cell labels as described herein can be used to select, track and kill cells transduced with a transgene and expressing the transgene. Cell labels can be used with any number of different transgenes. In this disclosure, chimeric antigen receptor transgenes are exemplified, but similar principles apply to the design, identification and selection of other transgenes expressed in transduced cells. In some embodiments, the transgene expresses an antigen receptor and / or another additional polypeptide. The antigen receptor can be, for example, an antibody, an engineered antibody (such as scFv, CAR, engineered TCR, TCR mimetic, or chimeric antibody-T cell receptor), or a chimeric signaling receptor. The antigen receptor can target an antigen of interest (e.g., a tumor antigen or an antigen of a pathogen). Antigens can include, but are not limited to, AFP (alpha-fetoprotein), αvβ6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (CC motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD123, CD138, CD171, CD352, CEA (carcinoembryonic antigen), claudin, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epidermal glycoprotein 2), EPG-40, ephrin B2, EPHa2 (ephrin receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP -α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (folate binding protein), FCRL5, FR-α (folate receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G protein coupled receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukemia) cell antigen A1), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight-melanoma associated antigen), IGF1R (insulin-like growth factor 1 receptor), Igκ, Igλ, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL-13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI-CAM), Liv-1, LRRC8A (leucine-rich repeat-containing protein 8 family member A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complex (e.g.,HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), connexin-4, NKG2D (natural killer group 2 member D) ligand, NY-ESO, carcinoembryonic antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen in melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), ROR1, ROR2, SIRPα (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (prostate six transmembrane epithelial antigen 1), survivin, TAG72 (tumor associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens. , Host cells and compositions: T lymphocyte populations.

[0131] The compositions described herein provide a genetically modified host cell with a vector and / or construct as described herein. In some embodiments, the host cell is a CD4+ and / or CD8+ T lymphocyte. In some embodiments, the host cell is a Treg cell. In some embodiments, the host cell is a precursor T cell. In some embodiments, the host cell is a hematopoietic stem cell.

[0132] T lymphocytes can be collected according to known techniques and enriched or exhausted by known techniques (such as affinity binding with antibodies, such as flow cytometry and / or immunomagnetic selection). After the enrichment and / or exhaustion step, the in vitro expansion of desired T lymphocytes can be carried out according to known techniques or variants thereof that are clear to those skilled in the art. In some embodiments, T cells are autologous T cells obtained from the patient.

[0133] The expanded T lymphocytes include CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T lymphocytes that may be specific for antigens present on human tumors or pathogens. The expanded T lymphocytes include Treg cells.

[0134] In some embodiments, the expansion method may further include the step of adding anti-CD3 antibody and / or anti-CD28 antibody to the culture medium. Optionally, the expansion method may further include the step of adding IL-2 and / or IL-15 to the culture medium.

[0135] After isolation of T lymphocytes, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, effector T cell and Treg cell subsets either before or after expansion. Composition

[0136] The present disclosure provides adoptive cell immunotherapy compositions, which include genetically modified cell preparations as described herein, such as genetically modified lymphocyte preparations. These cells are, for example, pluripotent cells (such as various progenitor cells or precursor cells of hematopoietic stem cells, hematopoietic lineages), and various immune cells (for example, people's autologous or allogeneic T cells, natural killer (NK) cells, dendritic cells or B cells). These cells can also be pluripotent stem cells (PSC), such as human embryonic stem cells and induced PSC, which can be used to produce therapeutic cell colonies. In some embodiments, pluripotent stem cells and pluripotent cells are differentiated into desired cell types in vitro before being implanted in patients.

[0137] In some embodiments, the genetically modified cell preparation is a T lymphocyte preparation. In some embodiments, the T lymphocyte preparation comprises a CD4+T cell with a chimeric receptor, and the chimeric receptor comprises an extracellular antibody variable domain, a spacer, a transmembrane domain, and an intracellular signaling domain of a T cell receptor specific for a ligand associated with a disease or disorder, and a cell label as described herein. In other embodiments, the adoptive cell immunotherapy composition further comprises a CD8+ cytotoxic T lymphocyte preparation modified by a chimeric receptor providing a cellular immune response, wherein the cytotoxic T lymphocyte preparation comprises a CD8+T cell with a chimeric receptor, and the chimeric receptor comprises an extracellular single-chain antibody, a spacer, a transmembrane domain, and an intracellular signaling domain of a T cell receptor specific for a ligand associated with a disease or disorder, and a cell label as described herein. In some embodiments, the chimeric receptor-modified T cell population of the present disclosure can persist in vivo for at least about 3 days or longer. In alternatives, each of these populations can be combined with each other or with other cell types to provide a composition. In some embodiments, the host cell is a Treg cell.

[0138] Embodiments include CD4 and / or CD8 host cells as described herein. In some embodiments, the host cell comprises an isolated nucleic acid (such as a nucleic acid encoding an isolated polypeptide) and a second nucleic acid encoding a second chimeric antigen receptor and a second cell tag, wherein the isolated polypeptide has at least 95% sequence identity with an IL5Rat polypeptide having a sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1 connected to a transmembrane domain, wherein the isolated polypeptide specifically binds to an antibody that binds to an epitope in domain I of IL5Ra. In some embodiments, the host cell is a Treg cell.

[0139] In other embodiments, the composition comprises a first host cell containing a first isolated nucleic acid (such as a nucleic acid encoding an isolated polypeptide) and a second host cell containing a second nucleic acid encoding a second chimeric antigen receptor and a second cell tag, wherein the isolated polypeptide has at least 95% sequence identity with an IL5Rat polypeptide having a sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1 connected to a transmembrane domain, wherein the isolated polypeptide specifically binds to an antibody that binds to an epitope in domain I of IL5Ra. In some embodiments, the first host cell and the second host cell can be the same or different types of host cells, for example, the first host cell can be a CD8 cell and the second host cell can be a CD4 cell. In some embodiments, the first host cell and the second host cell are each selected from: CD8 T cells, CD4 T cells, CD4 naive T cells, CD8 naive T cells, CD8 central memory cells, CD4 central memory cells, Treg cells, and combinations thereof.

[0140] In some embodiments, the CD4+T helper lymphocytes are selected from naive CD4+T cells, central memory CD4+T cells, effector memory CD4+T cells or a large number of CD4+T cells. In some embodiments, the CD4+ helper lymphocytes are naive CD4+T cells, wherein the naive CD4+T cells comprise CD45RO-, CD45RA+, CD62L+CD4+T cells.

[0141] In some embodiments, CD8+T cytotoxic lymphocytes are selected from naive CD8+T cells, central memory CD8+T cells, effector memory CD8+T cells or a large number of CD8+T cells. In some embodiments, CD8+ cytotoxic T lymphocytes are central memory T cells, wherein the central memory T cells include CD45RO+, CD62L+, CD8+T cells. In yet other embodiments, CD8+ cytotoxic T lymphocytes are central memory T cells and CD4+ helper T lymphocytes are naive or central memory CD4+T cells.

[0142] In some embodiments, Treg cells are CD4 + CD25 + CD127 lo . method

[0143] The present disclosure provides methods of preparing adoptive immunotherapy compositions and uses or methods of using these compositions for cellular immunotherapy in subjects suffering from a disease or disorder.

[0144] Embodiments include methods for making compositions comprising host cells as described herein. In some embodiments, the method includes introducing an isolated nucleic acid (such as a nucleic acid encoding an isolated polypeptide) into a host cell, wherein the isolated polypeptide has at least 95% sequence identity with an IL5Rat polypeptide having a sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1 connected to a transmembrane domain, wherein the isolated polypeptide specifically binds to an antibody that binds to an epitope in domain I of IL5Ra; and culturing the host cell in a culture medium comprising at least one growth factor. In some embodiments, the method further includes selecting the host cell for expression of IL5Rat before or after the culturing step or both before and after. In other embodiments, the manufacturing method further includes introducing a second nucleic acid encoding a second chimeric antigen receptor and a second cell tag into the host cell. In some embodiments, the method further includes selecting the host cell for expression of a second cell tag before or after the culturing step or both before and after. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a Treg cell.

[0145] In other embodiments, the method comprises introducing a first isolated nucleic acid (such as a nucleic acid encoding an isolated polypeptide) into a first host cell, the isolated polypeptide having at least 95% sequence identity to an IL5Ra polypeptide of the sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1 linked to a transmembrane domain, wherein the isolated polypeptide specifically binds to an antibody that binds to an epitope in domain I of IL5Ra; selecting a first host cell that expresses IL5Rat, introducing a second nucleic acid encoding a second chimeric antigen receptor and a second cell tag into a second host cell, selecting the second host cell for expression of the second cell tag, and optionally culturing the first host cell and the second host cell in a culture medium with at least one growth factor. In some embodiments, the composition comprises a first population of host cells and a second population of host cells.

[0146] In some embodiments, the method comprises introducing an isolated nucleic acid (such as a nucleic acid encoding an isolated polypeptide) into a host cell, wherein the isolated polypeptide has at least 95% sequence identity to an IL5Rat polypeptide having a sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1 linked to a transmembrane domain, wherein the isolated polypeptide has reduced binding to IL-5; and culturing the host cell in a medium comprising at least one growth factor. In some embodiments, the isolated polypeptide comprises a variant of domain 1 of IL5Ra having an amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 68, or SEQ ID NO: 69. In some embodiments, the isolated polypeptide comprises a variant of domain 2 of IL5Ra having an amino acid sequence of SEQ ID NO: 70, SEQ ID NO: 71, or SEQ ID NO: 72. In some embodiments, the isolated polypeptide comprises a variant of domain 3 of IL5Ra having an amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77. In some embodiments, the method further comprises selecting the host cell for expression of IL5Rat before or after the culturing step or both before and after. In other embodiments, the manufacturing method further comprises introducing a second nucleic acid encoding a second chimeric antigen receptor and a second cell tag into the host cell. In some embodiments, the method further comprises selecting the host cell for expression of a second cell tag before or after the culturing step or both before and after. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a Treg cell.

[0147] In some embodiments, the present disclosure provides a method of making a composition comprising obtaining modified naive, central memory or regulatory CD4+T cells, wherein the modified CD4+T lymphocyte preparation comprises CD4+T cells having a chimeric receptor comprising a ligand binding domain specific for a disease-associated antigen, a spacer domain, a transmembrane domain, and an intracellular signaling domain and a cell tag as described herein.

[0148] In other embodiments, the disclosure provides methods comprising obtaining modified CD8+ T cells, wherein the CD8 T lymphocyte preparation comprises CD8+ cells having a chimeric receptor comprising a ligand binding domain specific for a disease-associated antigen, a spacer domain, a transmembrane domain, and an intracellular signaling domain and a cell tag as described herein. In other embodiments, the CD8+ cells have a cytokine or chemokine receptor under the control of an inducible promoter.

[0149] The preparation of cells modified with chimeric receptors has been described above and in the Examples. The cells can be obtained from patients suffering from a disease or disorder or from healthy donors. The cells are prepared by stimulating T lymphocytes in vitro in the presence of an antigen. Subpopulations of cells can also be isolated as described herein and combined in the method of manufacture. The cell population is advantageously selected for expression of the IL5Rat tag described herein.

[0150] The present disclosure also provides a method for performing cellular immunotherapy in a subject with a disease or disorder, the method comprising administering a composition of cells (e.g., lymphocytes) expressing one or more chimeric antigen receptors and cell labels as described herein. In some embodiments, a method for performing cellular immunotherapy in a subject with a disease or disorder is provided, wherein the method comprises administering a composition of cells expressing one or more chimeric antigen receptors and cell labels.

[0151] In some embodiments, if the modified cells are no longer desired by the subject (e.g., a patient with a disease or disorder), an antibody that binds to a cell tag is administered. The antibody can bind to and kill the modified cells of the composition, for example, to avoid toxic and / or fatal side effects. In some embodiments, the antibody or antigen binding fragment preferably contains an Fc fragment to activate an immune response, such as ADCC, ADCP or CDC reaction. In other embodiments, the antibody or antigen binding fragment is conjugated to a cytotoxic agent. Cytotoxic agents include cantansinoid, calicheamicin and / or auristatin. In some embodiments, the cytotoxic agent comprises cantansinoid, calicheamicin and / or auristatin.

[0152] In some embodiments, the antibody is detectably labeled to allow tracking of modified cells in vivo. In some embodiments, when the antibody is used for in vivo detection, it is preferred that the antibody or antigen binding fragment lacks all or part of the Fc region to avoid ADCC reaction. Detectable labels include biotin, His tags, myc tags, radioactive labels and / or fluorescent labels. In some embodiments, detectable labels include biotin, His tags, myc tags, radioactive labels and / or fluorescent labels.

[0153] The subjects that can be treated by the present invention are generally humans and other primate subjects (such as monkeys and apes for veterinary purposes). The subject can be male or female, and can be of any suitable age, including infants, young children, teenagers, adults and elderly subjects. In some embodiments, the subject is a primate subject or a human.

[0154] Cells prepared as described above can be used in methods and compositions for adoptive immunotherapy according to known techniques, or variations thereof that will be apparent to those skilled in the art based on this disclosure.

[0155] A therapeutically effective number of modified cells is administered to a subject. As used herein, the term "therapeutically effective" refers to a cell quantity or pharmaceutical composition amount sufficient to treat, prevent and / or delay the onset or progression of one or more symptoms of a disease, disorder or condition when administered to a subject suffering from or susceptible to the disease, disorder and / or condition. The number of cells will depend on the end use to which the composition is intended, as will the cell types contained therein. For example, if cells specific to a particular antigen are desired, the population will contain greater than 70%, typically greater than 80%, 85% and 90%-95% of such cells or any percentage amount of cells within the range defined by any two of the above percentages.

[0156] The modified cells may be administered by a single infusion or by multiple infusions over a period of time. However, because different individuals are expected to have different responsiveness, the type and amount of cells infused, as well as the number of infusions and the time frame over which multiple infusions are given, are determined by the attending physician and can be determined by routine examination.

[0157] In some embodiments, the compositions as described herein are administered intravenously, intraperitoneally, intratumorally to the bone marrow, lymph nodes, and / or cerebrospinal fluid. In some embodiments, the chimeric receptor engineered compositions are delivered to the disease site (e.g., tumor or inflammation site). In some embodiments, the compositions as described herein are administered together with chemotherapeutic agents and / or immunosuppressants. Exemplary embodiments 1. A recombinant polypeptide comprising an extracellular region, a transmembrane region and optionally an intracellular region, wherein the extracellular region comprises an IL5 receptor alpha (IL5Ra) sequence linked to the transmembrane domain, wherein the recombinant polypeptide is incapable of playing a role in signal transduction. 2. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) of SEQ ID NO: 1. 3. A recombinant polypeptide according to embodiment 2, wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 124-242 (domain II) of SEQ ID NO:1. 4. A recombinant polypeptide according to embodiment 2 or 3, wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with 243-334 (domain III) of SEQ ID NO: 1. 5. A recombinant polypeptide according to embodiment 1, wherein the extracellular region comprises a truncated IL5Ra extracellular region, which comprises no more than a part or all of domain I, no more than a part or all of domain II and / or no more than a part or all of domain III of IL5Ra. 6. The recombinant polypeptide according to embodiment 1, wherein the extracellular region comprises a truncated IL5Ra extracellular region, wherein the truncated IL5Ra extracellular region comprises a part or all of domain I but does not comprise domain II and / or domain III of IL5Ra. 7. A recombinant polypeptide according to embodiment 1, wherein the extracellular region binds to benralizumab. 8. A recombinant polypeptide according to any preceding embodiment, wherein the transmembrane region comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with any of sequences 343-362 of SEQ ID NO: 1. 9. A recombinant polypeptide according to any one of embodiments 1 to 7, wherein the transmembrane region comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with an amino acid sequence selected from SEQ ID NOs: 3-12. 10. The recombinant polypeptide according to any preceding embodiment, wherein the recombinant polypeptide comprises an intracellular domain. 11. The recombinant polypeptide according to embodiment 10, wherein the intracellular domain comprises a truncated portion of the intracellular domain of IL5Ra, wherein the truncated portion has lost signal transduction ability. 12. The recombinant polypeptide of embodiment 10, wherein the intracellular domain consists of amino acids 363-370 of SEQ ID NO: 1. 13. The recombinant polypeptide of embodiment 10 or 12, wherein the intracellular domain comprises amino acids 363-366 of SEQ ID NO: 1. 14. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) or 32-242 (domains I and II) or 32-334 (domains I, II and III) of SEQ ID NO:1. 15. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-370 of SEQ ID NO: 1 (domains I, II and III, the transmembrane domain, and a fragment of the intracellular domain). 16. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with an IL5Ra polypeptide having a sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1. 17. The recombinant polypeptide according to any preceding embodiment, further comprising a signal peptide. 18. A recombinant polypeptide according to embodiment 17, wherein the signal peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with the amino acid sequence consisting of SEQ ID NO:58 and SEQ ID NO:15. 19. The recombinant polypeptide according to any preceding embodiment, further comprising a linker. 20. A recombinant polypeptide according to embodiment 19, wherein the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% amino acid sequence identity with any one of the amino acid sequences selected from SEQ ID NOs: 16-22. 21. A recombinant polypeptide according to embodiment 1, comprising an intracellular region, wherein the intracellular region has an amino acid sequence consisting of or essentially consisting of SEQ ID NO: 13, SEQ ID NO: 14; or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical thereto. 22. A recombinant polypeptide according to embodiment 1, which has an amino acid sequence comprising, consisting essentially of, or consisting of any one of SEQ ID NOs: 23-40; or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical thereto. 23. The recombinant polypeptide of embodiment 1, comprising at least a portion that is heterologous to IL5 Ra. 24. A nucleic acid molecule comprising a coding sequence for a recombinant polypeptide according to any one of the preceding embodiments. 25. The nucleic acid molecule of embodiment 24, comprising a nucleotide sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to any one of SEQ ID NOs: 41-57. 26. According to the nucleic acid molecule of embodiment 24, the nucleic acid molecule further comprises a coding sequence of a chimeric antigen receptor (CAR). 27. A nucleic acid molecule according to embodiment 26, wherein the coding sequences of the recombinant polypeptide and the CAR are operably linked to the same promoter so that the two coding sequences are co-transcribed. 28. The nucleic acid molecule of any one of embodiments 24-27, wherein the nucleic acid molecule is a viral vector, optionally a lentiviral or retroviral vector. 29. A cell comprising a nucleic acid molecule according to any one of embodiments 24-28. 30. A cell according to embodiment 29, wherein the cell is a human T cell. 31. A cell according to embodiment 30, wherein the cell is a human Treg cell. 32. A pharmaceutical composition comprising the cell according to embodiments 29 to 31, or the nucleic acid molecule according to any one of embodiments 21-28; and a pharmaceutically acceptable carrier. 33. A method of treating a patient in need thereof, the method comprising administering to the patient a cell according to embodiments 29 to 31, optionally wherein the cell is derived from the patient. 34. The method according to embodiment 33 comprises administering to the patient the cells according to embodiment 30 or 31, wherein the cells express a CAR specific for an antigen present in the disease. 35. The method according to embodiment 33 or 34, further comprising administering to the patient an effective amount of an antibody specific for IL5Ra once the patient has been treated, wherein the antibody elicits cytotoxicity against cells expressing the recombinant polypeptide, and optionally the antibody is IgG1 or IgG2. 36. A method for preparing a genetically engineered human cell, the method comprising providing an isolated human cell, and introducing a nucleic acid molecule according to any one of embodiments 24-28 into the human cell. 37. A method according to embodiment 36, wherein the human cells are human T cells. 38. A method according to embodiment 37, wherein the human cells are human Treg cells. 39. A cell population comprising a nucleic acid encoding a recombinant polypeptide comprising an extracellular region, a transmembrane region, and optionally an intracellular region, wherein the extracellular region comprises an IL5Ra sequence linked to the transmembrane domain. 40. A population according to embodiment 39, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) or 32-242 (domains I and II) or 32-334 (domains I, II and III) of SEQ ID NO:1. 41. A population according to embodiment 39, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-370 of SEQ ID NO:1 (domains I, II and III, the transmembrane domain, and a fragment of the intracellular domain). 42. A population according to embodiment 39, wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with an IL5Ra polypeptide having a sequence of amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 370 of SEQ ID NO: 1. 43. A population according to embodiment 39, wherein the recombinant polypeptide comprises SEQ ID NO: 23-40; or an amino acid sequence of any one of which is at least 80%, 85%, 90% or 95% identical thereto. 44. A population according to embodiment 39, wherein the nucleic acid further comprises a coding sequence for CAR. 45. A population according to embodiment 44, wherein the coding sequences of the recombinant polypeptide and the CAR are operably linked to the same promoter so that the two coding sequences are co-transcribed. 46. ​​A population according to embodiments 39 to 45, wherein the nucleic acid molecule is a viral vector, optionally a lentiviral or retroviral vector. 47. A population according to any one of embodiments 32 to 46, wherein the cell population is human T cells. 48. A population according to embodiment 47, wherein the cell population is human Treg cells. 49. A pharmaceutical composition comprising the population according to embodiments 39 to 48, and a pharmaceutically acceptable carrier. Further exemplary embodiments 1. A recombinant polypeptide comprising an extracellular region and a transmembrane region, wherein the extracellular region comprises a portion or all of the extracellular domain of IL5 receptor α (IL5Ra) and the extracellular region is connected to the transmembrane domain, wherein the recombinant polypeptide is unable to play a role in signal transduction, and optionally wherein the recombinant polypeptide has reduced binding to IL-5. 2. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) of SEQ ID NO: 1. 3. A recombinant polypeptide according to embodiment 2, wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 124-242 (domain II) of SEQ ID NO:1. 4. A recombinant polypeptide according to embodiment 2 or embodiment 3, wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 243-334 (domain III) of SEQ ID NO:1. 5. The recombinant polypeptide according to embodiment 1, wherein the extracellular region comprises a truncated IL5Ra extracellular region, and the truncated IL5Ra extracellular region comprises no more than part or all of the following: i) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69; ii) the amino acid sequence of SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72; and / or iii) the amino acid sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77. 6. A recombinant polypeptide according to embodiment 1, wherein the extracellular region comprises a truncated IL5Ra extracellular region, and the truncated IL5Ra extracellular region comprises part or all of the following: the amino acid sequence of SEQ ID NO:2, SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:68 or SEQ ID NO:69, but does not comprise the amino acid sequence of amino acids 124-342 of SEQ ID NO:1. 7. A recombinant polypeptide according to any one of embodiments 1-6, wherein the extracellular region binds to benralizumab. 8. A recombinant polypeptide according to any one of embodiments 1-7, wherein the transmembrane region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to amino acids 343-362 of SEQ ID NO:1. 9. A recombinant polypeptide according to any one of embodiments 1-7, wherein the transmembrane region comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:78 and SEQ ID NO:79. 10. The recombinant polypeptide of any one of embodiments 1-9, wherein the recombinant polypeptide further comprises an intracellular domain. 11. The recombinant polypeptide according to embodiment 10, wherein the intracellular domain comprises a truncated IL5Ra intracellular domain, optionally wherein the truncated IL5Ra intracellular domain does not associate with Janus kinase 2 (JAK2), optionally wherein the truncated IL5Ra intracellular domain has lost its signal transduction ability. 12. The recombinant polypeptide according to embodiment 11, wherein (i) the intracellular domain consists of amino acids 363-370 of SEQ ID NO: 1; or (ii) the intracellular domain comprises amino acids 363-366 of SEQ ID NO:1, but does not comprise the amino acid sequence of SEQ ID NO:13. 13. A recombinant polypeptide according to embodiment 10, wherein the length of the intracellular domain does not exceed 25, 30, 35, 40, 45 or 50 amino acids, and the intracellular domain comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85. 14. A recombinant polypeptide according to embodiment 1, wherein the extracellular domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) or amino acids 32-242 (domains I and II) or amino acids 32-334 (domains I, II and III) of SEQ ID NO:1. 15. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-370 of SEQ ID NO:1 (domains I, II and III, the transmembrane domain, and a fragment of the intracellular domain). 16. A recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 342, or 32 to 370 of SEQ ID NO:1. 17. The recombinant polypeptide according to any one of embodiments 1-16, further comprising a signal peptide. 18. A recombinant polypeptide according to embodiment 17, wherein the signal peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO:58, SEQ ID NO:15, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66. 19. The recombinant polypeptide according to any one of embodiments 1-18, further comprising a linker between the extracellular domain and the transmembrane domain. 20. A recombinant polypeptide according to embodiment 19, wherein the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22. 21. A recombinant polypeptide according to embodiment 10, wherein the intracellular domain consists of the amino acid sequence of SEQ ID NO:14, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85; or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical thereto. 22. The recombinant polypeptide according to embodiment 1, wherein the recombinant polypeptide comprises an amino acid sequence, wherein: i) an amino acid sequence selected from SEQ ID NOs: 23-40, or at least 80%, 85%, 90% or 95% identical thereto; or (ii) selected from SEQ ID NOs: 148-208, or an amino acid sequence at least 80%, 85%, 90% or 95% identical thereto; or (iii) an amino acid sequence selected from SEQ ID NO: 23-40 or selected from 148-208, or at least 80%, 85%, 90% or 95% identical thereto. 23. The recombinant polypeptide according to embodiment 1, comprising a heterologous region of at least 4 amino acids in length, wherein the amino acid sequence of the heterologous region is heterologous to IL5Ra, optionally wherein the amino acid sequence of the heterologous region is not present in the amino acid sequence of human IL5Ra of SEQ ID NO: 1. That is, the amino acid sequence of the heterologous region is not 100% identical to the fragment of human IL5Ra of SEQ ID NO: 1. 24. A nucleic acid molecule comprising a coding sequence for a recombinant polypeptide according to any one of the preceding embodiments. 25. The nucleic acid molecule of embodiment 24, wherein the nucleic acid molecule comprises a nucleotide sequence: (i) selected from SEQ ID NOs: 41-57 and 86, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; or (ii) selected from SEQ ID NOs: 87-147, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; or (iii) a nucleotide sequence selected from SEQ ID NOs: 41-57 and 86, or selected from SEQ ID NOs: 87-147, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto. 26. According to the nucleic acid molecule of embodiment 24, the nucleic acid molecule further comprises a coding sequence of a chimeric antigen receptor (CAR). 27. A nucleic acid molecule according to embodiment 26, wherein the coding sequences of the recombinant polypeptide and the CAR are operably linked to the same promoter so that the two coding sequences are co-transcribed. 28. The nucleic acid molecule of any one of embodiments 24-27, wherein the nucleic acid molecule is present in a viral vector, optionally wherein the viral vector is a lentiviral vector or a retroviral vector. 29. A cell comprising a nucleic acid molecule according to any one of embodiments 24-28. 30. A cell according to embodiment 29, wherein the cell is a human T cell. 31. A cell according to embodiment 30, wherein the cell is a human Treg cell. 32. A pharmaceutical composition comprising: (i) a cell according to any one of embodiments 29-31, or a nucleic acid molecule according to any one of embodiments 21-28; and (ii) a pharmaceutically acceptable carrier. 33. A method of treating a patient in need thereof, the method comprising administering to the patient a cell according to any one of embodiments 29 to 31, optionally wherein the cell is derived from the patient. 34. A method of treating a patient in need thereof, the method comprising administering to the patient a cell according to embodiment 30 or embodiment 31, wherein the cell expresses a CAR specific for an antigen present in the disease suffering from the patient. 35. The method of embodiment 33 or embodiment 34, further comprising administering to the patient an effective amount of an antibody specific for IL5Ra once the patient has been treated, wherein the antibody elicits cytotoxicity against cells expressing the recombinant polypeptide, optionally wherein the antibody is IgG1 or IgG2. 36. A method for preparing a genetically engineered human cell, the method comprising providing an isolated human cell, and introducing a nucleic acid molecule according to any one of embodiments 24-28 into the human cell. 37. A method according to embodiment 36, wherein the human cells are human T cells. 38. A method according to embodiment 37, wherein the human cells are human Treg cells. 39. The method of any one of embodiments 36-38, further comprising culturing the human cells under conditions such that the recombinant polypeptide is expressed on the surface of the genetically engineered human cells. 40. A pharmaceutical composition comprising: (i) a plurality of cells (eg, a cell population) according to any one of embodiments 29-31, and (ii) a pharmaceutically acceptable carrier. 41. A pharmaceutical composition comprising: (i) a plurality of the genetically engineered human cells (e.g., a cell population) produced by the method of any one of Embodiments 36-39, and (ii) a pharmaceutically acceptable carrier. 42. The recombinant polypeptide of any one of embodiments 1-23, wherein the recombinant polypeptide has reduced binding to IL-5. Example

[0158] Abbreviations: ADCC (antibody-dependent cell-mediated cytotoxicity); CAR (chimeric antigen receptor); CV (citrullinated vimentin); FI (fluorescence intensity); (IL-5Ra (interleukin-5 receptor alpha); IL-5Rat (truncated IL-5Ra); mAb (monoclonal antibody); MFI (median fluorescence intensity). Example 1: Expression of surface markers in primary Treg cells

[0159] Treg cells are purified from PBMC.CD4+ is enriched by negative selection from PBMC by magnetic cell sorting (Miltenyi Biotec).CD4 T cells are then stained with fluorescein-labeled mAbs specific to CD4, CD25 and CD127, and are sorted as CD4+CD25 high CD127 low cells by flow cytometry.In T cell culture medium (RPMI containing 10% FBS), in the presence of IL-2 (300U / ml), the primary Treg purified by Dynabead coated with anti-CD3 and anti-CD28 is amplified with a 1:2 ratio.Fresh culture medium containing IL-2 is added every 2 days, and cells are separated when needed.

[0160] On day 14 of expansion, cells were stained for surface protein expression. Two different donors were used. Donor B received Fc blocking during the staining protocol, while donor A did not. After staining, the cells were run on a flow cytometer. Anti-human VEGF receptor 2 therapeutic antibody (ramucirumab) and anti-human IL5RA therapeutic antibody (benralizumab) were purchased from Creative Biolabs. Each of these antibodies was used to stain primary Tregs, followed by washing and staining with a secondary anti-human Fab-PE antibody. The results are shown in Figure 2 middle.

[0161] Figure 2 Dot plot (1) shows unstained primary Tregs. Figure 2Dot plot (2) shows a positive control, where primary Tregs were stained with anti-CD25-PE antibody. As expected, the purified Treg cells were CD25 positive. Figure 2 The dot plot (3) shows cells stained with the secondary anti-human Fab-PE antibody alone. No background staining was detected. Figure 2 Scatter plots (4) and (5) show staining with anti-human VEGF receptor 2 therapeutic antibody (ramucirumab) and anti-human IL5RA therapeutic antibody (benralizumab), respectively. The results demonstrate that ramucirumab can bind to VEGF receptor 2 (which is on the surface of activated Tregs), and benralizumab cannot bind to activated Tregs due to the lack of IL5Ra expression on the surface of Treg cells. Therefore, this makes IL5Ra orthogonal to activated Tregs. Example 2: Expression of cell labels in cells

[0162] To test the expression of IL5Rat cell tag, the plasmid encoding IL5Rat cell tag was first transiently transfected into K562 cells. After successful transient transfection, the plasmid was made into lentivirus, in which the plasmid was transduced into Jurkat cells. Figure 3A and Figure 4A A diagram of the expression cassette is depicted. The promoter EF1a is followed by the anti-citrullinated vimentin (CV) chimeric antigen receptor (CAR). It is followed by the ribosomal skipping sequence P2A, a tag, and a stop codon. The end of the cassette is followed by the WPRE. In these experiments, cell cultures were transduced with plasmid pSB_0166 (a plasmid backbone designed for 3rd generation lentivirus). The IL5Rat tag in the pSB_0166 plasmid contains the entire extracellular domain of IL5Rat to the transmembrane domain to the first 4 amino acids of the intracellular domain:

[0163] Plasmids were transiently transfected into cell lines via electroporation. The protocol from Lonza was followed. The protocol was used for transient transfection. Lentivirus was prepared and titrated. The titrated virus was stained for both CAR expression via protein L staining and for the presence of the tag by anti-IL5Ra-PE antibody staining (data not shown). pSB_0166 can be prepared into advanced lentivirus.

[0164] After 48 hours, cells were stained and run on a flow cytometer using: CV peptide conjugated to biotin that had been bound to streptavidin-FITC for CAR detection; and commercial anti-IL5Ra-PE or benralizumab (Creative Biolab), followed by secondary anti-human Fab-PE for IL5Ra detection (IL5Rat tag in this case).

[0165] Figure 3A Shown is the expression cassette of the IL5Rat tag in plasmid pSB_0166, which was used for transient transfection of K562 cells. Figure 3B Shown are dot plots of K562 control cells that had been transiently transfected following staining with the full antibody cocktail. Figure 3C Shown is a scatter plot of transfected cells stained with CV-biotin and anti-human Fab-PE antibody conjugated to streptavidin-FITC for CAR detection, demonstrating that no staining of CAR was detected from the anti-human Fab-PE antibody. Figure 3D-Figure 3E Figures of transfected cells stained with: CV-biotin conjugated to streptavidin-FITC for CAR detection, and commercial anti-IL5Ra-PE antibody or benralizumab (CreativeBiolab), followed by secondary anti-human Fab-PE. pSB_0166 was equally effective for each staining convention. Therefore, the IL5Rat tag can be used as a cell marker for detecting transfected cells. Figure 4A An expression cassette for expressing a cell surface tag is shown. Figure 4B The expression of EGFRt tag is shown, and Figure 4C The expression of IL5Rat tag in transduced Jurkat cells is shown. The IL5Rat tag is expressed after transduction with a lentivirus generated from the pSB_0166 plasmid, and the EGFRt tag is expressed after transduction with a lentivirus generated from the same plasmid as pSB_0166 (the difference is that it contains the coding region of the conical EGFRt tag, rather than the coding region of the IL5Rat tag). Jurkat cells are transduced with each virus at an MOI of 3. On the 3rd day after transduction, each transduced cell is stained for the presence of anti-CV CAR and tags. Figure 4B and Figure 4C The scatter plot shows that the IL5Rat tag is expressed as well as the EGFRt tag.

[0166] In order to determine whether cells can be positively selected with IL5Rat labels, Jurkat cells were transduced with a virus prepared by pSB_0166 with an MOI of 0.5. On the 7th day after transduction, each transduced cell was placed by a positive selection process. IL5Rat labels were stained with anti-CD125-PE antibodies, and then anti-PE microbeads (Miltenyi Biotec) were applied. The stained cells were placed on LS columns (Miltenyi Biotech) for positive selection. Two days after positive selection, the cultures of cells before and after selection were stained for CAR and IL5Rat labels, and run on a flow cytometer. Figure 5 It was shown that the IL5Rat tag can be positively selected and a pure population of cells expressing the IL5Rat tag is generated.

[0167] Ablation of cells expressing IL5Rat labels will occur in the following manner: benralizumab binds to labels exposed on the cell surface, resulting in Fc receptor-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). In order to test this feature, target Jurkat cells (pSB_166) expressing IL5Rat labels are placed in ADCC reporter assays. ADCC reporter assays measure FcgR engagement associated with ADCC ability. ADCC reporter assays use stable effector Jurkat cell lines expressing human FcgRIIIa V158 and NFAT-induced luciferase. In the case of variable amounts of benralizumab or human IgG1 (negative control), the ratio of effector to target is 1: 1. Cells are incubated overnight, followed by addition of Bio-GloTM (Promega) to activate luciferase present in effector cells. Luminescence is measured on Molecular Devices SpectraMax iD3 plate reader. Figure 6 It was shown that expression of the IL5Rat tag after transduction with lentivirus generated from pSB_0166 activated effector cells in a dose-dependent manner, indicating that the IL5Rat tag is a suitable target for ablation via ADCC. Example 3: Expression of IL5Rat tag in cells

[0168] Different combinations of transmembrane domains and intracellular domains were tested by transducing Jurkat cells with lentivirus, comprising a bicistronic expression cassette for expressing CV-CAR and IL5Rat tags. The method used in this example is as described in Example 2 above. The amino acid sequence of an exemplary IL5Rat tag and the nucleotide sequence encoding the exemplary IL5Rat tag are shown in the SEQ ID NO in Table 3-1. In the following table, ID refers to the plasmid name, SEQ-N refers to the SEQ ID NO of the nucleotide sequence, SEQ-P refers to the SEQ ID NO of the amino acid sequence, and AA refers to the length of the IL5RAt tag. Table 3-1. Exemplary IL5Rat tag sequences

[0169] Fig. 7A and Fig. 8A Several different IL5Rat-tagged structures are shown. Figure 7B and Figure 8B The percentage of transduced Jurkat cells expressing different IL5Rat tags is shown. Figure 7C and Figure 8C Shown are the expression levels (median fluorescence intensity) of different IL5Rat tags on transduced Jurkat cells.

[0170] Fig.7D and Fig.8D Shown are the differences in ablative capacity of transduced Jurkat cells expressing different IL5Rat tags. In this experiment, ablative capacity was measured in an ADCC reporter assay using different amounts of benralizumab.

[0171] These results demonstrate that engineering of the transmembrane and intracellular domains of the IL5Rat tag can improve the functional properties of this type of cell surface tag.

[0172] Fig.9A and Fig. 9B Shown are the percentage of transduced Jurkat cells (pSB198 or pSB590) expressing the IL5Rat tag, and the expression level of the IL5Rat tag on the transduced Jurkat cells. Fig. 9C The difference in the ability of benralizumab to elicit ADCC of transduced Jurkat cells expressing the IL5Rat tag is shown. In short, these results indicate that inclusion of a chimeric HER2 transmembrane domain in the IL5Rat tag results in enhanced expression and benralizumab-mediated ADCC.

[0173] Fig.10 Binding of recombinant IL-5 to transduced Jurkat cells expressing various IL5Rat tags is shown. These results indicate that transduced cells expressing an IL5Rat tag with a mutant extracellular domain (pSB540, pSB541, pSB546, or pSB552) exhibit reduced IL-5 binding relative to transduced cells expressing an IL5Rat tag with a wild-type extracellular domain (pSB511 or pSB198).

[0174] Figure 11A-11C It is shown that transduced cells retain cell surface expression of the IL5Rat tag with the mutated extracellular domain despite reduced IL-5 binding.

[0175] Fig.12 Shown are differences in the ability of benralizumab to elicit ADCC of transduced Jurkat cells expressing an IL5Rat tag with a mutated extracellular domain.

[0176] Fig.13A The percentage of transduced Jurkat cells expressing different IL5Rat tags and the expression levels (median fluorescence intensity) of different IL5Rat tags on transduced Jurkat cells are shown. These results indicate that transduced cells expressing IL5Rat tags with mutant extracellular domains (pSB540) and / or heterologous transmembrane domains (pSB590 and pSB693) have higher levels of IL5Lat tag expression than transduced cells expressing control IL5Rat tags with wild-type transmembrane domains and homologous transmembrane domains (pSB198).

[0177] Fig. 13B Binding of recombinant IL-5 to transduced Jurkat cells expressing various IL5Rat tags is shown. These results indicate that transduced cells expressing an IL5Rat tag with a mutant extracellular domain (pSB540 or pSB693) exhibit reduced IL-5 binding relative to transduced cells expressing an IL5Rat tag with a wild-type extracellular domain (pSB198 or pSB590).

[0178] Fig. 13C Shown are differences in the ability of benralizumab to elicit ADCC of transduced Jurkat cells expressing various IL5Rat tags.

[0179] Example 4: Expression of CAR and cell surface markers in primary Treg cells

[0180] In this example, the sustained expression of CAR and cell surface tags (TAG) was evaluated in transduced human Treg cells subjected to a 14-day expansion protocol. The method used in this example is as described in Example 1 above.

[0181] Fig.14A and Fig. 14B Shown is maintenance of CAR and TAG expression in the expanded Treg population.

[0182] It should be understood that the description and the drawings are not intended to limit the present invention to the specific form disclosed, but rather, are intended to cover all modifications, equivalents and embodiments falling within the spirit and scope of the present invention as defined by the appended claims. In view of this description, further modifications and alternative embodiments of various aspects of the present invention will be clear to those skilled in the art. Therefore, this description and the drawings are only interpreted as illustrative and are intended to teach those skilled in the art the general manner of implementing the present invention. It should be understood that the forms of the present invention shown and described herein will be considered as examples of embodiments. Elements and materials may replace the elements and materials shown and described herein, parts and processes may be reversed or omitted, and certain features of the present invention may be used independently, all of which are as clear to those skilled in the art after benefiting from this description of the present invention. Without departing from the spirit and scope of the present invention as described in the following claims, the elements described herein may be changed. The titles used herein are only for organizational purposes and are not intended to be used to limit the scope of this specification.

[0183] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The present disclosure relates to the following embodiments: 1. A recombinant polypeptide comprising an extracellular region and a transmembrane region, wherein the extracellular region comprises a portion or all of the extracellular domain of IL5 receptor α (IL5Ra) and the extracellular region is connected to the transmembrane domain, wherein the recombinant polypeptide is unable to play a role in signal transduction, and optionally wherein the recombinant polypeptide has reduced binding to IL-5. 2. A recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) of SEQ ID NO: 1. 3. A recombinant polypeptide according to claim 2, wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 124-242 (domain II) of SEQ ID NO: 1. 4. A recombinant polypeptide according to claim 2, wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 243-334 (domain III) of SEQ ID NO: 1. 5. The recombinant polypeptide according to item 1, wherein the extracellular region comprises a truncated IL5Ra extracellular region, and the truncated IL5Ra extracellular region comprises no more than part or all of the following: i) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69; ii) the amino acid sequence of SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72; and / or iii) the amino acid sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77. 6. A recombinant polypeptide according to item 1, wherein the extracellular region comprises a truncated IL5Ra extracellular region, and the truncated IL5Ra extracellular region comprises part or all of the following: the amino acid sequence of SEQ ID NO:2, SEQ ID NO:60, SEQ ID NO:67, SEQID NO:68 or SEQ ID NO:69, but does not comprise the amino acid sequence of amino acids 124-342 of SEQ ID NO:1. 7. The recombinant polypeptide according to item 1, wherein the extracellular region binds to benralizumab. 8. A recombinant polypeptide according to claim 1, wherein the transmembrane region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to amino acids 343-362 of SEQ ID NO:1. 9. A recombinant polypeptide according to claim 1, wherein the transmembrane region comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:78 and SEQ ID NO:79. 10. The recombinant polypeptide according to item 1, wherein the recombinant polypeptide further comprises an intracellular domain. 11. A recombinant polypeptide according to item 10, wherein the intracellular domain comprises a truncated IL5Ra intracellular domain, optionally wherein the truncated IL5Ra intracellular domain does not associate with Janus kinase 2 (JAK2), optionally wherein the truncated IL5Ra intracellular domain has lost its signal transduction ability. 12. The recombinant polypeptide according to item 11, wherein (i) the intracellular domain consists of amino acids 363-370 of SEQ ID NO: 1; or (ii) the intracellular domain comprises amino acids 363-366 of SEQ ID NO:1, but does not comprise the amino acid sequence of SEQ ID NO:13. 13. A recombinant polypeptide according to claim 10, wherein the length of the intracellular domain does not exceed 25, 30, 35, 40, 45 or 50 amino acids, and the intracellular domain comprises the amino acid sequence of SEQ ID NO:14, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85. 14. A recombinant polypeptide according to claim 1, wherein the extracellular domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) or amino acids 32-242 (domains I and II) or amino acids 32-334 (domains I, II and III) of SEQ ID NO:1. 15. A recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-370 of SEQ ID NO: 1 (domains I, II and III, the transmembrane domain, and a fragment of the intracellular domain). 16. A recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 342, or 32 to 370 of SEQ ID NO: 1. 17. The recombinant polypeptide according to item 1, further comprising a signal peptide. 18. A recombinant polypeptide according to claim 17, wherein the signal peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO:58, SEQ ID NO:15, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66. 19. The recombinant polypeptide according to item 1, further comprising a linker between the extracellular domain and the transmembrane domain. 20. A recombinant polypeptide according to claim 19, wherein the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22. 21. A recombinant polypeptide according to claim 10, wherein the intracellular domain consists of the amino acid sequence of SEQ ID NO:14, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85; or an amino acid sequence that is at least 80%, 85%, 90% or 95% identical thereto. 22. The recombinant polypeptide according to item 1, wherein the recombinant polypeptide comprises an amino acid sequence, wherein: i) an amino acid sequence selected from SEQ ID NOs: 23-40, or at least 80%, 85%, 90% or 95% identical thereto; or (ii) selected from SEQ ID NOs: 148-208, or an amino acid sequence at least 80%, 85%, 90% or 95% identical thereto; or (iii) an amino acid sequence selected from SEQ ID NO: 23-40 or selected from 148-208, or at least 80%, 85%, 90% or 95% identical thereto. 23. A recombinant polypeptide according to item 1, comprising a heterologous region of at least 4 amino acids in length, wherein the amino acid sequence of the heterologous region is heterologous to IL5Ra, optionally wherein the amino acid sequence of the heterologous region is not present in the amino acid sequence of human IL5Ra of SEQ ID NO: 1. 24. A nucleic acid molecule comprising a coding sequence for a recombinant polypeptide according to any one of items 1 to 23. 25. A nucleic acid molecule according to item 24, wherein the nucleic acid molecule comprises a nucleotide sequence, wherein: (i) selected from SEQ ID NOs: 41-57 and 86, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; or (ii) selected from SEQ ID NOs: 87-147, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; or (iii) a nucleotide sequence selected from SEQ ID NOs: 41-57 and 86, or selected from SEQ ID NOs: 87-147, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto. 26. A nucleic acid molecule according to item 24, further comprising a coding sequence for a chimeric antigen receptor (CAR). 27. A nucleic acid molecule according to item 26, wherein the coding sequences of the recombinant polypeptide and the CAR are operably linked to the same promoter so that the two coding sequences are co-transcribed. 28. A nucleic acid molecule according to item 24, wherein the nucleic acid molecule is present in a viral vector, optionally wherein the viral vector is a lentiviral vector or a retroviral vector. 29. A cell comprising the nucleic acid molecule according to item 24. 30. A cell according to item 29, wherein the cell is a human T cell, optionally wherein the human T cell is a human Treg cell. 31. A pharmaceutical composition comprising: (i) the cell according to item 29; and (ii) a pharmaceutically acceptable carrier. 32. A pharmaceutical composition comprising: (i) a nucleic acid molecule according to item 21; and (ii) a pharmaceutically acceptable carrier. 33. A method of treating a patient in need thereof, the method comprising administering to the patient a cell according to item 29, optionally wherein the cell is derived from the patient. 34. A method for treating a patient in need thereof, the method comprising administering to the patient a cell according to claim 30, wherein the cell expresses a CAR specific for an antigen present in the disease suffering from the patient. 35. The method according to item 34, further comprising administering to the patient an effective amount of an antibody specific for IL5Ra once the patient has been treated, wherein the antibody elicits cytotoxicity against cells expressing the recombinant polypeptide, optionally wherein the antibody is IgG1 or IgG2. 36. A method for preparing a genetically engineered human cell, the method comprising providing an isolated human cell, and introducing the nucleic acid molecule according to item 24 into the human cell. 37. A method according to claim 36, wherein the human cells are human T cells. 38. A method according to claim 37, wherein the human cells are human Treg cells. 39. A method according to claim 36, further comprising culturing the human cells under conditions such that the recombinant polypeptide is expressed on the surface of the genetically engineered human cells. 40. A pharmaceutical composition comprising: (i) a plurality of cells according to item 29, and (ii) a pharmaceutically acceptable carrier. 41. A pharmaceutical composition comprising: (i) a plurality of said genetically engineered human cells produced by the method according to item 36, and (ii) a pharmaceutically acceptable carrier.

Claims

1. A recombinant polypeptide comprising an extracellular region and a transmembrane region, wherein the extracellular region comprises a portion or all of the extracellular domain of IL5 receptor α (IL5Ra) and the extracellular region is connected to the transmembrane domain, wherein the recombinant polypeptide is unable to play a role in signal transduction, and optionally wherein the recombinant polypeptide has reduced binding to IL-5.

2. The recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity with amino acids 32-123 (domain I) of SEQ ID NO: 1, optionally a) wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to amino acids 142-242 (domain II) of SEQ ID NO: 1; and / or b) wherein the recombinant polypeptide further comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to amino acids 243-334 (domain III) of SEQ ID NO:

1.

3. The recombinant polypeptide according to claim 1 or claim 2, wherein a) the extracellular region comprises a truncated IL5Ra extracellular region, wherein the truncated IL5Ra extracellular region comprises no more than part or all of the following: i) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69; ii) the amino acid sequence of SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:71 or SEQ ID NO:72; and / or iii) the amino acid sequence of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77; or b) the extracellular region comprises a truncated IL5Ra extracellular region, wherein the truncated IL5Ra extracellular region comprises part or all of the following: an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69, but does not comprise the amino acid sequence of amino acids 124-342 of SEQ ID NO: 1; or c) the extracellular region binds to benralizumab; or d) the extracellular region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to amino acids 32-123 (domain I) or amino acids 32-242 (domains I and II) or amino acids 32-334 (domains I, II and III) of SEQ ID NO:

1.

4. The recombinant polypeptide according to any one of claims 1 to 3, wherein a) the transmembrane region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to amino acids 343-362 of SEQ ID NO: 1; or b) the transmembrane region comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:78 and SEQ ID NO:

79.

5. The recombinant polypeptide according to any one of claims 1 to 4, wherein the recombinant polypeptide further comprises an intracellular domain, optionally wherein: a) the intracellular domain comprises a truncated IL5Ra intracellular domain, optionally wherein the truncated IL5Ra intracellular domain does not associate with Janus kinase 2 (JAK2), optionally wherein the truncated IL5Ra intracellular domain has lost its signal transduction ability, further optionally wherein (i) the intracellular domain consists of amino acids 363-370 of SEQ ID NO: 1; or (ii) the intracellular domain comprises amino acids 363-366 of SEQ ID NO: 1, but does not comprise the amino acid sequence of SEQ ID NO: 13; or b) the intracellular domain is no more than 25, 30, 35, 40, 45 or 50 amino acids in length, and the intracellular domain comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO: 85; or c) the intracellular domain consists of the amino acid sequence of SEQ ID NO:14, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, or an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical thereto.

6. The recombinant polypeptide according to any one of claims 1 to 5, a) wherein the recombinant polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to amino acids 32-370 (domains I, II and III, the transmembrane domain, and a fragment of the intracellular domain) of SEQ ID NO: 1; b) wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity to amino acids 32 to 123, or 32 to 242, or 32 to 334, or 32 to 342, or 32 to 370 of SEQ ID NO: 1; c) further comprises a signal peptide, optionally the signal peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO:58, SEQ ID NO:15, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66; and / or d) further comprises a linker between the extracellular domain and the transmembrane domain, optionally wherein the linker comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO:

22.

7. The recombinant polypeptide according to claim 1, wherein the recombinant polypeptide comprises the amino acid sequence: i) selected from the group consisting of SEQ ID NOs: 23-40, or an amino acid sequence at least 80%, 85%, 90% or 95% identical thereto; or (ii) selected from the group consisting of SEQ ID NOs: 148-208, or an amino acid sequence at least 80%, 85%, 90% or 95% identical thereto; or (iii) an amino acid sequence selected from SEQ ID NOs: 23-40 or from the group consisting of 148-208, or at least 80%, 85%, 90% or 95% identical thereto.

8. The recombinant polypeptide of claim 1, comprising a heterologous region of at least 4 amino acids in length, wherein the amino acid sequence of the heterologous region is heterologous to IL5Ra, optionally wherein the amino acid sequence of the heterologous region is not present in the amino acid sequence of human IL5Ra of SEQ ID NO:

1.

9. A nucleic acid molecule comprising a coding sequence for a recombinant polypeptide according to any one of claims 1 to 8, optionally a) wherein the nucleic acid molecule comprises the following nucleotide sequence: (i) selected from the group consisting of SEQ ID NOs: 41-57 and 86, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; or (ii) selected from the group consisting of SEQ ID NOs: 87-147, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; or (iii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 41-57 and 86, or selected from the group consisting of SEQ ID NOs: 87-147, or a nucleotide sequence at least 80%, 85%, 90%, 95% or 100% identical thereto; b) further comprises a coding sequence for a chimeric antigen receptor (CAR), further optionally wherein the coding sequence of the recombinant polypeptide and the CAR are operably linked to the same promoter such that the two coding sequences are co-transcribed; and / or c) wherein the nucleic acid molecule is present in a viral vector, optionally wherein the viral vector is a lentiviral vector or a retroviral vector.

10. A cell comprising the nucleic acid molecule according to claim 9, optionally wherein the cell is a human T cell, further optionally wherein the human T cell is a human Treg cell.

11. A pharmaceutical composition comprising: (i) the cell according to claim 10; and (ii) a pharmaceutically acceptable carrier.

12. A pharmaceutical composition comprising: (i) the nucleic acid molecule according to claim 9; and (ii) a pharmaceutically acceptable carrier.

13. The cell according to claim 10 for use in a method of treating a patient in need thereof, optionally wherein a) the cells are derived from the patient; and / or b) the cells express a CAR specific for an antigen present in the disease suffered by the patient, further optionally comprising administering to the patient an effective amount of an antibody specific for IL5Ra once the patient has been treated, wherein the antibody elicits cytotoxicity against cells expressing the recombinant polypeptide, optionally wherein the antibody is IgG1 or IgG2.

14. A method for preparing a genetically engineered human cell, the method comprising providing an isolated human cell, and introducing the nucleic acid molecule according to claim 9 into the human cell, optionally a) wherein the human cell is a human T cell, further optionally wherein the human cell is a human Treg cell; and / or b) further comprising culturing the human cells under conditions where the recombinant polypeptide is expressed on the surface of the genetically engineered human cells.

15. A pharmaceutical composition, comprising: (i) a) a plurality of cells according to claim 10, or b) a plurality of said genetically engineered human cells produced by the method according to claim 14; and (ii) a pharmaceutically acceptable carrier.

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