Cells for improved immunotherapy and uses thereof

Abstract

To provide cells for improved immunotherapy, and to provide uses thereof.SOLUTION: The presently disclosed subject matter provides cells and compositions for improved immunotherapy and methods of using such cells and compositions. It relates to cells comprising a ligand-recognizing receptor (e.g., an antigen-recognizing receptor, e.g., a chimeric antigen receptor (CAR) or a T-cell Receptor (TCR)) and an IgG-degrading enzyme or a fragment thereof. The IgG-degrading enzyme rapidly cleaves IgG. The IgG-degrading enzyme serves as a biomolecular shield against host humoral response. The cells have increased resistance to the host humoral response (e.g., an antibody-driven host humoral response), which allows for prolonged persistence of the cells, leading to enhanced activity of the cells.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 881,467, filed on August 1, 2019, the entire content of which is incorporated herein by reference.

[0002] Grant Information This invention was made with government support under grant number P30 CA008747 from the National Cancer Institute of the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application is being filed via EFS - Web and includes a sequence listing in ASCII format that is hereby incorporated by reference in its entirety. The ASCII copy created on July 28, 2020, is named "072734_1109_ST25.txt" and is 52,647 bytes in size.

[0004] 1. Introduction The subject matter of the present disclosure provides cells and compositions for improved immunotherapy and methods of using such cells and compositions. The subject matter of the present disclosure relates to cells comprising a ligand - recognizing receptor (e.g., an antigen - recognizing receptor, e.g., a chimeric antigen receptor (CAR) or a T - cell receptor (TCR)) and an IgG - degrading enzyme or a fragment thereof. The IgG - degrading enzyme rapidly cleaves IgG. The IgG - degrading enzyme serves as a biomolecular shield against the host humoral response. The cells increase resistance to the host humoral response (e.g., an antibody - driven host humoral response), thereby allowing for an extension of the persistence of the cells and resulting in an enhancement of cell activity.

Background Art

[0005] 2. Background of the Invention In synthetic immunology and synthetic biology, immune cells are utilized to kill tumor cells or treat other important diseases. Areas that have grown rapidly in synthetic immunology and biology are those in the use of adoptive cell transfer, stem cell transplantation, organ transplantation, CRISPR gene editing, gene therapy, and CAR-T cell therapy. In any situation where altered or engineered cells are introduced into a subject, the host (subject) may initiate an immune response against those cells or tissues, because the cells or tissues are foreign or contain foreign genes and proteins not normally found within the host. The results of this immune recognition can be neutralization of the therapeutic effect, tissue or cell rejection, and / or failure of the treatment intent. Therefore, there is a need for engineered cells with increased resistance to the host humoral response.

Summary of the Invention

Means for Solving the Problems

[0006] 3. Summary of the Invention The subject matter of the present disclosure provides cells comprising (a) a ligand recognition receptor and (b) an IgG-degrading enzyme or a fragment thereof. In certain embodiments, the IgG-degrading enzyme is secreted. In certain embodiments, the IgG-degrading enzyme is membrane-bound. In certain embodiments, the cells further comprise (c) a transmembrane domain attached to the IgG-degrading enzyme. The transmembrane domain may be attached to the C-terminus of the IgG-degrading enzyme. In certain embodiments, the transmembrane domain attached to the IgG-degrading enzyme comprises a CD8 polypeptide.

[0007] In certain embodiments, the IgG-degrading enzyme is selected from IgG-degrading enzyme of S. pyogenes (IdeS), IgG-degrading enzyme of S. equi subsp. zooepidemicus (IdeZ), IgG-degrading enzyme of S. equi subsp. equi (IdeE), endoglycosidase from Streptococcus pyogenes (EndoS), and streptococcal cysteine protease from Streptococcus pyogenes (SpeB).

[0008] In certain embodiments, the ligand recognition receptor is exogenous or endogenous. In certain embodiments, the ligand recognition receptor is recombinantly expressed. In certain embodiments, the ligand recognition receptor is expressed from a vector. In certain embodiments, the IgG degrading enzyme is expressed from a vector.

[0009] In certain embodiments, the cell is a responsive cell. In certain embodiments, the cell is a responsive cell, e.g., an immunoreactive cell. In certain embodiments, the cell is an activatable cell. In certain embodiments, the cell is selected from T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, stem cells, normal tissue cells (e.g., derived from kidney, liver, lung, bone marrow, or pancreas) and combinations thereof. In certain embodiments, the cell is a T cell.

[0010] In certain embodiments, the ligand recognition receptor binds to an antigen. The antigen may be a tumor antigen, a pathogen antigen, a normal cell antigen, an HLA antigen or an alloantigen. In certain embodiments, the antigen is a normal cell antigen. In certain embodiments, the alloantigen is a minor histocompatibility alloantigen.

[0011] In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is CD19.

[0012] In certain embodiments, the ligand recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the ligand recognition receptor is a CAR. In certain embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a single-chain variable fragment (scFv). In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide. In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling domain. In certain embodiments, the at least one co-stimulatory domain comprises a CD28 polypeptide, a 4-1BB polypeptide, or a combination thereof. In certain embodiments, the at least one co-stimulatory domain comprises a 4-1BB polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises two co-stimulatory signaling domains.

[0013] In certain embodiments, the IgG-degrading enzyme cleaves IgG, thereby preventing the IgG antibody from killing cells. In certain embodiments, the IgG-degrading enzyme cleaves IgG, thereby retaining the binding of the remaining fragment of IgG to cells and protecting the cells from one or more cytotoxic antibodies. In certain embodiments, the one or more cytotoxic antibodies bind to the same site as IgG and kill the cells. Thus, this process results in a protective shield.

[0014] The subject matter of the present disclosure also provides a composition comprising the cells described herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. In certain embodiments, the composition is for treating a neoplasm.

[0015] Furthermore, the subject matter of the present disclosure provides a method for generating the cells disclosed herein. In certain embodiments, the method includes introducing into a cell: (a) a first polynucleotide encoding a ligand recognition receptor, and (b) a second polynucleotide encoding an IgG degrading enzyme or a fragment thereof. In certain embodiments, (a) and / or (b) are genetically introduced into the cell. In certain embodiments, the first polynucleotide is operably linked, optionally, to a promoter element. In certain embodiments, the second polynucleotide is operably linked, optionally, to a promoter element. In certain embodiments, one or both of the first and second polynucleotides are contained within a vector. In certain embodiments, the first and second polynucleotides are contained within two different vectors. In certain embodiments, the vector is a retroviral vector. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, the vector is encoded within an mRNA molecule.

[0016] The subject matter of the present disclosure further provides a nucleic acid composition. In certain embodiments, the nucleic acid composition includes: (a) a first polynucleotide encoding a ligand recognition receptor and (b) a second polynucleotide encoding an IgG degrading enzyme or a fragment thereof. In certain embodiments, the first polynucleotide is operably linked to a promoter element. In certain embodiments, the second polynucleotide is operably linked to a promoter element. In certain embodiments, one or both of the first and second polynucleotides are contained within a vector. In certain embodiments, the first and second polynucleotides are contained within two different vectors. In certain embodiments, the vector is a retroviral vector. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, the vector is encoded within an mRNA molecule.

[0017] Also provided is a vector comprising the nucleic acid composition described herein.

[0018] The subject matter of the present disclosure further provides a kit comprising the cells described herein, the compositions described herein, the nucleic acid compositions described herein, or the vectors described herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm, a pathogen infection, and / or an autoimmune disorder.

[0019] The subject matter of the present disclosure also provides various methods. The subject matter of the present disclosure provides a method for reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells described herein, the compositions described herein, the nucleic acid compositions described herein, or the vectors described herein. In certain embodiments, the method reduces the number of tumor cells, decreases the tumor size, and / or eradicates the tumor in the subject.

[0020] The subject matter of the present disclosure provides a method for treating and / or preventing a neoplasm, a pathogen infection, and / or an autoimmune disorder. In certain embodiments, the method comprises administering to the subject an effective amount of the cells described herein, the compositions described herein, the nucleic acid compositions described herein, or the vectors described herein.

[0021] The subject matter of the present disclosure provides a method for prolonging the survival time of a subject having a neoplasm, a pathogen infection, and / or an autoimmune disorder. In certain embodiments, the method comprises administering to the subject an effective amount of the cells described herein, the compositions described herein, the nucleic acid compositions described herein, or the vectors described herein.

[0022] In certain embodiments, the neoplasm is selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, breast cancer, ovarian cancer, mesothelioma, glioblastoma, colorectal cancer, and pancreatic cancer.

[0023] The subject matter of the present disclosure provides a method for reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject receiving an organ transplant. In certain embodiments, the transplant is an allogeneic transplant (allograft). In certain embodiments, the subject is administered a cell, a composition, or a nucleic acid composition prior to the organ transplant.

[0024] The subject matter of the present disclosure further provides a method for reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject receiving cell therapy. In certain embodiments, the cells and / or tissues are autologous or allogeneic. In certain embodiments, the cells and / or tissues are used in cell therapy.

[0025] In certain embodiments, the method comprises administering an effective amount of a cell, a composition, a nucleic acid composition, or a vector described herein. 4. BRIEF DESCRIPTION OF THE DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]

Figure 1

[0027]

Figure 2

[0028]

Figure 3

[0029]

Figure 4

[0030]

Figure 5

[0031]

Figure 6

[0032]

Figure 7

[0033]

Figure 8

[0034]

Figure 9

[0035]

Figure 10

[0036]

Figure 11

[0037]

Figure 12

[0038] 5. DETAILED DESCRIPTION OF THE INVENTION The following detailed description, given by way of example and not intended to limit the subject matter of the present disclosure to the specific embodiments described, can be understood in conjunction with the accompanying drawings.

[0039] The subject matter of the present disclosure provides cells comprising genetically modified immunoreactive cells (e.g., T cells or NK cells) comprising a ligand recognition receptor (e.g., TCR or CAR) and an IgG-degrading enzyme or a fragment thereof, as well as compositions comprising such cells. The subject matter of the present disclosure also provides methods of generating such cells, as well as methods of using such cells and compositions comprising thereof. The subject matter of the present disclosure provides for treating and / or preventing neoplasms, other diseases / disorders, pathogen infections, and / or autoimmune disorders, for extending the survival time of subjects having neoplasms, pathogen infections, and / or autoimmune disorders, for reducing tumor burden in a subject, for treating and / or preventing autoimmune diseases, and / or for reducing and / or preventing antibody-mediated rejection of cells and / or tissues used in cell therapy in a subject, e.g., the subject receives an organ transplant or said cell therapy.

[0040] The subject matter of the present disclosure is based, at least in part, on the discovery that an IgG-degrading enzyme, e.g., IdeS, can deliver and cleave IgG, thereby increasing the resistance of the cells to the host humoral response, which results in an extended persistence of the cells and a more potent activity of the cells (e.g., anti-tumor activity). The extended persistence of the cells can also improve the cost-effectiveness of therapies comprising such cells, e.g., CAR-T cell therapy.

[0041] Non-limiting embodiments of the subject matter of the present disclosure are described herein and in the examples.

[0042] For the sake of clarity of the subject matter of the present disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definitions; 5.2. IgG-degrading enzymes; 5.3. Antigen ligand recognition receptors 5.4. Cells; 5.5. Compositions and vectors; 5.6. Polypeptides and analogs; 5.7. Administration; 5.8. Formulations; 5.9. Methods of use; and 5.10. Kits. 5.1. Definitions

[0043] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. The following references provide one of ordinary skill in the art with many general definitions of terms used in the subject matter of this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0044] As used herein, the terms “about” or “approximately” mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, “about” can mean within up to 20% of a given value, e.g., up to 10%, up to 5%, or up to 1%. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of the value, e.g., within 5-fold or within 2-fold.

[0045] The term "immunoresponsive cell" means a cell or precursor that functions in an immune response, or its progeny. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage or the myeloid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, dendritic cells, B cells, and stem cells that can differentiate into lymphoid cells (e.g., induced pluripotent stem cells). Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells that can differentiate into myeloid cells.

[0046] "Activating an immunoresponsive cell" means inducing signal transduction or a change in protein expression in a cell that results in the initiation of an immune response. For example, when the CD3 chains form clusters in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade occurs. In certain embodiments, when an endogenous TCR or an exogenous CAR binds to an antigen, formation of an immune synapse occurs, including clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the binding receptor. Such clustering of membrane-bound signal transduction molecules enables phosphorylation of the ITAM motifs contained within the CD3 chains. This phosphorylation then initiates a T cell activation pathway that ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors induce overall gene expression in T cells and increase IL-2 production for the proliferation and expression of the master regulator T cell protein to initiate a T cell-mediated immune response.

[0047] "Stimulating an immune-responsive cell" means a signal that results in a strong and persistent immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously via receptors including, but not limited to, CD28, CD137 (4-lBB), OX40, CD40, and ICOS. Administration of multiple stimulatory signals can be important for initiating a strong and long-term T cell-mediated immune response. T cells can be immediately inhibited and rendered unable to respond to antigens. The effects of these co-stimulatory signals can vary, but they generally result in increased gene expression and generate long-lived, proliferative, and anti-apoptotic T cells that strongly respond to antigens for complete and persistent eradication.

[0048] As used herein, the term "antigen recognition receptor" refers to a receptor capable of activating an immune or immune-responsive cell (e.g., a T cell) in response to binding to an antigen.

[0049] Antigen-binding fragments include F(ab’)2 and Fab. F(ab’)2 and Fab fragments lacking the Fc fragment of the intact antibody.

[0050] In certain embodiments, an antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H and a heavy chain constant region (C H ). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V L and a light chain constant C L region. The light chain constant region consists of one domain, C L . V H region and V L region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FR). Each V H and VL It is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0051] As used herein, "CDR" is defined as the amino acid sequence of the complementarity-determining region of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy-chain and three light-chain CDRs or CDR regions in the variable region. The CDRs provide the majority of the contact residues for the binding of the antibody to an antigen or epitope. In certain embodiments, the CDR regions are depicted using the Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0052] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the immunoglobulin heavy chain (V H ::V L ) and light chain (V H ) that are covalently linked to form a heterodimer. V L ) and the variable region of the light chain (V Hand V L is either directly joined or joined by a linker encoding a peptide (e.g., 10, 15, 20, 25 amino acids), whereby the N-terminus of V H is connected to the C-terminus of V L or the C-terminus of V H is connected to the N-terminus of V L The linker is usually glycine-rich with respect to flexibility and serine- or threonine-rich with respect to solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing the V H and V L coding sequences. See also U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publications 20050196754 and 20050196754. scFvs of antagonists having inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst See 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10):31-40). ScFvs of agonists with stimulatory activity are described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0053] As used herein, the term "affinity" means a measure of binding strength. Affinity can depend on the closeness of stereochemical fit between the antibody combining site and the epitope, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "avidity", which refers to the strength of antigen-antibody binding after reversible complex formation. Methods for calculating the affinity of an antibody for an antigen are known in the art and include, but are not limited to, various antigen-binding assays, such as functional assays (e.g., flow cytometry assays).

[0054] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to a transmembrane domain and an intracellular signaling domain capable of activating or stimulating an immune or immune response cell. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be derived from fusing the variable heavy and light chain regions of an antibody. Alternatively or additionally, the scFv can be derived from a Fab (obtained, for example, from a Fab library instead of being derived from an antibody). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain.

[0055] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule encoding a polypeptide of interest (e.g., an IL-36 polypeptide) or a fragment thereof. Such nucleic acid molecules need not be 100% identical or the same as the endogenous nucleic acid sequence, but can exhibit substantial identity. A polynucleotide having "substantial identity" or "substantial homology" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means to form pairs between complementary polynucleotide sequences (e.g., the genes described herein), or portions thereof, under various stringency conditions to form a double-stranded molecule. (See, for example, Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0056] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than about 75 mM trisodium citrate, such as less than about 500 mM NaCl and less than about 50 mM trisodium citrate, or less than about 250 mM NaCl and less than about 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, such as at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, at least about 37°C, or at least about 42°C. It is well known to those skilled in the art to vary additional parameters such as hybridization time, the concentration of a surfactant, such as sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA. Various levels of stringency, when required, are achieved by combining these various conditions. In certain embodiments, hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization is performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml of denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization is performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml of ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0057] In most applications, the wash steps after hybridization also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As noted above, wash stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, a stringent salt concentration for a wash step can be less than about 30 mM NaCl and less than about 3 mM trisodium citrate, such as less than about 15 mM NaCl and less than about 1.5 mM trisodium citrate. Stringent temperature conditions for a wash step typically include a temperature of at least about 25°C, at least about 42°C, or at least about 68°C. In certain embodiments, the wash step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the wash step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the wash step is performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those of skill in the art. Hybridization techniques are well known to those of skill in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0058] "Substantially identical" or "substantially homologous" means a polypeptide or polynucleotide that is at least about 50% homologous or identical to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such sequences are at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence or nucleic acid sequence used for comparison.

[0059] Sequence identity can be measured by using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program can be used, and probability scores between e-3 and e-100 indicate closely related sequences.

[0060] "Analog" means a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0061] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In certain embodiments, the ligand binds to a receptor on another cell, enabling cell-cell recognition and / or interaction.

[0062] As used herein, the term "constitutive expression" or "constitutively expressed" refers to expression under all physiological conditions or expression under such conditions.

[0063] "Disease" means any condition, disease or disorder that impairs or interferes with the normal function of a cell, tissue, or organ, such as a neoplasm, and infection of a cell by a pathogen.

[0064] "Effective amount" means an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to stop, alleviate, or inhibit the continuous growth, growth, or metastasis (e.g., invasion, or migration) of a neoplasm.

[0065] "Enhancing tolerance" means preventing the activity of autoreactive or immunoreactive cells that target a transplanted organ or tissue.

[0066] "Endogenous" means a polynucleotide or polypeptide that is normally expressed in a cell or tissue.

[0067] "Exogenous" means a polynucleotide or polypeptide that does not exist endogenously in a cell. Thus, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed intracellularly, such as foreign, heterologous, overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid means a nucleic acid that does not exist in a natural wild-type cell. For example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or a different sequence compared to its natural endogenous counterpart, may be introduced into the cell itself or its precursor by genetic manipulation, and may be linked to an alternative control sequence, such as a non-natural promoter or secretion sequence, as needed.

[0068] "Heterologous nucleic acid molecule or polypeptide" means a nucleic acid molecule (e.g., cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. This nucleic acid may be derived from another organism or may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0069] "Modulate" means to change positively or negatively. Exemplary modulation includes a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0070] "Increase" means to change positively by at least about 5%. The change may be up to about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0071] "Decrease" means to change negatively by at least about 5%. The change may be up to about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0072] The terms "isolated", "purified" or "biologically pure" refer to substances that contain varying degrees of the components that are normally associated with them as they are found in their natural state. "Isolating" indicates the degree of separation from the original source or environment. "Purifying" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein substantially contains no other substances such that any impurities do not substantially affect the biological properties of the protein and do not cause other harmful effects. That is, a nucleic acid or peptide is purified if, when produced by recombinant DNA techniques, it substantially contains no cell material, viral material, and culture medium, or, when chemically synthesized, it substantially contains no chemical precursors or other chemicals. Purity and homogeneity are typically determined using techniques of analytical chemistry, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may indicate that a nucleic acid or protein yields essentially one band on an electrophoretic gel. For proteins that may be subjected to modifications, such as phosphorylation or glycosylation, various modifications may occur to various isolated proteins, and these can be purified separately.

[0073] "Isolated cell" means a cell that is separated from the molecules and / or cell components that are naturally associated with the cell.

[0074] As used herein, the term "antigen-binding domain" refers to a domain that is capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0075] "Neoplasm" means a disease characterized by the pathological growth of cells or tissues and their subsequent migration or invasion into other tissues or organs. The growth of a neoplasm is typically uncontrolled and progressive and occurs under conditions that do not induce or cause the cessation of normal cell multiplication. Neoplasms can affect various cell types, tissues, or organs, including, but not limited to, those selected from the group consisting of the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or their tissue or cell types. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0076] "Receptor" means a polypeptide or a portion or fragment thereof that is present on the cell membrane and selectively binds to at least one ligand. In certain embodiments, the ligand is an antigen. The antigen can be a tumor antigen, a pathogen antigen, a normal cell antigen, an HLA antigen, or an alloantigen (e.g., a minor histocompatibility alloantigen).

[0077] "Recognize" means to selectively bind to a target, e.g., a ligand (e.g., an antigen). For example, a cell that recognizes a tumor (e.g., a T cell) can express a receptor (e.g., a TCR or a CAR) that binds to a tumor antigen.

[0078] As used herein, the term "ligand recognition receptor" refers to a receptor that is capable of recognizing a ligand.

[0079] "Reference" or "control" means a standard substance for comparison. For example, the level of scFv antigen binding by cells expressing a CAR and an scFv can be compared to the level of scFv antigen binding in corresponding cells expressing the CAR alone.

[0080] "Secreted" means a polypeptide that is released from a cell, for example, by a secretory pathway via the endoplasmic reticulum and Golgi apparatus, and transiently fuses at the cell plasma membrane and releases the polypeptide outside the cell as a vesicle.

[0081] "Specifically binds" means a polypeptide or fragment thereof that recognizes and binds to a target biomolecule (e.g., a polypeptide), but does not substantially recognize or bind to other molecules in a sample naturally containing the polypeptide of the present disclosure, e.g., a biological sample.

[0082] The term "tumor antigen", as used herein, refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on tumor cells as compared to normal cells or non-IS neoplastic cells. In certain embodiments, a tumor antigen can be any polypeptide expressed by a tumor that is capable of activating or inducing an immune response by an antigen recognition receptor (e.g., CD19, MUC-16), or capable of suppressing an immune response by receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0083] The terms "comprises", "comprising", and the like are intended to have the broad meaning given to them in U.S. patent law and can mean "includes", "including", and the like.

[0084] As used herein, "treatment" refers to a therapeutic intervention that attempts to alter the course of a disease in an individual or cell being treated, and can be implemented for prophylaxis or at any point during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequence of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and improving remission or prognosis. By preventing the progression of a disease or disorder, treatment can prevent worsening due to the disorder in an affected or diagnosed subject, or a subject suspected of having the disorder, but treatment can also prevent the onset of the disorder or the symptoms of the disorder in a subject at risk of or suspected of having the disorder.

[0085] "Individual" or "subject" as used herein refers to a human or non-human animal, e.g., a vertebrate such as a mammal. Mammals include, but are not limited to, humans, primates, farm animals, game animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and non-human primates such as apes and monkeys. The term "immunocompromised" as used herein refers to a subject having immunodeficiency. Subjects are highly susceptible to opportunistic infections caused by organisms that typically do not cause disease in people with a healthy immune system, but that can afflict people with a poor or suppressed immune system.

[0086] Other aspects of the subject matter of the present disclosure are described in the following disclosure and are within the scope of the subject matter of the present disclosure. 5.2. IgG-degrading enzyme

[0087] The cells of the present disclosure contain an IgG-degrading enzyme.

[0088] IgG-degrading enzyme can cleave IgG. IgG plays an important protective role in the human immune system, but is also associated with the etiology of diseases such as rheumatoid arthritis, myasthenia gravis, and systemic lupus, where the removal of IgG is used as a therapeutic means to treat these autoimmune diseases (Johansson et al., PLoS ONE (2008);3:1-6;Berta et al., The International Journal of Artificial Organs (1994);17:603-608, Stummvoll et al., Annals of the Rheumatic Diseases (2005); 64:1015-1021). Furthermore, host IgG plays an important role in allotransplantation, where antibody-mediated rejection of the allograft is caused by HLA donor incompatibility (Loupy et al., New England Journal of Medicine (2018);379:1150-1160).

[0089] IdeS has been evaluated in humans regarding desensitization prior to allotransplantation. In this study, 24 out of 25 patients were able to receive HLA-incompatible transplants after treatment with IdeS, which rapidly removed all donor-specific antibodies (Jordan et al., New England Journal of Medicine (2017);377:442-453;Lonze et al., Annals of Surgery (2018);268:488-496).

[0090] Studies have shown that IgG-degrading enzymes have positive therapeutic outcomes. For example, IdeS has been shown to have positive therapeutic outcomes in animal models of idiopathic thrombocytopenic purpura, Goodpasture's disease, and arthritis (Johansson et al., PLoS ONE (2008);3:1-6;Yang et al., Nephrology Dialysis Transplantation (2010);25:2479-2486;Nandakumar et al., Arthritis and Rheumatism (2007);56:3253-3260).

[0091] The IgG-degrading enzyme can cleave IgG, thereby preventing the IgG antibody from killing cells. Additionally or alternatively, the IgG-degrading enzyme can cleave IgG, thereby retaining the binding of the remaining fragments of IgG to cells and protecting the cells from one or more cytotoxic antibodies. In certain embodiments, one or more cytotoxic antibodies bind to the same epitope region as IgG or cross-compete with IgG for binding to the same epitope region, thereby killing the cells. Thus, this process results in a protective shield.

[0092] Using IgG-degrading enzymes, cells containing ligand recognition receptors (e.g., CAR or TCR) can be protected from the host humoral response. Non-limiting examples of host humoral responses include antibody-driven host immune responses (e.g., anti-CAR antibodies), host humoral responses directed against new amino acid sequences, host humoral responses to foreign sequences, host humoral responses to fusion point sequences, host humoral responses to alloantigens (e.g., minor histocompatibility alloantigens), host humoral responses to HLA antigens, host humoral responses to other alleles, host humoral responses to changes in protein or carbohydrate expression, host humoral responses to post-translational modifications of proteins, and host humoral responses induced by differences between the host and the injected cells. This can include existing responses or responses stimulated by the injection of cells. Protection from the host humoral response prevents cell death or neutralization and provides the cells with increased persistence, improved activity (e.g., anti-tumor activity, proliferation, cytokine secretion, involvement in cell lysis, or other functions specifically engineered into the cells). The increased persistence and function of the cells can also result in a reduction in the cost of any treatment involving the cells. For example, CAR-T cell therapy is associated with very high costs, e.g., a single injection can exceed hundreds of thousands of dollars (Lin, et al., Journal of Clinical Oncology (2018); 36:3192-3202). By improving the persistence of CAR-T cells, the cost-effectiveness of this type of treatment can be improved.

[0093] Non-limiting examples of IgG-degrading enzymes include the IgG-degrading enzyme of S. pyogenes (e.g., IdeS), the IgG-degrading enzyme of S. equi subsp. zooepidemicus (IdeZ), the IgG-degrading enzyme of S. equi subsp. equi (IdeE), endoglycosidase (EndoS) from Streptococcus pyogenes, and streptococcal cysteine protease (SpeB) from Streptococcus pyogenes.

[0094] IdeE and IdeZ are derived from Streptococcus equi (Lannergard et al., FEMS Microbiology Letters (2006);262:230-235). Each of IdeE and IdeZ cleaves the Fc region below the hinge region of IgG, where this region contains the site LLGGP.

[0095] EndoS is an endoglycosidase that removes the glycan sites on the gamma chain of IgG, thereby interfering with the interaction of IgG with Fc receptors (Collin et al., EMBO J. (2001);20(12):3046-3055).

[0096] In certain embodiments, the IgG degrading enzyme is capable of interfering with the interaction between IgG and Fc receptors. In certain embodiments, the IgG degrading enzyme is an endopeptidase, such as IdeS, IdeZ, IdeE, and SpeB. In certain embodiments, the IgG degrading enzyme is an IgG-specific endopeptidase, such as IdeS, IdeZ, and IdeE. In certain embodiments, the IgG degrading enzyme is an endoglycosidase, such as EndoS.

[0097] In certain embodiments, the IgG degrading enzyme is IdeS. Bacteria have evolved complex strategies to evade the human immune system, such as the release of proteolytic enzymes, to avoid opsonization and phagocytosis (Potempa et al., Biol Chem. (2012);393:873-888). Streptococcus pyogenes secretes an IgG degrading enzyme that cleaves IgG below the hinge region, generating Fab and Fc fragments.

[0098] IdeS is a cysteine protease with high specificity for immunoglobulin G, which does not cleave immunoglobulins A, M, E, and D (Von et al., EMBO Journal (2002);21:1607-1615, and Johansson et al., PLoS ONE (2008);3:1-6). Although IdeS is potentially immunogenic per se, this enzyme should protect itself from the host immune response for its original purpose. IdeS cleaves IgG below the hinge region, thereby releasing the Fc fragment while leaving the F(ab’)2 fragment intact (von Pawel-Rammingen et al., EMBO J. (2002);21(7):1607-15).

[0099] In certain embodiments, IdeS has an amino acid sequence having the GenBank accession number AEJ35177.1 (SEQ ID NO: 1) provided below, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical, or a fragment thereof (as used herein, homology and identity can be determined using standard software such as BLAST or FASTA), and / or, optionally, can include up to 1 or up to 2 or up to 3 conservative amino acid substitutions. In certain embodiments, IdeS comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 1 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, or at least about 300, and up to 341 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, IdeS comprises or has an amino acid sequence of amino acids 1-341, 30-341, 1-50, 50-100, 100-150, 150-200, or 200-341 of SEQ ID NO: 1. In certain embodiments, IdeS comprises or has amino acids 30-341 of SEQ ID NO: 1. SEQ ID NO: 1 is provided below.

Chemical formula

[0100] An exemplary nucleic acid sequence encoding amino acids 30-341 of SEQ ID NO: 1 is shown in SEQ ID NO: 2 provided below.

Chemical formula

[0101] In certain embodiments, the IgG-degrading enzyme is cell-bound (also referred to as "membrane-bound IgG-degrading enzyme"). See, for example, FIG. 1A. In a membrane-bound IgG-degrading enzyme, the enzyme is fused or attached to a transmembrane domain that enables the enzyme to bind or attach to the cell. See, for example, FIG. 1A. The transmembrane domain may be attached to the C-terminus or N-terminus of the IgG-degrading enzyme. In certain embodiments, the transmembrane domain is attached to the C-terminus of the IgG-degrading enzyme. See, for example, FIG. 1A.

[0102] The transmembrane domain may be a transmembrane domain of a molecule or protein or a portion thereof. The transmembrane domain may include a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a portion thereof), a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof), a CD3ζ polypeptide (e.g., the transmembrane domain of CD3ζ or a portion thereof), a CD4 polypeptide (e.g., the transmembrane domain of CD4 or a portion thereof), a 4-1BB polypeptide (e.g., the transmembrane domain of 4-1BB or a portion thereof), an OX40 polypeptide (e.g., the transmembrane domain of OX40 or a portion thereof), an ICOS polypeptide (e.g., the transmembrane domain of ICOS or a portion thereof), a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.

[0103] In certain embodiments, the transmembrane domain fused to the IgG-degrading enzyme is a CD8 polypeptide. In certain embodiments, the CD8 polypeptide comprises or has the amino acid sequence set forth in SEQ ID NO: 3 or amino acids 137-207 of SEQ ID NO: 27. SEQ ID NO: 3 is provided below.

Chem.

[0104] An exemplary nucleic acid sequence encoding the amino acids of SEQ ID NO: 3 is set forth in SEQ ID NO: 4 provided below.

Chem.

[0105] In certain embodiments, the IgG degrading enzyme is secreted from the cell (also referred to as "secreted IgG degrading enzyme"). See, for example, FIG. 1B. In a secreted IgG degrading enzyme, the enzyme is not fused or attached to a transmembrane domain, whereby the enzyme is secreted or released from the cell into the extracellular environment or in the vicinity of the cell. See, for example, FIG. 1B.

[0106] In certain embodiments, the IgG degrading enzyme is connected or fused to a signal peptide (also referred to as a "leader sequence"). As used herein, a "signal sequence" or "leader sequence" is a peptide sequence (e.g., about 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of a polypeptide or protein or a fragment thereof for directing its transport, e.g., for transporting an IgG degrading enzyme to the cell membrane or for transporting a ligand recognition receptor (e.g., a CAR) to the cell membrane.

[0107] Exemplary signal sequences include, but are not limited to, the CD4 signal peptide, the IgG heavy chain signal peptide, the IL-2 signal sequence (e.g., the human IL-2 signal peptide having the amino acid sequence shown in SEQ ID NO: 5 or the mouse IL-2 signal peptide having the amino acid sequence shown in SEQ ID NO: 6), the kappa signal sequence (e.g., the human kappa signal sequence having the amino acid sequence shown in SEQ ID NO: 7 or the mouse kappa signal sequence having the amino acid sequence shown in SEQ ID NO: 8), the CD8 signal sequence (e.g., the human CD8 signal peptide having the amino acid sequence shown in SEQ ID NO: 9 or the truncated human CD8 signal peptide having the amino acid sequence shown in SEQ ID NO: 10), the albumin signal sequence (e.g., the human albumin signal sequence having the amino acid sequence shown in SEQ ID NO: 11), and the prolactin signal sequence (e.g., the human prolactin signal sequence having the amino acid sequence shown in SEQ ID NO: 12). SEQ ID NOS: 5-12 are provided below.

Chemical Formula

[0108] In certain embodiments, the IgG-degrading enzyme is linked or fused to a CD8 signal sequence. In certain embodiments, the CD8 signal sequence comprises or has the amino acid sequence set forth in SEQ ID NO: 10.

[0109] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10 is set forth in SEQ ID NO: 13 provided below.

Chemical Formula

[0110] In certain embodiments, the IgG-degrading enzyme is expressed from a vector. Expression of the IgG-degrading enzyme can be detected by any suitable method including, but not limited to, immunoblotting, PCR, ELISA, mass spectrometry, and flow cytometry. 5.3. Ligand Recognition Receptor

[0111] The cells of the present disclosure include a ligand recognition receptor. Any receptor capable of binding to a ligand can be a ligand recognition receptor of the present disclosure. Non-limiting examples of ligand recognition receptors include antigen recognition receptors that bind to an antigen of interest, cell adhesion molecules, cytokine receptors (e.g., interleukin or cytokine receptors such as Fas ligand or TGFβ receptor, Trail, TCR, IgG, CAR, NK inhibitory receptors, growth factor receptors such as EGFR or FGFR, peptide ligands or adhesion molecules, carbohydrate receptors, G protein receptors, etc.), and Fc receptors. The receptor can be monovalent or multivalent. The ligand recognition receptor can be endogenous or exogenous. The ligand recognition receptor can be recombinantly expressed. In certain embodiments, the ligand recognition receptor is expressed from a vector.

[0112] In certain embodiments, the ligand recognition receptor is an antigen recognition receptor that binds to the antigen of interest. Non-limiting examples of antigen recognition receptors include chimeric antigen receptors (CARs), T cell receptors (TCRs), IgG, B cell receptors (BCRs), IgM, IgD, and IgE.

[0113] In certain embodiments, the ligand recognition receptor is a chimeric antigen receptor (CAR). In certain embodiments, the ligand recognition receptor is a T cell receptor (TCR).

[0114] In certain embodiments, the ligand recognition receptor binds to an antigen. The antigen can be a tumor antigen, a pathogen antigen, a normal cell antigen (e.g., for autoimmune diseases or organ transplants), an HLA antigen, or an alloantigen (e.g., a minor histocompatibility alloantigen). 5.3.1. Antigen

[0115] In certain embodiments, the ligand recognition receptor binds to an antigen that is a tumor antigen. Any tumor antigen (antigen peptide) can be used in the embodiments related to tumors described herein. The sources of the antigen include, but are not limited to, cancer proteins. The antigen can be expressed as a peptide or as a full-length protein or a portion thereof. The full-length protein or a portion thereof can be natural or mutagenized.Non-limiting examples of tumor antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD2, CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cells infected with cytomegalovirus (CMV) (e.g., cell surface antigens), HPV E6 or E7 peptides, EBV peptides, MAGE peptides, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, cancer-testis antigen NY-ESO-1, tumor fetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME, and ERBB.

[0116] In certain embodiments, the ligand recognition receptor binds to CD19. In certain embodiments, the ligand recognition receptor binds to the mouse CD19 polypeptide. In certain embodiments, the mouse CD19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14.

Chemical formula

[0117] In certain embodiments, the ligand recognition receptor binds to the human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 15.

Chemical formula

[0118] In certain embodiments, the ligand recognition receptor binds to the extracellular domain of the human or mouse CD19 protein.

[0119] In certain embodiments, for example, a ligand recognition receptor for use in treating and / or preventing, for example, a pathogen infection or other infectious disease in an immunocompromised subject binds to a pathogen antigen. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protists capable of causing disease.

[0120] Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 (also referred to as HDTV-III, LAVE or HTLV-III / LAV, or HIV-III); and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantaan virus, bunga virus, phlebovirus and Naira virus); Arenaviridae (hemorrhagic fever virus); Reoviridae (e.g., reovirus, orbivirus and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvovirida (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agent of non-A non-B hepatitis (class 1 = parenteral infection, class 2 = non-oral infection (i.e., hepatitis C));Examples include norwalk and related viruses and astroviruses.;

[0121] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A streptococcus), Streptococcus agalactiae (Group B streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli.

[0122] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), a viral antigen present in human papillomavirus (HPV), or a viral antigen present in influenza virus.

[0123] In certain embodiments, the ligand recognition receptor binds to alloantigens such as HLA molecules and minor histocompatibility alloantigens. 5.3.2 T cell receptor (TCR)

[0124] In certain embodiments, the ligand recognition receptor is a TCR. The TCR is a disulfide-bonded heterodimer protein consisting of two variable chains that are expressed as part of a complex with invariant CD3 chain molecules. The TCR is found on the surface of T cells and is responsible for the recognition of antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0125] Each chain of the TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity-determining regions (CDRs).

[0126] In certain embodiments, the TCR can form a receptor complex with three dimeric signaling modules CD3δ / ε, CD3γ / ε and CD247 ζ / ζ or ζ / η. When the TCR complex associates with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0127] In certain embodiments, the ligand recognition receptor is an endogenous TCR. In certain embodiments, the ligand recognition receptor is an exogenous TCR. In certain embodiments, the ligand recognition receptor is a recombinant TCR. In certain embodiments, the ligand recognition receptor is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is modified from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR is modified from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. 5.3.3. Chimeric Antigen Receptor (CAR)

[0128] In certain embodiments, the ligand recognition receptor is a CAR. A CAR is an engineered receptor that confers the specificity of interest onto immune effector cells. The specificity of monoclonal antibodies can be transplanted onto T cells using CARs, facilitated by the transfer of the coding sequences by retroviral vectors.

[0129] There are three generations of CARs. The "first-generation" CARs typically consist of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. The "first-generation" CARs provide de novo antigen recognition and, independent of HLA-mediated antigen presentation, in a single fusion molecule, activate CD4 + T cells and CD8 + T cells via their CD3ζ chain signaling domains. The "second-generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR, providing additional signals to the T cells. The "second-generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). The "third-generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen recognition receptor is a first-generation CAR. In certain embodiments, the antigen recognition receptor is a second-generation CAR.

[0130] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR (e.g., where an scFv or an analog thereof is embodied) binds to the antigen with a dissociation constant (K -7 ) of about 5×10 d M or less. In certain embodiments, K d is about 5×10 -7 M or less, about 1×10 -7 M or less, about 5×10 -8 M or less, about 1×10 -8 M or less, about 5×10 -9 M or less, about 1×10 -9 M or less, about 5×10 -10 M or less, about 1×10 -10 M or less, about 5×10 -11 M or less, about 1×10 -11 M or less, about 5×10 -12M or less, or about 1×10 -12 is M or less.

[0131] Binding of the extracellular antigen-binding domain (e.g., in an scFv or an analog thereof) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), surface plasmon resonance, Western blot assay, or other assays known in the art. Each of these assays generally detects the presence of a specific protein-antibody complex by using a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest. For example, an scFv can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). The radioisotope can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

[0132] According to the subject matter of the present disclosure, a CAR can include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain specifically binds to an antigen, e.g., a tumor antigen or a pathogen antigen. 5.3.3.1. Extracellular antigen-binding domain of CAR

[0133] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a cross-linked Fab, if desired. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an F(ab)2. In certain embodiments, any of the foregoing molecules is included within a fusion protein containing a heterologous sequence and can form the extracellular antigen-binding domain of the CAR.

[0134] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a mouse scFv. In certain embodiments, the extracellular antigen-binding domain of the CAR of the present disclosure comprises a scFv that binds to CD19.

[0135] In certain embodiments, the scFv comprises a heavy chain variable region (V H ) having the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the scFv comprises a light chain variable region (V L ) having the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the scFv comprises a V H having the amino acid sequence set forth in SEQ ID NO: 16 and a V L having the amino acid sequence set forth in SEQ ID NO: 17, and optionally (iii) a linker sequence between the V H and the V L , e.g., a linker peptide.

[0136] "Linker," as used herein, refers to a functional group (e.g., a chemical or a polypeptide) that covalently attaches two or more polypeptides or nucleic acids such that they are connected to each other. As used herein, "peptide linker" is used to link two proteins together (e.g., a V H domain and a V LRefers to one or more amino acids used (for concatenating domains).

[0137] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 18 provided below.

Chemical formula

[0138] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V that contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 16 H comprising. For example, the extracellular antigen-binding domain of the CAR comprises a V that contains an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 16 H comprising. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V that contains the amino acid sequence set forth in SEQ ID NO: 16 H comprising.

[0139] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V that contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 17 L comprising. For example, the extracellular antigen-binding domain of the CAR comprises a V that contains an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 17 L comprising. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V that contains the amino acid sequence set forth in SEQ ID NO: 17 Lincludes. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 16 H , and a V amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 17 L includes. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 16 H and a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 17 L includes.

[0140] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 19 or a conservative modification thereof H CDR1, a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 20 or a conservative modification thereof H CDR2, and a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 21 or a conservative modification thereof H CDR3. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 19 H CDR1, a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 20 H CDR2, and a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 21 H CDR3. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 22 or a conservative modification thereof L CDR1, a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 23 or a conservative modification thereof L CDR2, and a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 24 or a conservative modification thereof L CDR3. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 22 L CDR1, a V amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 23 LCDR2 and a V containing the amino acid sequence set forth in SEQ ID NO: 24 L CDR3. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V containing the amino acid sequence set forth in SEQ ID NO: 19 or a conservative modification thereof H CDR1, a V containing the amino acid sequence set forth in SEQ ID NO: 20 or a conservative modification thereof H CDR2, a V containing the amino acid sequence set forth in SEQ ID NO: 21 or a conservative modification thereof H CDR3, a V containing the amino acid sequence set forth in SEQ ID NO: 22 or a conservative modification thereof L CDR1, a V containing the amino acid sequence set forth in SEQ ID NO: 23 or a conservative modification thereof L CDR2, and a V containing the amino acid sequence set forth in SEQ ID NO: 24 or a conservative modification thereof L CDR3. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V containing amino acids having the sequence set forth in SEQ ID NO: 19 H CDR1, a V containing the amino acid sequence set forth in SEQ ID NO: 20 H CDR2, a V containing the amino acid sequence set forth in SEQ ID NO: 21 H CDR3, a V containing the amino acid sequence set forth in SEQ ID NO: 22 L CDR1, a V containing the amino acid sequence set forth in SEQ ID NO: 23 L CDR2, and a V containing the amino acid sequence set forth in SEQ ID NO: 24 L CDR3.

[0141] In certain embodiments, the extracellular antigen-binding domain comprises an scFv containing the amino acid sequence of SEQ ID NO: 25 and specifically binds to a human CD19 polypeptide (e.g., a human CD19 polypeptide containing the amino acid sequence set forth in SEQ ID NO: 15). In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25 is set forth in SEQ ID NO: 26.

[0142] SEQ ID NOs: 16, 17, and 19-26 are provided below.

Chemical formula

[0143] As used herein, the term "conservative array modification" refers to amino acid modifications that do not significantly affect and do not change the binding characteristics of the CARs of the present disclosure (e.g., the extracellular antigen-binding domain of the CAR) containing the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. The modifications can be introduced into the human scFv of the CARs of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid in the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutral charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Further, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; and nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retained function (i.e., the functions shown in (c) to (l) above) using the functional assays described herein. In certain embodiments, one or fewer, two or fewer, three or fewer, four or fewer, five or fewer residues within the specified sequence or within the CDR region are modified.

[0144] For a specific sequence (e.g., SEQ ID NO: 16 and 17), V has at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity H and / or V L amino acid sequences may contain substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the specified sequence(s), but retain the ability to bind to a target antigen (e.g., CD19). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a specific sequence (e.g., SEQ ID NO: 16 and 17). In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDRs of the extracellular antigen-binding domain (e.g., in the FR). In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V selected from the group consisting of SEQ ID NO: 16 and 17 (including post-translational modifications of those sequences (SEQ ID NO: 16 and 17)) H and / or V L sequence.

[0145] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0146] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Further, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0147] Alternatively or additionally, the amino acid sequences of the subject matter of the present disclosure can be further used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. The BLAST protein search can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., the sequences of the heavy and light chain variable regions of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402 can be utilized. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. 5.3.3.2. Transmembrane domain of CAR

[0148] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptor clusters and signals are transmitted to the cell. The transmembrane domain of the CAR can include a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a portion thereof), a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof), a CD3ζ polypeptide, a CD4 polypeptide (e.g., the transmembrane domain of CD4 or a portion thereof), a 4-1BB polypeptide (e.g., the transmembrane domain of 4-1BB or a portion thereof), an OX40 polypeptide (e.g., the transmembrane domain of OX4 or a portion thereof), an ICOS polypeptide (e.g., the transmembrane domain of ICOS or a portion thereof), a synthetic peptide (not based on proteins related to immune responses), or a combination thereof.

[0149] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of a CD8 polypeptide, such as the transmembrane domain of human CD8 or a portion thereof, or the transmembrane domain of mouse CD8. In certain embodiments, the CD8 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence provided herein as NCBI Reference No. NP_001139345.1 (SEQ ID NO: 27), or a fragment thereof (as used herein, homology can be determined using standard software such as BLAST or FASTA), and / or, optionally, may include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 27 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises or has the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 137-207, 137-209, 150-200, or 200-235 of SEQ ID NO: 25. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide that comprises or has amino acids 137-207 of SEQ ID NO: 27.

Chemical formula

[0150] An exemplary nucleotide sequence encoding amino acids 137-207 of SEQ ID NO: 27 is shown in SEQ ID NO: 28 provided below.

Chemical formula

[0151] In certain embodiments, the CD8 polypeptide comprises, or has, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having NCBI reference number AAA92533.1 (SEQ ID NO: 29) provided below, or a fragment thereof (homology herein can be determined using standard software such as BLAST or FASTA), and / or optionally may include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises, or has, an amino acid sequence that is a contiguous portion of SEQ ID NO: 27 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD8 polypeptide comprises, or has, the amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 29. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide that comprises, or has, the amino acid sequence of amino acids 151-219 of SEQ ID NO: 29. SEQ ID NO: 29 is provided below.

Chemical Formula

[0152] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide, e.g., the transmembrane domain of human CD28 or a portion thereof, or the transmembrane domain of mouse CD28. The CD28 polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having NCBI reference number NP_006130 (SEQ ID NO: 30), or a fragment thereof, and / or optionally may contain up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or has an amino acid sequence that is a continuous portion of SEQ ID NO: 30 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide comprises or has the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, or 200-220 of SEQ ID NO: 30. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide that comprises or has amino acids 153-179 of SEQ ID NO: 30.

[0153] SEQ ID NO: 30 is provided below:

Chemical formula

[0154] In certain non-limiting embodiments, the CAR further comprises a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be sufficiently flexible such that the antigen-binding domain can be oriented in various directions to facilitate antigen recognition. The spacer region can be a hinge region derived from IgG1, or the CH2CH3 region of an immunoglobulin and a portion of CD3, a portion of the CD28 polypeptide (e.g., the portion of SEQ ID NO: 30), a portion of the CD8 polypeptide (e.g., the portion of SEQ ID NO: 27 or 29), a variant form of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical thereto, or a synthetic spacer sequence. 5.3.3.3. Intracellular signaling domain of the CAR

[0155] In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that can activate or stimulate cells (e.g., cells of the lymphoid lineage, such as T cells). CD3ζ contains three ITAMs and, after antigen binding, transmits an activation signal to the cell (e.g., cells of the lymphoid lineage, such as T cells). The intracellular signaling domain of the CD3ζ chain is the major transmitter of signals from the endogenous TCR. In certain embodiments, the CD3ζ polypeptide comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to the sequence having NCBI Reference No. NP_932170 (SEQ ID NO: 31), or a fragment thereof, and / or may optionally include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 31 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1-164, 1-50, 50-100, 100-150, or 150-164 of SEQ ID NO: 31. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that comprises or has amino acids 52-164 of SEQ ID NO: 31.

[0156] SEQ ID NO: 31 is provided below:

Chemical formula

[0157] In certain embodiments, the CD3ζ polypeptide comprises, or has, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence having NCBI Reference No. NP_001106864.2 (SEQ ID NO: 32), or a fragment thereof, and / or, optionally, may contain up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide comprises, or has, an amino acid sequence that is a contiguous portion of SEQ ID NO: 32 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 90, or at least about 100, and up to 188 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD3ζ polypeptide comprises, or has, the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-142, 100-150, or 150-188 of SEQ ID NO: 32. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that comprises, or has, the amino acid sequence of amino acids 52-142 of SEQ ID NO: 32.

[0158] SEQ ID NO: 32 is provided below:

Chemical formula

[0159] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide that comprises, or has, the amino acid sequence shown in SEQ ID NO: 33 provided below.

Chemical formula

[0160] An exemplary nucleic acid sequence encoding SEQ ID NO: 33 is shown in SEQ ID NO: 34 provided below.

Chemical formula

[0161] In certain non-limiting embodiments, the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region. In certain embodiments, the co-stimulatory region comprises at least one co-stimulatory molecule or a portion thereof (e.g., the intracellular domain of a co-stimulatory molecule or a portion thereof). The co-stimulatory signaling region can provide optimal lymphocyte activation to the cell. As used herein, "co-stimulatory molecule" refers to cell surface molecules other than antigen recognition receptors or their ligands that are required for an efficient response of immune response cells to an antigen of interest. Non-limiting examples of co-stimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, CD2, and NKGD2. A co-stimulatory molecule is a protein expressed on the cell surface that can bind to a co-stimulatory ligand, which results in a co-stimulatory response upon binding to its receptor, i.e., an intracellular response that provides a stimulus provided when an antigen recognition receptor (e.g., CAR) binds to its target antigen. Co-stimulatory ligands include, but are not limited to, 4-1BB ligand (4-1BBL), CD80, CD86, CD70, OX40L, and ICOSLG. As an example, 4-1BBL can bind to 4-1BB to provide a co-stimulatory signal that, in combination with an activating signal, induces effector cell function of CAR-T cells. CARs comprising an intracellular signaling domain comprising a co-stimulatory signaling region comprising 4-1BB, ICOS, or DAP-10 are disclosed in U.S. 7,446,190, which is incorporated herein by reference in its entirety.

[0162] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes a 4-1BB polypeptide (e.g., the intracellular domain of 4-1BB or a portion thereof). The 4-1BB polypeptide can include, or have, an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the sequence having NCBI Reference No. NP_001552 (SEQ ID NO: 35), or a fragment thereof, and / or, optionally, can include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the 4-1BB polypeptide includes, or has, an amino acid sequence that is a contiguous portion of SEQ ID NO: 35 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 255 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the 4-1BB polypeptide includes, or has, the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO: 35. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of human 4-1BB or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes a 4-1BB polypeptide that includes, or has, amino acids 214-255 of SEQ ID NO: 35. SEQ ID NO: 35 is provided below.

Chemical formula

[0163] An exemplary nucleic acid sequence encoding amino acids 214-255 of SEQ ID NO: 35 is shown in SEQ ID NO: 36 provided below.

Chemical formula

[0164] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes a CD28 polypeptide (e.g., the intracellular domain of CD28 or a portion thereof). The CD28 polypeptide can include, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 30, or a fragment thereof, and / or, optionally, can include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide includes, or has, an amino acid sequence that is a contiguous portion of SEQ ID NO: 30 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CD28 polypeptide includes, or has, the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 180-220, or 200-220 of SEQ ID NO: 29 or SEQ ID NO: 30. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of human CD28 or a portion thereof. In certain embodiments, human CD28 has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, human CD28 has the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes a CD28 polypeptide that includes, or has, amino acids 180-220 of SEQ ID NO: 30.

[0165] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes an OX40 polypeptide (e.g., the intracellular domain of OX40 or a portion thereof). The OX40 polypeptide can include, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having NCBI Reference No. NP_003318.1 (SEQ ID NO: 37), or a fragment thereof, and / or, optionally, can include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the OX40 polypeptide includes, or has, an amino acid sequence that is a contiguous portion of SEQ ID NO: 37 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 277 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the OX40 polypeptide includes, or has, amino acids 1-220, 1-50, 50-100, 100-150, 150-200, or 200-277 of SEQ ID NO: 37. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of OX40 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of human OX40 or a portion thereof. In certain embodiments, human OX40 has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, human OX40 has the amino acid sequence set forth in SEQ ID NO: 37.

[0166] SEQ ID NO: 37 is provided below:

Chemical formula

[0167] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes an ICOS polypeptide (e.g., the intracellular domain of ICOS or a portion thereof). The ICOS polypeptide can include, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having NCBI reference number NP_036224.1 (SEQ ID NO: 38), or a fragment thereof, and / or may optionally include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the ICOS polypeptide includes, or has, an amino acid sequence that is a contiguous portion of SEQ ID NO: 38 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 199 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the ICOS polypeptide includes, or has, the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, or 150-199 of SEQ ID NO: 38. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of ICOS. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that includes the intracellular domain of human ICOS. In certain embodiments, human ICOS has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, human ICOS has the amino acid sequence set forth in SEQ ID NO: 38.

[0168] SEQ ID NO: 38 is provided below:

Chemical formula

[0169] In certain embodiments, the CAR comprises two costimulatory signaling domains, wherein the first costimulatory domain comprises the intracellular domain of 4-1BB or a portion thereof, and the second costimulatory domain comprises the intracellular domain of CD28 or a portion thereof.

[0170] In certain embodiments, the CAR of the present disclosure further comprises an inducible promoter for expressing a nucleic acid sequence in human cells. The promoter used for expressing the CAR gene can be a constitutive promoter such as the ubiquitin C (UbiC) promoter. 5.3.3.4. Exemplary CAR

[0171] In certain embodiments, the cell of the present disclosure comprises a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that binds to CD19, a transmembrane domain comprising a CD8 polypeptide (e.g., the transmembrane domain of human CD8 or a portion thereof), and an intracellular signaling domain comprising a CD3ζ polypeptide and a costimulatory signaling region comprising a 4-1BB polypeptide (e.g., the intracellular domain of human 4-1BB or a portion thereof).

[0172] In certain embodiments, the CAR is designated as "19BBz". In certain embodiments, the CAR (e.g., 19BBz) comprises a V H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a V H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, a V H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, a V L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22, a V L CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 23, a V LAn extracellular antigen-binding domain comprising a CDR3; a transmembrane domain comprising a CD8 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or amino acids 137-207 of SEQ ID NO: 27; an intracellular signaling domain comprising a CD3ζ polypeptide comprising the amino acid sequence shown in SEQ ID NO: 33, and a costimulatory signaling region comprising a 4-1BB polypeptide comprising amino acids 214-255 of SEQ ID NO: 35.

[0173] In certain embodiments, the CAR (e.g., 19BBz) comprises an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence shown in SEQ ID NO: 39 provided below.

Chemical formula

[0174] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 39 is shown in SEQ ID NO: 40 provided below.

Chemical formula

Chemical formula

[0175] In certain embodiments, the CAR (e.g., 19BBz) further comprises a CD8 signal peptide. In certain embodiments, the CD8 signal peptide comprises or has the amino acid sequence shown in SEQ ID NO: 10. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 10 is shown in SEQ ID NO: 41 provided below.

Chemical formula

[0176] The amino acid sequence of 19BBz comprising a CD8 signal peptide is shown in SEQ ID NO: 42 provided below.

Chemical formula

[0177] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is shown in SEQ ID NO: 43 provided below.

Chem.

Chem.

[0178] The subject matter of the present disclosure provides cells comprising (a) a ligand recognition receptor (e.g., the ligand recognition receptor disclosed in item 5.3), and (b) an IgG degrading enzyme (e.g., the IgG degrading enzyme disclosed in item 5.2). In certain embodiments, the ligand recognition receptor is capable of activating the cells. The cells can be transduced with the ligand recognition receptor and the IgG degrading enzyme such that the cells express the ligand recognition receptor and the IgG degrading enzyme simultaneously. In certain embodiments, the IgG degrading enzyme is attached to the cell surface. In certain embodiments, the IgG degrading enzyme is not attached to the cell surface and is delivered or released from the cell.

[0179] In certain embodiments, the cells further comprise a cleavable (e.g., self-cleavable) linker (e.g., a 2A peptide, e.g., a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide). In certain embodiments, the cells further comprise a P2A peptide. In certain embodiments, the P2A peptide is located between the ligand recognition receptor and the IgG degrading enzyme. In certain embodiments, the P2A peptide comprises or has the amino acid sequence shown in SEQ ID NO: 44 provided below:

Chem.

[0180] An exemplary nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 44 is shown in SEQ ID NO: 45 provided below.

Chemical Formula

[0181] In certain embodiments, the cells are responsive cells. In certain embodiments, the cells are responsive cells, such as, for example, immunoreactive cells. In certain embodiments, the cells are activatable cells. In certain embodiments, the cells are cells of the lymphoid lineage. In certain embodiments, the cells are cells of the myeloid lineage. In certain embodiments, the cells are derived from normal tissue, such as, for example, cells derived from the kidney, liver, lung, bone marrow, or pancreas.

[0182] Cells of the lymphoid lineage can result in, for example, antibody production, regulation of the cellular immune system, detection of foreign pathogenic agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells that can differentiate into lymphoid cells. The stem cells can be pluripotent stem cells (such as, for example, embryonic stem cells and induced pluripotent stem cells).

[0183] In certain embodiments, the cells are T cells. T cells can be lymphocytes that mature in the thymus and are mostly responsible for cell-mediated immunity. T cells are involved in the acquired immune system. The T cells of the subject matter of the present disclosure include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and type 2 effector memory T cells: for example, T EM cells and T EMRA cells, including), regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TIL), natural killer T cells (NK T cells), mucosa-associated invariant T cells, and γδ It can be any type of T cell, including but not limited to T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introducing antigen recognition receptors, such as CARs or TCRs. T cells can be CD4 + T cells or CD8 + T cells. In certain embodiments, the T cells are CD4 + T cells. In certain embodiments, the T cells are CD8 + T cells.

[0184] In certain embodiments, the cells are NK cells. Natural killer (NK) cells are part of cell-mediated immunity and can be lymphocytes that act in the innate immune response. NK cells do not require prior activation to exert a cytotoxic effect on target cells.

[0185] Examples of human lymphocyte types of the subject matter of the present disclosure include, but are not limited to, peripheral donor lymphocytes, such as Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, R.A., et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex containing α and β heterodimers), Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, Examples include those disclosed in G.A., et al. 2003 Blood 102:2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells).

[0186] Cells (e.g., T cells) may be autologous, allogeneic (e.g., allogeneic), and may be derived from engineered precursors or stem cells in vitro. In certain embodiments, the cells are allogeneic cells.

[0187] In certain embodiments, the cells are cells of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells that can differentiate into myeloid cells.

[0188] In certain embodiments, the cells of the present disclosure are used in therapy. In certain embodiments, the cells of the present disclosure are used in cell therapy. In certain embodiments, the cells of the present disclosure are used in gene therapy. In certain embodiments, the cells of the present disclosure are used in CRISPR gene therapy. The field of cell engineering is expanding, particularly because of the increasing popularity of the use of CRISPR-Cas9 technology and because the immunogenicity of cells is an important concern with respect to the introduction of multiple foreign proteins into cells. Immunogenic cells are rapidly eliminated from the patient, reducing their effectiveness (Porter et al., Science Translational Medicine (2015);7;Maude et al., N Engl J Med. (2014);371:1507-1517;Louis et al., Blood (2011);118:6050-6056). Gene therapy often involves inserting foreign genes into cells with viral genes or other foreign helper genes. Viral proteins, such as those derived from adeno-associated virus (AAV) used in hemophilia treatment and other genetic disorders, can persist in patients for months or years and serve as targets for the immune response.

[0189] The cells of the present disclosure can reduce the immunogenicity of foreign cells.

[0190] In certain embodiments, the cells of the present disclosure are used in immunotherapy. In certain embodiments, the cells of the present disclosure are used in adoptive cell transfer (ACT). A rapidly emerging and individualized type of immunotherapy is adoptive cell transfer (ACT), in which a patient's immune cells are used as tools to treat the patient's cancer (Kalos et al., Immunity (2013);39:49-60). T cells can be genetically engineered to recognize tumor cells, expanded in vitro, and then returned to the patient. There are several types of ACT, including chimeric antigen receptor (CAR) T cells, T cell receptor (TCR)-engineered T cells, and tumor-infiltrating lymphocytes (TIL) (Rosenberg et al., Nature Reviews Cancer (2008);8:299-308). In ACT, T cells can be genetically engineered to recognize tumor cells, expanded in vitro, and then returned to the patient.

[0191] CAR T cell therapy has advanced in clinical practice scenarios, including two therapies approved by the FDA in 2017 (Zheng et al., Drug Discovery Today (2018);23:1175-1182). Continued efforts in this field are targeting the current limitations of CAR T cell therapy to improve tumor trafficking and recognition, increase their proliferation and persistence, and enhance the inventors' control over their activity (Lim et al., Cell (2017);168:724-740). Improvements in ACT with reduced off-target effects and toxicity, as well as improved overall efficacy, are needed. Humoral responses in patients have been observed against the CAR T cells of the patients to whom they are administered. Such antibodies were against the CAR construct protein, and the proviral proteins derived from the retroviral vectors used for transduction (Kershaw et al., Clinical Cancer Research (2006);12:6106-6115;Lamers et al., Blood (2011);117:72-82;Jensen et al., Biology of Blood and Marrow Transplantation (2010);16:1245-1256.7-9). Since bacterial proteins are used for CAR T cell engineering by the use of CRISPR technology, and importantly, since allogeneic CAR T cells are becoming more prevalent, immunogenicity can also become a more common problem (Jung et al., Molecules and Cells (2018);41:717-723;Graham et al., Cells (2018);7:155).

[0192] The cells of the present disclosure can improve ACT efficacy and / or reduce toxicity associated with antigenicity against CAR T cells.

[0193] The cells of the present disclosure increase resistance to the humoral response, thereby enabling the extension of the peripheral persistence of CAR T cells, which in turn results in more potent activity (e.g., anti-tumor activity). The extension of cell persistence can also improve the cost-effectiveness of cell therapy (e.g., ACT, which usually involves very high costs).

[0194] Binding of antibodies to CAR-T cells results in lysis of CAR T cells by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thus potentially reducing the therapeutic effect. Anti-idiotypic antibodies have been shown to neutralize CAR T cell function (Lamers et al., Blood (2011);117:72-82). The limited peripheral persistence of CAR T cells also contributes to the cellular response, where CAR T cells are targeted by endogenous T cells (Lamers et al., Blood (2011);117:72-82; Jensen et al., Biology of Blood and Marrow Transplantation (2010);16:1245-1256). Epitopes responsible for anti-CAR immunity observed in a CAR T cell model targeting carbonic anhydrase IX (CAIX) have been identified and include peptide sequences derived from the complementarity-determining regions and framework regions of the CAR, as well as proviral sequences derived from the SFG retroviral vector (Lamers et al., Blood (2011);117:72-82). One approach taken to address this problem is to humanize the CAR to make it non-immunogenic (Gonzales et al., Tumor Biology (2005);26:31-43). However, this will not address the problem of immune responses specific to viral vectors or the problem of allogeneic cells. The cells of the present disclosure can overcome the humoral response to foreign cell therapies such as CAR T cell therapy and prevent the neutralization of cell activity by anti-cell antibodies.

[0195] There is prior art regarding the formation of anti-CAR antibodies in the literature, however, there are significant obstacles to the success of these therapies (Kershaw et al., Clinical Cancer Research (2006);12:6106-6115;Lamers et al., Blood (2011);117:72-82;Jensen et al., Biology of Blood and Marrow Transplantation (2010);16:1245-1256;Jung et al., Molecules and Cells (2018);41:717-723;Graham et al., Cells (2018);7:155).

[0196] The presently discloses cells comprising an IgG-degrading enzyme (e.g., IdeS) that serves as a biomolecular shield (e.g., potential antibody) against the host humoral response. Expression of the IgG-degrading enzyme in the cells provides protection from neutralizing anti-CAR antibodies, b) prolongs the persistence of the cells (e.g., engineered CAR T cells), and c) extends the therapeutic activity window, thereby resulting in overall higher efficacy. Other approaches in the field of CAR-T cells focus on the cellular immune response against CAR T cells, for example, by removing HLA I and TCR (Zhao et al., Journal of Hematology and Oncology (2018);11:1-9). However, to the inventors' knowledge, this is the first study directly aimed at addressing the antibody-driven host immune response. 5.5. Compositions and Vectors

[0197] The present discloses subject matter provides compositions comprising an IgG-degrading enzyme (e.g., as disclosed in item 5.2) and a ligand recognition receptor (e.g., as disclosed in item 5.3) disclosed herein. Cells comprising such compositions are also provided.

[0198] In certain embodiments, the IgG degrading enzyme is operably linked to a first promoter. In certain embodiments, the ligand recognition receptor is operably linked to a second promoter.

[0199] In certain embodiments, the composition further comprises a cleavable (e.g., self-cleavable) linker (e.g., a 2A peptide, e.g., a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide). In certain embodiments, the composition further comprises a P2A peptide. In certain embodiments, the P2A peptide is located between the ligand recognition receptor and the IgG degrading enzyme. In certain embodiments, the P2A peptide comprises or has the amino acid sequence set forth in SEQ ID NO: 43.

[0200] Furthermore, the present disclosure provides a nucleic acid composition comprising a first polynucleotide encoding an IgG degrading enzyme (e.g., as disclosed in item 5.2) and a second polynucleotide encoding a ligand recognition receptor (e.g., as disclosed in item 5.3) disclosed herein. Cells comprising such nucleic acid compositions are also provided.

[0201] In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the IgG degrading enzyme. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the ligand recognition receptor.

[0202] In certain embodiments, one or both of the first and second promoters are endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the CMV promoter, the SV40 promoter, the PGK promoter, and the metallothionein promoter.

[0203] In certain embodiments, the nucleic acid composition further comprises a cleavable (e.g., self-cleavable) linker (e.g., 2A peptide, e.g., P2A peptide, T2A peptide, E2A peptide, and F2A peptide). In certain embodiments, the nucleic acid composition further comprises a P2A peptide. In certain embodiments, the P2A peptide is positioned between the ligand recognition receptor and the IgG degrading enzyme. In certain embodiments, the P2A peptide comprises or has the nucleotide sequence set forth in SEQ ID NO: 45. The compositions and nucleic acid compositions can be administered to a subject and / or delivered intracellularly by methods known in the art or as described herein.

[0204] Genetic modification of cells (e.g., immune-responsive cells, e.g., T cells or NK cells) can be achieved by transducing a recombinant DNA construct into a substantially homogeneous cell composition. In certain embodiments, a retroviral vector (either a gammaretrovirus or a lentivirus) is used for the introduction of the nucleic acid composition into the cell. For example, a first polynucleotide encoding an IgG degrading enzyme and a second polynucleotide encoding a ligand recognition receptor can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from the long terminal repeat of the retrovirus, or from a promoter specific for the target cell type of interest. Non-viral vectors can be used as well.

[0205] For the initial genetic modification of cells to include a ligand recognition receptor (e.g., CAR or TCR), retroviral vectors are generally used for transduction, however, any other suitable viral vector or non-viral delivery system can also be used. The ligand recognition receptor and IgG degrading enzyme can be constructed in a single multicistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements that give rise to multicistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aftovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). The combination of a retroviral vector and a suitable packaging system is also suitable when the capsid protein is functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles, e.g., particles pseudotyped with VSVG, RD114, or GALV envelope and any others known in the art are also suitable.

[0206] Methods of transduction may include, for example, direct co-culture of cells with producer cells, such as by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with virus supernatant alone, or with concentrated vector stocks with or without appropriate growth factors and polycations, such as by the methods of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0207] Other transduction viral vectors may be used to modify the cells. In certain embodiments, the selected vector exhibits high efficiency of infection as well as stable integration and expression (e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, See 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus, or herpesvirus, such as Epstein-Barr virus (e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors have been particularly well developed and are used in clinical practice (Rosenberg et al., N. Engl. J. Med 323:370, 1990; U.S. Patent No. 5,399,346 to Anderson et al.).

[0208] Non-viral approaches can also be used for genetic modification of cells. For example, by administering nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990), nucleic acid molecules can be introduced into cells. Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for intracellular delivery of DNA. Transplantation of normal genes into diseased tissues of a subject can be achieved by introducing normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into the target tissue or systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0209] Any of the targeted genome editing methods can be used to deliver the IgG degrading enzymes and / or ligand recognition receptors disclosed herein to cells or subjects. In certain embodiments, the CRISPR system is used to deliver the IgG degrading enzymes and / or ligand recognition receptors disclosed herein. In certain embodiments, zinc finger nucleases are used to deliver the IgG degrading enzymes and / or ligand recognition receptors disclosed herein. In certain embodiments, the TALEN system is used to deliver the IgG degrading enzymes and / or ligand recognition receptors disclosed herein.

[0210] The clustered regularly interspaced short palindromic repeat (CRISPR) system is a genome editing tool found in prokaryotic cells. When utilized for genome editing, this system includes sections of Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, containing the region that binds to tracrRNA (generally in hairpin loop form) to form an active complex with Cas9 and the RNA used by Cas9 to guide it to the correct section of host DNA), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and a DNA repair template (DNA that guides the cellular repair process to allow insertion of a specific DNA sequence) as required. CRISPR / Cas9 often uses plasmids to transfect target cells. Since crRNA is the sequence used by Cas9 to identify and directly bind to the target DNA within the cell, it needs to be designed for each application. The repair template carrying the CAR expression cassette also needs to be designed for each application as it must overlap with the sequences on either side of the cleavage and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be ligated together with the Cas9 gene, made into a plasmid, and transfected into cells.

[0211] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining zinc finger DNA-binding domains with DNA cleavage domains. The zinc finger domains may be engineered to target specific DNA sequences, thereby enabling the zinc finger nucleases to target desired sequences within the genome. The DNA-binding domains of individual ZFNs typically contain multiple individual zinc finger repeats, each of which can recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger "modules" of known specificities. The most common cleavage domain in ZFNs is a non-specific cleavage domain derived from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying a CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. When the targeted sequence is cleaved by ZFNs, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template and then copies the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0212] Transcription activator-like effector nuclease (TALEN) is a restriction enzyme that can be engineered to cleave specific sequences of DNA. The TALEN system operates on much the same principle as ZFN. These are generated by combining a transcription activator-like effector DNA-binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid repeat motifs with two variable positions that have strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location within the genome. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any appropriate mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron construct). For example, if desired, the expression of the nucleic acid can be directed using enhancers known to preferentially direct gene expression in specific cell types. Enhancers that can be used include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, when genomic clones are used as therapeutic constructs, regulation can be mediated by cognate regulatory sequences containing any of the above promoters or regulatory elements, or, if desired, by regulatory sequences derived from a heterologous source.

[0213] The resulting cells can grow under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for various purposes.

[0214] Methods for delivering genome editing agents / systems can vary depending on the need. In certain embodiments, the components of a selected genome editing method are delivered as a nucleic acid composition (e.g., a DNA construct) in one or more plasmids. In certain embodiments, the components are delivered by a viral vector. General delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrodynamic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, viral vector envelope protein pseudotyping, replication-competent vector cis and trans acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0215] The compositions or nucleic acid compositions disclosed herein can be placed at any location within the genome. In certain embodiments, the composition or nucleic acid composition is placed at a site within the genome of a T cell. 5.6. Polypeptides and Analogs

[0216] For the desired purpose, for example, when expressed intracellularly, the polypeptides disclosed herein (e.g., CD19, 4-1BB, CD28, CD3ζ, and IgG degrading enzyme or fragments thereof) that are modified by methods for enhancing their anti-neoplastic and / or anti-tumor activities are also included in the subject matter of the present disclosure. The subject matter of the present disclosure provides methods for optimizing amino acid or nucleic acid sequences by causing changes in the sequence, as well as modified amino acid and nucleic acid sequences. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure further includes analogs of any naturally occurring polypeptide disclosed herein. An analog may differ from the naturally occurring polypeptides disclosed herein by differences in amino acid sequence, by post-translational modifications, or by both. An analog may be shown to be homologous to all or a portion of the naturally occurring amino acid sequence of the subject matter of the present disclosure at a rate of at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher. The length of the sequence comparison is at least 5, 10, 15 or 20 amino acid residues, e.g., at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Again, for an exemplary approach to determining the degree of identity, the BLAST program can be used, e -3 and e -100The probability scores between show closely related sequences. Modifications include in vivo and in vitro chemical derivatization of polypeptides, for example, acetylation, carboxylation, phosphorylation, or glycosylation, and such modifications can occur during the synthesis or processing of the polypeptide, or after treatment with isolated modifying enzymes. Analogs can differ from naturally occurring polypeptides also by changes in the primary sequence. These include both natural and induced genetic variants (e.g., resulting from random mutagenesis by irradiation or exposure to ethyl methyl sulfate, or by site-directed mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A

[0217] Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). For example, cyclic peptides, molecules, and analogs containing residues other than L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids (e.g., β or γ amino acids) are also included.

[0218] Non-protein analogs have chemical structures designed to mimic the functional activities of the proteins (e.g., IgG-degrading enzymes) disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and the synthesis of analogs can be carried out according to such methods by modifying the chemical structure so that the resulting analog, when expressed intracellularly, increases the anti-neoplastic activity of the original polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and changing the degree of saturation at specific carbon atoms of the reference polypeptide. In certain embodiments, the protein analogs are relatively resistant to in vivo degradation and provide a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the following examples. 5.7. Administration

[0219] Compositions containing the cells of the present disclosure may be administered systemically or directly to a subject to induce and / or enhance an immune response against an antigen and / or to treat and / or prevent a neoplasm, pathogen infection, or infectious disease. In certain embodiments, the cells, compositions, or nucleic acid compositions of the present disclosure are directly injected into the organ of interest (e.g., the organ affected by a neoplasm). Alternatively, the cells, compositions, or nucleic acid compositions of the present disclosure are indirectly administered to the organ of interest, for example, by administration to the circulatory system (e.g., the vasculature of a tumor). Expansion and differentiation agents can be administered before, during, or after the administration of the cells, compositions, or nucleic acid compositions to increase the production of cells (e.g., T cells (e.g., CTL cells) or NK cells) in vitro or in vivo.

[0220] The cells, compositions, or nucleic acid compositions of the present disclosure can be administered by any suitable route including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrathoracic, intraperitoneal, and topical. In certain embodiments, the cells, compositions, or nucleic acid compositions of the present disclosure are administered intraperitoneally to a subject. Typically, at least about 1×10 5 cells are administered and ultimately reach about 1×10 10 cells or more. The cells of the present disclosure can include a heterogeneous or purified population of cells. One of ordinary skill in the art can readily determine the percentage of the cells of the present disclosure in a population using various well-known methods such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing the cells of the present disclosure are from about 50% to about 55%, from about 5% to about 60%, and from about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. The dosage can be readily adjusted by one of ordinary skill in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like. The cells can be composed of various lineages of organs or tissues containing 10 9 or up to 10 11 cells.

[0221] The compositions of the present disclosure can be pharmaceutical compositions comprising the cells of the present disclosure or their precursors and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, the cells, or precursors, can be obtained from one subject and administered to the same subject or a different, compatible subject. Cells or their progeny derived from peripheral blood (e.g., in vivo, ex vivo or in vitro derived) can be administered by catheter administration, systemic injection, local injection, intravenous injection, or local injection including parenteral administration. When administering a therapeutic composition of the subject matter of the present disclosure (e.g., a pharmaceutical composition comprising the immunoreactive cells of the present disclosure), this can be formulated into an injectable unit dosage form (solution, suspension, emulsion). 5.8. Formulations

[0222] Compositions containing the cells of the present disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions (which can be buffered to a selected pH). Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to obtain a longer contact period with a particular tissue. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0223] Sterile injectable solutions can be prepared by incorporating the genetically modified immunoreactive cells, optionally with various amounts of other components, into a suitable solvent in the required amount. Such compositions may be mixed with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition may be lyophilized. The composition can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (such as methylcellulose), pH buffers, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the desired route of administration and the preparation. Standard texts such as "REMINGTON’S PHARMACEUTICAL SCIENCE", 17th edition, 1985, which are incorporated herein by reference, may be referred to in order to prepare suitable preparations without undue experimentation.

[0224] Various additives that enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolongation of absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with the genetically modified immunoreactive cells or their precursors.

[0225] The compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and tears. The desired isotonicity of the composition can be achieved using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be used, in particular, for buffers containing sodium ions.

[0226] The viscosity of the composition can be maintained at a selected level, if desired, using pharmaceutically acceptable thickening agents. For example, methylcellulose is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. Clearly, the choice of suitable carrier and other additives depends on the exact route of administration and the nature of the particular dosage form, e.g., whether the composition is to be formulated as a solution, suspension, gel, or another liquid form, such as a time release form or a liquid-filled form.

[0227] The amount of cells administered will vary for the subject being treated. In certain embodiments, about 10 4 ~ about 1010 between about 10 5 and about 10 9 are administered to a subject (e.g., a human subject). In certain embodiments, between about 10 6 and about 10 8 are administered to a subject (e.g., a human subject). More effective cells may be administered in even fewer numbers. In certain embodiments, at least about 1×10 4 to about 10 7 are administered to a subject (e.g., a human subject). In certain embodiments, between about 10 5 and about 10 7 are administered to a subject (e.g., a human subject). More effective cells may be administered in even fewer numbers. In certain embodiments, at least about 1×10 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , or about 5×10 8 are administered to a subject (e.g., a human subject). The exact determination of what is considered an effective dose may be based on factors specific to each subject, including their size, age, gender, weight, and condition of the particular subject. Dosages can be readily ascertained by one of ordinary skill in the art from the present disclosure and knowledge in the art.

[0228] One skilled in the art can readily determine the amounts of cells and, if necessary, additives, vehicles, and / or carriers in the composition and to be administered in the method. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of a 0.001 - 50% (by weight) solution in phosphate buffered saline, and the active ingredient is on the order of micrograms to milligrams, for example, about 0.0001 - about 5 wt%, about 0.0001 - about 1 wt%, about 0.0001 - about 0.05 wt% or about 0.001 - about 20 wt%, about 0.01 - about 10 wt%, or about 0.05 - about 5 wt%. For any composition to be administered to an animal or human, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice; the dosage of the composition(s) that elicits a suitable response, the concentration of the components therein, and the timing of administration of the composition(s). Such determinations do not require undue experimentation from the knowledge of one skilled in the art, the present disclosure, and the documents cited herein. And the time for sequential administration can be ascertained without using undue experimentation. 5.9. Method of Use

[0229] The subject matter of the present disclosure provides methods for administering the cells, compositions, or nucleic acid compositions of the present disclosure to a subject, for example, for treatment or therapy. Non-limiting examples of treatment or therapy include immunotherapy (e.g., adoptive cell transfer), cell therapy (or cellular therapy), stem cell transplantation, organ transplantation, gene therapy (e.g., CRISPR gene editing therapy), viral infusion (e.g., AAV), nucleic acids, free nucleic acids or more stabilized analogs, nanoparticles containing mRNA or stabilized mRNA, or organs or tissues containing engineered cells of the subject. The cells, compositions, and nucleic acid compositions of the present disclosure can be used in treatment, therapy, or medicine. In certain embodiments, increased resistance to the host humoral response, extended persistence of the cells, and / or attenuated immunogenicity of the foreign cells are desirable for treatment or therapy.

[0230] The subject matter of the present disclosure provides methods for treating and / or preventing neoplasms in a subject. The cells, compositions, and nucleic acid compositions of the present disclosure can be used to treat and / or prevent neoplasms in a subject. The cells, compositions, and nucleic acid compositions of the present disclosure can be used to extend the survival time of a subject suffering from a neoplasm.

[0231] The subject matter of the present disclosure provides methods for treating and / or preventing pathogen infections or other infectious diseases in a subject, such as an immunocompromised human subject. The cells, compositions, and nucleic acid compositions of the present disclosure can also be used to treat and / or prevent pathogen infections or other infectious diseases in a subject, such as an immunocompromised human subject. Such methods include administering, in an effective amount, a cell of the present disclosure, a composition of the present disclosure (e.g., a pharmaceutical composition), or a nucleic acid composition of the present disclosure to achieve a desired effect, even if it is alleviation of an existing condition or prevention of recurrence.

[0232] The subject matter of the present disclosure provides methods for treating and / or preventing autoimmune diseases in a subject. The cells, compositions, and nucleic acid compositions of the present disclosure can also be used to treat and / or prevent autoimmune diseases in a subject. Such methods include administering, in an effective amount, a cell of the present disclosure, a composition of the present disclosure (e.g., a pharmaceutical composition), or a nucleic acid composition of the present disclosure to a subject having an autoimmune disease.

[0233] The subject matter of the present disclosure provides a method for reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject who has received an organ transplant. The cells, compositions, and nucleic acid compositions of the present disclosure can also be used to reduce and / or prevent antibody-mediated rejection of cells and / or tissues in a subject who has received an organ transplant. Such methods include administering, in an effective amount, a cell of the present disclosure, a composition of the present disclosure (e.g., a pharmaceutical composition), or a nucleic acid composition of the present disclosure to a subject who has received an organ transplant.

[0234] The subject matter of the present disclosure provides a method for reducing and / or preventing antibody-mediated rejection of autologous or allogeneic cells and / or tissues in a subject, wherein the subject undergoes cell therapy. The cells, compositions, and nucleic acid compositions of the present disclosure can also be used to reduce and / or prevent antibody-mediated rejection of cells and / or tissues, wherein the subject undergoes cell therapy. Such methods include administering, in an effective amount, a cell of the present disclosure, a composition of the present disclosure (e.g., a pharmaceutical composition), or a nucleic acid composition of the present disclosure to a subject undergoing cell therapy.

[0235] In the treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be given in a single or series of administrations. The effective amount can be given by bolus or by continuous perfusion.

[0236] An "effective amount" (i.e., a "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result upon treatment. The effective amount can be administered to the subject in one or more doses. For a treatment, the effective amount is an amount sufficient to reduce, alleviate, stabilize, reverse, or delay the progression of the disease or, alternatively, to reduce the pathological consequences of the disease. The effective amount is generally determined by a physician in each individual case and is within the skill of the art. When determining an appropriate dosage to achieve an effective amount, typically several factors are considered. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells being administered.

[0237] In adoptive immunotherapy using T cells specific for an antigen, cell doses in the range of about 10 6 ~10 10 (e.g., about 10 9 ) are typically infused. Upon administration of the cells of the present disclosure to a host and subsequent differentiation, T cells that are specifically directed against a specific antigen are induced. neoplasm

[0238] The subject matter of the present disclosure provides a method for treating and / or preventing neoplasms in a subject. The method can include administering to a subject having a neoplasm an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure.

[0239] Non-limiting examples of neoplasms include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, pharyngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various cancers (including prostate cancer and small cell lung cancer). Suitable cancers further include any known in the field of oncology and are not limited to the following: astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, anaplastic glioma, epithelioma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell lung adenocarcinoma and large cell lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma, and their liver metastases, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland cancer, papillary carcinoma, sebaceous adenocarcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchiogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, anaplastic glioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease, breast tumors (e.g., ductal and lobular adenocarcinoma), squamous cell carcinoma and adenocarcinoma of the cervix, uterine epithelial cancer and ovarian epithelial cancer, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B cell lymphoma and T cell lymphoma (nodular and diffuse) plasmacytoma, acute leukemia and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the neoplasm is selected from blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and pharyngeal cancer. In certain embodiments, the cells, compositions, nucleic acid compositions of the present disclosure can be used to treat and / or prevent blood cancers (e.g., leukemia, lymphoma, and myeloma) or ovarian cancer that are not amenable to conventional therapeutic interventions.In certain embodiments, the cells, compositions, and nucleic acid compositions of the present disclosure can be used to treat and / or prevent solid tumors. In certain embodiments, the cells, compositions, and nucleic acid compositions of the present disclosure can be used to treat and / or prevent neoplasms selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, breast cancer, ovarian cancer, mesothelioma, glioblastoma, colorectal cancer, and pancreatic cancer. Autoimmune diseases

[0240] The subject matter of the present disclosure provides methods for treating and / or preventing autoimmune diseases in a subject. The method can include administering to a subject having an autoimmune disease an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure. Non-limiting examples of autoimmune diseases include rheumatoid arthritis, myasthenia gravis, systemic lupus, Graves' disease, Hashimoto's thyroiditis, systemic sclerosis, biliary cirrhosis, celiac disease, axonal neuropathy, inflammatory myopathy, cerebellar degeneration, type 1 diabetes, and polymyositis.

[0241] In the United States, there are over 20 million patients with autoimmune disorders. Many of these, such as lupus and myasthenia gravis, involve an attack by the patient's own antibodies on tissue components, DNA, and cells. https: / / www.google.com / search?client=firefox-b-1-d&q=incidence+outimmune+disease. There are few effective or curative approaches to these diseases. IgG plays an important protective role in the human immune system but is also associated with the etiology of diseases such as rheumatoid arthritis, myasthenia gravis, and systemic lupus, where the removal of IgG is used as a therapeutic means to treat these autoimmune diseases (Johansson et al., PLoS ONE (2008);3:1-6;Berta et al., The International Journal of Artificial Organs (1994); 17:603-608; Stummvoll et al., Annals of the Rheumatic Diseases (2005); 64:1015-1021). The IgG-degrading enzyme contained in the cells of the present disclosure depletes functional IgG that attacks host cells, thereby being able to treat autoimmune diseases. Antibody-mediated rejection

[0242] The subject matter of the present disclosure provides a method for reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject. In certain embodiments, the subject receives an organ transplant. In certain embodiments, the transplant is an allogeneic transplant (allograft). In certain embodiments, the subject is administered a cell, composition, or nucleic acid composition of the present disclosure prior to an organ transplant. In certain embodiments, the subject receives cell therapy, e.g., cells and / or tissues (e.g., autologous or allogeneic cells and / or tissues) are used in the cell therapy.

[0243] The method may include administering to the subject an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure.

[0244] Solid organ transplants, such as those related to the kidney, liver, lung, heart, and other organs, are used in the United States in more than 36,000 patients per year, and more than 100,000 people are waiting for a transplant. https: / / www.organdonor.gov / statistics-stories / statistics.html. These organs are matched to patients as well as possible, but immunosuppression in patients with severe and sometimes fatal outcomes is frequent and lifelong. The cost in the United States is $100 billion. Host IgG plays an important role in allotransplantation, where antibody-mediated rejection of the allograft is caused by HLA donor incompatibility (Loupy et al., New England Journal of Medicine (2018);379:1150-1160). IdeS has been evaluated in humans with respect to desensitization prior to allotransplantation. In this study, 24 out of 25 patients were able to receive HLA-incompatible transplants after treatment with IdeS, which rapidly removed all donor-specific antibodies (Jordan et al., New England Journal of Medicine (2017);377:442-453;Lonze et al., Annals of Surgery (2018);268:488-496). The IgG-degrading enzyme contained in the cells of the present disclosure can deplete functional IgG (e.g., host IgG), attack donor organ cells, thereby reducing and / or preventing antibody-mediated rejection associated with organ transplantation.

[0245] The subject may have an advanced form of the disease, in which case the purpose of the treatment may include reducing or reversing disease progression and / or alleviating side effects. The subject may have a medical history of a previously treated condition, in which case the treatment objective typically includes reducing or delaying the risk of recurrence.

[0246] Human subjects suitable for treatment typically include two treatment groups that can be identified by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are subjects with clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or x-ray; their own positive biochemical or histopathological markers are insufficient to identify this population). The pharmaceutical composition is administered to these subjects to induce an anti-tumor response for the purpose of alleviating these conditions. Ideally, a reduction in tumor mass results, but any clinical improvement constitutes a benefit. Clinical improvements include a decrease in the risk or rate of progression, or a reduction in the pathological outcome of the tumor.

[0247] Suitable subjects for the second group are known in the art as the "adjuvant group". These are individuals who have a history of neoplasm but were responsive to another treatment modality. Previous treatments may include, but are not limited to, surgical resection, radiation therapy, and traditional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected of having a risk of disease progression, either near the original tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made based on features observed before and after the initial treatment. These features are known in the clinical art and are preferably defined for different neoplasms. Typical features of the high-risk subgroup are those in which the tumor invades adjacent tissues or shows lymph node involvement.

[0248] Another group has a genetic predisposition to neoplasm but has not yet manifested clinical signs of neoplasm. For example, women who are positive in a test for a gene mutation associated with breast cancer and are still of childbearing age may wish to receive one or more of the cells described in the present invention in a prophylactic treatment to prevent the appearance of neoplasm until they are suitable for prophylactic surgery.

[0249] As a result of the expression of an IgG-degrading enzyme that enhances a ligand recognition receptor (e.g., an antigen recognition receptor that binds to a tumor antigen) and the activity of a cell (e.g., antitumor activity), the adoptively transferred cells are given an increased selective cytolytic activity at the tumor site. Further, subsequent to their localization and their proliferation against a tumor or a viral infection, the cells (e.g., T cells) convert the tumor or viral infection site into a highly conductive environment for a wide range of immune cells (tumor infiltrating lymphocytes, NK cells, NKT cells, dendritic cells, and macrophages) involved in a physiological antitumor or antiviral response.

[0250] Furthermore, the subject matter of the present disclosure provides a method for treating and / or preventing a pathogen infection (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoan infection) in a subject, e.g., a subject in an immunocompromised state. The method can include administering to a subject having a pathogen infection an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure. Exemplary viral infections susceptible to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

[0251] Further modifications can be introduced into the cells of the present disclosure (e.g., T cells) to avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as the risk of graft-versus-host disease (GvHD), or the risk of a similar outcome to GvHD when healthy tissue expresses the same target antigen as tumor cells. A potential solution to this problem is to engineer a suicide gene into the immune-responsive cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is the EGFRt polypeptide. The EGFRt polypeptide may enable T cell elimination by administration of an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt may be covalently conjugated upstream of the antigen recognition receptor of the CAR of the present disclosure. The suicide gene may be included within a vector containing the nucleic acid encoding the CAR of the present disclosure. In this method, administration of a prodrug (e.g., AP1903, which can activate iCasp-9) designed to activate the suicide gene during malignant T cell transformation (e.g., GVHD) induces apoptosis in suicide gene-activated CAR-expressing T cells. Incorporation of the suicide gene into the CAR of the present disclosure provides an additional level of safety along with the ability to eliminate the majority of CAR T cells within a very short period of time. The cells of the present disclosure (e.g., T cells) incorporating the suicide gene can be preemptively eliminated at a given time point after CAR T cell infusion or eradicated at the time of the earliest signs of toxicity. 5.10. Kits

[0252] The subject matter of the present disclosure provides kits for treating and / or preventing neoplasms, or pathogen infections, or autoimmune diseases in a subject, as well as kits for reducing and / or preventing antibody-mediated rejection in a subject who undergoes an organ transplant. In certain embodiments, the kit comprises an effective amount of a cell of the present disclosure, a composition of the present disclosure, or a nucleic acid composition of the present disclosure. In certain embodiments, the kit comprises a sterile container; such container can be in the form of a box, an ampule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container forms known in the art. Such container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding a medicament. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding an antigen recognition receptor (e.g., CAR or TCR) for an antigen of interest and an isolated nucleic acid molecule encoding an IL-36 polypeptide in an expressible (and secretable) form (these may be contained in the same or different vectors as needed).

[0253] If desired, the cell, composition, or nucleic acid composition is provided with instructions for administering the cell, composition, or nucleic acid composition to a subject who has or is at risk of having or developing a neoplasm, a pathogen infection, or an autoimmune disease, or a subject who undergoes an organ transplant. The instructions generally include information about the use of the cell, composition, or nucleic acid composition for the treatment or prevention of a neoplasm or a pathogen infection, or an autoimmune disease. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosing schedule and administration for the treatment or prevention of a neoplasm, a pathogen infection, or an immune disorder or its symptoms; cautions for use; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or inside the container, or as a separate sheet, pamphlet, card, or folder supplied with the container.

Examples

[0254] 6. Examples In the practice of the present disclosure, unless otherwise specified, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art, are used. Such techniques are fully described in the literature such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and can thus be considered in making and practicing the subject matter of the present disclosure. Particularly useful techniques for specific embodiments are discussed in the following sections.

[0255] The following examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the cells and compositions of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention. (Example 1) Generation and in vitro activity Overview

[0256] CAR T cells expressing two forms of IdeS were generated, namely, a membrane-bound form (IdeS expressed as a surface membrane-bound form) and a secreted form (IdeS secreted from the cells). As shown below, in both forms, IdeS successfully cleaved IgG in vitro. Results Generation of constructs and stable lines

[0257] CAR T cell therapy targeting CD19, an antigen of B cell tumors, has recently been approved by the FDA (Zheng et al., Drug Discovery Today (2018);23:1175-1182). CD19 is specific to B cells and is not normally expressed on other cells or tissues, making it an ideal antigen. CD19 CAR with a 4-1BB / CD3ζ signaling domain has been well characterized in vitro and in vivo in patients (Brentjens et al., Sci Transl Med. (2013);5:177ra38; Pegram et al., Blood (2012);119:4133-4141; Kalos et al., Science Translational Medicine (2011);3:1-11).

[0258] As shown in Figure 1, constructs of the membrane-bound form (referred to as "IdeS-tm"; see Figure 1A) and the secreted form (referred to as "IdeS-sec"; see Figure 1B) were generated. T cells were engineered to express a CD19-targeting CAR containing an intracellular signaling domain comprising a 4-1BB polypeptide and a CD3ζ polypeptide, and a transmembrane domain comprising a CD8 polypeptide. The CAR is designated as "19BBz".

[0259] The design of the construct was based on methods previously reported prior to using the SFG gamma-retroviral vector (Riviere et al., Proceedings of the National Academy of Sciences of the United States of America (1995);92:6733-7). The CD8 signal peptide sequence was used to transport IdeS to the cell membrane (see FIGS. 1A and 1B). To generate a membrane-bound version of the enzyme, the transmembrane domain of CD8 was incorporated at the C-terminus of the enzyme. Subsequently, the CAR construct was added after the self-cleaving peptide 2A. As shown in FIGS. 1A and 1B, the CAR construct contains the CD8 signal peptide sequence, an antigen-specific scFv (in this case, anti-CD19), the CD8 transmembrane domain, and the 4-1BB / CD3ζ intracellular signaling domain. The process of generating T cells expressing CAR has been previously described (Brentjens et al., Sci Transl Med. (2013);5:177ra38;Parente-Pereira et al., Journal of Biological Methods (2014);1:7). The construct was used to transfect the H29 retroviral packaging cell line. Viruses derived from H29 cells were used to generate a stable PG13 retroviral packaging cell line. PG13 cells produce gibbon ape leukemia virus (GALV) particles, which were used to transduce either cell lines or primary cells (Parente-Pereira et al., Journal of Biological Methods (2014);1:7). Peripheral blood mononuclear cells (PBMCs) were transduced to generate CAR T cells. Additionally, a Galv9-producing cell line was used to produce the virus. Expression and activity in vitro

[0260] The stable expression of IdeS was tested using immunoblotting in a stable T cell line of a model such as Jurkat cells. Since the C-terminal HA tag was included in the IdeS construct, the cell lysates and supernatants of this cell line were tested using anti-HA antibody. As shown in Figure 2, the expression of IdeS can be successfully adapted to mammalian cells by transient transfection of HEK293t cells. The function of CAR T cells was evaluated with respect to IdeS activity and also with respect to CAR activity.

[0261] The enzymatic activity of IdeS was tested by evaluating the degree of IgG cleavage. SDS-PAGE assay and ELISA-based assay were used. Figures 3A - 3C show the results of the SDS-PAGE assay. In the SDS-PAGE assay, HEK 293t cells were transiently transfected with IdeS-tm. 48 hours after transfection, IgG was added to the wells of a 24-well plate and then removed at various time points and quenched using Laemmli buffer. See Figure 3A. The cleavage of human polyclonal IgG was followed over time and detected using the SDS-PAGE assay, which was visualized with both Coomassie and immunoblotting (Figures 3B and 3C). As shown in Figures 3B and 3C, IdeS expressed from HEK293t cells was active in vitro.

[0262] Figures 4A and 4B show the results of the ELISA-based assay. Cleavage of IgG by IdeS was detected using a modified ELISA-based assay (Jaernum et al., Molecular Cancer Therapeutics (2017);16:1887-1897). Cleavage ELISA was validated using recombinant IdeS (see Figure 4A). HEK293t cells were transfected with the secreted version of IdeS (“IdeS-sec”). Expression of the enzyme was verified by testing the supernatant by immunoblot using anti-HA (see Figure 4B). By applying cleavage ELISA, it was confirmed that IdeS-sec efficiently cleaved human polyclonal IgG at various time points (see Figure 4B).

[0263] To evaluate whether IdeS could successfully cleave antibodies bound to the cell surface, a co-culture experiment was designed. In the co-culture experiment, IdeS-expressing cells were incubated with Raji cells expressing CD20 and the antibody rituximab that binds to Raji cells. However, IdeS cleaved the antibody and released the Fc fragment. As shown in Figure 5, HEK293t cells secreting IdeS successfully cleaved the Fc fragment of rituximab bound to Raji cells.

[0264] Specific lysis by CAR T cells was tested by examining activity against target cells expressing luciferase as previously disclosed in Dao et al. Science Translational Medicine (2013);5:1-11. CD19 +The Raji cell line was used as a tumor model, and specific lysis of Raji cells was measured. Raji cells were modified to express firefly luciferase, enabling a luciferase-based killing assay. As shown in Figure 6, both IdeS-tm 19BBz cells and IdeS-sec 19BBz cells killed Raji cells in vitro to a similar extent as 19BB cells without IdeS. Thus, addition of IdeS to CAR T cells does not impair the killing activity of CAR T cells.

[0265] Furthermore, it was investigated whether IdeS-expressing cells could be protected from complement-dependent cytotoxicity (CDC). Untransduced T cells, IdeS-tm 19BBz T cells, IdeS-sec 19BBz T cells, and 19BBz T cells without IdeS were treated with various concentrations of rabbit anti-thymocyte globulin (ATG), then rabbit serum was added and incubated for 1 hour. Cell viability was measured by Cell Titer Glo. As shown in Figure 7, both IdeS-tm 19BBz T cells and IdeS-sec 19BBz T cells cleaved and removed the Fc fragment of IgG, thus avoiding CDC.

[0266] Furthermore, it was investigated whether IdeS-expressing cells could be protected from antibody-dependent cell-mediated cytotoxicity (ADCC). IdeS-tm 19BBz T cells, IdeS-sec 19BBz T cells, and 19BBz T cells without IdeS were treated with various doses of anti-thymocyte globulin (ATG), and then treated with human PBMCs. Cytotoxicity was 51 determined using a 51Cr release assay. As shown in Figure 10, both IdeS-tm 19BBz T cells and IdeS-sec 19BBz T cells were protected from lysis compared to 19BBz T cells without IdeS.

[0267] Furthermore, the inventors investigated whether IdeS-expressing cells could cleave IgG in the sera from kidney transplant patients and protect against complement-dependent cytotoxicity (CDC). As shown in Figure 11A, flow cytometry indicated that sera from a kidney transplant patient (Patient 2) containing rejection-causing anti-HLA antibodies bound to A02+ cells. As shown in Figure 11B, sera from Patient 2 were cleaved by A02+ IdeS-tm 19BBz T cells and IdeS-sec 19BBz T cells, as verified by flow cytometry. As shown in Figure 11C, A02+ IdeS-tm 19BBz T cells and IdeS-sec 19BBz T cells were also protected from complement killing (CDC) mediated by the sera of Patient 2 (right). Mechanism of action

[0268] Next, the inventors investigated how IdeS-expressing cells shield themselves from potential antibodies. Figure 8 shows one proposed mechanism of action. As shown in Figure 8, IgG antibodies bind to cell surface antigens and receptors, leading to cell death by CDC, ADCC, and opsonization. IdeS cleaves IgG below the hinge region, releasing the Fc fragment. IdeS-expressing cells remain coated with F(ab’)2 fragments, preventing further antibody binding.

[0269] It is important to understand the cleavage mechanism of the secreted enzyme with respect to the membrane-bound enzyme. The membrane-bound form allows for more local activity; however, the data shown in Figure 5 suggest that the secreted form is more effective at cleaving antibodies in the trans configuration. The mechanism of cis or trans cleavage by IdeS-expressing cells is also evaluated. For this purpose, rabbit anti-mouse antibodies are used that target the murine portion of the scFv. It is evaluated whether the membrane-bound version or the secreted version of IdeS is more advantageous.

[0270] An important aspect of the mechanism of this enzyme is that since it cleaves IgG below the hinge region, the F(ab’)2 fragment remains intact and can thus remain bound to the cell surface. Using recombinant IdeS, it was observed that increasing the concentration of IdeS removed the Fc fragment from the cell surface, while the F(ab’)2 fragment remained bound to the surface. As shown in Figure 9, CAR T cells expressing IdeS cleaved the IgG Fc and maintained the F(ab’)2 shield. After IgG(ATG) was added to IdeS-expressing cells (either transmembrane or secreted form), the IgG remained bound to the cells as Fab form and the Fc was released. The lack of Fc eliminated the function, but the bound Fab prevented new cytotoxic IgG from binding and thus functioned as a shield. This did not occur for cells without IdeS.

[0271] This suggests that the remaining F(ab’)2 fragments create a shield around the cells, hiding them from potential future humoral responses. This hypothesis is tested by using membrane-bound IdeS and cell-secreted IdeS in the stable system described above. Anti-Fab specific antibodies or anti-Fc specific antibodies are used to detect which part of the IgG remains bound to the cells. The kinetics of this process are also examined to understand how long the F(ab’)2 fragments remain bound after cleavage and whether they are removed by competition with intact IgG. Conclusion

[0272] The data of the inventors shown in this example demonstrate that CAR T cells expressing IdeS were successfully expressed in mammalian cells and showed effective cleavage activity in vitro. Furthermore, even when IdeS was included in CAR T cells, the in vitro killing activity of the CAR was not impaired. Additionally, CAR-T cells expressing IdeS were protected against complement-dependent cytotoxicity (CDC). (Example 2) Activity in vivo Overview

[0273] In this example, the in vivo activity of CAR T cells expressing IdeS of Example 1 is investigated. The IgG cleavage activity and cytotoxic activity of these cells are investigated in an in vivo mouse model. These cells are tested in a system in which they are targeted by an antibody, reproducing a model of humoral immunogenicity against CAR T cells. In this model, the persistence of the cells is tested along with the tumor cell killing efficiency against normal CAR T cells. One important concern in these experimental designs is that IdeS does not cleave mouse IgG. Thus, NSG mice, a highly immunodeficient mouse model compatible with additional heterologous antibodies (e.g., rabbit or human on which IdeS acts, and potentially engrafted immune effector cells), are used. Cell persistence in vivo

[0274] The persistence in vivo of CAR T cells expressing IdeS is tested and compared with CAR T cells without IdeS. NSG mice are injected intraperitoneally (IP) with CAR T cells expressing IdeS or CAR T cells without IdeS. Then, the mice are injected IP with a human antibody targeting the CAR T cells, such as an anti-CD3, anti-MHC class I antibody, or an antibody targeting the CAR. Since IdeS completely cleaves all isotypes of rabbit IgG and human IgG, antibodies derived from rabbit or human are used (Johansson et al., PLoS ONE (2008);3:1-6; Yang et al., Nephrology Dialysis Transplantation (2010);25:2479-2486; Wang et al., Experimental Neurology (2017);291:134-140). IdeS-expressing cells are expected to cleave the antibody below the hinge and thus release the Fc fragment. Ascites and peripheral blood collected from the mice are analyzed at various time points by immunoblot or ELISA previously shown (Rafiq et al., Nature Biotechnology (2018);36:847-858) to evaluate IgG cleavage (shown in Figures 3 and 4). The presence of CAR T cells is determined by collecting ascites and analyzing by flow cytometry using the anti-CD19 CAR and anti-HA tag present in the IdeS construct. To investigate whether the Fab fragment remains bound to the cell surface (as a shield against further binding of functional IgG), residual binding is analyzed by flow cytometry using an anti-Fab specific antibody. The presence of remaining available CAR T cell surface targets (i.e., CD3 or MHC I) is also evaluated. Similar experiments are re conducted by intravenous (IV) injection of the cells and antibodies. In vivo efficacy

[0275] CD19 +The Raji cell line is used as a tumor model to measure specific lysis of Raji cells. Raji cells are modified to express firefly luciferase, enabling luciferase-based killing assays and in vivo bioluminescence imaging of tumors (Koneru et al., Oncoimmunology (2015);4:e994446). Raji cells are injected IP into NSG mice, followed by injection of CAR-T cells expressing IdeS or CAR T cells without IdeS, and an antibody targeting CAR T cells that do not bind to Raji cells (e.g., anti-CD3 or anti-mouse antibody) is injected to target the CAR. Tumor growth and disease progression are evaluated using bioluminescence imaging. The persistence of CAR T cells is also evaluated in this system using flow cytometry or by imaging of CAR T cells carrying alternative luminescent probes. CAR T cells engineered to express Gaussia luciferase can be used orthogonally to firefly luciferase (Santos et al., Nature Medicine (2009);15:338-344). CAR T cells carrying luminescent probes enable tracking and monitoring of CAR T cell persistence over time, while tumor tracking is also possible. Similar experiments are performed by IV injection of cells and antibodies. (Example 3)

[0276] Human T cells were transduced with 19BBz without IdeS CAR (lanes 1-2 from the left) or IdeS-tm 19BBz CAR (transmembrane form) (lanes 3-5 from the left) and IdeS-sec 19BBz CAR (secreted form) (lanes 6-8 from the left). 2×10 6Individual CAR T cells were injected i.p. into NSG mice, and 24 hours later, human polyclonal IgG was also injected i.p. Cleavage of IgG was evaluated by performing i.p. washes using PBS, purifying the samples using magnetic protein G beads, and analyzing by Western blot using an anti-human Fc-specific HRP secondary antibody. As shown in Figure 12, the uncleaved heavy chain was observed around 55 kDa (lane 9), while the cleaved Fc fragment was present around 25 kDa (arrow). Embodiments of the subject matter of the present disclosure

[0277] From the foregoing, it will be apparent that changes and modifications may be made to the subject matter of the present disclosure in order to adopt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

[0278] As used herein, a recitation of elements in any definition of a variable includes that variable's definition as any single element or combination of recited elements (or sub-combination). As used herein, a recitation of an embodiment includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0279] All patents and publications referred to in this specification are hereby incorporated by reference as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. In certain embodiments, for example, the following items are provided. (Item 1) (a) A ligand recognition receptor, and (b) An IgG degrading enzyme or a fragment thereof A cell comprising. (Item 2) The cell according to item 1, wherein the IgG degrading enzyme is secreted. (Item 3) The cell according to item 1, wherein the IgG degrading enzyme is membrane-bound. (Item 4) (c) The cell according to item 3, further comprising a transmembrane domain attached to the IgG-degrading enzyme. (Item 5) The cell according to item 4, wherein the transmembrane domain is attached to the C-terminus of the IgG-degrading enzyme. (Item 6) The cell according to item 4 or 5, wherein the transmembrane domain attached to the IgG-degrading enzyme comprises a CD8 polypeptide and, optionally, the transmembrane portion of CD8. (Item 7) The cell according to any one of items 1 to 6, wherein the IgG-degrading enzyme is selected from the IgG-degrading enzyme of S. pyogenes (IdeS), the IgG-degrading enzyme of S. equi subsp. zooepidemicus (IdeZ), the IgG-degrading enzyme of S. equi subsp. equi (IdeE), the endoglycosidase (EndoS) derived from Streptococcus pyogenes, and the streptococcal cysteine protease (SpeB) derived from Streptococcus pyogenes. (Item 8) The cell according to any one of items 1 to 7, wherein the ligand recognition receptor is exogenous or endogenous. (Item 9) The cell according to any one of items 1 to 8, wherein the ligand recognition receptor is recombinantly expressed. (Item 10) The cell according to any one of items 1 to 9, wherein the ligand recognition receptor is expressed from a vector. (Item 11) The cell according to any one of items 1 to 10, wherein the IgG-degrading enzyme is expressed from a vector. (Item 12) The cell according to any one of items 1 to 11, which is a responsive cell or an activatable cell. (Item 13) The cell according to any one of items 1 to 12, which is an immunoreactive cell. (Item 14) The cell according to any one of items 1 to 13, selected from the group consisting of T cells, natural killer (NK) cells, B cells, macrophages, monocytes, dendritic cells, stem cells, and normal tissue cells. (Item 15) The cell according to any one of items 1 to 14, which is a T cell. (Item 16) The cell according to any one of items 1 to 15, wherein the ligand recognition receptor binds to an antigen. (Item 17) The cell according to item 16, wherein the antigen is selected from tumor antigens, pathogen antigens, normal cell antigens, HLA antigens, and allogeneic antigens. (Item 18) The cell according to item 16 or 17, wherein the antigen is a tumor antigen. (Item 19) The cell according to item 17 or 18, wherein the tumor antigen is CD19. (Item 20) The cell according to item 16 or 17, wherein the antigen is a normal cell antigen. (Item 21) The cell according to item 16 or 17, wherein the antigen is an HLA antigen or an allogeneic antigen. (Item 22) The cell according to item 21, wherein the allogeneic antigen is a minor histocompatibility allogeneic antigen. (Item 23) The cell according to any one of items 1 to 22, wherein the ligand recognition receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). (Item 24) The cell according to any one of items 1 to 23, wherein the ligand recognition receptor is a CAR. (Item 25) The cell according to item 24, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. (Item 26) The cell according to item 25, wherein the extracellular antigen-binding domain of the CAR comprises a single-chain variable fragment (scFv). (Item 27) The cell according to item 25 or 26, wherein the transmembrane domain comprises a CD8 polypeptide . (Item 28) The cell according to any one of items 25 to 27, wherein the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide (Item 29) The cell according to any one of items 25 to 28, wherein the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling domain (Item 30) The cell according to item 29, wherein the at least one co-stimulatory domain comprises a CD28 polypeptide, a 4-1BB polypeptide, or a combination thereof (Item 31) The cell according to item 29 or 30, wherein the at least one co-stimulatory domain comprises a 4-1BB polypeptide (Item 32) The IgG-degrading enzyme (a) cleaves IgG, thereby preventing the IgG antibody from killing the cell and / or (b) cleaves IgG, thereby retaining the binding of the remaining fragment of the IgG to the cell, protecting the cell from one or more cytotoxic antibodies, and optionally, the one or more cytotoxic antibodies bind to the same epitope region as the IgG and kill the cell. The cell according to any one of items 1 to 31 (Item 33) A composition comprising the cell according to any one of items 1 to 32 (Item 34) The composition according to item 33, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. (Item 35) The composition according to item 33 or 34, which is for treating a neoplasm (Item 36) A method for generating cells, comprising the step of introducing into a cell (a) a first polynucleotide encoding a ligand recognition receptor, and (b) a second polynucleotide encoding an IgG degrading enzyme or a fragment thereof, wherein each of the first and second nucleic acid sequences is operably linked to a promoter element as required. (Item 37) The method according to item 36, wherein one or both of the first and second polynucleotides are contained in a vector. (Item 38) The method according to item 37, wherein the vector is a retroviral vector or a lentiviral vector, or is encoded in an mRNA molecule. (Item 39) A nucleic acid composition comprising (a) a first polynucleotide encoding a ligand recognition receptor and (b) a second polynucleotide encoding an IgG degrading enzyme or a fragment thereof. (Item 40) The nucleic acid composition according to item 39, wherein the first polynucleotide is operably linked to a promoter element. (Item 41) The nucleic acid composition according to item 39 or 40, wherein the second polynucleotide is operably linked to a promoter element. (Item 42) The nucleic acid composition according to any one of items 39 to 41, wherein one or both of the first and second polynucleotides are contained in a vector. (Item 43) The nucleic acid composition according to item 42, wherein the vector is a retroviral vector or a lentiviral vector, or is encoded in an mRNA molecule. (Item 44) A vector comprising the nucleic acid composition according to any one of items 39 to 43. (Item 45) A kit comprising a cell according to any one of items 1 to 32, a composition according to any one of items 33 to 35, a nucleic acid composition according to any one of items 39 to 43, or a vector according to item 44. (Item 46) The kit according to item 45, further comprising written instructions for treating and / or preventing a neoplasm, a pathogen infection, and / or an autoimmune disorder. (Item 47) A method for reducing tumor burden in a subject, the method comprising administering to the subject an effective amount of a cell according to any one of items 1 to 32, a composition according to any one of items 33 to 35, a nucleic acid composition according to any one of items 39 to 43, or a vector according to item 44. (Item 48) The method according to item 47, wherein in the subject, the number of tumor cells is reduced, the tumor size is decreased, and / or the tumor is eradicated. (Item 49) A method for treating and / or preventing a neoplasm, a pathogen infection, and / or an autoimmune disease, the method comprising administering to a subject an effective amount of a cell according to any one of items 1 to 32, a composition according to any one of items 33 to 35, a nucleic acid composition according to any one of items 39 to 43, or a vector according to item 44. (Item 50) A method for prolonging the survival time of a subject having a neoplasm, a pathogen infection, and / or an autoimmune disease, the method comprising administering to the subject an effective amount of a cell according to any one of items 1 to 32, a composition according to any one of items 33 to 35, a nucleic acid composition according to any one of items 39 to 43, or a vector according to item 44. (Item 51) The method according to any one of items 44 to 47, wherein the tumor or neoplasm is selected from acute myeloid leukemia (AML), lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma, non-Hodgkin lymphoma, Hodgkin lymphoma, breast cancer, ovarian cancer, mesothelioma, glioblastoma, colorectal cancer, and pancreatic cancer. (Item 52) The method according to item 49 or 50, wherein the autoimmune disease is selected from rheumatoid arthritis, myasthenia gravis, systemic lupus, Graves' disease, Hashimoto's thyroiditis, systemic sclerosis, biliary cirrhosis, celiac disease, axonal neuropathy, inflammatory myopathy, cerebellar degeneration, type 1 diabetes, and polymyositis. (Item 53) A method for reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject receiving an organ transplant, the method comprising administering an effective amount of the cells according to any one of items 1 to 32, the composition according to any one of items 33 to 35, the nucleic acid composition according to any one of items 39 to 43, or the vector according to item 44. (Item 54) The method according to item 53, wherein the transplant is an allogeneic transplant (allograft). (Item 55) The method according to item 53 or 54, wherein the subject is administered the cells, composition, or nucleic acid composition before the organ transplant. (Item 56) A method for reducing and / or preventing antibody-mediated rejection of cells or tissues used in a subject receiving cell therapy, the method comprising administering an effective amount of the cells according to any one of items 1 to 32, the composition according to any one of items 33 to 35, the nucleic acid composition according to any one of items 39 to 43, or the vector according to item 44. (Item 57) The method according to item 56, wherein the cells and / or tissues are included in the cell therapy. (Item 58) The method according to item 56 or 57, wherein the cells and / or tissues are autologous or allogeneic. (Item 59) The cell according to any one of Items 1 to 32 for use in treatment. (Item 60) The cell according to any one of Items 1 to 32 for use in reducing tumor burden. (Item 61) The cell according to any one of Items 1 to 32 for use in treating and / or preventing neoplasms, pathogen infections, and / or autoimmune disorders. (Item 62) The cell according to any one of Items 1 to 32 for use in extending the survival time of a subject having a neoplasm, pathogen infection, and / or autoimmune disease. (Item 63) The cell according to any one of Items 1 to 32 for use in reducing and / or preventing antibody-mediated rejection of cells and tissues in a subject receiving an organ transplant. (Item 64) The cell according to any one of Items 1 to 32 for use in reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject receiving cell therapy. (Item 65) The composition according to any one of Items 33 to 35 for use in treatment. (Item 66) The composition according to any one of Items 33 to 35 for use in reducing tumor burden. (Item 67) The composition according to any one of Items 33 to 35 for use in treating and / or preventing neoplasms, pathogen infections, and / or autoimmune diseases. (Item 68) The composition according to any one of Items 33 to 35 for use in extending the survival time of a subject having a neoplasm, pathogen infection, and / or autoimmune disease. (Item 69) The composition according to any one of Items 33 to 35 for use in reducing and / or preventing antibody-mediated rejection of cells and tissues in a subject receiving an organ transplant. (Item 70) The composition according to any one of Items 33 to 35 for use in reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject receiving cell therapy. (Item 71) The nucleic acid composition according to any one of Items 39 to 43 for use in treatment. (Item 72) The nucleic acid composition according to any one of Items 39 to 43 for use in reducing tumor burden. (Item 73) The nucleic acid composition according to any one of Items 39 to 43 for use in treating and / or preventing neoplasms, pathogen infections, and / or autoimmune diseases. (Item 74) The nucleic acid composition according to any one of Items 39 to 43 for use in prolonging the survival time of a subject having a neoplasm, pathogen infection, and / or autoimmune disease. (Item 75) The nucleic acid composition according to any one of Items 39 to 43 for use in reducing and / or preventing antibody-mediated rejection of cells and tissues in a subject receiving organ transplantation. (Item 76) The nucleic acid composition according to any one of Items 39 to 43 for use in reducing and / or preventing antibody-mediated rejection of cells and / or tissues in a subject receiving cell therapy.

Claims

【Claim 1】 The invention described in the specification.

Images