Therapeutic t cell products

Abstract

The present invention discloses immune cells modified to increase the expression or activity of SERPINB9. In addition, compositions comprising such cells and methods of using such cells and compositions are also disclosed.

Description

[0001] This application claims priority to US 63 / 330718, filed April 13, 2022, and US 63 / 446520, filed February 17, 2023, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field

[0002] The present disclosure relates to the field of molecular biology, and more particularly to cell therapy. The present disclosure also relates to medical treatment and prevention methods.

[0003] background

[0004] Adoptive cell therapy has emerged as a powerful and effective treatment for various cancers. For example, T cells expressing the CD19 chimeric antigen receptor (CAR) are highly effective against B-cell malignancies, while virus-specific T cells (VSTs) have shown great potential in treating patients with Epstein-Barr virus (EBV)-associated lymphomas and viral infections in hematopoietic stem cell transplant (HSCT) recipients.

[0005] To avoid graft-versus-host disease (GVHD) or graft rejection, most cell therapies are autologous. However, many challenges are associated with these highly personalized treatments because they are generated from cells derived from patients with cancer or genetic diseases. In addition, high costs and long "sample-to-treatment" times limit the use of autologous cell therapies.

[0006] On the other hand, off-the-shelf third-party derived cells can provide a readily available source of therapy to patients at the point of care and overcome many of the challenges associated with custom cell products. However, adverse immune responses to allogeneic third-party derived therapeutic cells limit bioavailability and therapeutic efficacy. Efforts have been made to increase the persistence of therapeutic products and reduce their allogeneic elimination, such as through the expression of allogeneic defense receptors (ADRs) that target and kill alloreactive host T cells and NK cells (1,2), genetic knockout of HLA molecules (3) and CD52 (4,5), and cell encapsulation methods to hide therapeutic agents from the host immune system (6). However, many of these approaches have adverse effects on the host, such as the elimination of alloreactive or pathogen-specific immune cells that may lead to opportunistic infections. There remains a strong need for strategies to protect therapeutic cells without compromising host immunity.

[0007] Granzyme B (GzmB) is a serine protease that is a key cytotoxic molecule used by T cells or natural killer (NK) cells to clear allogeneic cells or pathogen-infected cells (7-9). In addition, granzyme B produced after intrinsic stimulation of therapeutic cells (i.e., intrinsic granzyme B) is also associated with steady-state cell death (Bird et al., Cell Death and Differentiation (2014) 21, 876-887). GzmB cleaves its target proteins, such as the pro-apoptotic Bc-2 family member Bid or apoptotic caspase substrates (e.g., DNA-PK, PARP, and NuMA), leading to mitochondrial instability and caspase activation, respectively, thereby causing target cell apoptosis (10, 11). Wild-type SERPINB9 (also referred to herein as SB9 (WT)) inhibits GzmB by acting as a pseudosubstrate that forms a stable covalent bond with GzmB, preventing the activation of apoptosis (12). The glutamic acid at position (340E) of the SB9 reaction center loop (RCL), while crucial for its specificity for GzmB, limits its interaction with other caspases involved in Fas-mediated apoptosis (13). Substitution of the glutamic acid for aspartic acid (E340D), resulting in the variant SB9(CAS) used herein, has been shown to broaden the inhibitory effects of SB9 on GzmB- and Fas-mediated apoptosis (13). Wild-type SB9 has also been observed to be sensitive to reactive oxygen species (ROS), and conversion of C341S and C342S to produce functional SB9, thereby forming the variant SB9(ROS) used herein, which resists ROS inactivation while maintaining inhibition of GzmB (14).

[0008] Overview

[0009] In a first aspect, the present disclosure provides an immune cell comprising a modification that increases the expression or activity of SERPINB9. Specifically, the present disclosure provides an immune cell comprising a modification that increases the expression or activity of SERPINB9, the immune cell being used in a treatment or prevention method by adoptive cell transfer.

[0010] In some embodiments, the immune cell comprises an exogenous nucleic acid encoding a SERPINB9 polypeptide. In some embodiments, the exogenous nucleic acid encoding a SERPINB9 polypeptide is an expression vector or is contained in an expression vector; optionally, the expression vector is a retroviral expression vector. In some embodiments, the SERPINB9 polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a variant thereof having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, 4, 5, 6, or 7.

[0011] In some embodiments, the immune cell is an effector immune cell, optionally wherein the effector immune cell is a T cell or a natural killer (NK) cell.

[0012] In some embodiments, the immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain that binds to a cancer-associated antigen, and the cancer-associated antigen is selected from the group consisting of: CD30, CD19, CD20, CD22, B7H3, hepatocyte growth factor receptor, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA; optionally, wherein the CAR comprises an antigen binding domain that binds to CD30.

[0013] In some embodiments, the immune cell is a virus-specific T cell or an activated T cell (ATC).

[0014] In certain embodiments, the immune cell is a virus-specific T cell. In certain embodiments, the virus-specific T cell is specific for a virus selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV) or varicella-zoster virus (VZV). In certain embodiments, the virus is EBV.

[0015] The present disclosure also provides a pharmaceutical composition comprising the immune cells according to the present disclosure and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0016] The present disclosure also provides an immune cell or pharmaceutical composition according to the present disclosure for use in a medical treatment or prevention method.

[0017] The present disclosure also provides uses of the immune cells or pharmaceutical compositions of the present disclosure for preparing a medicament for use in a medical treatment or prevention method. The present disclosure also provides a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the immune cells or pharmaceutical compositions of the present disclosure.

[0018] The present disclosure also provides a method for reducing the activity of a serine protease or caspase in a cell, comprising modifying the cell to increase the expression or activity of SERPINB9.

[0019] The present disclosure also provides a method of increasing the resistance of a cell to serine protease or caspase activity, comprising modifying the cell to increase the expression or activity of SERPINB9.

[0020] The present disclosure also provides a method of increasing resistance of a cell to granzyme B-mediated cell killing, comprising modifying the cell to increase expression or activity of SERPINB9.

[0021] The present disclosure also provides a method of increasing resistance of cells to death receptor-mediated apoptosis, comprising modifying the cells to increase expression or activity of SERPINB9.

[0022] In some embodiments according to aspects of the present disclosure, modifying a cell to increase expression or activity of SERPINB9 comprises introducing a nucleic acid encoding a SERPINB9 polypeptide into the cell. In some embodiments, the nucleic acid encoding the SERPINB9 polypeptide is an expression vector or is contained in an expression vector; optionally, wherein the expression vector is a retroviral expression vector. In some embodiments, the SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a variant thereof having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, 4, 5, 6, or 7.

[0023] In some embodiments, the cell is an effector immune cell, optionally wherein the effector immune cell is a T cell or a natural killer (NK) cell.

[0024] In some embodiments, the cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain that binds to a cancer-associated antigen, and the cancer-associated antigen is selected from the group consisting of: CD30, CD19, CD20, CD22, B7H3, hepatocyte growth factor receptor, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA; optionally, wherein the CAR comprises an antigen binding domain that binds to CD30.

[0025] In certain embodiments, the immune cell is a virus-specific T cell. In certain embodiments, the virus-specific T cell is specific for a virus selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV) or varicella-zoster virus (VZV). In certain embodiments, the virus is EBV.

[0026] The present disclosure also provides an immune cell for treating or preventing cancer, wherein:

[0027] The immune cell comprises a nucleic acid encoding a CAR comprising: (i) an antigen binding domain that binds to CD30 or CD19; (ii) a transmembrane domain; and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); and

[0028] The immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0029] The present disclosure also provides the use of immune cells in preparing a medicament for treating or preventing cancer, wherein:

[0030] The immune cell comprises a nucleic acid encoding a CAR comprising: (i) an antigen binding domain that binds to CD30 or CD19; (ii) a transmembrane domain; and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); and

[0031] The immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0032] The present invention also provides a method for treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of immune cells, wherein:

[0033] The immune cell comprises a nucleic acid encoding a CAR comprising: (i) an antigen binding domain that binds to CD30 or CD19; (ii) a transmembrane domain; and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); and

[0034] The immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0035] In some embodiments, the immune cells are virus-specific T cells, optionally wherein the immune cells are Epstein-Barr virus (EBV)-specific T cells.

[0036] The present disclosure also provides immune cells for treating or preventing cancer, wherein:

[0037] The immune cells are virus-specific T cells, optionally wherein the immune cells are Epstein-Barr virus (EBV)-specific T cells; and

[0038] The immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0039] The present disclosure also includes the use of immune cells in the preparation of a medicament for treating or preventing cancer, wherein:

[0040] The immune cells are virus-specific T cells, optionally wherein the immune cells are Epstein-Barr virus (EBV)-specific T cells; and

[0041] The immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0042] The present disclosure also provides a method of treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of immune cells, wherein:

[0043] The immune cells are virus-specific T cells, optionally wherein the immune cells are Epstein-Barr virus (EBV)-specific T cells; and

[0044] The immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0045] In some embodiments, the immune cell comprises a nucleic acid encoding a CAR comprising: (i) an antigen binding domain that binds to CD30 or CD19; (ii) a transmembrane domain; and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM).

[0046] In some embodiments according to aspects of the present disclosure, the subject (ie, the subject being treated / to be treated) is allogeneic with respect to the immune cells (ie, the immune cells being administered / to be administered according to treatment / prevention).

[0047] In some embodiments according to aspects of the present disclosure, the immune cell comprises an exogenous nucleic acid encoding a SERPINB9 polypeptide. In some embodiments, the exogenous nucleic acid encoding the SERPINB9 polypeptide is an expression vector or is contained in an expression vector; alternatively, the expression vector is a retroviral expression vector. In some embodiments, the SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a variant thereof having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1, 4, 5, 6, or 7.

[0048] illustrate

[0049] The present disclosure is based on the unexpected discovery of the inventors that upregulating the expression / activity of SERPINB9 in immune cells increases their persistence / survival in the presence of allogeneic effector immune cells. This is believed to be a result of inhibiting the activity of granzyme B in such cells. Therefore, upregulating the expression / activity of SERPINB9 in cells used in methods of treating disease by adoptive cell transfer increases their persistence in a subject, particularly in the context of allogeneic transplantation.

[0050] It is shown herein that cells modified to upregulate SERPINB9 expression / activity proliferate / expand in vitro to a similar extent as equivalent cells lacking such modifications, and when such cells are effector immune cells, they exhibit similar effector activity as equivalent cells lacking modifications that upregulate SERPINB9 expression / activity. Cells modified in this manner also have comparable toxicity profiles (i.e., toward allogeneic recipient subjects) compared to equivalent cells lacking such modifications.

[0051] SERPINB9

[0052] SERPINB9 (also known as cytoplasmic antiproteinase 3 (CAP-3 / CAP3) or peptidase inhibitor 9 (PI-9) is a protein identified by UniProt P50453. Human SERPINB9 has the amino acid sequence shown in SEQ ID NO: 1. SERPINB9 is a member of the Serpin family of protease inhibitors. SERPINB9 is an intracellular inhibitor of the cytotoxic lymphocyte serine protease granzyme B.

[0053] The structure and function of SERPINB9 are reviewed in, for example, Kaiserman et al., Cell Death and Differentiation (2010) 17(4): 586-595, Bird et al., Mol Cell Biology (1998), 18(11): 6387-6398, and Bird et al., Cell Death and Differentiation (2014) 21, 876-887, all of which are incorporated herein by reference in their entirety. SERPINB9 is present in the nucleus and cytoplasm of cells and is endogenously expressed in immune privileged sites (e.g., placenta and lung), T cells, antigen presenting cells, and hematopoietic stem cells. In T cells, SERPINB9 is reported to prevent fratricide caused by misdirection of granzyme B at the immune synapse. SERPINB9 also inhibits the activity of serine proteases (e.g., granzyme B) against cells that produce the enzyme. That is, SERPINB9 protects activated effector immune cells expressing serine proteases (eg, granzyme B) from the action of the serine proteases they produce—ie, SERPINB9 protects cells from autolysis by self-derived serine proteases (eg, granzyme B).

[0054] Serine protease inhibitors present an exposed reactive center loop (RCL) to their target serine proteases, which then cleave the peptide bond between the serine protease inhibitor's two P1 and P1' residues. This cleavage triggers a conformational change in the serine protease inhibitor, irreversibly trapping the protease in a covalently bound complex. Residues surrounding P1 contribute to protease binding, and mutations in the serine protease inhibitor RCLs have been shown to abolish inhibition and / or alter target specificity.

[0055] The RCL of human SERPINB9 is formed from positions 334 to 348 of SEQ ID NO: 1, as shown in SEQ ID NO: 2. The P1 residue of human SERPINB9 is the glutamic acid residue at position 340 of SEQ ID NO: 1, and the P1′ residue of human SERPINB10 is the cysteine ​​residue at position 341 of SEQ ID NO: 1.

[0056] The T327R and E340A modifications in human SERPINB9 have been shown to abolish the ability of SERPINB9 to inhibit granzyme B in vitro (see Bird et al., Mol Cell Biol (1998), 18(11): 6387-6398). The E340A modification removes a critical glutamic acid residue at P1, and the T327R modification disrupts the mobility of the serpin loop and the conserved proximal hinge domain required for inhibitory function.

[0057] Wild-type SERPINB9 has also been reported to inhibit caspase activity (e.g., see Annand et al., Journal of Biological Chemistry (1999) 342(Part 3): 655-665). The E340D modification has been shown to reduce the inhibitory effect of SERPINB9 on granzyme B, but broaden its target specificity and increase its inhibitory activity on caspases, thereby inhibiting Fas-mediated apoptosis (see Bird et al., Mol. Cell Biol. (1998), 18(11): 6387-6398).

[0058] The C341S and C342S modifications have been shown to reduce reactive oxygen species (ROS)-mediated inactivation of SERPINB9 while retaining serine protease inhibitory activity (see Mangan et al., J. Biol. Chem. (2016) 291(7):3626-38).

[0059] In this specification, "SERPINB9" refers to SERPINB9 from any species, including isoforms, fragments, variants or homologs from any species.

[0060] "Fragment" generally refers to a portion of a reference protein. The minimum length that a fragment of SERPINB9 can have is one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids, and the maximum length that can have is one of 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids. "Isoform" generally refers to a variant of a reference protein expressed in the same species as the reference protein. "Homologue" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologues include orthologues.

[0061] A "variant" generally refers to a protein whose amino acid sequence comprises one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of a reference protein, but which retains a substantial degree of amino acid sequence identity (e.g., at least 70%) with the amino acid sequence of the reference protein.

[0062] In some embodiments, SERPINB9 is a SERPINB9 from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, or human) and / or a rodent (e.g., rat or mouse). An isoform, fragment, variant, or homolog of SERPINB9 can optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature SERPINB9 isoform from a given species (e.g., human).

[0063] In some embodiments, the SERPINB9 is human SERPINB9. In some embodiments, the SERPINB9 is cynomolgus monkey SERPINB9. In some embodiments, the SERPINB9 is mouse SERPINB9 (also known as "SPI6").

[0064] The isoform, fragment, variant or homolog may optionally be a functional isoform, fragment, variant or homolog, e.g., having the functional activity of a reference SERPINB9 (e.g., human SERPINB9) as determined by an appropriate assay for functional activity, e.g., protease inhibition (e.g., serine protease (e.g., granzyme B) and / or cysteine ​​protease (e.g., caspase) inhibition).

[0065] In some embodiments, SERPINB9 comprises, or consists of, an amino acid sequence that has at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, such as ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0066] Aspects and embodiments of the present disclosure relate to SERPINB9 variants. As referred to herein, a "SERPINB9 variant" refers to a SERPINB9 that comprises one or more (eg, one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) modifications relative to a reference SERPINB9.

[0067] "Modification" refers to the difference relative to a reference amino acid sequence. The reference amino acid sequence can be an amino acid sequence encoded by the most common nucleotide sequence of a gene encoding the protein of interest. In the embodiments herein (and in the more general technical field), "modification" may also be referred to as "substitution" or "mutation."

[0068] In a preferred embodiment, a SERPINB9 variant according to the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1 and comprises one or more (e.g., one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) modifications relative to SEQ ID NO: 1.

[0069] Modification generally includes replacing amino acid residues with non-identical " replacement " amino acid residues. The replacement amino acid residue according to the modification of the present disclosure can be a naturally occurring amino acid residue (i.e., encoded by a genetic code), which is different from the amino acid residue at the relevant position of the amino acid sequence before modification, and is selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). In some embodiments, the replacement amino acid residue of modification can be a non-natural amino acid residue, i.e., an amino acid residue except the amino acid residue described in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman et al., Methods in Enzymology, 202 (1991), 301-336.

[0070] The SERPINB9 variants according to the present disclosure may comprise modifications at specified positions in the amino acid sequence of SERPINB9.

[0071] In the present invention, when referring to positions of human wild-type SERPINB9 (i.e., the amino acid sequence of SEQ ID NO: 1), corresponding positions in human wild-type SERPINB9 homologous sequences are also considered. Positions corresponding to those positions determined in human wild-type SERPINB9 can be determined by sequence alignment, which can be performed using, for example, ClustalOmega ( J. 2005, Bioinformatics 21, 951-960) sequence alignment software.

[0072] In some embodiments, the SERPINB9 variants according to the present disclosure comprise modifications at one or more of the following positions (numbered relative to SEQ ID NO: 1): 340, 341, and 342. In some embodiments, the SERPINB9 variants comprise modifications at position 340. In some embodiments, the SERPINB9 variants comprise modifications at position 341 and / or position 342. In some embodiments, the SERPINB9 variants comprise modifications at positions 340, 341, and / or 342.

[0073] In some embodiments, the SERPINB9 variant comprises 340D. In some embodiments, the SERPINB9 variant comprises 341S. In some embodiments, the SERPINB9 variant comprises 342S.

[0074] In some embodiments, the SERPINB9 variant comprises the amino acid sequence shown in SEQ ID NO: 3, wherein the amino acid sequence is different from SEQ ID NO: 2. In some embodiments, the SERPINB9 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, wherein the amino acid sequence is different from SEQ ID NO: 1.

[0075] In some embodiments, a SERPINB9 according to the present disclosure comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, 4, 5, 6 or 7, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%. In some embodiments, a SERPINB9 according to the present disclosure comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 4, 5, 6 or 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 4, 5, 6 or 7, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%. In some embodiments, a SERPINB9 according to the present disclosure comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 4, 5 or 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 4, 5 or 7, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0076] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0077] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO:4, or the amino acid sequence has at least 70% amino acid sequence identity to SEQ ID NO:4, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0078] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO:5, or the amino acid sequence has at least 70% amino acid sequence identity to SEQ ID NO:5, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0079] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO:6, or the amino acid sequence has at least 70% amino acid sequence identity to SEQ ID NO:6, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0080] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO:7, or the amino acid sequence has at least 70% amino acid sequence identity to SEQ ID NO:7, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%.

[0081] In some embodiments, SERPINB9 (or a SERPINB9 variant) according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 1. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence different from the amino acid sequence of SEQ ID NO: 1.

[0082] In some embodiments, SERPINB9 (or a SERPINB9 variant) according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 5. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence different from the amino acid sequence of SEQ ID NO: 5.

[0083] In some embodiments, SERPINB9 (or a SERPINB9 variant) according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 6. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence different from the amino acid sequence of SEQ ID NO: 6.

[0084] In some embodiments, SERPINB9 (or a SERPINB9 variant) according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 7. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence different from the amino acid sequence of SEQ ID NO: 7.

[0085] In some embodiments, SERPINB9 (or a SERPINB9 variant) according to the present disclosure does not comprise or consist of the amino acid sequence of SEQ ID NO: 47. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence different from the amino acid sequence of SEQ ID NO: 47.

[0086] In some embodiments, one or more amino acids of the amino acid sequence (such as SERPINB9 or SERPINB9 variant) mentioned herein are replaced by another amino acid. Replacement includes replacing amino acid residues with different " replacement " amino acid residues. According to the replacement amino acid residue of the disclosure, it can be a naturally occurring amino acid residue (i.e., encoded by a genetic code), which is different from the amino acid residue at the relevant position of the equivalent amino acid sequence that is not substituted, and is selected from: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val). In some embodiments, replacement amino acid can be a non-naturally occurring amino acid residue, i.e., an amino acid residue except the amino acid residue described in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman et al., Methods in Enzymology, 202 (1991), 301-336.

[0087] In some embodiments, the substitution may be biochemically conservative. In some embodiments, rows 1 to 5 of the following table provide the amino acid to be substituted, and the substituted amino acid is another different amino acid given in the same row:

[0088] OK Shared attributes amino acids 1 hydrophobicity Met, Ala, Val, Leu, Ile, Trp, Tyr, Phe, Norleucine 2 Neutral hydrophilicity Cys,Ser,Thr,Asn,Gln 3 Acidic or negatively charged Asp,Glu 4 Alkaline or positively charged His,Lys,Arg 5 Directional influence Gly,Pro

[0089] For example, in some embodiments, where a Met residue is substituted, the replacement amino acid can be selected from Ala, Val, Leu, He, Trp, Tyr, Phe, and norleucine.

[0090] In some embodiments, the replacement amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in the substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces:

[0091]

[0092] That is, in some embodiments, a non-polar amino acid is replaced by a different non-polar amino acid. In some embodiments, a polar amino acid is replaced by a different polar amino acid. In some embodiments, a polar acidic amino acid is replaced by a different polar acidic amino acid. In some embodiments, a polar basic amino acid is replaced by a different polar basic amino acid. In some embodiments, a neutral amino acid is replaced by a different neutral amino acid. In some embodiments, a positively charged amino acid is replaced by a different positively charged amino acid. In some embodiments, a negatively charged amino acid is replaced by a different negatively charged amino acid.

[0093] In some embodiments, the substitution may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of the antigen binding molecules comprising the substitution, compared to an equivalent unsubstituted molecule.

[0094] Aspects and embodiments of the present disclosure relate to SERPINB9 polypeptides.A SERPINB9 polypeptide according to the present disclosure may be a SERPINB9 or SERPINB9 variant according to any of the embodiments described above.

[0095] The present disclosure also provides a fusion polypeptide comprising the SERPINB9 polypeptide of the present invention and another polypeptide of interest. The amino acid sequence of the SERPINB9 polypeptide and the amino acid sequence of another polypeptide of interest can be provided.

[0096] According to these aspects and embodiments, the polypeptide of interest can be a molecule for guiding the activity of immune cells for cells expressing a given target antigen. In some embodiments, the molecule for guiding the activity of immune cells for cells expressing a given target antigen can be a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0097] According to these embodiments, the SERPINB9 polypeptide and another polypeptide of interest can be connected by a connecting sequence. In some embodiments, the connecting sequence can be a cleavable connexon. That is, the connecting sequence can include an amino acid sequence that can be cleaved. For example, the connecting sequence can include a sequence that can serve as a substrate for an enzyme that can cut a peptide bond (i.e., a cleavage site). Many such cleavage sites are well known and usable to those skilled in the art of molecular biology. In some embodiments, the cleavable connexon can include a self-cleavage site. The self-cleavage site can automatically cut without enzyme treatment. The 2A cleavage site includes a typical "NPGP" motif, which is cut at the "G / P" position. The 2A cleavage site includes P2A, T2A, E2A, and F2A self-cleavage sites. The connecting sequence comprising the 2A self-cleavage site is referred to herein as the 2A connexon. Some constructs described in the experimental examples use the P2A self-cleavage site (i.e., in the P2A connexon). Some constructs described in the experimental examples use the T2A self-cleavage site (i.e., in the T2A connexon).

[0098] In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[SERPINB9 polypeptide]-[another polypeptide of interest]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[SERPINB9 polypeptide]-[cleavable linker]-[another polypeptide of interest]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[SERPINB9 polypeptide]-[cleavable linker]-[CAR]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[SERPINB9 polypeptide]-[2A linker]-[CAR]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[SERPINB9 polypeptide]-[2A linker]-[CAR]-[2A linker]-[another polypeptide of interest]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[another polypeptide of interest]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[another polypeptide of interest]-[cleavable linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[CAR]-[cleavable linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[CAR]-[2A linker]-[SERPINB9 polypeptide]-[2A linker]-[another polypeptide of interest]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[CAR]-[2A linker]-[SERPINB9 polypeptide]-[2A linker]-[another polypeptide of interest]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[CAR]-[2A linker]-[another polypeptide of interest]-[2A linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[another polypeptide of interest]-[2A linker]-[SERPINB9 polypeptide]-[2A linker]-[CAR]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure: N-terminus-[...]-[another polypeptide of interest]-[2A linker]-[CAR]-[2A linker]-[SERPINB9 polypeptide]-[...]-C-terminus.

[0099] As used herein to represent polypeptide structures, "[...]" indicates that other polypeptides / protein domains / regions of interest may optionally be present. For example, in the structure described in the previous paragraph, other polypeptides / protein domains / regions of interest may optionally be present upstream of the SERPINB9 polypeptide and before the N-terminus of the fusion polypeptide. In addition, as used herein to represent polypeptide structures, "-" indicates an optional linker sequence. For example, in the final structure described in the previous paragraph, a linker sequence may optionally be provided between the SERPINB9 polypeptide and the 2A linker.

[0100] In some embodiments, a polypeptide according to the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 43, 44, 45 or 46, such as one of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100%.

[0101] Agents for increasing SERPINB9 expression or activity

[0102] Aspects and embodiments of the present disclosure relate to agents for increasing the expression or activity of SERPINB9. As used herein, "agents for increasing the expression or activity of SERPINB9" refer to agents for increasing the expression (i.e., gene and / or protein expression) or activity of SERPINB9. Agents that upregulate SERPINB9 expression and / or activity may also be referred to as agents for increasing / enhancing the expression or activity of SERPINB9. Such agents may also be referred to as "SERPINB9 agonists" or "SERPINB9 enhancers."

[0103] As used herein, "expression" can be gene expression or protein expression. Gene expression includes transcription from DNA into RNA, and can be measured by various methods well known to those skilled in the art, such as measuring mRNA levels by quantitative real-time PCR (qRT-PCR), or by reporter gene-based methods. Similarly, protein expression can be measured by various methods well known to the art, such as by antibody-based methods, such as by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter gene-based methods.

[0104] Upregulation of SERPINB9 expression can be characterized by an increase in the level of RNA encoding SERPINB9 and / or an increase in the level of SERPINB9 protein compared to the absence of such upregulation. Similarly, upregulation of SERPINB9 activity can be characterized by an increase in the level of the relevant activity compared to the absence of such upregulation.

[0105] Therefore, agents for increasing the expression or activity of SERPINB9 include agents that increase the gene and / or protein expression level of SERPINB9, as well as agents that increase the activity level of SERPINB9. It will be understood that the "increase" mentioned in the preceding sentence refers to the expression level of SERPINB9 or the level of related SERPINB9 activity observed in the absence of the agent.

[0106] As used herein, the "activity" of SERPINB9 can refer to the inhibition of proteases. The activity of SERPINB9 can be the inhibition of serine proteases (e.g., granzyme B) and / or cysteine ​​proteases (e.g., caspase). The inhibition of serine proteases (e.g., granzyme B) can include binding to and / or inactivating the serine protease.

[0107] In some embodiments, agents for increasing the expression or activity of SERPINB9 according to the present disclosure (ie, SERPINB9 agonists) exhibit one or more of the following properties:

[0108] increasing expression of SERPINB9 (e.g., gene and / or protein expression);

[0109] Increased levels of RNA encoding SERPINB9;

[0110] increasing transcription of a nucleic acid encoding SERPINB9;

[0111] Reduced degradation of RNA encoding SERPINB9;

[0112] Increased levels of SERPINB9 protein;

[0113] reducing the degradation of SERPINB9 protein; or

[0114] Increased levels correlate with SERPINB9 activity.

[0115] It will be appreciated that a given SERPINB9 agonist can exhibit the multiple characteristics enumerated in the preceding paragraph. Suitable tests can be used to assess whether a given SERPINB9 agonist has the characteristics enumerated in the preceding paragraph. For example, these tests can be, for example, in vitro tests, alternatively cell-based tests or cell-free tests. In some embodiments, these tests can be, for example, in vivo tests, i.e., carried out in non-human animals. In some embodiments, these tests can be, for example, in vitro tests, i.e., carried out on cells / tissues / organs obtained from a subject.

[0116] When the test is a cell-based test, it can include treating cells with a given reagent to determine whether the reagent shows one or more listed characteristics. The material of a detectable entity label can be used to test for ease of detection. The test can be included in assessing the listed characteristics after treating cells with a series of given reagents (e.g., dilution series) of quantity / concentration. The cells used in such a cell-based test can express SERPINB9.

[0117] Increase the expression of SERPINB9 or the reagent of activity can use the test including detection and / or quantitative coding SERPINB9 RNA level to identify, the expression of described increase SERPINB9 or the reagent of activity can increase SERPINB9 gene expression and / or increase the RNA level of coding SERPINB9 and / or increase the nucleic acid transcription of coding SERPINB9 and / or reduce the RNA degradation of coding SERPINB9.Such test can include by RT-qPCR, Northern blot etc. quantitative coding SERPINB9 RNA, these are the technology well known to those skilled in the art.These methods can use the primer and / or probe of detection and / or quantitative coding SERPINB9 RNA.Such test can include contacting the cell of in vitro culture with the SERPINB9 agonist of hypothesis, then (such as after appropriate time period, i.e., the time period that is enough to observe the change of RNA level of coding SERPINB9) measure the RNA level of coding SERPINB9. Such an assay may further comprise comparing the levels of RNA encoding SERPINB9 in cells treated with a putative SERPINB9 agonist to the levels of RNA encoding SERPINB9 detected under control conditions, wherein cells of the same type are placed under the same conditions except that they are not treated with the putative SERPINB9 agonist or are treated with a negative control agent known not to affect the levels of RNA encoding SERPINB9.

[0118] Increased transcription of nucleic acids encoding SERPINB9 may be the result of promoting the assembly and / or activity of factors required for transcription of the DNA encoding SERPINB9. Reduced degradation of RNA encoding SERPINB9 may be the result of reduced enzymatic degradation of RNA encoding SERPINB9, such as the result of RNA interference (RNAi), and / or the result of increased stability of RNA encoding SERPINB9.

[0119] In the present invention, "contacting" a cell with a given reagent (e.g., a hypothetical SERPINB9 agonist) can include applying the reagent to the cell and / or mixing the reagent with the cell. In some embodiments, the SERPINB9 agonist is provided to the cell together with one or more other reagents to promote the introduction of the SERPINB9 agonist into the cell and / or promote the uptake of the SERPINB9 agonist by the cell. For example, in embodiments where the SERPINB9 agonist is one or more nucleic acids or is encoded by one or more nucleic acids, the cell can be contacted with the nucleic acid and an agent that promotes the introduction of the nucleic acid into the cell, for example, by transfection or transduction.

[0120] Increase the expression of SERPINB9 or the reagent of activity can be identified using a test including detection of SERPINB9 protein levels, the expression of the described increase SERPINB9 or the reagent of activity can increase SERPINB9 protein levels and / or reduce the degradation of SERPINB9 protein and / or increase the mRNA translation of encoding SERPINB9, such as using technology well known to those skilled in the art, such as methods (western blotting, ELISA, immunohistochemistry, etc.) based on antibodies or reporter genes. These methods can use antibodies specific to SERPINB9. Such a test can include contacting cells cultured in vitro with a hypothetical SERPINB9 agonist, and then (such as after an appropriate time period, i.e., a time period sufficient to observe changes in SERPINB9 protein levels) measuring SERPINB9 protein levels. Such an assay may further comprise comparing the levels of SERPINB9 protein in cells treated with a putative SERPINB9 agonist to the levels of RNA encoding SERPINB9 detected under control conditions, wherein cells of the same type are placed under the same conditions except that they are not treated with the putative SERPINB9 agonist or are treated with a negative control agent that is known not to affect SERPINB9.

[0121] An increase in SERPINB9 protein levels can be the result of, for example, increased levels of RNA encoding SERPINB9, increased post-transcriptional processing of RNA encoding SERPINB9, or decreased degradation of SERPINB9 protein.

[0122] Increase the expression of SERPINB9 or the reagent of activity can be identified using a test including detecting the level of SERPINB9 related functions, and the reagent that increases the expression of SERPINB9 or activity can increase a functional level of SERPINB9 (such as a function of SERPINB9 described above). The detection of a given functional level can include detecting and / or quantifying the related substance of this function. Such a test can include contacting cells cultured in vitro with a hypothetical SERPINB9 agonist, and then measuring the level of the related function and / or its related substance (such as after an appropriate time period, i.e., a time period sufficient to observe changes in the level of the related function and / or its related substance). Such a test can further include comparing the functional level of SERPINB9 detected under the control conditions with the functional level of SERPINB9 in the cell treated with the hypothetical SERPINB9 agonist, wherein the cells of the same type are under the same conditions, except that they are not treated with the hypothetical SERPINB9 agonist, or are treated with a negative control reagent known to not affect the related functional level of SERPINB9.

[0123] In some embodiments, a SERPINB9 agonist according to the present disclosure is capable of increasing the expression (e.g., gene and / or protein expression) of SERPINB9 / levels of RNA encoding SERPINB9 / transcriptions of nucleic acids encoding SERPINB9 / protein levels of SERPINB9 / levels of SERPINB9 activity-associated substances by more than 1-fold, for example, by ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold relative to the level observed in the absence of the SERPINB9 agonist or in the presence of the same amount of a control agent known not to have such agonist activity in a given assay.

[0124] In some embodiments, a SERPINB9 agonist according to the present disclosure is capable of reducing degradation of SERPINB9 protein / degradation of RNA encoding SERPINB9 to less than 1-fold, for example, one of ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold relative to the level observed in the absence of the SERPINB9 agonist or in the presence of the same amount of a control agent known not to have such agonist activity in a given assay.

[0125] In some embodiments, an agent that increases the expression or activity of SERPINB9 according to the present disclosure is or comprises a nucleic acid encoding SERPINB9, such as described herein.

[0126] Nucleic acids and vectors

[0127] The present disclosure provides one or more nucleic acids encoding the polypeptides of the present disclosure. For example, the present disclosure provides one or more nucleic acids encoding the SERPINB9 polypeptide. Also provided is one or more nucleic acids encoding an agent for increasing the expression or activity of SERPINB9.

[0128] In some embodiments, the nucleic acid comprises DNA and / or RNA, or is composed of DNA and / or RNA. In some embodiments, the nucleic acid can be a vector or multiple vectors, or can be contained in a vector or multiple vectors. That is, the nucleotide sequence of the nucleic acid can be contained in a vector. SERPINB9 / agents for increasing the expression or activity of SERPINB9 can be produced in the cell by transcribing from a vector encoding a peptide / polypeptide and subsequently translating the transcribed RNA.

[0129] Thus, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids according to the present disclosure. The vector can facilitate the delivery of a nucleic acid comprising / encoding a SERPINB9 polypeptide. The vector can be an expression vector comprising the elements required to express a nucleic acid comprising / encoding a SERPINB9 polypeptide.

[0130] As used herein, a "vector" is a nucleic acid molecule that serves as a means for introducing exogenous nucleic acid into a cell. The vector can be a vector for intracellular expression of a nucleic acid. Such a vector can include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0131] The term "operably linked" can include situations where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked by placing the expression of the nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence is capable of affecting the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[0132] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., retroviral vectors, such as gamma-retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, such as SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), such as those described in Maus et al., Annual Review of Immunology (2014) 32: 189-225, or Morgan and Boyerinas, Biomedicines (2016) 4: 9, both of which are incorporated herein by reference in their entirety.

[0133] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is an MLV-derived vector. In some embodiments, the vector is a SFG vector.

[0134] In some embodiments, the vector can be a eukaryotic vector, such as a vector comprising elements required for expression of nucleic acid from a vector in a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, such as one comprising a cytomegalovirus (CMV) or SV40 promoter to drive expression. In some embodiments, the vector comprises a cell or tissue specific promoter, such as an immune cell specific promoter.

[0135] In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ to which the nucleic acid is desired to be delivered. In some embodiments, the vector is selected based on the tropism of the cell type / tissue / organ to which SERPINB9 is desired to be expressed. For example, it may be desirable to deliver the nucleic acid encoding the SERPINB9 polypeptide to an immune cell (e.g., an effector immune cell, such as a T cell or NK cell).

[0136] In some embodiments, nucleic acids according to the present disclosure include modifications to incorporate one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest (e.g., an immune cell). In some embodiments, nucleic acids according to the present disclosure are linked (e.g., chemically conjugated) to one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest.

[0137] The nucleic acid modification and composition that promote nucleic acid targeting delivery to the cell type of interest and / or tissue are described in, for example, Journal of Controlled Release (2015) 203 of Lorenzer et al.: 1-15, which is incorporated herein by reference in its entirety. The portion that promotes delivery to the cell type of interest or tissue and / or being taken in by it can be selectively combined with the target cell type / tissue of interest. The portion can promote the cell membrane of the cell in the target cell type of interest and / or tissue. The portion can be combined with the molecule expressed on the cell surface of the target cell type / tissue of interest. The portion can promote the internalization (for example, by endocytosis) of the target cell type / tissue of interest to nucleic acid. In certain embodiments, the nucleic acid also encodes another polypeptide of interest, such as a molecule (such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR)), which is used to guide the activity of immune cells to cells expressing a given target antigen.

[0138] The nucleic acid may comprise a nucleotide sequence encoding a SERPINB9 polypeptide as described herein and a nucleotide sequence encoding another polypeptide of interest. The nucleotide sequence encoding the SERPINB9 polypeptide and the nucleotide sequence encoding the other polypeptide of interest may be in the same reading frame and may not contain a stop codon between these nucleotide sequences.

[0139] In some embodiments, the nucleic acid / vector according to the present disclosure is a polycistronic (e.g., bicistronic, tricistronic, etc.); that is, in some embodiments, the vector encodes an mRNA with multiple protein coding regions. In some embodiments, the vector is bicistronic. In some embodiments, the vector includes nucleic acids encoding internal ribosome entry sites (IRES). In some embodiments, the vector includes nucleic acids allowing SERPINB9 polypeptides and another polypeptide of interest (e.g., CAR) to be translated separately from the same RNA transcript.

[0140] In some embodiments, the nucleic acid encodes a fusion polypeptide as described herein, eg, a fusion polypeptide comprising a SERPINB9 polypeptide as described herein and a CAR as described herein.

[0141] The nucleic acids and vectors according to the present disclosure can be provided in purified or isolated form, i.e., separated from other nucleic acids or naturally occurring biological materials. Applications of SERPINB9 and SERPINB9 expression / activity enhancers The SERPINB9 polypeptides according to the present disclosure, nucleic acids / vectors encoding the SERPINB9 polypeptides according to the present disclosure, and agents for increasing SERPINB9s expression or activity according to the present disclosure can be used in various applications.

[0142] Example 2.3 of the present invention demonstrates that immune cells modified to upregulate SERPINB9 expression / activity are less susceptible to elimination by alloreactive immune cells than equivalent cells lacking such modifications. Increased SERPINB9 expression / activity appears to protect immune cells from the cytolytic activity of allogeneic effector immune cells, particularly granzyme B-mediated cell lysis.

[0143] The SERPINB9 polypeptide according to the present disclosure, the nucleic acid / vector encoding the SERPINB9 polypeptide according to the present disclosure, and the agent for increasing the expression or activity of SERPINB9s according to the present disclosure can be used in the following methods, and / or in methods comprising the following:

[0144] Increase the intracellular expression level of SERPINB9 (i.e., gene or protein expression);

[0145] Reduce the activity of intracellular serine proteases (such as granzyme B);

[0146] Reducing the activity of intracellular caspases (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10);

[0147] increasing the resistance of cells to (and / or decreasing the susceptibility of cells to) cell killing by serine proteases (e.g., granzyme B);

[0148] increasing the resistance of a cell to (and / or decreasing the susceptibility of a cell to) cell killing by a cell expressing a serine protease (e.g., a cell expressing granzyme B; e.g., an effector immune cell);

[0149] Reducing the rate of cell killing by cells expressing serine proteases (e.g., cells expressing granzyme B; e.g., effector immune cells);

[0150] Increase the resistance of cells to (and / or reduce the susceptibility of cells to) death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis);

[0151] Increased persistence / survival / proliferation of cells in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells);

[0152] Increased persistence / survival / proliferation of cells in allogeneic subjects;

[0153] reducing allograft rejection in a subject;

[0154] increasing the anti-cancer activity of cells in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells);

[0155] increasing the anti-cancer activity of cells in an allogeneic subject; or

[0156] Increases cell persistence / survival / proliferation under conditions of chronic antigen exposure.

[0157] "Decrease" or "increase" according to the preceding paragraph can be relative to the level of the relevant property normally exhibited by cells of that type (e.g., a decrease or increase relative to a reference value of the level of the relevant property of cells of that type), for example, in the absence of treatment with the preparation of the present disclosure. The properties identified in the preceding paragraph can be assessed using appropriate methods well known to those skilled in the art.

[0158] The gene expression level of SERPINB9 in the cell and the SERPINB9 protein level in the cell can be assessed, for example, as described above. Appropriate tests can be used to assess the activity level of a given enzyme (e.g., protease, serine protease (e.g., granzyme B), cysteine ​​protease (e.g., caspase, e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) in the cell to detect and / or quantify its activity level. Such tests can include measuring the substrate level of the relevant enzyme and / or measuring the level of enzyme activity product over time. These methods can include applying the substrate of the enzyme to the cell and detecting and / or quantifying the level of substrate level and / or enzyme-mediated substrate processing product after a given time period. Such tests can use labeled substances to detect and / or quantify substrate and / or enzyme activity product, or can use reagents for detecting and / or quantifying substrate and / or enzyme activity product. Such tests can also use colorimetric substrates or reaction products.

[0159] For example, the activity of granzyme B can be assessed using the Granzyme B Detection Kit (Cat. No. BML-AK711-0001) from Enzo Life Sciences, Inc., which uses the colorimetric substrate IEPD-pNA. Cleavage of the p-nitroaniline (pNA) group from IEPD-pNA increases the absorbance at 405 nm, thereby enabling the detection and quantification of granzyme B activity. Other suitable assays for detecting and quantifying granzyme B activity are well known in the art and include, for example, the Granzyme B Activity Detection Kit (Cat. No. MAK176) from Sigma-Aldrich. Granzyme B activity can also be assessed as described by Bird et al., Molecular and Cellular Biology (1998), 18(11): 6387-98, which is incorporated herein by reference above.

[0160] To further illustrate, the activity of caspases can be assessed, for example, using the Caspase-Glo assay kit from Promega, which uses a luminescent caspase substrate and luciferase. Cleavage of the substrate releases aminoluciferin, a substrate for luciferase. The action of luciferase on aminoluciferin results in the generation of light, enabling the detection and quantification of caspase activity.

[0161] In the present invention, "serine protease" is an enzyme that cleaves peptide bonds in polypeptides, wherein serine acts as a nucleophilic amino acid in the active site of the enzyme. "Serine protease activity" refers to the catalytic action of a serine protease on the cleavage of peptide bonds in polypeptides. For example, serine proteases are reviewed in Patel, Allergol. Immunopathol. (Madr). (2017) 45(6): 579-591, which is incorporated herein by reference in its entirety.

[0162] In preferred embodiments, the serine protease is a granzyme. Granzymes are serine proteases contained in the cytoplasmic granules of effector immune cells, such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Granzymes, including granzymes A, B, H, K, and M, are reviewed, for example, in Chowdhury and Lieberman, Annu. Rev. Immunol. (2008) 26:389-420 (incorporated herein by reference in its entirety). In preferred embodiments, the granzyme according to the present disclosure is granzyme B. Thus, in some embodiments, the serine protease is granzyme B. The biology of granzyme B is reviewed, for example, in Lord et al., Immunol. Rev. (2003) 193:31-38 and Trapani et al., Curr. Opin. Immunol. (2003) 15:533-43. Granzyme B cleaves peptides after aspartate residues and induces apoptosis by activating caspases such as the executioner caspase-3. Human granzyme B also activates cell death by directly cleaving Bid and ICAD, activating the same mitochondrial and DNA damage pathways, respectively.

[0163] As used herein, "cysteine ​​protease" is an enzyme that cleaves peptide bonds in polypeptides, wherein cysteine ​​acts as a nucleophilic amino acid in the enzyme's active site. "Cysteine ​​protease activity" refers to the catalysis of peptide bond cleavage in polypeptides by cysteine ​​proteases. Cysteine ​​proteases are reviewed, for example, in Verma et al., Front. Pharmacol. (2016) 7:107, which is incorporated herein by reference in its entirety.

[0164] In a preferred embodiment, the cysteine ​​protease is a caspase. Caspase is a cysteine-dependent, aspartic acid-guided protease that plays a role in programmed cell death including apoptosis and pyroptosis. For example, in Julien and Wells' Cell Death & Differentiation (2017) 24: 1380-1389 (incorporated herein by reference in its entirety), caspase is reviewed, including caspase-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -12 and -14. In some embodiments, according to the caspase disclosed herein, it is a caspase involved in apoptosis, for example, selected from caspase-2, -8, -9, -10, -3, -6 and -7. In some embodiments, caspase is an initial caspase, for example, selected from caspase-2, -8, -9 and -10. In some embodiments, the caspase is an executioner caspase, for example selected from caspase-3, -6, and -7. In some embodiments, the caspase according to the present disclosure is a caspase involved in pyroptosis, for example selected from caspase-1, -4, -5, and -12.

[0165] The resistance / susceptibility of cells to cell killing by serine proteases (e.g., granzyme B), the resistance / susceptibility of cells to cell killing by cells expressing a serine protease (e.g., cells expressing granzyme B; e.g., effector immune cells), and the rate of cell killing by cells expressing a serine protease (e.g., cells expressing granzyme B; e.g., effector immune cells) can be assessed using assays that include detecting and / or quantifying cell lysis / cell killing of cells.

[0166] In some embodiments, a serine protease (e.g., granzyme B) may be expressed by a cell being evaluated for resistance / susceptibility to cell killing by a serine protease (e.g., granzyme B). In some embodiments, a serine protease (e.g., granzyme B) may be expressed by a cell different from the cell being evaluated for resistance / susceptibility to cell killing by a serine protease (e.g., granzyme B). In some embodiments, the cell expressing the serine protease (e.g., granzyme B) is allogeneic with respect to the cell. That is, the cell expressing granzyme B may be obtained / derived from a subject different from the subject from which the test cell was obtained / derived (e.g., a genetically non-identical subject). In some embodiments, the cell expressing the serine protease (e.g., granzyme B) may be autologous or homologous with respect to the cell. That is, the cell expressing granzyme B may be obtained / derived from the same or genetically identical subject as the subject from which the test cell was obtained / derived.

[0167] In the present invention, "death receptor-mediated apoptosis" refers to programmed cell death (i.e., apoptosis) of cells expressing death receptors induced by death receptor activation. For example, death receptor-mediated apoptosis is reviewed in Green's Cold Spring Harb Perspect Biol. (2022) 14: a041053, which is incorporated herein by reference in its entirety.

[0168] Death receptors belong to the tumor necrosis factor / nerve growth factor superfamily. They are type I transmembrane proteins with a conserved cytoplasmic death domain (DD). Death receptors are activated after binding to their cognate ligands. Upon activation, the DD promotes homotypic interactions with adaptor proteins through its death domain motif, activating the caspase cascade and ultimately leading to apoptosis. In some embodiments, the death receptor is selected from: Fas (also known as CD95 and APO-1), tumor necrosis factor receptor-1 (TNFR1), TRAIL receptor-1 (also known as DR4), TRAIL receptor-2 (also known as DR5), death receptor 3 (DR3), death receptor 6 (DR6), nerve growth factor receptor (NGFR) and ectodermal dysplasia protein A receptor (EDAR). In some embodiments, the death receptor is Fas.

[0169] In the present invention, "Fas-mediated apoptosis" refers to programmed cell death (i.e., apoptosis) induced by activation of Fas receptors in cells expressing the Fas receptor. For example, Timmer et al., J. Pathol. (2002), 196(2): 125-34, provide a review of Fas-mediated apoptosis, which is incorporated herein by reference in its entirety. Fas-mediated apoptosis generally involves cross-linking of Fas by its ligand, FasL (e.g., expressed on the surface of effector immune cells), activating the caspase cascade and ultimately leading to apoptosis.

[0170] The resistance / sensitivity of cells to apoptosis mediated by death receptors (e.g., Fas-mediated apoptosis) can be assessed using tests that include detecting and / or quantifying death receptor-mediated apoptosis. Such tests can include contacting cells with reagents that activate apoptosis mediated by death receptors (e.g., ligands of a given death receptor or cells expressing a ligand of a given death receptor), and detecting and / or quantifying apoptosis of the cells. For example, such tests can include contacting cells with reagents that activate Fas-mediated apoptosis (e.g., FasL or cells expressing FasL), and detecting and / or quantifying apoptosis of the cells. Detecting / quantifying apoptosis can include analysis of the expression level or activity of one or more caspases, and / or can include detecting and / or quantifying viable, dead, and / or apoptotic cells. Detecting / quantifying apoptosis can include detecting and / or quantifying one or more apoptosis markers (e.g., phosphatidylserine (PS) exposure, Bcl-2 family proteins (e.g., Bax, Bak, Bid) activation, reactive oxygen species (ROS) generation, caspase activation, mitochondrial membrane permeability, or DNA fragmentation).

[0171] Cell killing can be studied using any of the methods reviewed in, for example, Zaritskaya et al., Expert Rev Vaccines (2011), 9(6): 601-616, which is incorporated herein by reference in its entirety. Examples of in vitro assays for cytotoxicity / cell killing include release assays, e.g. 51 Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released in lysed cells. Cell killing of a given effector immune cell type against a given test cell type can be analyzed, for example, by co-culturing the test cells with effector immune cells and measuring the number / ratio of test cells that survive / die (e.g., lysed) after an appropriate period of time. Other suitable assays include the xCELLigence real-time cell lysis in vitro potency assay described by Cerignoli et al. in PLoS One. (2018) 13(3): e0193498 (incorporated herein by reference in its entirety). Increased resistance to cell killing and / or decreased sensitivity to cell killing of cells expressing granzyme B (e.g., effector immune cells) relative to a reference level of cell killing (e.g., for that cell type) can be determined by detecting a decrease in the number / proportion of dead (e.g., lysed) test cells and / or an increase in the number / proportion of viable (e.g., viable, non-lysed) test cells after a given period of time.

[0172] The cell killing rate of a given test cell can be determined by analyzing cell lysis over time, for example, by determining the number / proportion of test cells that are lysed and / or not lysed at different time points. For example, such analysis can be performed using the xCELLigence system described in Example 1.6 herein. A decrease in the cell killing rate relative to a reference rate of cell killing (e.g., for that cell type) can be determined by detecting a decrease in the number / proportion of test cells that die (e.g., lyse) and / or an increase in the number / proportion of test cells that survive (e.g., survive, not lysed) per unit time.

[0173] The persistence / survival / proliferation / expansion of a given cell / its population can be assessed in vitro by measuring or monitoring the number or proportion of such cells over time. Cell proliferation / expansion can be studied by analyzing cell division or cell number over a period of time. For example, cell division can be analyzed in vitro by analyzing incorporated 3H-thymidine or by a CFSE dilution assay as described by Fulcher and Wong in Immunol Cell Biol. (1999) 77(6):559-564, which is incorporated herein by reference in its entirety. Proliferating cells can also be identified by analyzing incorporated 5-ethynyl-2'-deoxyuridine (EdU) by appropriate assays, such as those described by Buck et al. in Biotechnology, June 2008; 44(7):927-9 and by Sali and Mitchison in Proceedings of the National Academy of Sciences, February 19, 2008; 105(7):2415-2420, which are incorporated herein by reference in their entirety.

[0174] The level of cell proliferation or population expansion of a given cell type can also be assessed by counting the number of the relevant cell type at one or more defined time points, such as after culture under certain conditions.

[0175] The increase in persistence / survival can be determined by detecting a greater number / ratio of viable cells (e.g., live, un-lyzed) after a given time. The above-mentioned cell killing assay can also be used to assess the persistence / survival of cells in the presence of allogeneic effector immune cells. For example, the persistence / survival of a given test cell type in the presence of allogeneic effector immune cells can be analyzed by co-culturing the test cells with allogeneic effector immune cells and measuring the number / ratio of test cells that survive / die (e.g., lyse) after an appropriate time period. Relative to a reference level (e.g., for the cell type), the increase in persistence / survival can be determined by detecting a decrease in the number / ratio of test cells that die (e.g., lyse) and / or an increase in the number / ratio of test cells that survive (e.g., survive, un-lyse) after a given time.

[0176] Suitable assays for assessing proliferation / expansion / persistence / survival in the presence of allogeneic effector immune cells include, for example, a mixed lymphocyte reaction (MLR) assay, such as that described in Example 1.5 herein.

[0177] According to the assays described herein, the given / prescribed time period described prior to assessing the level of the characteristic of interest can be any suitable time period that allows for a meaningful comparative analysis of the level of the characteristic of interest between the test cells and the control in the assay. In some embodiments, the level of the characteristic of interest is assessed at or after a time sufficient for the characteristic of interest to reach or have reached a maximum level in the assay. In some embodiments, the given / prescribed time period is one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, the given / prescribed time period is one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. As used herein, when a given cell is referred to herein as "allogeneic" relative to a reference cell, it is obtained / derived from a subject other than the subject from which the reference cell was obtained / derived. Thus, the allogeneic effector immune cells referred to in the preceding paragraph are obtained / derived from a subject other than the subject from which the test cell was obtained / derived. In some embodiments, the allogeneic cells comprise MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules) that are different from the MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules) encoded by the MHC / HLA genes of the reference cells.

[0178] The survival / persistence of a given cell or cell population in a subject (e.g., an allogeneic subject) can be assessed using methods such as labeling the cells with a detectable marker or reporter gene and introducing them into the subject, and monitoring their survival over time. Such methods include, for example, labeling the cells with firefly luciferase and measuring firefly luciferase activity by bioluminescence imaging at different time points, such as after administration of D-luciferin (e.g., as described by Prescher and Contag in Curr. Opin. Chem. Biol. (2010) 14(1):80-9, which is incorporated herein by reference in its entirety). An increase in persistence / survival relative to a reference level (e.g., for that cell type) can be determined by detecting an increase in the number / ratio of test cells in the subject after a given period of time.

[0179] As used herein, when a subject is referred to herein as "allogeneic" relative to a reference cell, the subject is a subject other than the subject from which the reference cell was obtained / derived. Thus, the allogeneic subject referred to in the preceding paragraph is a subject other than the subject from which the test cell was obtained / derived. In some embodiments, the allogeneic subject comprises MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules) that are different from the MHC / HLA molecules (e.g., MHC class I α and / or MHC class II molecules) encoded by the MHC / HLA genes of the reference cell.

[0180] Suitable assays for assessing the survival / persistence of a given cell or cell population in an allogeneic subject (i.e., allogeneic to the test cells) can be performed in an alternative allogeneic subject. An alternative allogeneic subject can be established by injecting allogeneic immune cells (i.e., allogeneic to the test cells) into a non-allogeneic subject, for example, by injecting allogeneic effector immune cells into MHC knockout mice. Thus, suitable assays for assessing the survival / persistence of a given cell or cell population in an allogeneic subject can include co-infusing the given cell or cell population with allogeneic immune cells (i.e., allogeneic to the test cells) into MHC knockout mice. In some embodiments, the survival / persistence of a given cell or cell population in an allogeneic subject (e.g., an alternative allogeneic subject) can be assessed essentially as described in Examples 1.10 and 4.

[0181] The above-mentioned methods for assessing the persistence / survival / proliferation of cells in allogeneic subjects can also be used to assess allogeneic transplant rejection in subjects. The SERPINB9 polypeptides according to the present disclosure and other agents for increasing SERPINB9 expression or activity can also be used to reduce / prevent transplant rejection. Graft rejection refers to the destruction of transplanted cells / tissues / organs by the recipient's immune system after transplantation. When graft rejection is an allogeneic transplant, it can be referred to as allogeneic transplant rejection. Increased expression or activity of SERPINB9 in cells confers resistance to depletion mediated by serine proteases (e.g., granzyme B), which is present between cells expressing granzyme B in the recipient subject (e.g., effector immune cells).

[0182] The anti-cancer activity of a given cell type or population of such cells can be assessed by, for example, analyzing cell killing and / or its correlation with cancer cells by such cells.

[0183] For example, detecting and / or quantifying cell killing of cancer cells can be used to analyze anticancer activity in vitro.The cell killing assays described above can be used to assess cell killing of cancer cells.

[0184] For example, in the presence of allogeneic effector immune cells, test cells can be cultured with allogeneic effector immune cells and cancer cells, and the number / ratio of surviving and / or dying (e.g., lysed) cancer cells can be monitored over time to assess the anti-cancer activity of a given test cell type. After a given period of time, an increase in anti-cancer activity can be determined by detecting a decrease in the number / ratio of surviving cancer cells and / or an increase in the number / ratio of dying (e.g., lysed) cancer cells (e.g., relative to the level of anti-cancer activity exhibited by cells of this type without treatment with the product of the present disclosure).

[0185] Suitable assays for assessing the anti-cancer activity of a cell or cell population include, for example, a mixed lymphocyte reaction (MLR) assay. In some embodiments, the anti-cancer activity of a cell or cell population can be assessed essentially as described in Example 1.5 herein.

[0186] For example, anti-cancer activity can also be analyzed in vivo using assays that detect and / or quantify cell killing of cancer cells in a subject.

[0187] For example, the anti-cancer activity of a given test cell type can be assessed by an experiment in which the test cells are given to a subject suffering from cancer and the number / ratio of cancer cells, cancer burden and / or tumor volume are monitored over time. By detecting the reduction in the number / ratio of cancer cells in the subject after a given time period, the reduction in cancer burden and / or tumor volume, the increase in anti-cancer activity (e.g., relative to the anti-cancer activity level shown by the type of cell, e.g., in the absence of treatment with the product of the present disclosure) can be determined. In some embodiments, such experiments can use cancer cells labeled with a detectable marker or a reporter gene, and the anti-cancer activity in vivo can be assessed by monitoring the number / ratio of cells over time. Such methods include, for example, labeling cells with firefly luciferase and measuring firefly luciferase activity at different time points by bioluminescent imaging, e.g., after administering D-luciferin (e.g., as described in Prescher and Contag, Curr. Opin. Chem. Biol. (2010) 14 (1): 80-9, which is incorporated herein by reference in its entirety).

[0188] In some embodiments, the anti-cancer activity of a cell or cell population can be assessed essentially as described in Example 1.10 herein.

[0189] Under conditions of chronic antigen exposure, by subjecting the cells to continuous antigen stimulation, and over time or after a given period of time, measuring or monitoring the quantity or ratio of such cells, the persistence / survival / proliferation of cells or cell groups in vitro can be assessed. For example, the persistence / survival / proliferation of a given test cell type can be analyzed by continuously co-culturing the test cells with cancer cells (e.g., by re-plating the test cells with fresh cancer cells for multiple rounds of tumor stimulation) and monitoring the quantity / ratio of the test cells that survive and / or die (e.g., crack) after an appropriate period of time. By detecting a reduction in the number / ratio of dead (e.g., cracked) test cells and / or an increase in the number / ratio of surviving (e.g., surviving, not cracked) test cells after a given period of time, an increase (e.g., relative to the level shown by the type of cells, e.g., in the absence of treatment with a product of the present invention) the persistence / survival of a given test cell type can be determined.

[0190] In some embodiments, "conditions of chronic antigen exposure" may refer to multiple rounds (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds) of stimulation of immune cells with antigens or cells comprising / expressing antigens, wherein the immune cells comprise specific receptors (e.g., CAR comprising an antigen binding domain that binds to an antigen and / or TCR bound to a peptide complex MHC comprising an antigen peptide). In some embodiments, "conditions of chronic antigen exposure" may refer to multiple (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) additions of antigens or cells comprising / expressing antigens, wherein the immune cells comprise specific receptors (e.g., CAR comprising an antigen binding domain that binds to an antigen and / or TCR bound to a peptide complex MHC comprising an antigen peptide) added to cultured immune cells. In some embodiments, rounds of stimulation / addition of antigens with antigens are performed at regular time intervals, for example, every 1, 2, 3, 4, or 5 days. In some embodiments, rounds of stimulation / addition of antigens with antigens are performed at regular time intervals, for example, every 2-3 days. In some embodiments, "antigen-exposed conditions" can refer to stimulating immune cells in an in vivo model that is transplanted with tumors / tumor cells containing / expressing an antigen, wherein the immune cells contain specific receptors (e.g., a CAR containing an antigen binding domain that binds to the antigen and / or a TCR that binds to a peptide complex MHC containing the antigenic peptide).

[0191] In some embodiments, the persistence / survival / proliferation of cells or cell populations under conditions of chronic antigen exposure can be assessed essentially as described in Example 1.9 and / or Example 1.10 "Activation-Induced Cell Death (AICD) Model" herein.

[0192] In some embodiments, in the methods and uses according to the present disclosure, the SERPINB9 polypeptides, nucleic acids, vectors or agents for increasing the expression or activity of SERPINB9 according to the present disclosure can increase the expression level of SERPINB9 in cells (i.e., gene or protein expression) / the resistance of cells to cell killing mediated by serine proteases (e.g., granzyme B) / the resistance of cells to cell killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells) / the resistance of cells to apoptosis mediated by death receptors (e.g., Fas-mediated apoptosis) / the persistence, survival or proliferation of cells in the presence of allogeneic effector immune cells / the persistence, survival or proliferation of cells in allogeneic subjects / the The anti-cancer activity in the presence of allogeneic effector immune cells / the anti-cancer activity of the cells in an allogeneic subject / the persistence, survival or proliferation of the cells under conditions of chronic antigen exposure is increased to greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold or ≥10-fold, relative to the level of the relevant property normally exhibited by cells of that type (e.g., a reference value for the level of the relevant property of cells of that type, i.e., in the absence of treatment with the product of the present disclosure).

[0193] In some embodiments, in the methods and uses according to the present disclosure, the SERPINB9 polypeptides, nucleic acids, vectors or agents for increasing the expression or activity of SERPINB9 according to the present disclosure can increase the activity of serine proteases (e.g., granzyme B) in cells / the activity of caspases (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8 or -10) in cells / the sensitivity of cells to cell killing mediated by serine proteases (e.g., granzyme B) / the sensitivity of cells to cell killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells) / the sensitivity of cells to apoptosis mediated by death receptors (e.g., Fas-mediated apoptosis) Sensitivity / cell killing rate by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / allograft rejection in a subject is reduced to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the level of the relevant property typically exhibited by that type of cell (e.g., a reference value for the level of the relevant property of that type of cell).

[0194] Cells modified to increase the expression or activity of SERPINB9

[0195] The present disclosure provides a cell modified to increase SERPINB9 expression (i.e., gene and / or protein expression) or activity. It should be understood that when cells are referred to herein in the singular (i.e., "a cell / the cell"), plural numbers / cell populations of such cells are also contemplated.

[0196] Cells with increased SERPINB9 expression (i.e., gene and / or protein expression) or activity can be characterized by a level of expression of SERPINB9 or a level of activity of SERPINB9 that is higher than the expression level / activity typically exhibited by cells of that type. Cells with increased SERPINB9 expression or activity can be characterized by a level of expression of SERPINB9 or a level of activity of SERPINB9 that is higher than a reference value for the expression level / activity of that type of cell.

[0197] Cells with increased SERPINB9 expression (ie, gene and / or protein expression) or activity can be the result of treatment / modification as described herein.

[0198] In some embodiments, the cell is a cell that has been treated / modified to increase the level or activity of SERPINB9 protein in the cell. In some embodiments, the cell is a cell that has been treated / modified to increase the expression of SERPINB9 gene and / or protein.

[0199] The present disclosure provides a cell comprising or expressing a SERPINB9 polypeptide as described herein. The present disclosure also provides a cell comprising or expressing a nucleic acid (e.g., an exogenous nucleic acid) encoding the SERPINB9 polypeptide of the present invention. The present disclosure also provides a cell comprising or expressing a vector encoding the SERPINB9 polypeptide of the present invention.

[0200] As used herein, an "exogenous" nucleic acid refers to a nucleic acid that is not endogenous to the cell containing the exogenous nucleic acid. The exogenous nucleic acid may not be encoded by the genome of the subject from which the cell was obtained / derived. A cell containing an exogenous nucleic acid may be the result of a modification as described herein, for example, by introducing a nucleic acid / vector encoding a SERPINB9 polypeptide into the cell.

[0201] In some embodiments, the cell is a cell into which a nucleic acid / vector encoding a SERPINB9 polypeptide of the present disclosure has been introduced. Such cells can be characterized by a level of expression of SERPINB9 or a level of activity of SERPINB9 that is higher than the expression level / activity displayed by an equivalent cell without the introduction of the nucleic acid / vector.

[0202] In some embodiments, the cell is a cell that has been treated with an agent that increases the expression or activity of SERPINB9 of the present disclosure. Such cells can be characterized by a level of expression of SERPINB9 or a level of activity of SERPINB9 that is higher than the expression level / activity exhibited by an equivalent cell that has not been treated with the agent.

[0203] The expression level of SERPINB9 can be measured by appropriate methods well known to those skilled in the art. SERPINB9 gene expression level can be analyzed using a test comprising detecting and / or quantitatively encoding SERPINB9 RNA levels. Such tests can include quantifying the RNA encoding SERPINB9 by RT-qPCR, Northern blotting, etc. The method can use primers and / or probes for detecting and / or quantitatively encoding SERPINB9 RNA. SERPINB9 protein levels can be analyzed using a test comprising detecting and / or quantitatively encoding SERPINB9 protein levels. Such tests include, for example, methods based on antibodies / reporter molecules (Western blotting, ELISA, immunohistochemistry / cytochemistry, etc.), and for example, antibodies specific to SERPINB9 can be used.

[0204] The activity level of SERPINB9 can be measured using an appropriate test for the activity. Such tests include tests for analyzing the inhibition of serine protease (e.g., granzyme B) activity. Such tests may include assessing the activity of serine proteases, for example, as described above. In some embodiments, for example, the activity of SERPINB9 may be assessed as described in Bird et al., Mol Cell Biol. (1998), 18 (11): 6387-98, which is incorporated herein by reference.

[0205] In some embodiments, the SERPINB9 expression level or activity level shown by cells with increased SERPINB9 expression (i.e., gene and / or protein expression) or activity according to the present disclosure can be more than 1 times the expression level or activity level normally shown by cells of that type (e.g., a reference value for the expression level or activity level of cells of that type), for example, ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times, or ≥10 times.

[0206] In some embodiments, the expression level or activity level of SERPINB9 shown by a cell comprising or expressing a nucleic acid (e.g., an exogenous nucleic acid) or vector encoding a SERPINB9 polypeptide can be more than 1 times the expression level or activity level shown by an equivalent cell that does not comprise the nucleic acid / vector, for example, ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times, or ≥10 times.

[0207] In some embodiments, the expression level or activity level of SERPINB9 shown in cells treated with an agent for increasing the expression or activity of SERPINB9 according to the present disclosure can be more than 1 times the expression level or activity level shown in equivalent cells not treated with the agent, for example, ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times. In some embodiments, the expression level of SERPINB9 shown in cells treated with an agent for increasing the expression or activity of SERPINB9 according to the present disclosure can be more than 1.3 times the expression level shown in equivalent cells not containing the nucleic acid / vector (basically determined by analysis as described in Example 1.7), for example ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times.

[0208] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal of the order Rodentia), cat, dog, pig, sheep, goat, cattle (including dairy cows, such as dairy cows, or any animal of the order Artiodactyla), horse (including any animal of the family Equine), donkey and non-human primate). In a preferred embodiment, the cell is a human cell.

[0209] In preferred embodiments, the cell is an immune cell. The immune cell can be a cell of hematopoietic origin, such as a neutrophil, an eosinophil, a basophil, a dendritic cell, a lymphocyte or a monocyte. Lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells or innate lymphocytes (ILCs), or their precursor cells (such as thymocytes or pre-B cells). The immune cell can express CD3 polypeptides (such as CD3γ, CD3ε, CD3ζ or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4 or CD8. In some embodiments, the immune cell is a T cell, such as a CD3+ T cell. In some embodiments, the T cell is CD3+, CD4+ T cell. In some embodiments, the T cell is CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (such as a cytotoxic T lymphocyte (CTL)). In some embodiments, the immune cell is a T cell or a NK cell.

[0210] In a preferred embodiment, the cell is an effector immune cell. As used herein, "effector immune cell" can be an immune cell that displays effector function. The effector immune cell can be a CD8+T cell, a CD8+ cytotoxic T lymphocyte (CD8+CTL), a CD4+T cell, a CD4+T helper cell, a NK cell, a cell that produces IFNγ, a memory T cell, a central memory T cell, an antigen-experienced T cell, or a CD45RO+T cell. The effector immune cell can have one or more of the following characteristics: granzyme B expression, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granulysin expression, and / or FAS ligand (FASL) expression. In some embodiments, the effector immune cell according to the present disclosure is a cell that expresses granzyme B.

[0211] The immune cells may be derived from any suitable source. The immune cells may be obtained / isolated from a subject, for example, a subject as described herein. The immune cells may be suitable for administration to a subject according to the therapeutic / preventive interventions disclosed herein. The cells may be obtained / isolated from a subject to be treated according to the therapeutic / preventive interventions disclosed herein. The cells may be obtained / isolated from a subject other than the subject to be treated according to the therapeutic / preventive interventions disclosed herein. The cells may be obtained / isolated from a healthy subject, for example, a subject known not to suffer from a disease / disorder.

[0212] The present disclosure also provides a method for producing the cells of the present disclosure, comprising introducing the nucleic acid or vector of the present disclosure into the cells. In some embodiments, introducing the nucleic acid or vector of the present disclosure into the cells comprises transformation, transfection, electroporation or transduction (e.g., retroviral transduction).

[0213] Transfection involves the process of introducing nucleic acids into cells using means other than viral infection and is therefore a non-viral method. Transfection can be performed by physical / mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection, and laser irradiation) or chemical methods (liposome-based or non-liposome-based). Liposome-based transfection reagents are chemical substances that can form positively charged lipid aggregates that can then fuse with the phospholipid bilayer of the cell to facilitate the entry of exogenous genetic material. Examples of liposome-based transfection reagents include, but are not limited to and Non-liposomal transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers, and non-liposomal lipids. An example of a non-liposomal transfection reagent is polyethyleneimine (PEI).

[0214] Electroporation can be performed as described in Koh et al., Molecular Therapy - Nucleic Acids (2013) Vol. 2, e114, which is incorporated herein by reference in its entirety.

[0215] Transduction is a process in which a nucleic acid is introduced into a cell by a virus or a viral vector. Therefore, in some embodiments, the nucleic acid is contained in a viral vector, or the vector is a viral vector. For example, Simmons and Alberola-Ila described the transduction of immune cells with a viral vector in Methods Mol Biol. (2016) 1323: 99-108, which is incorporated herein by reference in its entirety. Reagents can be used in the methods disclosed herein to improve transduction efficiency. Hexamethylammonium bromide (polybrene) is a cationic polymer that is commonly used to improve transduction efficiency by neutralizing the charge repulsion between viral particles and sialic acid residues expressed on the cell surface. Other reagents commonly used to enhance transduction include, for example, poloxamer-based reagents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), as well as SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0216] In some embodiments, the method comprises centrifuging cells into which one desires to introduce a nucleic acid of the present disclosure in the presence of a cell culture medium containing a viral vector comprising the nucleic acid (referred to in the art as "centrifugal transfection").

[0217] In some embodiments, the method further comprises culturing the cells under conditions suitable for expression of the nucleic acid / vector by the cells.

[0218] Those skilled in the art are familiar with methods for culturing immune cell groups in vitro / ex vivo (including generation and / or expansion). Suitable culture conditions (i.e., cell culture medium, additives, stimulation, temperature, gaseous environment), cell number, culture cycle, and methods for introducing nucleic acids encoding polypeptides of interest into cells can be determined by reference to, for example, Hombach et al., Journal of Immunology (2001) 167: 6123-6131, Ramos et al., J. Clin. Invest. (2017) 127 (9): 3462-3471, WO 2021 / 245249 A1, WO 2021 / 222927 A1, WO 2021 / 222928 A1, WO 2021 / 222929 A1, WO 2015 / 028444 A1, or WO 2016 / 008973 A1, all of which are incorporated herein by reference in their entirety.

[0219] Conveniently, cell cultures according to the present disclosure can be maintained at 37° C. in a humidified atmosphere containing 5% CO 2 . Cells in cell culture can be established and / or maintained at any suitable density, which can be readily determined by one skilled in the art.

[0220] Cell culture can be carried out in any container suitable for the culture volume, such as in the wells of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, cells are cultured in a bioreactor, such as the bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, which is incorporated herein by reference in its entirety. In some embodiments, cells are cultured in a GRex cell culture container, such as a GRex culture flask or a GRex 100 bioreactor.

[0221] In some embodiments, the methods are performed in vitro.The present disclosure also provides cells obtained or obtainable according to the methods of the present disclosure, and of course populations of such cells.

[0222] In some embodiments, the cells are cells for use in a medical treatment or prevention method. "Cells for use in a medical treatment or prevention method" refers to cells suitable for use in a medical treatment or prevention method. Such cells may not contain certain agents / contaminants that would make them unsuitable for such use.

[0223] In some embodiments, the cell is a cell for medical treatment or prevention methods, and the medical treatment or prevention is carried out by adoptive cell transfer (ACT). Adoptive cell transfer includes administering cells / cell groups to a subject to treat / prevent a disease / disorder. In particular, adoptive cell transfer generally involves administering immune cells (e.g., T cells) to a subject to provide a colony of immune cells for treating / preventing a disease / disorder, or to increase the number of such cells in the subject. In some embodiments, the adoptively transferred cell comprises a molecule for directing an immune cell against a molecule that contains / expresses a given target antigen (e.g., a disease-associated antigen).

[0224] Adoptive cell transfer can involve isolating / obtaining cells (e.g., immune cells) from a subject, for example, by drawing a blood sample from which the cells can be isolated. The cells are then typically modified and / or expanded and administered to the same subject (in the case of autologous / homologous cell adoptive transfer) or to a different subject (in the case of allogeneic cell adoptive transfer). The treatment is generally intended to provide a subject with a cell population having certain desired characteristics, or to increase the frequency of cells having such characteristics in the subject.

[0225] Cells used in methods of medical treatment or prevention by adoptive cell transfer may comprise / express a molecule that directs the cells to defend against the activity of cells expressing a given target antigen.

[0226] In some embodiments, the cell comprises a T cell receptor (TCR) that directs the cell against the activity of cells presenting TCR-specific MHC-peptide complexes. In some embodiments, the TCR is encoded by the genome of the subject from whom the cell was obtained / derived. In some embodiments, the TCR is encoded by a nucleic acid that has been introduced into the cell.

[0227] In some embodiments, the cell includes a chimeric antigen receptor (CAR), which is used to guide the activity of cells against cells expressing CAR-specific antigens. In some embodiments, CAR is encoded by the nucleic acid introduced into the cell.

[0228] In some embodiments, the immune cell is an immune cell that is engineered to express a molecule that is used to guide the immune cell to resist the activity of cells expressing a given target antigen (e.g., CAR engineered immune cells or TCR engineered immune cells) and / or immune cells that are specific for disease-associated antigens (e.g., immune cells that are specific for pathogens, such as virus-specific immune cells). In some embodiments, the immune cell is an immune cell that is specific for disease-associated antigens and is engineered to express a molecule that is used to guide the immune cell to resist the activity of cells containing / expressing a given target antigen. In some embodiments, the immune cell is a virus-specific, CAR-engineered immune cell.

[0229] In some embodiments, the immune cell is engineered to express a chimeric antigen receptor (CAR; i.e., the immune cell is a CAR-engineered immune cell), or is engineered to express a T cell receptor (TCR; i.e., the immune cell is a TCR-engineered immune cell). TCR-engineered immune cells are described, for example, in Zhao et al., Front. Immunol. (2021) Dec 30; 12: 658753, which is incorporated herein by reference in its entirety. TCR-engineered immune cells can be modified to express a TCR specific for a given MHC-peptide complex, for example, by introducing into the cell a nucleic acid encoding a TCR component polypeptide.

[0230] In some embodiments, the immune cell comprises / expresses a TCR that is encoded by a non-endogenous nucleic acid (ie, a nucleic acid that is not encoded by the cell's genome prior to introduction of nucleic acid encoding the component polypeptides of the TCR into the cell).

[0231] In some embodiments, the method of the present disclosure includes introducing (i) a nucleic acid encoding a molecule that is used to guide immune cells to resist the activity of cells containing / expressing a given target antigen (e.g., CAR or TCR); and (ii) a nucleic acid encoding a SERPINB9 polypeptide into an immune cell. According to such embodiments, the nucleic acids of (i) and (ii) can be introduced into the cell simultaneously or sequentially. When the nucleic acids of (i) and (ii) are introduced simultaneously, they can be introduced in the form of a nucleic acid (e.g., a vector) comprising both nucleic acids of (i) and (ii). Alternatively, the nucleic acids of (i) and (ii) can be contained in separate nucleic acids (e.g., separate vectors). When the nucleic acids of (i) and (ii) are introduced sequentially, the method may include: (a) introducing the nucleic acid of (i) or (ii) into the immune cell; and (b) subsequently (e.g., after a determined time period, e.g., after 12 hours to 14 days, e.g., after a certain time period of 1 to 7 days, 2 to 5 days, or 3 to 4 days), introducing another nucleic acid (i.e., the nucleic acid not introduced into the cell in (a)) into the immune cell.

[0232] The TCR-engineered immune cell can comprise a TCR that is specific for any MHC-peptide complex of interest. In some embodiments, the TCR of the TCR-engineered immune cell is specific for an MHC-peptide complex that comprises a peptide of a disease-associated antigen.

[0233] Immune cells (T cells) engineered to express a TCR specific for a particular MHC-peptide complex can be directed to kill cells expressing that MHC-peptide complex. Binding of the TCR-engineered T cell to its cognate MHC-peptide complex triggers intracellular signaling and thereby activates the T cell. The activated TCR-engineered T cell is stimulated to divide and produce factors that cause the cell expressing the MHC-peptide complex to be killed.

[0234] In some embodiments, cells according to the present disclosure (i.e., cells comprising / expressing SERPINB9 as described herein, or cells comprising / expressing a nucleic acid / vector of SERPINB9 as described herein) do not comprise a chimeric HLA auxiliary receptor (CHAR) for directing the activity of cells against alloreactive T cells. CHAR and cells expressing CHAR are described in, for example, US2021 / 0238255 A1, which is incorporated herein by reference in its entirety.

[0235] In some embodiments, cells according to the present disclosure (i.e., cells comprising / expressing SERPINB9 as described herein, or cells comprising / expressing nucleic acids / vectors of SERPINB9 as described herein) are not modified to comprise / express CHAR. In some embodiments, the cells do not comprise a nucleic acid encoding CHAR (e.g., an exogenous nucleic acid). In some embodiments, the methods according to the present disclosure do not include introducing a nucleic acid encoding CHAR (e.g., an exogenous nucleic acid) into the cell.

[0236] In some embodiments, the cells according to the present disclosure (i.e., cells comprising / expressing SERPINB9 as described herein, or cells comprising / expressing a nucleic acid / vector of SERPINB9 as described herein) are not modified to express or overexpress an intracellular FLICE inhibitory protein (cFLIP) or a variant thereof. Human cFLIP is a protein recognized by UniProt O15519-1. Variants of cFLIP include polypeptides having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to the amino acid sequence of human cFLIP. In some embodiments, variants of cFLIP may comprise a K167R substitution relative to UniProt O15519-1.

[0237] In some embodiments, the cell does not comprise a nucleic acid (eg, an exogenous nucleic acid) encoding cFLIP or a variant thereof. In some embodiments, methods according to the present disclosure do not comprise introducing a nucleic acid (eg, an exogenous nucleic acid) encoding cFLIP or a variant thereof into the cell.

[0238] As used herein, a "disease associated antigen" refers to an antigen whose presence is indicative of a given disease / disease state, or whose elevated antigen levels are positively correlated with a given disease / disease state. The disease associated antigen may refer to an antigen whose expression is associated with the development, progression, or severity of symptoms of a given disease. The disease associated antigen may be associated with the cause or pathology of the disease, or may be abnormally expressed as a result of the disease. The disease associated antigen may be an antigen of an infectious source or pathogen, a cancer associated antigen, or an autoimmune disease associated antigen.

[0239] In some embodiments, the disease-associated antigen is an antigen of a pathogen. The pathogen can be a prokaryotic organism (bacteria), a eukaryotic organism (e.g., protozoa, worms, fungi), a virus, or a prion. In some embodiments, the pathogen is an intracellular pathogen. In some embodiments, the pathogen is a virus, such as the virus described above. In some embodiments, the pathogen is a bacterium. The bacterium can be gram-positive or gram-negative. In some embodiments, the present invention relates to bacteria of the genus Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter pylori, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio and Yersinia. In some embodiments, pathogen is a protozoan. In some embodiments, the present invention relates to protozoans of the genus Entamoeba, Plasmodium, Giardia, Trypanosoma, Leishmania, Bethnosella and Toxoplasma. In some embodiments, pathogen is a fungus. In particular, the present disclosure contemplates fungi of the genera Candida, Aspergillus, Blastomyces, Coccidioides, Sporothrix, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys, Rhizopus, Mucor, Cunninghamella, Aphidomonas, Trichophyton, Microsporum, Epidermophyton, Fusarium, and Aspergillus.

[0240] In some embodiments, the disease-associated antigen is a cancer-associated antigen. In some embodiments, the cancer-associated antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of cancer. The cancer-associated antigen may be associated with the cause or pathology of the cancer, or may be abnormally expressed as a result of the cancer. In some embodiments, the cancer-associated antigen refers to an antigen whose expression level (e.g., at the RNA and / or protein level) is upregulated by cancer cells, for example, compared to comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type).

[0241] In some embodiments, the cancer associated antigen may be preferentially expressed by cancer cells rather than by comparable non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, the cancer associated antigen may be the product of a mutated proto-oncogene or a mutated tumor suppressor gene. In some embodiments, the cancer associated antigen may be a product of overexpression of an intracellular protein, a cancer antigen produced by an oncovirus, a carcinoembryonic antigen, or a cell surface glycolipid or glycoprotein.

[0242] In Kufe DW, Pollock RE, Weichselbaum RR, et al., eds., Holland-Frei Cancer Medicine, 6th ed. Hamilton (Ontario): BC Decker; in mid-2003, a review of cancer-associated antigens was given in Zarour HM, DeLeo A, Finn OJ, et al., Classes of tumor antigens. Cancer-associated antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; oncoviral antigens such as HPV E6 and E7; overexpressed proteins: BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, HER2 / neu, telomerase, mesothelin, SAP-1, survivin; cancer-testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-1 / 2, MC1R, prostate-specific antigen; mutant antigens: β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; post-translationally modified antigens: MUC1, idiotypic antigens: Ig, TCR. Other cancer-associated antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose-regulated protein 78 (GRP78), vimentin, nucleolin, fetal-acinar pancreatic protein (FAPP), placental-like 2 alkaline phosphatase (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7) and cyclophilin B. In some embodiments, the cancer-associated antigen is a cancer-associated antigen described by Zhao and Cao in Front Immunol. 2019; 10: 2250, which is incorporated herein by reference in its entirety. In some embodiments, the cancer-associated antigen is selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY and PSCA. In some embodiments, the cancer-associated antigen is an antigen expressed by hematological malignancy cells. In some embodiments, the cancer-associated antigen is selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, and BCMA. In some embodiments, the cancer-associated antigen is an antigen expressed by solid tumor cells.In some embodiments, the cancer-associated antigen is selected from the group consisting of mesothelin, EGFR, GPC3, MUCl, HER2, GD2, CEA, EpCAM, LeY, and PSCA.

[0243] In some embodiments, the immune cell is an immune cell engineered with a chimeric antigen receptor (CAR). In some embodiments, the immune cell comprises / expresses CAR. Chimeric antigen receptor (CAR) is a recombinant receptor molecule that provides antigen binding and T cell activation functions. The structure and modification of CAR are reviewed in, for example, Dotti et al. in Immunol Rev (2014) 257 (1), which is incorporated herein by reference in its entirety.

[0244] The immune cells expressing CAR may contain or express nucleic acids encoding CAR according to the present disclosure. It is understood that cells expressing CAR contain the CAR it expresses. It is also understood that cells expressing nucleic acids encoding CAR also express and contain CAR encoded by the nucleic acid. CAR comprises an antigen binding domain connected to a signaling domain via a transmembrane domain. An optional hinge region or Spacer domain can provide a gap between the antigen binding domain and the transmembrane domain and can act as a flexible linker. When expressed by the cell, the antigen binding domain is located in the extracellular space, while the signaling domain is located within the cell.

[0245] Immune cells (usually T cells, but also other immune cells such as NK cells) engineered to express CARs specific for specific target antigens can be directed to kill cells expressing the target antigen. The binding of CAR-expressing T cells (CAR-T cells) to their specific target antigens triggers intracellular signaling and thus activates T cells. Activated CAR-T cells are stimulated to divide and produce factors that cause cells expressing the target antigen to be killed.

[0246] The antigen binding domain mediates the binding to the target antigen specifically recognized by CAR. The “antigen binding domain” refers to a domain that can bind to the target antigen. The antigen binding domain of CAR can be an antigen binding domain of an antibody that is specific for the antigen targeted by CAR. For example, the antigen binding domain of CAR can include the amino acid sequence of the complementary determining region (CDR) of the antibody that specifically binds to the target antigen. The antigen binding domain of CAR can include or consist of the light chain and heavy chain variable region amino acid sequences of the antibody that specifically binds to the target antigen. The antigen binding domain can be provided as a single chain variable fragment (scFv) comprising the light chain sequence and heavy chain variable region amino acid sequence of the antibody. The antigen binding domain of CAR can target antigens based on other proteins: protein interactions, such as ligand: receptor binding; for example, a CAR targeting IL-13Rα2 has been developed using an antigen binding domain based on IL-13 (see, for example, Kahlon et al., 2004 Cancer Res 64(24):9160-9166).

[0247] The antigen binding domain of the CAR according to the present disclosure can be specific for any antigen, for example, a disease-associated antigen as described herein. It is understood that the antigen binding domain of the CAR is specific for an antigen expressed by a cell that wishes to direct the activity of the cell expressing the CAR. In the example of a T cell expressing a CAR, the CAR may comprise an antigen binding domain that binds to an antigen expressed by a target cell that wishes to direct the activity of a T cell effector. In a preferred embodiment, the antigen binding domain of the CAR according to the present disclosure binds to a cancer-associated antigen. In a more preferred embodiment, the antigen binding domain of the CAR according to the present disclosure binds to CD30.

[0248] The antigen-binding domains according to the present disclosure can be derived from antibodies / antibody fragments (e.g., Fv, scFv, Fab, single-chain Fab (scFab), single-domain antibodies (e.g., VhH), etc.) directed against disease-associated antigens, or another disease-associated antigen-binding molecule (e.g., target antigen-binding peptide or target nucleic acid aptamer, target ligand or other target molecule). In some embodiments, the antigen-binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody that can specifically bind to a disease-associated antigen. In some embodiments, the domain capable of binding to the target antigen comprises or consists of a peptide / polypeptide that binds to a disease-associated antigen, such as those reviewed in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082-1101, e.g., a peptide aptamer, thioredoxin, a monoclonal antibody, anticalin, a Kunitz domain, AviMer, knottin, Fynomer, AtriMer, DARPin, affibody, nanobody (i.e., a single domain antibody (sdAb)), avidin, armadillo repeat protein (ArmRP), OBody, or fibronectin, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0249] The antigen binding domain of CAR in the present disclosure can be derived from the VH and VL of antibodies that can specifically bind to disease-associated antigens. Antibodies typically contain six complementary determining regions (CDRs); three in the heavy chain variable region (VH): HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable region (VL): LC-CDR1, LC-CDR2, and LC-CDR3. These six CDRs together define the paratope of the antibody, that is, the part that the antibody binds to the target antigen. The VH and VL regions contain framework regions (FRs) on both sides of each CDR to provide a scaffold for the CDRs. From N-terminus to C-terminus, VHs have the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus; VLs have the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0250] As long as the antigen binding domain can be connected to the other domains of CAR, VH and VL sequences can be provided in any suitable form. Considered forms related to the antigen binding domains of the present disclosure include those described by Carter in "Nat. Rev. Immunol" 2006, 6:343-357, such as scFv, dsFV, (scFv) 2 double antibodies, three antibodies, four antibodies, Fab, small antibodies and F (ab) 2 forms.

[0251] In some embodiments, the antigen binding domain comprises the complementary determining regions (CDRs) of an antibody / antibody fragment that can bind to a disease-associated antigen. In some embodiments, the antigen binding domain comprises the VH and VL of an antibody / antibody fragment that can bind to a disease-associated antigen. The portion consisting of the VH and VL of an antibody may also be referred to herein as a variable fragment (Fv). VH and VL can be provided on the same polypeptide chain and connected by a linker sequence; such portions are referred to as single-chain variable fragments (scFvs). Suitable linker sequences for preparing scFv are known to those skilled in the art and may comprise serine and glycine residues. In some embodiments, the antigen binding domain comprises or consists of an Fv that can bind to a disease-associated antigen. In some embodiments, the antigen binding domain comprises or consists of an scFv that can bind to a disease-associated antigen. In some embodiments, the antigen binding domain is a ligand derived from a disease-associated antigen.

[0252] In some embodiments, the antigen binding domain of a CAR according to the present disclosure binds to CD30 (i.e., is a domain that binds CD30). In some embodiments, the antigen binding domain of a CAR according to the present disclosure binds to CD19 (i.e., is a domain that binds CD19).

[0253] In some embodiments, the CD30-binding domain is derived from an antigen-binding portion of an anti-CD30 antibody. In some embodiments, the CD30-binding domain according to the present disclosure comprises the CDRs of an anti-CD30 antibody. In some embodiments, the CD30-binding domain according to the present disclosure comprises the VH and VL regions of an anti-CD30 antibody. In some embodiments, the CD30-binding domain according to the present disclosure comprises an scFv comprising the VH and VL regions of an anti-CD30 antibody. Such anti-CD30 antibodies include HRS3 and HRS4 (e.g., as described in Hombach et al., Scand J Immunol. (1998) 48(5):497-501), HRS3 derivatives described in Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12):847-860, BerH2 (MBL International, Cat. No. K0145-3, RRID:AB_590975), SGN-30 (also known as cAC10, e.g., as described in Forero-Torres et al., Br J Haematol (2009) 146:171-179), MDX-060 (e.g., as described in Ansell et al., J Clin Oncol (2007) 25:2764-2769; also known as 5F11, islatuzumab), and MDX-1401 (e.g., as described in Cardarelli et al., Clin Cancer Res. (2009) 15(10): 3376-3383), and anti-CD30 antibodies described in WO 2020 / 068764 A1, WO 2003 / 059282 A2, WO 2006 / 089232 A2, WO 2007 / 084672 A2, WO 2007 / 044616 A2, WO 2005 / 001038 A2, US2007 / 166309A1, US2007 / 258987A1, WO 2004 / 010957A2 and US2005 / 009769A1.

[0254] In some embodiments, the domain that binds to CD19 is derived from the antigen binding portion of an anti-CD19 antibody. In some embodiments, the domain that binds to CD19 according to the present disclosure comprises the CDRs of an anti-CD19 antibody. In some embodiments, the domain that binds to CD19 according to the present disclosure comprises the VH region and VL region of an anti-CD19 antibody. In some embodiments, the domain that binds to CD19 according to the present disclosure comprises an scFv comprising the VH and VL regions of an anti-CD19 antibody. Such anti-CD19 antibodies include FMC63 (e.g., as described in Zola et al., Immunol Cell Biol (1991) 69:411-422), HD37 (e.g., as described in Pezzutto et al., J Immunol (1987) 138:2793-2799), tafacitinib (also known as MOR208 or XmAB5574), and the anti-CD19 antibodies described in WO 2009 / 054863 A2, WO 2010 / 053716 A1, WO 2006 / 089133 A2, and WO 2021 / 173471 A1.

[0255] There are several different conventions for defining antibody CDRs and FRs, such as VBASE2 as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and Retter et al., Nucl. Acids Res. (2005) 33(Suppl 1):D671-D674. The CDRs and FRs of the heavy chain variable region (VH) and light chain variable region (VL) of the antibodies described herein are defined according to VBASE2.

[0256] In some embodiments, the CD30-binding domain comprises:

[0257] VH comprising the following CDRs:

[0258] HC-CDR1 having the amino acid sequence of SEQ ID NO: 15

[0259] HC-CDR2 having the amino acid sequence of SEQ ID NO: 16

[0260] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 17,

[0261] or a variant in which one, two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are substituted with another amino acid;

[0262] as well as

[0263] A VL comprising the following CDRs:

[0264] LC-CDR1 having the amino acid sequence of SEQ ID NO: 18

[0265] LC-CDR2 having the amino acid sequence of SEQ ID NO: 19

[0266] LC-CDR3 having the amino acid sequence of SEQ ID NO: 20,

[0267] Alternatively, the variant may comprise a variant in which one, two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0268] In some embodiments, the CD30-binding domain comprises:

[0269] A VH comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO: 21.

[0270] as well as

[0271] A VL comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO: 22.

[0272] In some embodiments, the CD30 binding domain may comprise or consist of a single-chain variable fragment (scFv) as described herein, comprising a VH sequence and a VL sequence. The VH sequence and VL sequence may be covalently linked. In some embodiments, the VH and VL sequences are linked by a flexible linker sequence, for example, as described herein. The flexible linker sequence may be attached to the ends of the VH and VL sequences, thereby linking the VH and VL sequences. In some embodiments, the VH and VL sequences are linked by a linker sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 23.

[0273] In some embodiments, the CD30 binding domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:24.

[0274] In some embodiments, the CD30-binding domain is capable of binding to CD30, e.g., the extracellular domain of CD30. In some embodiments, the CD30-binding domain is capable of binding to an epitope of CD30 bound by the antibody HRS3, e.g., within the region of amino acid positions 185-335 of human CD30 numbered according to SEQ ID NO: 10, as set forth in SEQ ID NO: 12 (Schlapschy et al., Protein Engineering, Design and Selection (2004) 17(12):847-860, incorporated herein by reference in its entirety).

[0275] In some embodiments, the CD19 binding domain comprises:

[0276] VH comprising the following CDRs:

[0277] HC-CDR1 having the amino acid sequence of SEQ ID NO: 58

[0278] HC-CDR2 having the amino acid sequence of SEQ ID NO: 59

[0279] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 60,

[0280] or a variant in which one, two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are substituted with another amino acid;

[0281] as well as

[0282] A VL comprising the following CDRs:

[0283] LC-CDR1 having the amino acid sequence of SEQ ID NO: 61

[0284] LC-CDR2 having the amino acid sequence of SEQ ID NO: 62

[0285] LC-CDR3 having the amino acid sequence of SEQ ID NO: 63,

[0286] Alternatively, the variant may comprise a variant in which one, two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0287] In some embodiments, the CD19 binding domain comprises:

[0288] A VH comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO: 64.

[0289] as well as

[0290] A VL comprising, or consisting of, an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence of SEQ ID NO: 65.

[0291] In some embodiments, the domain that binds to CD19 may comprise or consist of a single-chain variable fragment (scFv) as described herein, comprising a VH sequence and a VL sequence. The VH sequence and the VL sequence may be covalently linked. In some embodiments, the VH and VL sequences are connected by, for example, a flexible linker sequence as described herein. The flexible linker sequence may be connected to the ends of the VH and VL sequences, thereby connecting the VH and VL sequences. In some embodiments, VH and VL are connected by a linker sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 66.

[0292] In some embodiments, the CD 19 binding domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:67.

[0293] In some embodiments, the CD19 binding domain is capable of binding CD19, such as the extracellular domain of CD19.

[0294] In some embodiments, the antigen binding domain (and therefore CAR) is multispecific. The "multispecific" refers to that the antigen binding domain shows specific binding to more than one target. In some embodiments, the antigen binding domain is a bispecific antigen binding domain. In some embodiments, the antigen binding molecule includes at least two different antigen binding moieties (i.e., at least two antigen binding moieties, for example, including different VHs and VLs). The respective antigen binding moieties of the multispecific antigen binding domain can be connected, for example, by a connecting sequence. The antigen binding domain can be combined with at least two different target antigens, and is therefore at least bispecific. The term "bispecific" means that the antigen binding domain can specifically bind to at least two different antigenic determinants. At least one target antigen of the multispecific antigen binding domain / CAR can be CD30. At least one target antigen of the multispecific antigen binding domain / CAR can be CD19. It is understood that the antigen binding domain (e.g., multispecific antigen binding domain) according to the present disclosure includes an antigen binding moiety that can bind to an antigen binding domain-specific target. For example, an antigen-binding domain capable of binding to CD30 and an antigen other than CD30 may comprise: (i) an antigen-binding portion capable of binding to CD30, and (ii) an antigen-binding portion capable of binding to a target antigen other than CD30. In further embodiments, an antigen-binding domain capable of binding to CD19 and an antigen other than CD19 may comprise: (i) an antigen-binding portion capable of binding to CD19, and (ii) an antigen-binding portion capable of binding to a target antigen other than CD19.

[0295] According to the present disclosure, CARs include a transmembrane domain. The transmembrane domain refers to a three-dimensional structure formed by a thermodynamically stable amino acid sequence in a biological membrane (such as a cell membrane). For the purposes of the present disclosure, the transmembrane domain can be an amino acid sequence that spans the cell membrane of a cell expressing CAR. The transmembrane domain of CAR is located between the antigen binding domain and the signaling domain of CAR. The transmembrane domain is used to anchor CAR to the cell membrane of a cell expressing CAR, so that the antigen binding domain is located in the extracellular space and the signaling domain is located in the cell. The transmembrane domain of CAR can be derived from the transmembrane region sequence of a cell membrane-bound protein (such as CD28, CD8, etc.).

[0296] As used herein, polypeptides, domains, and amino acid sequences that are "derived from" a reference polypeptide / domain / amino acid sequence have at least 60% sequence identity to the amino acid sequence of the reference polypeptide / domain / amino acid sequence, and preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Polypeptides, domains, and amino acid sequences that are "derived from" a reference polypeptide / domain / amino acid sequence preferably retain the functional and / or structural properties of the reference polypeptide / domain / amino acid sequence.

[0297] For example, an amino acid sequence derived from the intracellular domain of CD28 may comprise an amino acid sequence having 60% amino acid sequence identity, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the intracellular domain of CD28, e.g., as set forth in SEQ ID NO: 32. Furthermore, the amino acid sequence derived from the intracellular domain of CD28 preferably retains the functional properties of the amino acid sequence of SEQ ID NO: 32, i.e., the ability to activate CD28-mediated signaling.

[0298] The amino acid sequence of a given polypeptide or its domain can be retrieved from databases known to those skilled in the art, including GenBank, EMBL, and UniProt, or determined from nucleic acid sequences retrieved from databases known to those skilled in the art.

[0299] The membrane-spanning domain may comprise or consist of an amino acid sequence forming a hydrophobic alpha helix or beta-barrel structure. The amino acid sequence of the membrane-spanning domain of the present disclosure CAR may be the amino acid sequence of the membrane-spanning domain of a protein comprising a membrane-spanning domain, or may be derived from the amino acid sequence. The membrane-spanning domain is recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, protein information resource library, protein database, Ensembl and InterPro, and / or can be identified / predicted using, for example, amino acid sequence analysis tools such as TMHMM (Krogh et al., 2001, J Mol Biol 305: 567-580).

[0300] In some embodiments, the amino acid sequence of the transmembrane domain of the CAR of the present disclosure can be the amino acid sequence of the transmembrane domain of a protein expressed on the cell surface, or be derived from the amino acid sequence. In some embodiments, the protein expressed on the cell surface is a receptor or ligand, such as an immune receptor or ligand. In some embodiments, the amino acid sequence of the transmembrane domain can be or be derived from ICOS, ICOSL, CD86, CTLA-4, CD28, CD80, MHC class I α, MHC class II α, MHC Amino acid sequence of the transmembrane domain of one of class II beta, CD3ε, CD3δ, CD3γ, CD3-ζ, TCRα, TCRβ, CD4, CD8α, CD8β, CD40, CD40L, PD-1, PD-L1, PD-L2, 4-1BB, 4-1BBL, OX40, OX40L, GITR, GITRL, TIM-3, galectin-9, LAG3, CD27, CD70, LIGHT, HVEM, TIM-4, TIM-1, ICAM1, LFA-1, LFA-3, CD2, BTLA, CD160, LILRB4, LILRB2, VTCN1, CD2, CD48, 2B4, SLAM, CD30, CD30L, DR3, TL1A, CD226, CD112, and CD276. In some embodiments, the transmembrane domain is the amino acid sequence of the transmembrane domain of CD28, CD3-ζ, CD8α, CD8β or CD4 or is derived from the amino acid sequence. In some embodiments, the transmembrane domain is the amino acid sequence of the transmembrane domain of CD28 or is derived from the amino acid sequence.

[0301] In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:26.

[0302] In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:27.

[0303] In some embodiments, the transmembrane domain comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:28.

[0304] The chimeric antigen receptor of the present disclosure comprises a signal domain. The signal domain provides a sequence for initiating intracellular signal transduction for cells expressing CAR. The signal domain comprises the amino acid sequence required for activating immune cell function. The signal domain of CAR may comprise the amino acid sequence of the CD3-ζ intracellular domain, which provides an immunoreceptor tyrosine-based activation motif (ITAMs) for phosphorylation and activation of cells expressing CAR. Signal domains comprising other protein sequences containing ITAMs are also used in CARs, such as domains comprising FcγRI containing ITAM regions (Haynes et al., 2001, J Immunol 166(1): 182-187). CARs comprising a signal domain derived from the CD3-ζ intracellular domain are generally referred to as first generation CARs. To provide the costimulatory signals necessary to enhance immune cell activation and effector function, the signal domain of CARs generally also comprises the signal domain of costimulatory proteins (e.g., CD28, 4-1BB, etc.). CARs having a signal domain containing an additional costimulatory sequence are generally referred to as second generation CARs. In some cases, CARs are engineered to provide costimulation of different intracellular signal transduction pathways. For example, CD28 co-stimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, while 4-1BB co-stimulation triggers signal transduction through the TNF receptor-associated factor (TRAF) adaptor protein. Therefore, the signaling domain of CARs sometimes contains a co-stimulatory sequence derived from the signaling domain of more than one co-stimulatory molecule. CARs containing signaling domains with multiple co-stimulatory sequences are generally referred to as third-generation CARs.

[0305] The signaling domain comprises an ITAM-containing sequence. The ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). The ITAMs comprise the amino acid sequence YXXL / I (SEQ ID NO: 29), where "X" represents any amino acid. In ITAM-containing proteins, the sequence according to SEQ ID NO: 29 is typically separated by 6 to 8 amino acids; i.e., YXXL / I(X) 6-8YXXL / I (SEQ ID NO: 30). When a phosphate group is added to the tyrosine residue of an ITAM by a tyrosine kinase, a signaling cascade is initiated in the cell. In some embodiments, the signaling domain comprises one or more copies of the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments, the signaling domain comprises at least 1, 2, 3, 4, 5, or 6 copies of the amino acid sequence of SEQ ID NO: 30. In some embodiments, the signaling domain comprises at least 1, 2, or 3 copies of the amino acid sequence of SEQ ID NO: 30.

[0306] In some embodiments, the signaling domain comprises an amino acid sequence that is or is derived from an amino acid sequence containing an ITAM sequence of a protein having an ITAM amino acid sequence. In some embodiments, the signaling domain comprises an amino acid sequence that is or is derived from an amino acid sequence of the intracellular domain of one of CD3-ζ, FcγRI, CD3ε, CD3δ, CD3γ, CD79α, CD79β, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIV, or DAP12. In some embodiments, the signaling domain comprises an amino acid sequence that is the intracellular domain of CD3-ζ or is derived from the intracellular domain of CD3-ζ. In some embodiments, the signal domain comprises an amino acid sequence comprising, or consisting of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:31.

[0307] The signaling domain may further comprise one or more costimulatory sequences. The costimulatory sequence is an amino acid sequence that provides costimulation for cells expressing the CAR disclosed herein. After binding to the target antigen, costimulation can promote the proliferation and survival of cells expressing CAR, and can also promote the production, differentiation, cytotoxicity and memory formation of cytokines in cells expressing CAR. The molecular mechanism of T cell costimulation was reviewed in Chen and Flies' Nat Rev Immunol 13(4):227-242 published in 2013.

[0308] The costimulatory sequence can be the amino acid sequence of a costimulatory protein, or the amino acid sequence derived from a costimulatory protein. In some embodiments, the costimulatory sequence is the amino acid sequence of the intracellular domain of a costimulatory protein, or is derived from the amino acid sequence. When CAR binds to the target antigen, the costimulatory sequence provides costimulation for the cells expressing CAR, and this costimulation is the same as the costimulatory type provided by the costimulatory protein that produces the costimulatory sequence when it binds to its cognate ligand. For example, in the case where a signal domain containing a costimulatory sequence derived from CD28 is included in CAR, binding to the target antigen triggers signal transduction in cells expressing CAR, and this signal transduction is the same as the signal transduction type triggered when CD80 and / or CD86 binds to CD28. Therefore, the costimulatory sequence is able to transmit the costimulatory signal of the costimulatory protein that produces the costimulatory sequence.

[0309] In some embodiments, the costimulatory protein can be a member of the B7-CD28 superfamily (e.g., CD28, ICOS), or a member of the TNF receptor superfamily (e.g., 4-1BB, OX40, CD27, DR3, GITR, CD30, HVEM). In some embodiments, the costimulatory sequence is or is derived from the amino acid sequence of the intracellular domain of one of CD28, 4-1BB, ICOS, CD27, OX40, HVEM, CD2, SLAM, TIM-1, CD30, GITR, DR3, CD226, and LIGHT. In some embodiments, the costimulatory sequence is the intracellular domain of CD28 or the intracellular domain derived from CD28.

[0310] In some embodiments, the signaling domain comprises more than one non-overlapping costimulatory sequence. In some embodiments, the signaling domain comprises 1, 2, 3, 4, 5, or 6 costimulatory sequences. Multiple costimulatory sequences can be provided in series.

[0311] Can be by analyzing the relevant factors (for example, due to the signal transduction mediated by costimulatory protein, expression / activity up-regulated or down-regulated factor) etc. of the signal transduction mediated by given costimulatory protein to study whether a given amino acid sequence can start the signal transduction mediated by given costimulatory protein.Costimulatory protein up-regulates the expression of genes that promote cell growth, effector function and survival through a variety of transduction pathways.For example, CD28 and ICOS conduct signals by phosphatidylinositol 3 kinases (PI3K) and AKT, to up-regulate the expression of genes that promote cell growth, effector function and survival by NF-κB, mTOR, NFAT and AP1 / 2.CD28 also activates AP1 / 2 by CDC42 / RAC1, and activates ERK1 / 2 by RAS, and ICOS activates C-MAF.4-1BB, OX40 and CD27 raise TNF receptor-associated factors (TRAFs), and conduct signals by MAPK pathway and PI3K.

[0312] In some embodiments, the signaling domain comprises a costimulatory sequence that is CD28 or a costimulatory sequence derived from CD28. In some embodiments, the signaling domain comprises a costimulatory sequence that comprises, or consists of, an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32.

[0313] Kofler et al. describe a CD28 intracellular domain variant in Mol. Ther. (2011) Vol. 19: 760-767, in which the lck kinase binding site is mutated to reduce the induction of IL-2 production when CAR is connected, thereby minimizing the inhibition of CAR-T cell activity mediated by regulatory T cells. The amino acid sequence of the CD28 intracellular domain variant is shown in SEQ ID NO: 33. In some embodiments, the signaling domain comprises a costimulatory sequence comprising or consisting of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33.

[0314] In some embodiments, the signal domain comprises, or consists of, an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:34.

[0315] The chimeric antigen receptor of the present disclosure may include a hinge region or a spacer domain. The hinge region / spacer domain may provide a spacer between the antigen binding domain and the transmembrane domain and may act as a flexible linker. Such a domain may be a flexible domain or include a flexible domain, allowing the binding portion to face in different directions, which may be, for example, a CH1-CH2 hinge region derived from IgG. The presence, absence, and length of the hinge region have been shown to affect the function of CAR (e.g., reviewed in Dotti et al., Immunol Rev (2014) 257 (1)).

[0316] In some embodiments, the CAR comprises a hinge region comprising an amino acid sequence that is or is derived from the CH1-CH2 hinge region of human IgG1, a hinge region derived from CD8α as described in WO 2012 / 031744 A1, or a hinge region derived from CD28 as described in WO 2011 / 041093A1. In some embodiments, the CAR comprises a hinge region derived from the CH1-CH2 hinge region of human IgG1. In some embodiments, the hinge region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35 or 36, or consists of, or consists of.

[0317] In some embodiments, the CAR comprises a hinge region comprising or consisting of an amino acid sequence that is or is derived from the CH2-CH3 hinge region (i.e., Fc region) of human IgG1. In some embodiments, the hinge region comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37, or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0318] Hombach et al., Gene Therapy (2010) 17:1206-1213, describe a CH2-CH3 region variant for reducing activation of cells expressing FcγRs, such as monocytes and NK cells. The amino acid sequence of the CH2-CH3 region variant is set forth in SEQ ID NO:38. In some embodiments, the hinge region comprises or consists of an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:38.

[0319] In some embodiments, the hinge region comprises or consists of: an amino acid sequence that is or is derived from the CH1-CH2 hinge region of human IgG1; and an amino acid sequence that is or is derived from the CH2-CH3 hinge region (i.e., Fc region) of human IgG1. In some embodiments, the hinge region comprises or consists of an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39, or consists of.

[0320] According to the present disclosure, CARs may include additional, i.e., amino acid sequences in addition to those amino acid sequences forming antigen binding domains, optional hinge regions / Spacer domains, transmembrane domains, and signal domains. The CAR may also include a signal peptide (also referred to as a leader sequence or signal sequence). The signal peptide is typically composed of a sequence of 5-30 hydrophobic amino acids that form a single α helix. Secretory proteins and proteins expressed on the cell surface typically include signal peptides. The signal peptides of many proteins are known and recorded in databases such as GenBank, UniProt, and Ensembl, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011, Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008, Bioinformatics 24: 2172-2176).

[0321] The signal peptide may be present at the N-terminus of the CAR and may be present in newly synthesized CARs. The signal peptide provides efficient transport of the CAR to the cell surface. The signal peptide is removed by cleavage and is therefore not present in mature CARs expressed on the cell surface.

[0322] In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:40.

[0323] In some embodiments, the CAR comprises one or more connecting sequences between different domains (i.e., antigen binding domains, hinge regions, transmembrane domains, signaling domains). In some embodiments, the CAR comprises one or more connecting sequences between the subsequences of the domains (e.g., between the VH and VL of the antigen binding domains).

[0324] Linker sequences are well known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. The flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Flexible linkers are well known to those skilled in the art and several flexible linkers are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues. In some embodiments, the linker sequence contains at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and / or serine residues. In some embodiments, the linker sequence contains at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence contains or consists of glycine and serine residues. In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) copies of the sequence motif G3S (e.g., in tandem). In some embodiments, the linker sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) copies of the sequence motif G4S (e.g., in tandem). In some embodiments, the linker sequence is 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids in length. In some embodiments, the signal peptide comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0325] In some embodiments, the CAR of the present disclosure further comprises an epitope tag, for example, for ease of identification. Suitable tags are well known in the art and include, for example, His (e.g., (His) 6), c-Myc, GST, MBP, CBP, FLAG, HA, E, and C tags. Such tags can be provided at the N-terminus and / or C-terminus of the CAR.

[0326] In an embodiment of the present disclosure, the CAR of the present disclosure comprises or consists of the extracellular portion of the anti-CD30 HRS3 scFv domain linked to the CH2-CH3 derived spacer domain / hinge region of human IgG1, the transmembrane domain and intracellular domain of CD28, and the intracellular domain of CD3ζ.

[0327] In some embodiments, the CAR is selected from Hombach et al. Cancer Res. (1998) 58(6): 1116-9, Hombach et al. Gene Therapy (2000) 7: 1067-1075, Hombach et al. J Immunother. (1999) 22(6): 473-80, Hombach et al. Cancer Res. (2001) 61: 1976-1982, Hombach et al. J Immunol (2001) 167: 6123-6131, Savoldo et al. Blood (2007) 110(7): 2620-30, Koehler et al. Cancer Res. (2007) 67(5): 2265-2273, Di Stasi et al. Blood (2009) 113(25):6392-402, Hombach et al. Gene Therapy (2010) 17:1206-1213, Chmielewski et al. Gene Therapy (2011) 18:62-72, Kofler et al. Mol. Ther. (2011) 19(4):760-767, Gilham, Abken and Pule in Trends in Mol. Med. (2012) 18(7):377-384, Chmielewski et al. Gene An embodiment of a CD30-specific CAR described in WO 2021 / 245249 A1, WO 2021 / 222927 A1, WO 2021 / 222928 A1, WO 2021 / 222929 A1, WO 2015 / 028444 A1, or WO 2016 / 008973 A1, all of which are incorporated herein by reference in their entirety.

[0328] In some embodiments of the invention, the CAR comprises, or consists of:

[0329] an antigen binding domain comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24;

[0330] a hinge region comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39 or 49;

[0331] a transmembrane domain comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26; and

[0332] A signal domain comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34.

[0333] In some embodiments of the invention, the CAR comprises, or consists of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 41, 42, 50 or 51.

[0334] In some embodiments, the CAR is a CD19-specific CAR.

[0335] In some embodiments of the invention, the CAR comprises, or consists of:

[0336] an antigen binding domain comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67;

[0337] a hinge region comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 68;

[0338] a transmembrane domain comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26; and

[0339] A signal domain comprising, or consisting of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34.

[0340] In some embodiments of the invention, the CAR comprises, or consists of, an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 69 or 70.

[0341] The method for producing immune cells expressing CAR is well known to technicians. They generally involve modifying immune cells to express / include CAR, for example, by introducing nucleic acids encoding CAR into immune cells. According to methods well known to technicians, immune cells can be modified to include / express CAR as described herein or nucleic acids encoding CAR. The method generally includes nucleic acid transfer to achieve permanent (stable) or transient expression of the transferred nucleic acid. The method may also include culturing cells under conditions suitable for cell expression of CAR. In some embodiments, cells into which nucleic acids encoding CAR have been introduced are cultured to expand their number.

[0342] Suitable methods for modifying cells include the use of genetic engineering platforms, such as gamma-retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery and RNA transfection, such as Maus et al. in "Annu Rev Immunol" (2014) 32: 189-225, which is incorporated herein by reference in its entirety. The method also includes, for example, those described in Wang and Rivière's "Mol Ther Oncolytics." (2016) 3: 16015, which is incorporated herein by reference in its entirety. Suitable methods for introducing nucleic acid / vector into cells include introducing nucleic acid or vector encoding CAR into cells by transformation, transfection, electroporation or transduction (e.g., retroviral transduction), for example, as described herein. In some embodiments, the immune cells expressing CAR according to the present disclosure are produced substantially as described in Example 1.4 herein.

[0343] In particular, the present disclosure contemplates the production of CAR-expressing immune cells according to the methods described in WO 2021 / 245249 A1, WO 2021 / 222927 A1, WO 2021 / 222928 A1, and WO 2021 / 222929 A1, all of which are incorporated herein by reference in their entirety.

[0344] In some embodiments, the immune cells of the present disclosure are virus-specific immune cells. As used herein, "virus-specific immune cells" refer to immune cells that are specific for viruses. Virus-specific immune cells express / comprise receptors (preferably T cell receptors) that can recognize peptides of viral antigens (e.g., when presented by MHC molecules). Virus-specific immune cells can express / comprise such receptors due to the expression of endogenous nucleic acids encoding such antigen receptors, or express / comprise such receptors due to being engineered to express such receptors. The virus-specific immune cells preferably express / comprise TCRs that are specific for peptides of viral antigens.

[0345] In some embodiments, the immune cells are virus-specific T cells. The virus-specific T cells can respond to T cell-specific viral antigens or respond to cells containing / expressing viruses / antigens and display certain functional properties of T cells. In some embodiments, the properties are functional properties associated with effector T cells (e.g., cytotoxic T cells).

[0346] In some embodiments, virus-specific T cells can exhibit one or more of the following characteristics: cytotoxicity against cells containing / expressing T cell-specific viruses / viral antigens; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression and / or FAS ligand (FASL) expression in response to T cell-specific virus / viral antigen stimulation or exposure to cells containing / expressing T cell-specific viruses / viral antigens.

[0347] When presented by appropriate MHC molecules, virus-specific T cells express / comprise TCRs that can recognize peptides of the T cell-specific viral antigens. The virus-specific T cells can be CD4+ T cells and / or CD8+ T cells.

[0348] The virus to which the virus-specific immune cells are directed can be any virus. For example, the virus can be a double-stranded DNA virus (e.g., adenovirus, herpesvirus, poxvirus), a single-stranded RNA virus (e.g., parvovirus), a double-stranded RNA virus (e.g., reovirus), (+) a single-stranded RNA virus (e.g., picornavirus, togavirus), (-) a single-stranded RNA virus (e.g., orthomyxovirus, rhabdovirus), a single-stranded RNA-retrovirus (e.g., retrovirus) or a double-stranded DNA-retrovirus (e.g., hepadnavirus). In particular, the present disclosure contemplates viruses of the Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepatoviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae. In some embodiments, the virus is selected from Epstein-Barr virus, adenovirus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, hepatitis B virus, parvovirus B19, human astrovirus, norovirus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, forest encephalitis virus, rubella virus, hepatitis E virus, human immunodeficiency virus, influenza virus, Lassa virus, Crimean-Congo hemorrhagic fever virus, Hantavirus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, picornavirus, respiratory syncytial virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, Colorado tick-borne fever virus, and Banna virus.

[0349] In some embodiments, the virus is selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV) or varicella-zoster virus (VZV).

[0350] In some embodiments, the virus-specific immune cells can be specific for peptides / polypeptides of a virus selected from, for example, Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV) or herpes simplex virus (HSV).

[0351] Herein, T cells that are specific for viral antigens may be referred to as virus-specific T cells (VSTs). T cells that are specific for a particular viral antigen may be described as being specific for the related virus; for example, T cells that are specific for EBV antigens may be referred to as EBV-specific T cells, or "EBVSTs."

[0352] Thus, in some embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVST), adenovirus-specific T cells (AdVST), cytomegalovirus-specific C cells (CMVST), human papillomavirus (HPVST), influenza virus-specific T cells, measles virus-specific T cells, hepatitis B virus-specific T cells (HBVST), hepatitis C virus-specific T cells (HCVST), human immunodeficiency virus-specific T cells (HIVST), lymphocytic chorioplexitis virus-specific T cells (LCMVS), herpes simplex virus-specific T cells (BKVST), or varicella-zoster virus-specific T cells (VZVST). ST).

[0353] In some preferred embodiments, the virus-specific immune cells are specific for the peptides / polypeptides of the EBV antigens. In preferred embodiments, the virus-specific immune cells are Epstein-Barr virus-specific T cells (EBVST). "EBV-specific immune cells" as used herein refer to immune cells that are specific for Epstein-Barr virus (EBV). The EBV-specific immune cells express / comprise receptors (preferably T cell receptors) that can recognize EBV antigen peptides (for example, when presented by MHC molecules). The EBV-specific immune cells preferably express / comprise TCRs that are specific for the EBV antigen peptides presented by MHC I.

[0354] EBV-specific immune cells can be specific to any EBV antigen, for example, to EBV antigens as described herein. EBV-specific immune cell populations, or compositions comprising multiple EBV-specific immune cells, can include immune cells specific to one or more EBV antigens. In some embodiments, the EBV antigen is an EBV latent antigen, such as a III type latent antigen (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, BARF1, EBNA2, EBNA3A, EBNA3B, or EBNA3C), a II type latent antigen (e.g., EBNA1, EBNA-LP, LMP1, LMP2A, LMP2B, or BARF1), or a type I latent antigen (e.g., EBNA1 or BARF1). In some embodiments, the EBV antigen is an EBV lytic antigen, such as an immediate-early lytic antigen (e.g., BZLF1, BRLF1, or BMRF1), an early lytic antigen (e.g., BMLF1, BMRF1, BXLF1, BALF1, BALF2, BARF1, BGLF5, BHRF1, BNLF2A, BNLF2B, BHLF1, BLLF2, BKRF4, BMRF2, FU, or EBNA1-FUK), or a late lytic antigen (e.g., BALF4, BILF1, BILF2, BNFR1, BVRF2, BALF3, BALF5, BDLF3, or gp350).

[0355] The virus-specific immune cells can be produced by methods well known to those skilled in the art. The method may include presenting viral antigen peptides: in the presence of antigen presenting cells (APCs) presenting MHC complexes, culturing an immune cell population (e.g., a heterogeneous immune cell population, such as peripheral blood mononuclear cells; PBMCs) having antigen-specific receptors (TCRs), under conditions providing appropriate costimulation and signal amplification to cause activation and amplification. APCs can be infected with encoding viruses, or can contain / express viral antigens / peptides and present viral antigen peptides in the context of MHC molecules. Stimulation causes T cell activation and promotes cell division (proliferation), thereby producing and / or amplifying viral antigen-specific T cell populations. The process of T cell activation is well known to those skilled in the art and is described in detail, for example, in "Immunobiology" 5th edition (Janeway CA Jr, Travers P, Walport M et al., New York: Garland Science (2001), Chapter 8), which is incorporated herein by reference in its entirety.

[0356] Compared to the cell population before stimulation, the cell population obtained after stimulation is enriched with virus-specific T cells (that is, in the cell population after stimulation, virus-specific T cells are present at a higher frequency). In this way, virus-specific T cell populations are expanded / produced from heterogeneous T cell populations with different specificities. By stimulation and subsequent cell division, virus-specific T cell populations can be produced from single T cells. By stimulating and subsequently dividing cells in the virus-specific T cell population, existing virus-specific T cell populations can be expanded. In some embodiments, virus-specific immune cells of the present disclosure are produced substantially by as described in Example 1.4 herein.

[0357] Aspects and embodiments of the present disclosure are particularly related to EBV-specific immune cells. For example, methods for producing / amplifying EBV-specific immune cell populations are described in WO 2013 / 088114 A1, Lapteva and Vera's Stem Cells Int. (2011): 434392, Straathof et al.'s Blood (2005) 105(5): 1898-1904, WO 2017 / 202478 A1, WO 2018 / 052947 A1, and WO 2020 / 214479 A1, all of which are incorporated herein by reference in their entirety.

[0358] In particular, the present disclosure contemplates the production of virus-specific immune cells (e.g., EBV-specific immune cells) according to the methods described in WO 2021 / 222927 A1, WO 2021 / 222928 A1, and WO 2021 / 222929 A1, all of which are incorporated herein by reference in their entirety. Aspects and embodiments of the present disclosure relate to virus-specific immune cells expressing CAR. In some embodiments, the immune cells are EBV-specific immune cells expressing CAR. In some embodiments, the immune cells are virus-specific immune cells expressing CD30-specific CAR. In some embodiments, the immune cells are EBV-specific immune cells expressing CD30-specific CAR. In some embodiments, the immune cells are virus-specific immune cells expressing CD19-specific CAR. In some embodiments, the immune cells are EBV-specific immune cells expressing CD19-specific CAR. Methods for producing such cells are known in the art and are described in WO 2021 / 222927 A1, WO 2021 / 222928 A1 and WO 2021 / 222929 A1, all of which are incorporated herein by reference in their entirety.

[0359] In some embodiments, the immune cells according to the present disclosure are activated T cells (ATC). As used herein, "activated T cells" or "ATC" refer to T cells whose signaling is activated by the CD3-TCR complex. In some embodiments, the activated T cells have a wide range of specificities (i.e., the activated T cells respond to a variety of different antigens).

[0360] In some embodiments, the activated T cells can exhibit certain functional properties of T cells. In some embodiments, the properties are functional properties associated with effector T cells, such as cytotoxic T cells. In some embodiments, activated T cells can exhibit one or more of the following properties: proliferation, expression of one or more characteristic surface markers of T cell activation (e.g., CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, CD134), expression of growth factors (e.g., IL-2), IFNγ expression, CD107a expression, TNFα expression, GM-CSF expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression.

[0361] The activated T cells may be CD4+ T cells and / or CD8+ T cells.

[0362] The activated T cells can be produced by methods well known to those skilled in the art. These methods may include stimulating peripheral blood mononuclear cells (PBMCs) with agonistic anti-CD3 and agonistic anti-CD28 antibodies in the presence of one or more cytokines (e.g., IL-2, IL-7, IL-15) to nonspecifically activate T cells in vitro. In some embodiments, the activated T cells can be produced as described in Example 1.4 herein.

[0363] In some embodiments, cells modified to increase the expression or activity of SERPIN B9 are not toxic (i.e., substantially not toxic) to allogeneic cells (e.g., allogeneic T cells). In some embodiments, cells modified to increase the expression or activity of SERPIN B9 are not toxic to allogeneic T cells. In some embodiments, cells modified to increase the expression or activity of SERPIN B9 are not toxic to allogeneic NK cells.

[0364] In some embodiments, the "allogeneic cells" are cells that are HLA mismatched with the test cells (e.g., cells modified to increase the expression or activity of SERPINB9). In some embodiments, the "allogeneic cells" are PBMCs that are HLA mismatched with the test cells. In some embodiments, the "allogeneic cells" are alloreactive T cells that are HLA mismatched with the test cells, such as alloreactive T cells generated by activating PBMCs from HLA mismatched donors with CD3 and CD28. In some embodiments, the "allogeneic cells" do not include tumor / disease cells.

[0365] As used herein, a cell that is "HLA-mismatched" relative to a reference cell / subject can be: (i) <8 / 8 (i.e., 0 / 8, 1 / 8, 2 / 8, 3 / 8, 4 / 8, 5 / 8, 6 / 8, or 7 / 8) matched on HLA-A, -B, -C, and -DRB1; or (ii) <10 / 10 (i.e., 0 / 10, 1 / 10, 2 / 10, 3 / 8, 4 / 8, 5 / 8, 6 / 8, or 7 / 8) matched on HLA-A, -B, -C, -DRB1, and -DQB1. or (iii) a match of <12 / 12 (i.e., 0 / 12, 1 / 12, 2 / 12, 3 / 12, 4 / 12, 5 / 12, 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, or 11 / 12) on HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1. As used herein, a cell that is "HLA matched" relative to a reference cell / subject can be: (i) 8 / 8 matched at HLA-A, -B, -C, and -DRB1; or (ii) 10 / 10 matched at HLA-A, -B, -C, -DRB1, and -DQB1; or (iii) 12 / 12 matched at HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1.

[0366] In some embodiments, cells that are "not toxic" or "substantially not toxic" to a particular type of cell (e.g., allogeneic T cells / NK cells) may have a level of cell killing / cytolysis / cytotoxicity to the particular type of cell that is similar to (e.g., ≥0.5-fold and ≤2-fold, e.g., ≥0.55-fold and ≤1.9-fold, ≥0.6-fold and ≤1.8-fold, ≥0.65-fold and ≤1.7-fold, ≥0.7-fold and ≤1.6-fold, ≥0.75-fold and ≤1.5-fold, ≥0.8-fold and ≤1.4-fold, ≥0.85-fold and ≤1.3-fold, ≥0.9-fold and ≤1.2-fold, or ≥0.95-fold and ≤1.1-fold) or lower than (i.e., <1-fold) the level exhibited by a reference cell type that is known not to kill / cause cell lysis / exhibit cytotoxicity to the particular type of cell in the same assay.

[0367] The level of killing of allogeneic cells can be assessed using a suitable test. Suitable tests for assessing the level of killing of allogeneic cells include, for example, mixed lymphocyte reaction (MLR) assays, such as those described in Example 1.5. Suitable tests for assessing the level of killing of allogeneic cells include, for example, in vivo assays, such as those described in Example 1.10. In some embodiments, allogeneic cells or allogeneic cell subtypes are identified by expression or non-expression of one or more cell markers CD3, CD4, CD8, HLA-A2 and / or HLA-A3, TCRαβ, CD45, CD19, CD30.

[0368] Properties of cells containing / expressing the polypeptides, nucleic acids and vectors of the present disclosure

[0369] Cells with increased SERPINB9 expression or activity (e.g., cells comprising or expressing a nucleic acid / vector of the present disclosure, or cells treated with an agent of the present disclosure for increasing SERPINB9 expression or activity) may have one or more of the following:

[0370] reduced activity of serine proteases (e.g., granzyme B);

[0371] decreased caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity;

[0372] Increased resistance to (and / or decreased sensitivity to) cell killing by serine proteases (e.g., granzyme B);

[0373] Increased resistance to (and / or decreased sensitivity to) killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells);

[0374] Reduced killing rate of cells expressing serine proteases (e.g., cells expressing granzyme B; e.g., effector immune cells);

[0375] Increased resistance to (and / or decreased sensitivity to) death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis);

[0376] have similar proliferation / expansion in vitro (e.g. when cultured alone, i.e. as a monoculture);

[0377] similar effector activity (i.e., in embodiments where the cell is an effector immune cell);

[0378] Increased persistence / survival / proliferation in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells);

[0379] increased persistence / survival / proliferation in allogeneic subjects;

[0380] reduced rejection when administered as an allograft;

[0381] Increased anti-cancer activity in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells);

[0382] increased anti-cancer activity in allogeneic subjects;

[0383] increased persistence / survival / proliferation under conditions of chronic antigen exposure; or

[0384] Similar expression of one or more markers of immune cell exhaustion (ie, in embodiments where the cells are effector immune cells).

[0385] It will be appreciated that a given cell may exhibit a variety of characteristics as described in the preceding paragraph. According to the preceding paragraph, the level of a given characteristic that is "reduced," "increased," or "similar" can be the level of related characteristics typically displayed relative to that type of cell (e.g., a reduction or increase relative to a reference value of the level of related characteristics of that type of cell). For cells comprising or expressing nucleic acids / vectors of the present invention, the level of a given characteristic described in the preceding paragraph that is "reduced," "increased," or "similar" can be the level of related characteristics displayed relative to equivalent cells that do not comprise the nucleic acid / vector. For cells treated with an agent disclosed herein for increasing SERPINB9 expression or activity, the level of a given characteristic described in the preceding paragraph that is "reduced," "increased," or "similar" can be the level of related characteristics displayed relative to equivalent cells that are not treated with the agent. The characteristics determined in the preceding paragraph can be evaluated as described above.

[0386] Relative to a reference level, the level of a given property that is "increased" can be greater than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, relative to a reference level. Relative to a reference level, the level of a given property that is "reduced" can be less than 1 times the reference level, for example, one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times. Relative to a reference level, the level of a given characteristic that is "similar" can be one of ≥0.5-fold and ≤2-fold, such as ≥0.55-fold and ≤1.9-fold, ≥0.6-fold and ≤1.8-fold, ≥0.65-fold and ≤1.7-fold, ≥0.7-fold and ≤1.6-fold, ≥0.75-fold and ≤1.5-fold, ≥0.8-fold and ≤1.4-fold, ≥0.85-fold and ≤1.3-fold, ≥0.9-fold and ≤1.2-fold, or ≥0.95-fold and ≤1.1-fold, of the reference level. The effector activity of effector immune cells can be assessed using an appropriate test for effector activity. In some embodiments, the effector activity is selected from: cytotoxicity against cells containing / expressing an antigen, wherein the effector immune cell comprises a molecule (e.g., TCR, CAR) for directing the activity of the effector immune cell against cells containing / expressing the antigen; proliferation, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression and / or FAS ligand (FASL) expression in response to stimulation of the antigen or cells containing / expressing the antigen, wherein the effector immune cell comprises a molecule (e.g., TCR, CAR) for directing the activity of the effector immune cell against cells containing / expressing the antigen. For example, if the cell is an effector immune cell comprising a CAR specific for CD30, the effector activity can be selected from: cytotoxicity against cells comprising / expressing CD30; proliferation in response to stimulation with CD30 or cells comprising / expressing CD30, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression and / or FAS ligand (FASL) expression.As another example, if the cell is an effector immune cell that comprises a TCR specific for an MHC-peptide complex of a peptide comprising an EBV antigen, the effector activity can be selected from: cytotoxicity against cells comprising / expressing the MHC-peptide complex; proliferation in response to stimulation with the MHC-peptide complex or cells comprising / expressing the MHC-peptide complex, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granzyme expression, granulysin expression and / or FAS ligand (FASL) expression.

[0387] The gene and / or protein expression of the immune cell (e.g., effector immune cell) exhaustion marker of a given cell can be as described herein (mRNA levels are measured by quantitative real-time PCR (qRT-PCR); assessed by antibody-based methods, such as Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, or ELISAPOT). Immune cell exhaustion markers include, for example, immune checkpoint molecules (e.g., PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, and BTLA), CD160, and CD244. In a preferred embodiment, the immune cell exhaustion marker is selected from PD-1, LAG-3, and TIM-3. In some embodiments, the cell surface expression of one or more immune cell exhaustion markers can be assessed, for example, by flow cytometry.

[0388] In some embodiments, in the same assay, cells with increased SERPINB9 expression or activity (e.g., cells comprising or expressing a nucleic acid / vector of the present disclosure, or cells treated with an agent of the present disclosure for increasing SERPINB9 expression or activity) exhibit serine protease (e.g., granzyme B) activity levels / caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity levels / sensitivity to cell killing by serine proteases (e.g., granzyme B) / sensitivity to killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells) / sensitivity to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) The sensitivity to immunoglobulin / antigen / antigen-derived proteins of the present invention / the killing rate of cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / the level of rejection when administered as an allograft is less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the level of the relevant property typically exhibited by that type of cells (e.g., a reference value for the level of the relevant property of that type of cells).

[0389] In some embodiments, in the same assay, the serine protease (e.g., granzyme B) activity level of cells containing or expressing a nucleic acid / vector of the present disclosure / caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity level / sensitivity to cell killing by a serine protease (e.g., granzyme B) / sensitivity to killing by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / sensitivity to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / sensitivity to cell killing by a serine protease (e.g., granzyme B; e.g., effector immune cells) / sensitivity to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / sensitivity to cell killing by a serine protease (e.g., granzyme B; e.g., effector immune cells) The killing rate of cells (such as effector immune cells) / the level of rejection when administered as an allograft is less than 1 times the level of the relevant property displayed by equivalent cells not comprising the nucleic acid / vector, for example, one of ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times, ≤0.05 times, or ≤0.01 times.

[0390] In some embodiments, in the same assay, the serine protease (e.g., granzyme B) activity level / caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity level / sensitivity to cell killing by serine proteases (e.g., granzyme B) / sensitivity to killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells) / sensitivity to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / sensitivity to expression of serine proteases (e.g., granzyme B; e.g., effector immune cells) of cells treated with an agent for increasing the expression or activity of SERPINB9 of the present invention are as follows: The killing rate of cells that are capable of killing an immune cell (e.g., granzyme B; e.g., effector immune cells) / level of rejection when administered as an allograft is less than 1-fold, e.g., one of ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the relevant property exhibited by equivalent cells not treated with the agent.

[0391] In some embodiments, in the same assay, cells with increased SERPINB9 expression or activity (e.g., cells comprising or expressing a nucleic acid / vector of the present disclosure, or cells treated with an agent of the present disclosure for increasing SERPINB9 expression or activity) exhibit resistance to cell killing by serine proteases (e.g., granzyme B) / resistance to killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells) / resistance to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / persistence, survival, or proliferation in the presence of allogeneic effector immune cells / persistence, survival, or proliferation in allogeneic subjects / in allogeneic cells. The anti-cancer activity in the presence of somatic effector immune cells / the anti-cancer activity in allogeneic subjects / the persistence, survival or proliferation level under chronic antigen exposure conditions is more than 1 times the level of the relevant property normally displayed by this type of cells (e.g., a reference value of the level of the relevant property of this type of cells), for example, ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times.

[0392] In some embodiments, in the same assay, the resistance of cells comprising or expressing a nucleic acid / vector of the present disclosure to cell killing by a serine protease (e.g., granzyme B) / resistance to killing by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / persistence, survival, or proliferation in the presence of allogeneic effector immune cells / resistance to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / persistence, survival, or proliferation in allogeneic subjects / anti-cancer activity in the presence of allogeneic effector immune cells / anti-cancer activity in allogeneic subjects is determined. The level of anti-cancer activity / persistence, survival or proliferation under conditions of chronic antigen exposure is greater than 1-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold or ≥10-fold, relative to the level of the relevant property exhibited by an equivalent cell not comprising the nucleic acid / vector.

[0393] In some embodiments, in the same assay, cells treated with an agent for increasing the expression or activity of SERPINB9 of the present disclosure exhibit resistance to cell killing by serine proteases (e.g., granzyme B) / resistance to killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells) / resistance to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / persistence, survival, or proliferation in allogeneic subjects / anti-cancer activity in the presence of allogeneic effector immune cells / in allogeneic subjects. The anti-cancer activity / level of persistence, survival or proliferation under conditions of chronic antigen exposure is greater than one-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold or ≥10-fold, the level of the relevant property exhibited by equivalent cells not treated with the agent.

[0394] In some embodiments, cells with increased SERPINB9 expression or activity (e.g., cells comprising or expressing a nucleic acid / vector of the present disclosure, or cells treated with an agent disclosed herein for increasing the expression or activity of SERPINB9) exhibit increased resistance (and / or decreased sensitivity) to cell killing by a serine protease (e.g., granzyme B) and increased resistance (and / or decreased sensitivity) to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis). In some embodiments, cells with increased SERPINB9 expression or activity (e.g., cells comprising or expressing a nucleic acid / vector of the present disclosure, or cells treated with an agent disclosed herein for increasing the expression or activity of SERPINB9) do not exhibit increased resistance (and / or decreased sensitivity) to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis).

[0395] In some embodiments, cells treated with an agent disclosed herein for increasing the expression or activity of SERPINB9 survive / persist for at least 3 days (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days) in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells). In some embodiments, cells treated with an agent disclosed herein for increasing the expression or activity of SERPINB9 survive / persist for at least 11 days (e.g., at least 11, 12, 13, 14, or 15 days) in an allogeneic subject.

[0396] In some embodiments, in the same assay, the proliferation or expansion level / effector activity level / expression level of one or more immune cell exhaustion markers of cells with increased SERPINB9 expression or activity (e.g., cells comprising or expressing a nucleic acid / vector of the present disclosure, or cells treated with an agent of the present disclosure for increasing the expression or activity of SERPINB9) is ≥0.5 times and ≤2 times, such as ≥0.55 times and ≤1.9 times, ≥0.6 times and ≤1.8 times, ≥0.65 times and ≤1.7 times, ≥0.7 times and ≤1.6 times, ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times, of the relevant characteristic level normally exhibited by cells of this type (e.g., the reference value of the relevant characteristic level of cells of this type).

[0397] In some embodiments, the level of proliferation or expansion / level of effector activity / level of expression of one or more immune cell exhaustion markers of cells comprising or expressing a nucleic acid / vector of the present disclosure is ≥0.5-fold and ≤2-fold, e.g., ≥0.55-fold and ≤1.9-fold, ≥0.6-fold and ≤1.8-fold, ≥0.65-fold and ≤1.7-fold, ≥0.7-fold and ≤1.6-fold, ≥0.75-fold and ≤1.5-fold, ≥0.8-fold and ≤1.4-fold, ≥0.85-fold and ≤1.3-fold, ≥0.9-fold and ≤1.2-fold, ≥0.95-fold and ≤1.1-fold, of the level of the relevant property displayed by an equivalent cell that does not comprise the nucleic acid / vector in the same assay.

[0398] In some embodiments, in the same assay, the level of proliferation or expansion / level of effector activity / level of expression of one or more immune cell exhaustion markers of cells treated with an agent for increasing the expression or activity of SERPINB9 of the present disclosure is ≥0.5 times and ≤2 times the level of the relevant property displayed by equivalent cells not treated with the agent, for example, ≥0.55 times and ≤1.9 times, ≥0.6 times and ≤1.8 times, ≥0.65 times and ≤1.7 times, ≥0.7 times and ≤1.6 times, ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times.

[0399] The present disclosure relates particularly to SERPINB9 variants, nucleic acids / vectors encoding such SERPINB9 variants, and cells comprising such SERPINB9 variants / nucleic acids / vectors. In some embodiments, such cells may have one or more of the following properties relative to the level of the relevant property exhibited by an equivalent cell comprising a wild-type human SERPINB9 polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 1), or an equivalent cell containing / expressing a nucleic acid / vector encoding a wild-type human SERPINB9 polypeptide (e.g., having the amino acid sequence of SEQ ID NO: 1):

[0400] reduced activity of serine proteases (e.g., granzyme B);

[0401] decreased caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity;

[0402] Increased resistance to (and / or decreased sensitivity to) cell killing by serine proteases (e.g., granzyme B);

[0403] Increased resistance to (and / or decreased sensitivity to) killing by cells expressing serine proteases (e.g., granzyme B; e.g., effector immune cells);

[0404] Reduced killing rate of cells expressing serine proteases (e.g., cells expressing granzyme B; e.g., effector immune cells);

[0405] Increased resistance to (and / or decreased sensitivity to) death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis);

[0406] have similar proliferation / expansion in vitro (e.g. when cultured alone, i.e. as a monoculture);

[0407] similar effector activity (i.e., in embodiments where the cell is an effector immune cell);

[0408] Increased persistence / survival / proliferation in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells);

[0409] increased persistence / survival / proliferation in allogeneic subjects;

[0410] reduced rejection when administered as an allograft;

[0411] Increased anti-cancer activity in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells);

[0412] increased anti-cancer activity in allogeneic subjects;

[0413] increased persistence / survival / proliferation under conditions of chronic antigen exposure; or

[0414] Similar expression of one or more markers of immune cell exhaustion (ie, in embodiments where the cells are effector immune cells).

[0415] It will be appreciated that a given cell may exhibit a variety of the characteristics listed in the preceding paragraph. The characteristics identified in the preceding paragraph may be assessed as described above.

[0416] In some embodiments, in the same assay, cells comprising a SERPINB9 variant of the present disclosure (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7) have less serine protease (e.g., granzyme B) activity level / caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity level / sensitivity to cell killing by a serine protease (e.g., granzyme B) / sensitivity to killing by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / sensitivity to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / killing rate of cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / rejection level when administered as an allograft than cells comprising a wild-type human SERPINB9 variant (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7). NO:1) is 1-fold, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, as compared to the level of the relevant property exhibited by the same type of cell compared to the amino acid sequence of NO:1). In some embodiments, in the same assay, cells comprising / expressing a nucleic acid / vector encoding a SERPINB9 variant of the present disclosure (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7) have lower levels of serine protease (e.g., granzyme B) activity / levels of caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) activity / sensitivity to cell killing by a serine protease (e.g., granzyme B) / sensitivity to killing by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / sensitivity to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / killing rate of cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / levels of rejection when administered as an allograft than cells comprising / expressing a nucleic acid / vector encoding a wild-type human SERPINB9 (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7). NO:1) is expressed as 1-fold, for example, ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to the level of the relevant property exhibited by the same type of cell compared to ...In some embodiments, in the same assay, the resistance of cells comprising a SERPINB9 variant of the present disclosure (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7) to cell killing by a serine protease (e.g., granzyme B) / resistance to killing by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / resistance to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / persistence, survival, or proliferation in allogeneic subjects / anti-cancer activity in the presence of allogeneic effector immune cells / anti-cancer activity in allogeneic subjects / persistence, survival, or proliferation levels under chronic antigen exposure conditions are greater than those of cells comprising a wild-type human SERPINB9 variant (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7) in the same assay. NO:1) is more than 1-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, as compared to the level of the relevant property displayed by the same type of cell. In some embodiments, in the same assay, the resistance of cells comprising / expressing a nucleic acid / vector encoding a SERPINB9 variant of the present disclosure (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7) to cell killing by a serine protease (e.g., granzyme B) / resistance to killing by cells expressing a serine protease (e.g., granzyme B; e.g., effector immune cells) / resistance to death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis) / persistence, survival, or proliferation in allogeneic subjects / anti-cancer activity in the presence of allogeneic effector immune cells / anti-cancer activity in allogeneic subjects / persistence, survival, or proliferation levels under chronic antigen exposure conditions is greater than that of cells comprising / expressing a nucleic acid / vector encoding a wild-type human SERPINB9 variant (e.g., having the amino acid sequence of SEQ ID NO: 5, 6, or 7) in the same assay. NO:1) is expressed as a nucleic acid / vector having an amino acid sequence as described herein (e.g., an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein, wherein the amino acid sequence as described herein is expressed as a nucleic acid / vector having an amino acid sequence as described herein,

[0417] In some embodiments, in the same assay, the level of proliferation or expansion / level of effector activity / level of expression of one or more immune cell exhaustion markers of cells comprising a SERPINB9 variant of the present disclosure (e.g., having an amino acid sequence of SEQ ID NO: 5, 6 or 7) is ≥0.5 times and ≤2 times the level of the relevant property displayed by the same type of cells comprising wild-type human SERPINB9 (e.g., having an amino acid sequence of SEQ ID NO: 1), for example, ≥0.55 times and ≤1.9 times, ≥0.6 times and ≤1.8 times, ≥0.65 times and ≤1.7 times, ≥0.7 times and ≤1.6 times, ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times.

[0418] In some embodiments, in the same assay, the level of proliferation or expansion / level of effector activity / level of expression of one or more immune cell exhaustion markers of cells comprising / expressing a nucleic acid / vector encoding a SERPINB9 variant of the present disclosure (e.g., having an amino acid sequence of SEQ ID NO: 5, 6 or 7) is ≥0.5 times and ≤2 times, for example, ≥0.55 times and ≤1.9 times, ≥0.6 times and ≤1.8 times, ≥0.65 times and ≤1.7 times, ≥0.7 times and ≤1.6 times, ≥0.75 times and ≤1.5 times, ≥0.8 times and ≤1.4 times, ≥0.85 times and ≤1.3 times, ≥0.9 times and ≤1.2 times, ≥0.95 times and ≤1.1 times the level of the relevant property displayed by the same type of cells comprising / expressing a nucleic acid / vector encoding wild-type human SERPINB9 (e.g., having an amino acid sequence of SEQ ID NO: 1).

[0419] Specific exemplary cells

[0420] The present disclosure contemplates the following specific exemplary cells:

[0421] (1) An effector immune cell (eg, a T cell or a NK cell) comprising an exogenous nucleic acid encoding a SERPINB9 polypeptide.

[0422] (2) An effector immune cell (e.g., a T cell or a NK cell), comprising:

[0423] (i) an exogenous nucleic acid encoding a SERPINB9 polypeptide; and

[0424] (ii) a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain that binds to CD30.

[0425] (3) An effector immune cell (e.g., a T cell or a NK cell), comprising:

[0426] (i) an exogenous nucleic acid encoding a SERPINB9 polypeptide; and

[0427] (ii) a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain that binds to CD19.

[0428] (4) A virus-specific T cell (eg, an Epstein Barr virus (EBV)-specific T cell) comprising an exogenous nucleic acid encoding a SERPINB9 polypeptide.

[0429] (5) A virus-specific T cell (e.g., an Epstein Barr virus (EBV)-specific T cell), comprising:

[0430] (i) an exogenous nucleic acid encoding a SERPINB9 polypeptide; and

[0431] (ii) a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain that binds to CD30.

[0432] (6) A virus-specific T cell (e.g., an Epstein Barr virus (EBV)-specific T cell), comprising:

[0433] (i) an exogenous nucleic acid encoding a SERPINB9 polypeptide; and

[0434] (ii) a nucleic acid encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain that binds to CD19.

[0435] Composition

[0436] The present invention also provides a composition comprising the polypeptide, nucleic acid, expression vector and cell of the present invention.

[0437] The polypeptides, nucleic acids, expression vectors, and cells of the present invention can be formulated into pharmaceutical compositions or medicaments for use in therapeutic and / or preventive methods, and can include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The polypeptides, nucleic acids, expression vectors, and cells of the present invention can be formulated for diagnostic and / or prognostic applications.

[0438] The compositions of the present invention may include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, glucose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silicon dioxide, stearic acid, vegetable stearic acid), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium oxide).

[0439] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc. that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human subject) without producing excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. According to the present invention, each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of the composition must also be "acceptable," meaning compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings or sweeteners can be found in standard pharmaceutical texts, for example, Remington's "The Science and Practice of Pharmacy" (ed. A. Adejare), 23rd edition (2020), Academic Press.

[0440] The composition can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal administration. In some embodiments, the pharmaceutical composition / medicament can be formulated for administration by injection or infusion, or by ingestion.

[0441] Suitable preparations can include the relevant components in a sterile or isotonic medium. Medicines and pharmaceutical compositions can be prepared in liquid (including gel) forms. Liquid preparations can be formulated to be administered to a selected area of ​​the human or animal body by injection or infusion (e.g., by catheter).

[0442] In some embodiments, the composition is formulated for injection or infusion, eg, injection or infusion into a target vessel or tissue / organ.

[0443] The present invention also provides a method for preparing a pharmaceutical composition, which preparation method may include one or more steps selected from the following groups: producing the polypeptide, nucleic acid, expression vector or cell described herein; isolating the polypeptide, nucleic acid, expression vector or cell described herein; and / or mixing the polypeptide, nucleic acid, expression vector or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[0444] Therapeutic and prophylactic applications according to the present disclosure

[0445] Immune cells modified to increase the expression or activity of SERPINB9 as described herein, and pharmaceutical compositions comprising such cells, can be used in therapeutic and / or preventive methods.

[0446] The present disclosure provides a method for treating / preventing a disease / disorder in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the immune cells or pharmaceutical composition of the present disclosure.

[0447] The present disclosure also provides methods for using the immune cells or pharmaceutical compositions of the present disclosure in treating / preventing diseases. The present disclosure also provides methods for using the immune cells or pharmaceutical compositions of the present disclosure in preparing a medicament for treating / preventing a disease / disorder.

[0448] It will be understood that these methods generally comprise administering to a subject a population of immune cells of the present disclosure. In some embodiments, the immune cells may be administered in the form of a pharmaceutical composition comprising such cells.

[0449] In particular, the use of the immune cells of the present disclosure in methods for treating / preventing diseases / conditions by adoptive cell transfer (ACT) is contemplated. The disease / condition to be treated / prevented can be any disease / condition that would benefit from the adoptive transfer of immune cells. In some embodiments, the disease / condition to be treated / prevented by adoptive cell transfer can be, for example, a T cell dysfunction, cancer, an infectious disease, or an autoimmune disease.

[0450] The immune cells and compositions of the present disclosure can be used in methods involving allogeneic transplantation, for example, for treating / preventing a disease / disorder in a subject.

[0451] As used herein, "allogeneic transplantation" refers to the transplantation of cells, tissues, or organs that are genetically different from the recipient into a recipient. The cells, tissues, or organs can be from or derived from cells, tissues, or organs from a donor subject that is genetically different from the recipient. Allogeneic transplantation is distinct from autologous transplantation, which refers to the transplantation of cells, tissues, or organs from a donor that is genetically identical to the recipient.

[0452] The immune cells and compositions disclosed herein can be used to reduce / prevent the deleterious effects of alloreactive immune responses (particularly T and / or NK cell-mediated alloreactive immune responses) on allogeneic transplants. Following adoptive transfer, the immune cells disclosed herein are less susceptible / more resistant to the recipient's T and / or NK cell-mediated alloreactive immune responses, thereby exhibiting higher persistence / survival rates in the recipient subject following transfer, as well as superior therapeutic / preventive effects.

[0453] In particular, the immune cells and compositions of the present disclosure are contemplated for use in the preparation and administration of "off-the-shelf" materials for use in therapeutic and prophylactic methods involving the administration of allogeneic materials.

[0454] It will be appreciated that adoptive transfer of allogeneic immune cells is a form of allogeneic transplantation. In some embodiments, the immune cells are used as therapeutic / prophylactic agents in methods for treating / preventing a disease / disorder by allogeneic transplantation.

[0455] The administration of the immune cells and compositions of the present disclosure preferably uses a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to show a therapeutic or prophylactic benefit to the subject. The actual dosage, rate of administration, and time course will depend on the nature and severity of the disease / disorder and the specific ingredients administered. Therapeutic prescriptions, such as dosage determinations, are the responsibility of practicing physicians and other doctors, and generally take into account the disease / disorder to be treated, the individual condition of the subject, the delivery site, the method of administration, and other factors known to practitioners. Examples of the above techniques and experimental procedures can be found in Remington's "The Science and Practice of Pharmacy" (Edited by A. Adejare), 23rd edition (2020), Academic Press.

[0456] Multiple dosages can be provided. Multiple dosages can be separated by predetermined time intervals, and the time interval can select any one of the following time periods: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours or longer, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days, or 1, 2, 3, 4, 5 or 6 months. For example, the drug can be administered once every 7, 14, 21 or 28 days (plus or minus 3, 2 or 1 day).

[0457] In some embodiments, the treatment may also include other therapeutic or preventive interventions, such as chemotherapy, immunotherapy, radiotherapy, surgery, vaccination, and / or hormone therapy. Such other therapeutic or preventive interventions may occur before, during, and / or after the treatment encompassed by the present disclosure, and the implementation of the other therapeutic or preventive interventions may be through different routes of administration as a treatment regimen of the present disclosure.

[0458] Drug administration can be alone or in combination with other treatments, either simultaneously or sequentially depending on the disease to be treated.The immune cells and compositions described herein can be administered simultaneously or sequentially with another therapeutic intervention.

[0459] Simultaneous administration refers to the administration of two or more therapeutic interventions together, for example, as a pharmaceutical composition containing both active agents (i.e., in a combined preparation), or immediately after each other, and optionally by the same route of administration, for example, to the same artery, vein or other blood vessel.

[0460] Sequential administration refers to the administration of one therapeutic intervention and, after a given time interval, the separate administration of one or more further therapeutic interventions. It is not required that these treatments be administered by the same route, although this is the case in certain embodiments. The time interval can be any time interval.

[0461] For example, adoptive transfer of immune cells is described in Kalos and June (2013), Immunity 39(1):49-60; and Davis et al. (2015), Cancer J. 21(6):486-491, both of which are incorporated herein by reference in their entirety. A skilled artisan will be able to determine appropriate reagents and procedures for adoptive transfer of immune cells based on the present disclosure, for example, with reference to Dai et al., 2016 J Nat Cancer Inst 108(7):djv439, which is incorporated herein by reference in its entirety.

[0462] Some aspects and embodiments of the present disclosure include modifying immune cells to increase the expression or activity of SERPINB9.

[0463] In some embodiments, the method comprises:

[0464] (a) modifying immune cells to increase the expression or activity of SERPINB9, and

[0465] (b) administering the modified immune cells to a subject.

[0466] As described above, such modification may include treating the cell with an agent that increases SERPINB9 expression or activity. In some embodiments, modifying the immune cell to increase SERPINB9 expression or activity includes introducing a nucleic acid encoding a SERPINB9 polypeptide into the immune cell.

[0467] In some embodiments, the immune cell of (a) comprises a molecule that directs the immune cell against the activity of cells comprising / expressing a given target antigen, such as a disease-associated antigen.

[0468] In some embodiments, the immune cell of (a) comprises a TCR specific for an MHC-peptide complex comprising a disease-associated antigenic peptide. In some embodiments, the immune cell of (a) is a virus-specific immune cell, such as described herein.

[0469] In some embodiments, the immune cell of (a) comprises a CAR specific for a disease-associated antigen.

[0470] In some embodiments, the immune cell of (a) is an immune cell expressing a CAR, such as described herein.

[0471] In some embodiments, the method comprises:

[0472] (a) modifying an immune cell to contain / express a molecule for directing the activity of the immune cell against cells containing / expressing a given target antigen,

[0473] For example, disease-associated antigens (e.g., TCR or CAR);

[0474] (b) modifying immune cells to increase the expression or activity of SERPINB9; and

[0475] (c) administering the modified immune cells to a subject.

[0476] In some embodiments, the method comprises:

[0477] (a) isolating or obtaining virus-specific immune cells;

[0478] (b) modifying an immune cell to contain / express a molecule for directing the activity of the immune cell against cells containing / expressing a given target antigen,

[0479] For example, disease-associated antigens (e.g., TCR or CAR);

[0480] (c) modifying immune cells to increase the expression or activity of SERPINB9; and

[0481] (d) administering the modified immune cells to a subject.

[0482] In some embodiments, the method comprises:

[0483] (a) isolating immune cells (e.g., PBMCs) from a subject;

[0484] (b) generating / expanding virus-specific immune cell populations;

[0485] (b) Modifying virus-specific immune cells to contain / express a molecule that directs the immune cells to defend against cells containing / expressing a given target antigen.

[0486] activity of cells, such as disease-associated antigens (e.g., TCR or CAR);

[0487] (c) modifying immune cells to increase the expression or activity of SERPINB9; and

[0488] (d) administering the modified immune cells to a subject.

[0489] In a preferred embodiment, the object of producing immune cells is different from the object of administering cells (i.e., adoptive transfer can be allogeneic cells). In some embodiments, the object of producing immune cells is the same object as the object of administering cells (i.e., adoptive transfer can be autologous / autogenic cells). In some embodiments, virus-specific immune cells are modified to increase the expression or activity of SERPINB9, and immune cells are modified to include / express a molecule for guiding immune cells to resist the activity of cells comprising / expressing a given target antigen, such as disease-associated antigens (e.g., TCR or CAR). For example, in an embodiment, immune cells are modified with nucleic acids, and the nucleic acids include a nucleotide sequence encoding a SERPINB9 polypeptide and a nucleotide sequence encoding a molecule, which is used to guide immune cells to resist the activity of cells comprising / expressing a given target antigen, such as disease-associated antigens (e.g., TCR or CAR).

[0490] In some embodiments, the method may include one or more of the following:

[0491] obtaining a blood sample from the subject;

[0492] isolating immune cells (e.g., PBMCs) from a blood sample obtained from a subject;

[0493] Generation / expansion of virus-specific immune cell populations (e.g., by culturing PBMCs in the presence of cells containing / expressing viral antigens / peptides (e.g., APCs), or by culturing PBMCs in the presence of virus-infected cells (e.g., APCs);

[0494] Cultivation of virus-specific immune cells in vitro or ex vivo cell culture;

[0495] According to the present disclosure, virus-specific immune cells are modified to express / contain a CAR, or to express / contain a nucleic acid encoding a CAR (e.g., by transducing a viral vector encoding such a CAR or a viral vector containing such a nucleic acid);

[0496] According to the present disclosure, virus-specific immune cells expressing / comprising CAR, or expressing / comprising nucleic acid encoding CAR, are cultured in in vitro or ex vivo cell culture;

[0497] According to the present disclosure, collecting / isolating virus-specific immune cells that express / contain CAR, or express / contain nucleic acid encoding CAR;

[0498] According to the present disclosure, virus-specific immune cells expressing / comprising CAR, or expressing / comprising nucleic acid encoding CAR, are formulated to obtain a pharmaceutical composition, for example, by mixing the cells with a pharmaceutically acceptable adjuvant, diluent or carrier;

[0499] According to the present disclosure, virus-specific immune cells expressing / comprising CAR, or expressing / comprising nucleic acid encoding CAR, or pharmaceutical compositions comprising such cells are administered to a subject.

[0500] Treatment and / or prevention methods may effectively slow down the development / progress of the disease / illness, alleviate the symptoms of the disease / illness, or reduce the pathological abnormalities of the disease / illness. These methods may effectively prevent the progression of the disease / illness, such as preventing the disease / illness from worsening or slowing down the rate of development of the disease / illness. In some embodiments, these methods can lead to an improvement in the disease / illness, such as a reduction in the severity of the symptoms of the disease / illness, or a reduction in some other aspects related to the severity / activity of the disease / illness. In some embodiments, these methods can prevent the disease / illness from developing to a later stage (e.g., a chronic stage or metastasis).

[0501] It is understood that, according to the present disclosure, the therapeutic and preventive applications of CAR-expressing immune cells can be extended to the treatment / prevention of any disease / disorder that would benefit from a reduction in the number / activity of cells expressing / overexpressing the CAR target antigen. Similarly, it is understood that, according to the present disclosure, the therapeutic and preventive applications of virus-specific immune cells can be extended to the treatment / prevention of any disease / disorder that would benefit from a reduction in the number / activity of cells infected with a virus (or containing / expressing a viral antigen).

[0502] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is a disease / disorder in which a virus that is specific to immune cells is pathologically implicated. That is, in some embodiments, the disease / disorder is a disease / disorder caused or exacerbated by a viral infection, a disease / disorder for which a viral infection is a risk factor, and / or a disease / disorder in which a viral infection is positively correlated with the onset, development, progression, and / or severity of the disease / disorder.

[0503] In some embodiments, according to the present disclosure, the disease / disorder to be treated / prevented is pathologically related to the target antigen of the CAR. That is, in some embodiments, the disease / disorder is a disease / disorder caused or exacerbated by the expression / overexpression of the target antigen, a disease / disorder for which the expression / overexpression of the target antigen is a risk factor, and / or a disease / disorder in which the expression / overexpression of the target antigen is positively correlated with the onset, development, progression, or severity of the disease / disorder.

[0504] The disease / disorder may be a disease / disorder in which CD30 or cells expressing / overexpressing CD30 are pathologically associated, for example, cells expressing / overexpressing CD30 are positively correlated with the onset, development or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder, or CD30 expression / overexpression is a risk factor for the onset, development and progression of the disease / disorder.

[0505] The disease / disorder may be a disease / disorder in which CD19 or cells expressing / overexpressing CD19 are pathologically associated, for example, cells expressing / overexpressing CD19 are positively correlated with the onset, development or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder, or CD19 expression / overexpression is a risk factor for the onset, development and progression of the disease / disorder.

[0506] According to the present disclosure, the disease / disorder to be treated / prevented can be a disease / condition characterized by EBV infection. For example, the disease / disorder can be a disease / disorder in which EBV or cells infected with EBV are pathologically associated, such as a disease / disorder in which EBV infection is positively correlated with the onset, development or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder, or in which EBV infection is a risk factor for the onset, development and progression of the disease / disorder.

[0507] The treatment may be intended to achieve one or more of the following: reduce viral load, reduce the number / ratio of virus-positive cells (such as EBV-positive cells), reduce the number / ratio of cells expressing / overexpressing CAR target antigens (such as CD30-expressing cells, CD19-expressing cells), reduce the activity of virus-positive cells (such as EBV-positive cells), delay / prevent the onset / progression of disease / disorder symptoms, reduce the severity of disease / disorder symptoms, reduce the survival / growth of virus-positive cells (such as EBV-positive cells), reduce the survival / growth of cells expressing / overexpressing CAR target antigens (such as CD30-expressing cells, CD19-expressing cells), or improve the survival rate of the subject.

[0508] In some embodiments, the subject can be selected for treatment as described herein based on detection of a virus (e.g., EBV), cells infected with a virus (e.g., EBV), or cells expressing / overexpressing a CAR target antigen (e.g., CD30, CD19), for example, on the cell surface, or in an organ / tissue affected by a disease / disorder (e.g., an organ / tissue exhibiting symptoms of a disease / disorder), or by detecting virus-positive cancer cells (e.g., EBV-positive cancer cells) or detecting cancer cells expressing / overexpressing a CAR target antigen (e.g., CD30, CD19). Disease / disorder may affect any tissue or organ or organ system. In some embodiments, disease / disorder may affect several tissues / organs / organ systems.

[0509] In some embodiments, a subject can be selected for treatment / prevention according to the present disclosure based on a determination that the subject is infected with EBV or contains cells infected with EBV. In some embodiments, a subject can be selected for treatment / prevention according to the present disclosure based on a determination that the subject contains cells that express / overexpress CD30, such as cancer cells that express / overexpress CD30. In some embodiments, a subject can be selected for treatment / prevention according to the present disclosure based on a determination that the subject contains cells that express / overexpress CD19, such as cancer cells that express / overexpress CD19.

[0510] In some embodiments, prior to administering the virus-specific immune cells expressing / comprising a CAR described herein (or expressing / comprising a nucleic acid encoding such a CAR), the subject is subjected to lymphodepleting chemotherapy.

[0511] That is, in some embodiments, the method of treating / preventing a disease / disorder according to the present disclosure comprises: (i) performing lymphodepleting chemotherapy on a subject, and (ii) subsequently administering virus-specific immune cells that express / comprising a CAR of the present disclosure (or express / comprising a nucleic acid encoding a CAR of the present disclosure).

[0512] As used herein, "lymphocyte-depleting chemotherapy" refers to treatment with a chemotherapeutic agent that results in depletion of lymphocytes (e.g., T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors) in a subject. A "lymphocyte-depleting chemotherapeutic agent" refers to a chemotherapeutic agent that results in lymphocyte depletion.

[0513] Lymphodepleting chemotherapy and its use in adoptive cell transfer therapy are described in Klebanoff et al., Trends Immunol. (2005) 26(2):111-7, and Muranski et al., Nat Clin Pract Oncol. (2006) (12):668-81. The entire contents of both are incorporated herein by reference. The goal of lymphodepleting chemotherapy is to deplete the recipient's endogenous lymphocyte population.

[0514] In the context of adoptive transfer of immune cells for treatment of disease, lymphodepleting chemotherapy is often administered prior to adoptive cell transfer to allow the recipient to accept the adoptively transferred cells. Lymphodepleting chemotherapy is thought to promote the persistence and activity of adoptively transferred cells by creating a permissive environment, for example by eliminating cells expressing immunosuppressive cytokines and creating the "lymphoid space" required for the expansion and activity of adoptively transferred lymphocytes.

[0515] Commonly used chemotherapy drugs in lymphodepleting chemotherapy include fludarabine, cyclophosphamide, bendamustine, and pentostatin.

[0516] In some embodiments, the disease to be treated / prevented of the present disclosure is cancer. Cancer may refer to any unwanted cell proliferation (or any disease exhibiting unwanted cell proliferation), tumor, or neoplasm. Cancer may be benign or malignant, and may be primary or secondary (metastatic). A tumor or neoplasm may be any abnormal growth or proliferation of cells and may be located in any tissue. Cancer tissue or cells may be from, for example, the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxillary, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testicle, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, and / or leukocytes. Tumors may be neural or non-neural tumors. Nervous system tumors may originate from the central nervous system or peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-nervous system cancers / tumors may originate from any other non-nervous tissue, such as melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0517] In some embodiments, the cancer is selected from the group consisting of solid cancer, blood cancer, stomach cancer (e.g., gastric cancer, gastric adenocarcinoma, gastrointestinal adenocarcinoma), liver cancer (hepatocellular carcinoma, bile duct cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma), oral cancer (e.g., oropharyngeal cancer (e.g., oropharyngeal cancer), oral cancer, laryngeal cancer, nasopharyngeal cancer, esophageal cancer), colorectal cancer (e.g., colorectal tumors), colon cancer, colon cancer, cervical cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma), bladder cancer, urothelial carcinoma, skin cancer, Skin cancer (e.g., melanoma, advanced melanoma), renal cell carcinoma (e.g., renal cell carcinoma), ovarian cancer (e.g., ovarian tumor), mesothelioma, breast cancer, brain cancer (e.g., glioblastoma), prostate cancer, pancreatic cancer, myeloid malignancies, lymphocytic malignancies, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), lymphoma, non-Hodgkin lymphoma (NHL), thymoma, or multiple myeloma (MM).

[0518] In some embodiments, the cancer is a cancer that is pathologically associated with virus-specific immune cells. That is, in some embodiments, the cancer is a cancer caused or exacerbated by a viral infection, a cancer for which a viral infection is a risk factor, and / or a cancer in which a viral infection is positively correlated with the initiation, development, progression, severity, or metastasis of the cancer.

[0519] EBV infection is associated with various cancers, as described by Jha et al., Front Microbiol, (2016) 7: 1602. This is incorporated herein by reference in its entirety.

[0520] In some embodiments, cancer to be treated / prevented is an EBV-associated cancer. In some embodiments, cancer is a cancer caused or aggravated by EBV infection, and EBV infection is a cancer that is a risk factor for cancer and / or EBV infection is positively correlated with the generation, development, progression, severity or metastasis of cancer. Cancer may be characterized by EBV infection, and for example, cancer may comprise cells that infect EBV. This cancer may be referred to as an EBV-positive cancer.

[0521] According to the present disclosure, EBV-related cancers that can be treated / prevented include B cell-related cancers, such as Burkitt's lymphoma, post-transplant lymphoproliferative disorder (PTLD), central nervous system lymphoma (CNS lymphoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma and EBV-related lymphoma associated with immunodeficiency (including, for example, EBV-positive lymphoma associated with X-linked lymphoproliferative disease, EBV-positive lymphoma associated with HIV infection / AIDS and oral hairy leukoplakia), and epithelial cell-related cancers, such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC).

[0522] In some embodiments, the cancer is selected from lymphoma (eg, EBV-positive lymphoma), head and neck squamous cell carcinoma (HNSCC; eg, EBV-positive HNSCC), nasopharyngeal carcinoma (NPC; eg, EBV-positive NPC), and gastric cancer (GC; eg, EBV-positive GC).

[0523] In some embodiments, the cancer is a cancer that is pathologically associated with the target antigen of the CAR. That is, in some embodiments, the cancer is a cancer caused or exacerbated by the expression of the target antigen, a cancer for which the expression of the target antigen is a risk factor, and / or a cancer in which the expression of the target antigen is positively correlated with the occurrence, development, progression, severity, or metastasis of the cancer. The cancer may be characterized by the expression of the target antigen, for example, the cancer may contain cells that express the target antigen. Such cancers may be referred to as target antigen-positive cancers.

[0524] A cancer that is "positive" for a target antigen may be one that contains cells that express the target antigen (e.g., on the cell surface). A cancer that is "positive" for a target antigen may overexpress the target antigen. Overexpression of the target antigen can be determined by detecting gene or protein expression levels of the target antigen that are greater than the expression levels of corresponding non-cancerous cells / non-tumor tissue.

[0525] In some embodiments, the target antigen is a cancer cell antigen as described herein. In some embodiments, the target antigen is CD30. In some embodiments, the cancer is a cancer pathologically associated with CD30. That is, in some embodiments, the cancer is a cancer that causes or exacerbates CD30 expression, a cancer for which CD30 expression is a risk factor, and / or a cancer in which CD30 expression is positively correlated with the development, development, progression, severity, or metastasis of the cancer. The cancer may be characterized by CD30 expression, for example, the cancer may comprise cells that express CD30. Such cancers may be referred to as CD30-positive cancers.

[0526] A CD30-positive cancer may be a cancer comprising cells that express CD30 (e.g., cells that express the CD30 protein on their surface). A CD30-positive cancer may overexpress CD30. Overexpression of CD30 can be determined by detecting a level of CD30 gene or protein expression that is greater than the expression level of a corresponding non-cancerous cell / non-tumor tissue. A CD30-positive cancer may be a cancer comprising cells CD30-positive cancer is described in van der Weyden et al., Blood Cancer Journal (2017) 7:e603 and Muta and Podack, Immunol Res (2013), 57(1-3):151-8, both of which are incorporated herein by reference in their entirety. CD30 is expressed in small subsets of activated T and B lymphocytes and in various lymphoid neoplasms, including classical Hodgkin lymphoma and anaplastic large cell lymphoma. Variable expression of CD30 is also observed in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), adult T-cell leukemia / lymphoma, cutaneous T-cell lymphoma (CTCL), extranodal NK-T-cell lymphoma, various B-cell non-Hodgkin lymphomas (including diffuse large B-cell lymphoma, especially EBV-positive diffuse large B-cell lymphoma), and advanced systemic mastocytosis. CD30 expression has also been observed in some non-hematopoietic malignancies, including germ cell tumors and testicular embryonal carcinoma.

[0527] The transmembrane glycoprotein CD30 is a member of the tumor necrosis factor receptor superfamily (Falini et al., Blood (1995) 85(1): 1-14). Members of the TNF / TNF receptor (TNF-R) superfamily coordinate immune responses at multiple levels, and CD30 plays a role in regulating the function or proliferation of normal lymphocytes. CD30 was originally described as an antigen recognized by the monoclonal antibody Ki-1, which was generated by immunizing mice with the HL-derived cell line L428 (Muta and Podack, Immunol Res (2013) 57: 151-158). CD30 antigen expression has been used to identify ALCL and Reed-Sternberg cells in Hodgkin's disease (Falini et al., Blood (1995) 85(1): 1-14). Because CD30 is widely expressed in lymphoma malignant cells, it is a potential target for the development of antibody-based immunotherapy and cell therapy. Importantly, under physiological conditions, CD30 is not normally expressed in normal tissues and is therefore notably absent on resting mature or precursor B or T cells (Younes and Ansell, Semin Hematol (2016) 53:186-189). Brentuximab is an antibody-drug conjugate targeting CD30 that was initially approved for the treatment of CD30-positive HL ( US Drug Package Insert 2018). Trial data for brentuximab support CD30 as a therapeutic target for the treatment of CD30-positive lymphomas, although there are concerns about toxicity associated with its use.

[0528] Hodgkin lymphoma (HL) is a rare malignancy involving the lymph nodes and lymphatic system. The incidence of HL is bimodal, with most patients diagnosed between the ages of 15 and 30 years, followed by another peak in adults aged 55 years or older. An estimated 8,110 new cases (3,540 in women and 4,570 in men) and 1,000 deaths from the disease will occur in the United States in 2019 (American Cancer Society 2019). Based on cases from 2012 to 2016 in the National Cancer Institute's Surveillance, Epidemiology, and End Results database, the incidence of HL in pediatric patients in the United States is as follows: 1-4 years: 0.1; 5-9 years: 0.3; 10-14 years: 1.3; and 15-19 years: 3.3 per 100,000 (SEER Cancer Statistical Reviews, 1975-2016). The World Health Organization (WHO) classification divides HL into two main types: classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). In Western countries, cHL accounts for 95% of all HL cases, and NLPHL accounts for 5% (2019 National Comprehensive Cancer Network Guidelines).

[0529] First-line chemotherapy for patients with advanced cHL is associated with a cure rate of 70% to 75% (Karantanos et al., Blood Lymphat Cancer (2017) 7:37-52). Salvage chemotherapy followed by autologous stem cell transplantation (ASCT) is commonly used for patients who relapse after initial treatment. Unfortunately, up to 50% of patients with cHL experience disease relapse after ASCT. The median overall survival for patients who relapse after ASCT is approximately 2 years (Alinari Blood (2016) 127:287-295). Despite aggressive combination chemotherapy, 10%-40% of patients do not respond to salvage chemotherapy, and there are no randomized clinical trial data to support ASCT for nonresponders. For patients who do not respond to salvage chemotherapy, relapse after ASCT, or are not suitable for this approach, the prognosis remains grim, and new treatments are urgently needed (Keudell British Journal of Haematology (2019) 184:105–112).

[0530] While the majority of the pediatric population (children, adolescents, and young adults) can be cured with currently available therapies, a small proportion of patients may have refractory or relapsed disease, and newer therapies with acceptable safety profiles and greater efficacy and benefit are needed (Flerlage et al., Blood (2018) 132: 376-384; Kelly, Blood (2015) 126: 2452-2458; McClain and Kamdar, UpToDate 2019; Moskowitz, ASCO Educational Book (2019) 477-486). HL patients who receive high-dose chemotherapy in childhood often experience long-term treatment-related sequelae, such as cardiac, pulmonary, gonadal, and endocrine toxicities, as well as second malignancies (Castellino et al., Blood (2011) 117(6): 1806-1816).

[0531] In some embodiments, the CD30-positive cancer can be selected from the group consisting of solid cancer, hematological cancer, hematopoietic malignancy, Hodgkin's lymphoma (HL), anaplastic large cell lymphoma (ALCL), ALK-positive anaplastic T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma (e.g., PTCL-NOS), T-cell leukemia, T-cell lymphoma, cutaneous T-cell lymphoma (CTCL), NK-T cell lymphoma (e.g., extranodal NK-T cell lymphoma), non-Hodgkin's lymphoma (NHL), B-cell non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (e.g., diffuse large B-cell lymphoma-NOS), primary mediastinal B-cell lymphoma, EBV-positive B-cell lymphoma, EBV-positive diffuse large B-cell lymphoma, advanced systemic mastocytosis, germ cell tumor, and testicular embryonal carcinoma. In some embodiments, the target antigen is a cancer cell antigen as described herein. In some embodiments, the target antigen is CD19. In some embodiments, the cancer is a cancer pathologically associated with CD19. That is, in some embodiments, the cancer is a cancer that causes or exacerbates CD19 expression, a cancer for which CD19 expression is a risk factor, and / or a cancer in which CD19 expression is positively correlated with the development, development, progression, severity, or metastasis of the cancer. The cancer may be characterized by CD19 expression, for example, the cancer may contain cells that express CD19. Such a cancer may be referred to as a CD19-positive cancer.

[0532] A CD19-positive cancer may be a cancer that contains cells that express CD19 (e.g., cells that express the CD19 protein on their surface). A CD19-positive cancer may overexpress CD19. Overexpression of CD19 can be determined by detecting CD19 gene or protein expression levels that are higher than those in corresponding non-cancerous cells / non-tumor tissue.

[0533] CD19 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. CD19 is classified as a type I transmembrane protein with a single transmembrane domain, a cytoplasmic C-terminus, and an extracellular N-terminus. CD19 was originally identified as the B4 antigen of human B lymphocytes by using an anti-B4 monoclonal antibody (mAb) against CD19. CD19 is expressed throughout B cell development until terminal plasma cell differentiation. It is not expressed in stem cells or most other normal cell types. CD19 enhances B cell antigen receptor signaling by increasing the activity of phosphoinositide-3-kinase and Bruton's tyrosine kinase (Wang et al., Exp Hematol Oncol (2012) 1(1):36, Fujimoto et al., Semin Immunol (1998) 10:267-277), which plays a crucial role in tumor cell proliferation and survival (Seda et al., Eur J Haematol (2015) 94(3):193-205). CD19 is widely and uniformly expressed in different B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) (Olejniczak et al., Immunol Invest (2006) 35(1):93-114; Schuurman et al., Am J Pathol (1988) 131:102-111).

[0534] Non-Hodgkin lymphoma (NHL) is one of the most common cancers in the United States, accounting for approximately 4% of all cancers. It is estimated that by 2023, approximately 80,550 people (44,880 men and 35,670 women) will be diagnosed with NHL in the United States, and approximately 20,180 people (11,780 men and 8,400 women) will die from this cancer (American Cancer Society 2023). The World Health Organization (WHO) classification divides NHL into two main groups: B-cell-derived and T-cell / natural killer (NK) cell-derived. These two main groups are further divided into subtypes. Regarding the subtypes of B cell origin, WHO provides further classification subtypes: precursor B lymphocytic leukemia / lymphoma and peripheral B cell neoplasms (including further classification subtypes B cell chronic lymphocytic leukemia / small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma / immunocytic neoplasm, mantle cell lymphoma, follicular lymphoma, extranodal marginal zone mucosa-associated lymphoid tissue (MALT) type B cell lymphoma, lymph node marginal zone B cell lymphoma (± monocytoid B cells), splenic marginal zone lymphoma (± villous lymphocytes), hairy cell leukemia, plasmacytoma / plasma cell myeloma, diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma)

[0535] In real-life practice and in most clinical trials, NHL histologic subtypes are broadly categorized into indolent, aggressive, and very aggressive groups based on their typical clinical behavior. Indolent B-cell lymphomas account for 35% to 40% of non-Hodgkin lymphomas (NHL), with survival typically measured in years. The most common subtypes include follicular lymphoma (FL), chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), a small subset of mantle cell lymphoma (MCL), extramedullary, nodal, and splenic marginal zone lymphoma (MZL), and lymphoplasmacytic lymphoma (LPL) (Swerdlow et al., Blood (2016) 127:2375-90; Rizvi et al., Blood (2006) 107:1255-64). Aggressive subtypes can result in survival of several months if left untreated, but with adequate treatment, a significant proportion of patients may achieve definitive remission and cure. The most common subtype is large B-cell lymphoma, which includes anaplastic and primary mediastinal lymphomas, as well as various diffuse large B-cell lymphomas (DLBCL). Highly aggressive subtypes account for approximately 5% of NHL cases and, if left untreated, may have survival rates measurable in just a few weeks. However, with aggressive treatment using high-intensity chemotherapy regimens, cure is possible.

[0536] Over the past few decades, chemotherapy, radiotherapy, and immunotherapy have been used alone or in combination to treat B-cell NHL. Treatment outcomes can vary depending on clinical behavior, whether indolent or aggressive, and patients may experience various relapse patterns requiring subsequent salvage therapy. Poor prognosis still affects a large proportion of patients with mature B-cell lymphoma, and new treatment strategies should be considered to improve objective response and survival (Jiang et al., Expert Rev Hematol (2017) 10:405-15; Gisselbrecht et al., Br J Haematol (2018) 182:633-43; El-Mallawany et al., Clin Adv Hematol Oncol (2015) 13:113-23; Mei et al., Clin Lymphoma Myeloma Leuk (2018) 18:26-33; Shanbhag et al., CA Cancer J Clin (2018) 68:116-32; Biccler et al., Leuk Lymphoma (2018) doi:10.1080 / 10428194.2018.1540044; Barth et al., Br J Haematol (2019) doi: 10.1111 / bjh.15783).

[0537] In some embodiments, the CD19-positive cancer can be selected from the group consisting of non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), chronic lymphoma (CLL), marginal zone B-cell lymphoma (MZBL), extranodal marginal zone mucosa-associated lymphoid tissue (MALT)-type B-cell lymphoma, nodal marginal zone B-cell lymphoma (±monocytoid B cells), splenic marginal zone lymphoma (±villous lymphocytes), leukemia, hairy cell leukemia (HCL), hairy cell leukemia variant (HCL-v), acute lymphoblastic leukemia (ALL), Philadelphia chromosome-positive ALL (Ph+ALL) and Philadelphia chromosome-negative ALL (Ph-ALL), B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma / immunocytic neoplasm, plasmacytoma / plasma cell myeloma, and Burkitt's lymphoma.

[0538] In some embodiments, the cancer is selected from the group consisting of: CD30 positive cancer, CD19 positive cancer, EBV associated cancer, hematological cancer, myeloid hematological malignancy, hematopoietic malignancy, lymphocytic hematological malignancy, myelodysplastic syndrome, leukemia, T cell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B cell non-Hodgkin lymphoma, diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, EBV associated lymphoma, EBV positive B cell lymphoma, EBV positive diffuse large B cell lymphoma, EBV positive lymphoma associated with X-linked lymphoproliferative disorder, HIV infection / AIDS associated EBV positive lymphoma, oral hairy leukoplakia, Burkitt lymphoma, post-transplant lymphoproliferative disorder, central nervous system lymphoma, anaplastic large cell lymphoma, T cell lymphoma, ALK Positive anaplastic T-cell lymphoma, ALK-negative anaplastic T-cell lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, NK-T-cell lymphoma, extranodal NK-T-cell lymphoma, thymoma, multiple myeloma, solid cancer, epithelial cell cancer, gastric cancer, gastric tumor, gastric adenocarcinoma, gastrointestinal adenocarcinoma, liver cancer, hepatocellular carcinoma, bile duct cancer, head and neck cancer, head and neck squamous cell carcinoma, oral cancer, oropharyngeal cancer, oral tumor, laryngeal cancer, nasopharyngeal cancer, esophageal cancer, colorectal cancer , colorectal cancer, colon cancer, colon cancer, cervical cancer, prostate cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, bladder cancer, urothelial cancer, skin cancer, melanoma, advanced melanoma, renal cell carcinoma, renal cell tumor, ovarian cancer, ovarian tumor, mesothelioma, breast cancer, brain cancer, glioblastoma, prostate cancer, pancreatic cancer, mastocytosis, advanced systemic mastocytosis, germ cell tumor, or testicular embryonal carcinoma.

[0539] In some embodiments, the cancer may be a recurrent cancer. As used herein, a "recurrent" cancer refers to a cancer that responds to treatment (e.g., a first-line treatment for the cancer) but subsequently reappears / progresses, e.g., after a period of remission. For example, a recurrent cancer may be a cancer whose growth / progression was inhibited by treatment (e.g., a first-line treatment for the cancer) and which subsequently reappears / progresses.

[0540] In some embodiments, the cancer may be a refractory cancer. As used herein, a "refractory" cancer refers to a cancer that has not responded to treatment (e.g., a first-line treatment for the cancer). For example, a refractory cancer may be a cancer that has not been inhibited by treatment (e.g., a first-line treatment for the cancer). In some embodiments, a refractory cancer may be a cancer that has not shown a partial or complete response to treatment in a subject receiving cancer treatment.

[0541] In embodiments where the cancer is anaplastic large cell lymphoma, the cancer may be relapsed or refractory to chemotherapy, brentuximab, or crizotinib. In embodiments where the cancer is peripheral T-cell lymphoma, the cancer may be relapsed or refractory to chemotherapy or brentuximab. In embodiments where the cancer is extranodal NK-T cell lymphoma, the cancer may be relapsed or refractory to chemotherapy (with or without asparaginase) or brentuximab. In embodiments where the cancer is diffuse large B-cell lymphoma, the cancer may be relapsed or refractory to chemotherapy (with or without rituximab) or CD19 CAR-T therapy. In embodiments where the cancer is primary mediastinal B-cell lymphoma, the cancer may be relapsed or refractory to chemotherapy, immune checkpoint inhibitors (e.g., PD-1 inhibitors), or CD19 CAR-T therapy.

[0542] Treating cancer according to the methods of the present disclosure can achieve one or more of the following therapeutic effects: reducing the number of cancer cells in a subject, reducing the size of a cancerous tumor / lesion in a subject, inhibiting (e.g., preventing or slowing) the growth of cancer cells / lesions in a subject, inhibiting (e.g., preventing or slowing) the growth of a cancerous tumor / lesion in a subject, inhibiting (e.g., preventing or slowing) the development / progression of cancer (e.g., to an advanced stage or metastasis), reducing the severity of cancer symptoms in a subject, improving the survival rate (e.g., progression-free survival or overall survival) of a subject, reducing the number or activity of cancer cells in a subject, and / or reducing the cancer burden in a subject.

[0543] Subjects can be evaluated according to the modified response evaluation criteria: Lugano classification (as described by Cheson et al., J Clin Oncol (2014) 32:3059-3068, incorporated herein by reference in its entirety) to determine their response to treatment. In some embodiments, treatment of a subject according to the methods of the present disclosure achieves one of the following: complete response, partial response, or stable disease.

[0544] In some embodiments, treatment of cancer further includes chemotherapy and / or radiation therapy.

[0545] Chemotherapy and radiotherapy refer to the use of drugs or ionizing radiation to treat cancer (e.g., radiotherapy using X-rays or gamma rays). Drugs can be chemical entities, such as small molecule drugs, antibiotics, DNA intercalators, protein inhibitors (e.g., kinase inhibitors), or biological agents, such as antibodies, antibody fragments, aptamers, nucleic acids (e.g., DNA, RNA), peptides, polypeptides, or proteins. The drug can be formulated into a pharmaceutical composition or medicament. The preparation can include one or more drugs (e.g., one or more active agents) and one or more pharmaceutically acceptable diluents, excipients, or carriers.

[0546] Chemotherapy may involve the use of more than one drug. Drugs may be given alone or in combination with other treatments, either at the same time or in sequence, depending on the condition being treated.

[0547] Chemotherapy can be administered by one or more routes of administration, such as parenteral, intravenous, oral, subcutaneous, intradermal, or intratumoral.

[0548] Chemotherapy may be administered according to a treatment plan. A treatment plan is a predetermined chemotherapy schedule, plan, regimen, or timetable that may be prepared by a physician or doctor and customized for the patient being treated. A treatment plan may indicate one or more of the following: the type of chemotherapy to be administered to the patient; the dose of each drug or radiation; the intervals between doses; the length of each treatment; and the number and nature of any treatment breaks, if any. For combination therapy, a single treatment plan may be provided that indicates how each drug will be administered.

[0549] Chemotherapy drugs can be selected from: abemaciclib, abiraterone acetate, abiraterone (methotrexate), albumin-paclitaxel (paclitaxel-albumin stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, acalabrutinib, AC-T, Adcetris (brentuximab), ADE, emtansine trastuzumab, doxorubicin (doxorubicin hydrochloride), afatinib maleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Atelor (imiquimod), aldesleukin, alectinib (arentinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), afatinib maleate, selegiline (everolimus), aletinib (netupitant and palonosetron hydrochloride), alemtuzumab (imiquimod), aletinib (pemetrexed disodium), aletinib (pemetrexed disodium), aletinib (pemetrexed disodium), aletinib (alectinib), alemtuzumab, alemtuzumab (pemetrexed disodium), aletinib (alectinib), alemtuzumab, alemtuzumab (alemtuzumab), ... Lekepa (copanlicoxib hydrochloride), Akron (melphalan hydrochloride) for injection, Akron tablets (melphalan), Aloxi (palonosetron hydrochloride), Brigatinib (brigatinib), Ambroxol (chlorambucil), Chlorambucil (chlorambucil), Amifostine, Aminopyruvic acid, Anastrozole, Apretan, Aledia (pamidronate disodium), Arimidex (anastrozole), Aromestane (exemestane), Nelarabine (nilarabine), Arsenic trioxide, Arzerra (ofatumumab), Asparaginase, Atezolizumab, Avastin (bevacizumab), Avelumab, Axicabtagene Cioleucel, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becon (carmustine), belodac (belinostat), belinostat, bendamustine hydrochloride, BEP, Bespona (enoxitumomab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (belintosumab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib (cabozantinib malate), cabozantinib malate, CAF, Calquence (acalabrutinib), Campath (alemtuzumab), Camposar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil - topical), carboplatin, CARBOPLATIN-TAXOL, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant human papillomavirus bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, clarithromycin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF,Cobimetinib, Cometriq (cabozantinib malate), Copalixib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (actinomycin D), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Daco gen (decitabine), actinomycin D, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomal, decitabine, defibrotide sodium, Defitelio (defibrotide sodium salt), degarelix, denileukin, denosumab, DepoCyt (cytarabine liposomal), dexamethasone, dexazolidine hydrochloride, dexximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, dox SL (doxorubicin hydrochloride liposomal), DTIC Dome (dacarbazine), durvalumab, Efudex (fluorouracil topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotizumab, Eloxatin (oxaliplatin), eltrombopag, Emend (afoptin), Empliciti (elotizumab), enalapril mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase), Ethyol (amifostine), Etopophos (etoposide phosphate), Etoposide, etoposide phosphate, Evacet (liposomal doxorubicin hydrochloride), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU topical), Fareson (toremifene), Farydak (panopistat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), Fludarabine phosphate, Fluoroplex (fluorouracil topical), fluorouracil injection, fluorouracil topical, Flutamide, Folex (methotrexate), Folex PFS (methotrexine), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexed), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil9 (recombinant HPV vaccine-free), Gazyva (otuzumab),Gefitinib, gemcitabine hydrochloride, cisplatin-cisplatin, cisplatin-oxaliplatin, oxaliplatin, ogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib maleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), carboxypeptidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant vaccine, inactivated HPV vaccine, recombinant, HPV quadrivalent vaccine, reconstituted vaccine, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibrutinib, ibrutinib, ICE, Iclusig (bonatinib hydrochloride), idarubicin hydrochloride, idarubicin hydrochloride, Idelalisib, Idhifa (enalapril mesylate), Ifex (ifosfamide), ifosfamide, ifosfamide (Helosin), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), Imiquimod, Imlygic (talimogene laherparepvec), Inlyta (axitinib), olgaituzumab, interferon alfa-2b, recombinant, interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2a), iodine I 131 Tocilizumab and tocilizumab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan liposomal hydrochloride, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (trastuzumab), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (Tisagenl ecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib dimesylate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, folinic acid, Leukeran (chlorambucil), leuprorelin acetate, Leustatin (clalibine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprorelin acetate),Lupron Depot (leuprorelin acetate), Lupron Depot Ped (leuprorelin acetate); Lynparza (olaparib), Marqibo (liposomal vincristine sulfate), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexine), midothalene, mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobipro (plerixafor), Mustargen (nitrogen mustard hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemocilizumab), nanoparticle paclitaxel (albumin-stabilized nanoparticle formulation of paclitaxel), and Navelbine (vinorelbine tartrate), nesituzumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate) , Nplate (romiplastin), obinutuzumab, Odomzo (sonidegi), OEPA, ofatumumab, OFF, olaparib, olaramumab, homoharringtonine, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (liposomal irinotecan hydrochloride), Ontak (denileukin-2 injection), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride Joan and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Pegylated Interferon Alpha-2b, PEG-Intron (Pegylated Interferon Alpha-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide),Ponatinib hydrochloride, Portrazza (netotuzumab), pratrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag), propranolol hydrochloride, Purinethol (mercaptopurine), Purixan (mercaptopurine), radium 223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) 9-valent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and human hyaluronidase), Rituximab, Rituximab and human hyaluronidase, Rolapitant hydrochloride, Romidepsin, Romiplostim, Rubidomycin (daunorubicin hydrochloride), Rucaparib Camsylate, Rucaparib Camsylate, Ruxolitinib phosphate, Rydapt (midostaurin), Sclerosant Intrapleural Aerosol (talc), Sipuleucel-T, Somatuline Depot (lanreotide acetate), Sonidegi, Sorafenib Tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc (Talc), Steritalc (Talc), Stivarga (regorafenib), Sunitinib Malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alfa-2b), Sylvant (siltuximab), Synribo (omacetin succinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotinib), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, Tisagenlecleucel, Tolak (fluorouracil - topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus),Tositumomab and iodine I 131 Tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib dimesylate), Unituxin (enzalutamide), uridine triacetate, VAC, rubicin, Valstar (rubicin), vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abecib), Viadur (leuprorelin acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, liposomal vincristine sulfate, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxaze (carboxypeptidase), Vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (liposomal daunorubicin hydrochloride and cytarabine), Wellcovorin (leucovorin), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Y Escarta (Akirensel), Yondelis (Trabectedin), Zaltrap (Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexaroxazolidinone Hydrochloride), Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalis), Zykadia (Ceritinib), and Zytiga (Abiraterone Acetate).

[0550] EBV infection has also been associated with the development / progression of several autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus (SLE; see, e.g., Ascherio and Munger Curr Top Microbiol Immunol, (2015); 390(Pt 1):365-85), and the EBV antigen EBNA2 has recently been shown to be associated with genetic regions that are considered risk factors for the development of SLE, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis, and celiac disease (Harley et al., Nat Genet. (2018) 50(5):699-707).

[0551] Thus, in some embodiments, the disease / disorder to be treated / prevented by the present disclosure is selected from the group consisting of: autoimmune disease, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis, and celiac disease.

[0552] Object

[0553] According to various aspects of the present disclosure, the subject can be any animal or human. The subject is preferably a mammal, preferably a human. The subject can be a non-human mammal, but preferably a human. The subject can be male or female. The subject can be a patient. The subject may have been diagnosed with a disease or condition described herein (e.g., cancer) and is in need of treatment (e.g., cancer), may be suspected of having such a disease / condition, or may be at risk of developing / infecting such a disease / condition.

[0554] In embodiments of the present disclosure, the subject is preferably a human subject. In some embodiments, according to the treatment or prevention methods of the present disclosure, the subject to be treated is a subject suffering from or at risk of developing a disease / disorder as described herein. In embodiments of the present disclosure, a subject can be selected for treatment according to a method based on the characteristics of specific markers of such disease / disorder.

[0555] According to the present disclosure, the subject may be an allogeneic subject of interest for the therapeutic intervention. According to the present disclosure, the subject to be treated / prevented may be genetically different from the subject providing the immune cells. According to the present disclosure, the subject to be treated / prevented may not be HLA-matched to the subject providing the immune cells. According to the present disclosure, the subject to be treated / prevented may be HLA-matched to the subject providing the immune cells.

[0556] According to the present disclosure, the subject to which cells are administered may be allogeneic / non-autologous relative to the source of the cells. The subject to which cells are administered may be different from the subject from which the cells were obtained to prepare the administered cells. The subject to which cells are administered may be genetically different from the subject from which the cells were obtained to prepare the administered cells.

[0557] The subject to which the cells are administered may contain MHC / HLA genes encoding MHC / HLA molecules that are different from the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from which the cells were obtained to prepare the administered cells. The subject to which the cells are administered may contain MHC / HLA genes encoding MHC / HLA molecules that are the same as the MHC / HLA molecules encoded by the MHC / HLA genes of the subject from which the cells were obtained to prepare the administered cells.

[0558] In some embodiments, the object of administering cells is HLA matched with the object from which cells are obtained to prepare the object of administering cells. In some embodiments, the object of administering cells is HLA matched with the object from which cells are obtained to prepare the object of administering cells, or HLA matched completely. In some embodiments, the matching degree of the object on HLA-a, -B, -C and -DRB1 is ≥4 / 8 (i.e. 4 / 8, 5 / 8, 6 / 8, 7 / 8 or 8 / 8). In some embodiments, the matching degree of the object on HLA-a, -B, -C, -DRB1 and -DQB1 is ≥5 / 10 (i.e. 5 / 10, 6 / 10, 7 / 10, 8 / 10, 9 / 10 or 10 / 10). In some embodiments, the matching degree of the object on HLA-a, -B, -C, -DRB1, -DQB1 and -DPB1 is ≥6 / 12 (i.e. 6 / 12, 7 / 12, 8 / 12, 9 / 12, 10 / 12, 11 / 12 or 12 / 12). In some embodiments, the subject has an 8 / 8 match for HLA-A, -B, -C, and -DRB1. In some embodiments, the subject has an 8 / 8 match for HLA-a, -B, -C, -DRB1, and -DQB1. In some embodiments, the subject has an 10 / 10 match for HLA-a, -B, -C, -DRB1, -DQB1, and -DPB1. In some embodiments, the subject has an 12 / 12 match for HLA-a, -B, -C, -DRB1, -DQB1, and -DPB1.

[0559] Sequence identity

[0560] As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to the nucleotides / amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity between the sequences. Pairwise and multiple sequence alignments to determine percent sequence identity between two or more amino acid or nucleic acid sequences can be achieved using various means known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega ( J., Bioinformatics (2005)

[0561] 21951-960), T-coffee (Notredame et al., J. Mol. Biol. (2000) 302205-217), Kalign (Lassmann and Sonnhammer, BMC Bioinformatic (2005) 6298), and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30(4)772-780) software. When using such software, it is preferred to use the default parameters, such as gap penalty and gap extension penalty.

[0562] sequence

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569] ***

[0570] The features disclosed in the above description, the following claims, or the accompanying drawings, in their specific form or as means for performing the disclosed functions or methods or processes for obtaining the disclosed results, may be used, as appropriate, to implement the present invention in various forms, either alone or in any combination of these features. The exemplary embodiments described herein are illustrative and non-restrictive, and equivalent modifications and variations will be apparent to those skilled in the art when given the present invention. The present invention includes the combination of the described aspects and preferred features, unless such combination is clearly not permitted or explicitly avoided.

[0571] For the avoidance of doubt, any theoretical explanations provided herein are intended to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0572] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0573] Aspects and embodiments of the present invention will now be described by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated herein by reference.

[0574] In this specification, including the following claims, unless the context requires otherwise, the words "comprise" and "comprising", and variations such as "including", "comprising", "containing", and "containing", will be understood to imply the inclusion of a stated integer or stages or groups of integers but not the exclusion of any other integer or groups of integers.

[0575] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the from particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another embodiment. The term "about" in relation to a value is optional, for example, to mean + / - 10%.

[0576] When a nucleic acid sequence is disclosed herein, its reverse complement is also expressly contemplated.

[0577] The methods described herein may preferably be performed in vitro.The term "in vitro" is intended to encompass procedures performed on cells in culture, whereas the term "in vivo" is intended to encompass procedures performed on intact multicellular organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0579] Implementations and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings.

[0580] Figure 1 . Schematic diagram of the production process of activated TCR knockout (KO) cells as therapeutic transplant cells.

[0581] Figure 2 . Schematic diagram of the production process of virus-specific cells (VSTs) as therapeutic transplant cells.

[0582] Figure 3 Schematic diagram of the production process for CD30 knockout (KO) cells, allogeneic T (allo-T) cells, as an example of generating allo-T cells that do not express the CAR-targeted antigen.

[0583] Figures 4A to 4C.Line graphs and bar graphs show that T cells from two different donors, transduced with GFP or Serpin-GFP, expanded and expressed SERPINB9 (SB9). The fold change in T cell proliferation during the culture period starting from the day of transduction was confirmed by trypan blue staining and cell number counting using a hemocytometer. Transduced T cells were collected 5 days after transduction, and the percentage of GFP expression to the total number of CD3-positive cells and the intracellular SB9 expression (median fluorescence intensity) of GFP-positive cells were determined by flow cytometry. (4A) Cumulative fold change in cell number over time. (4B) GFP expression as a percentage of the total number of CD3-positive cells. (4C) Intracellular SB9 expression in GFP-positive cells.

[0584] Figures 5A to 5C Line and bar graphs show CD30 amplification and expression. CAR (IgG1 spacer) and SERPINB9 (SB9) expression in T cells from two different donors transduced with GFP-CAR or Serpin-CAR. T cell proliferation fold change during the culture period starting from the day of transduction was confirmed by trypan blue staining and cell number counting using a hemocytometer. GFP-transduced T cells were used as a control. Transduced T cells were collected 7 days after transduction, and the percentage of CD30.CAR expression in the total number of CD3-positive cells and the intracellular SB9 expression (median fluorescence intensity) of CD30.CAR-positive cells were determined by flow cytometry. (5A) Cumulative fold change of cell number over time. (5B) Percentage of CD30.CAR expression in the total number of CD3-positive cells. (5C) Intracellular SB9 expression in CD30.CAR-positive cells.

[0585] Figure 6 The figure shows the cytolytic effect of KM-H2 cells by T cells transduced with GFP, CD30.CAR (IgG1 spacer), or SERPINB9 and CD30.CAR (IgG1 spacer) prepared from two different donors. CD30.CAR-positive T cells were normalized to 70% of the total culture cells (in the GFP-CD30.CAR and Serpin-CD30.CAR conditions). Transduced T cells (effector cells) were co-cultured with CD30-expressing KM-H2 cells (target cells) at effector:target ratios of 1:1 and 2:1, and KM-H2 cytolysis was measured over 48 hours using xCELLigence real-time cell analysis software (Agilent). GFP-transduced T cells served as a negative control.

[0586] Figures 7A to 7ESchematic diagram and line graph show that SERPINB9 expression protects transplanted T cells from in vitro immune rejection. (7A) Mixed lymphocyte reaction (MLR) assay and schematic diagram of the protocol used. Using HLA-mismatched host PBMCs, GFP-transduced TCR knockout (KO) transplanted T cells (negative control) or Serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:10 ratio (PBMC mixed lymphocyte assay), and cell counts were determined over 12 days. MLR assays were performed using 10 ng / mL of IL-7 and IL-15, respectively, and expansion cultures were performed as needed. (7B) Cell counts of transplanted T cells cultured alone. (7C) Cell counts of transplanted T cells cultured in a PBMC MLR. (7D) Cell counts of host CD3-positive T cells. (7E) Cell counts of host CD3-negative, CD56-positive natural killer (NK) cells. All data are mean ± SD.

[0587] Figures 8A to 8E Schematic diagram, line graph, and bar graph demonstrate that SERPINB9 expression protects transplanted CD30.CAR (IgG1 spacer) T cells from T cell-mediated in vitro immune rejection. (8A) Mixed lymphocyte reaction (MLR) assay and schematic diagram of the protocol used. Using HLA-mismatched CD30 KO alloreactive T cells, GFP-transduced TCR knockout (KO) transplanted T cells (negative control) or Serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:10 ratio (CD30 KO allo-T cell mixed lymphocyte assay). HLA-mismatched CD30 KO alloreactive T cells recognized and killed transplanted cells, and cell numbers were measured within 3 days. MLR assays were performed using 10 ng / mL of IL-7 and IL-15. (8B) Cell number of transplanted T cells cultured alone. (8C) Cell number of transplanted T cells cultured in an MLR. (8D) Number of transplanted T cells after 3 days of culture in MLR. (8E) Number of host CD3-positive T cells cultured in MLR. All data are mean ± SD.

[0588] Figures 9A to 9K Schematic diagrams, graphs, and bar graphs show that overexpression of SB9 (CAS) in CD30.CAR-transplanted T cells does not affect CAR cytolytic function and is superior in protecting transplanted cells from allogeneic rejection. (9A) Graph showing CD30.CAR (IgG1 spacer) ATC-mediated cytolysis of KM-H2 cells using the xCELLigence system. (9B) Schematic diagram of the in vitro co-culture setup of 9C-9F, in which TCRαβKO CD30.CAR (IgG1 spacer) ATCs and host CD30 KO ATCs were mixed at a 1:1 ratio, and host CD30 KO ATCs can recognize and kill transplanted cells. Detection was performed using 10 ng / mL of IL-7 and IL-15.

[0589] (9C, 9D, and 9E) Line graphs show the cell populations of representative graft-host pairs, including monocultured graft T cells (C), cocultured graft T cells (D), and host T cells (E). (9F) Bar graph shows host-mediated graft killing, as determined by Calculated, where each dot represents an independent graft-host pairing and the bars are medians (n ​​= 6). P values ​​were obtained by Tukey's post hoc one-way ANOVA. *, P = 0.0189; **, P = 0.0034. (9G)

[0590] Schematic diagram of the in vitro co-culture setup of 9H-9K, in which CD30.CAR (4-1BB spacer) EBVST and host CD30 KO ATC was mixed at a ratio of 1:1-4, and host CD30 KO ATCs can recognize and kill transplanted cells. Detection was performed using 10 ng / mL of IL-7 and IL-15. (9H, 9I, and 9J) Line graphs show cell numbers for representative graft-host pairs, including monocultured graft T cells (H), cocultured graft T cells (I), and host T cells (J). (9K) Bar graphs show host-mediated graft killing, where each dot represents an individual graft-host pair and the bar graphs represent the median (n = 4). The minimum graft-to-host ratio was selected to achieve ≥80% host-mediated graft killing of CD30.CAR (4-1BB spacer) EBVSTs on day 4. P values ​​were obtained by Tukey's post hoc one-way ANOVA. *, P = 0.0150; **, P < 0.01.

[0591] Figures 10A to 10E Schematic diagram, images, and bar graphs showing TCRαβ expressing different forms of SB9 KO Manufacturing process and characteristics of CD30.CAR (IgG1 spacer) ATCs. (10A) Schematic diagram showing TCRαβ expressing different forms of SB9. KOManufacturing timeline for CD30.CAR ATCs, where transduction was performed using retrovirus encoding a bicistronic construct (top) or retrovirus encoding CAR and SB9, respectively (bottom). (10B) Bar graphs show SB9 expression as determined by intracellular staining and flow cytometry. (10C and 10D) Bar graphs show the percentage of CD30.CAR expression on CD3-positive T cells (C) and the geometric mean fluorescence intensity (MFI) (D). (10E) Images show flow cytometric analysis of CD3 wild-type (WT) T cells or TCRαβ modified using Crispr gene editing technology. KO Expression of TCRαβ in T cells after gene repression.

[0592] Figure 11A and 11B Schematic diagram and image showing the production of alloreactive CD30 proteins that recognize and kill transplanted cells (-T) KO Host cells. (11A) Schematic diagram showing the manufacturing timeline for allo-T. (11B) Images showing CD30 expression on allo-T cells by staining with two different antibody clones, BY88 and BerH8, followed by flow cytometric analysis.

[0593] Figures 12A to 12KSchematic diagram, SDS-PAGE, and bar graphs show the manufacturing process and characteristics of CD30.CAR (4-1BB spacer) EBVSTs expressing different forms of SB9. (12A) Schematic diagram showing the manufacturing timeline for CD30.CAR EBVSTs expressing different forms of SB9. (12B) SDS-PAGE analysis shows SB9 expression in a representative donor, where the top band (approximately 62 kDa) represents preformed GzmB and SB9 complexes in the cytoplasm and the bottom band (approximately 43 kDa) represents functional SB9 monomers. Transduction was performed with low (10 ng / mL) or high (100 ng / mL) concentrations of IL-15, indicated by L and H, respectively. (12C) Bar graph showing SB9 monomer density analysis normalized to CD30.CAR EBVST control and calnexin loading control. (12D and 12E) Histograms show the percentage of CD30.CAR expression on CD3-positive T cells (D) and the geometric mean fluorescence intensity (MFI) (E), n = 4 donors. (12F) Histograms show the percentage of IFNγ and / or TNFα produced by CD3-positive T cells in response to EBV peptides, as analyzed by intracellular cytokine staining and flow cytometry, n = 4 donors. (12G, 12H, 12I, 12J, and 12K) Histograms show the CD4 / CD8 subsets (G), memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: CD45RA-expressing T effector memory) (H), PD-1, Tim-3, Lag-3 expression (I), CD39 expression (J), and CD25 expression (K) of CD30.CAR EBVST from representative donors.

[0594] Figures 13A to 13J Schematic diagram, line graph, and bar graph show that overexpression of SB9 (CAS) in non-CAR transplanted T cells (ATCs and EBVSTs) is more advantageous in protecting transplanted cells from allogeneic rejection. (13A) Schematic diagram of the in vitro co-culture setup of 13B-13E, in which TCRαβ was transplanted at a ratio of 1:10-20. KO ATCs were mixed with HLA-mismatched host PBMCs. IL-7 and IL-15 were used for detection at 10 ng / mL each. (13B, 13C, and 13D) Line graphs show the number of cells from representative graft-host pairs, including monocultured graft T cells (B), cocultured graft T cells (C), and host T and NK cells (D). (13E) Bar graphs show host-mediated graft killing, as determined by Calculated, where each dot represents an independent graft-host pairing and the bars are medians (n ​​= 5). P values ​​were obtained by Tukey's post hoc one-way ANOVA. **, P = 0.0016; ***, P < 0.001. (13F) Schematic diagram of the in vitro co-culture setup of 13G-13J, in which EBVST and alloreactive CD30 KO Host ATC was mixed at a ratio of 1:1-4, allogeneic reactive CD30 KO Host ATC can recognize and kill transplanted cells. Detection was performed using 10 ng / mL of IL-7 and IL-15. (13G, 13H, and 13I) Line graphs show the number of cells from representative graft-host pairs, including monocultured graft T cells (G), cocultured graft T cells (H), and host T cells (I). (13J) Bar graph shows host-mediated graft killing, as determined by Calculated, where each dot represents an independent graft-host pair and the bars are medians (n=2). Statistics were not performed because n<3.

[0595] Figures 14A-14K Schematic diagrams, images, bar graphs, and line charts show that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVSTs protects CD30.CAR EBVSTs from allogeneic rejection and enhances anti-tumor efficacy in vivo or in vitro. (14A) (B) and (C) Schematic diagram of an in vitro tri-culture setup in which engineered NALM6 (truncated positive for CD30 and HLA I and II) KO ), transplanted CD30.CAR EBVST and host CD30 KO ATC was mixed at a ratio of 1:1:1-4, and host CD30 KO ATC can recognize and kill transplanted cells. Detection was performed in the absence of cytokines. (14B) The bar graph shows host-mediated transplant killing, which is Calculated, where each dot represents an independent graft-host pairing and the bars are medians (n=5). The minimum graft-to-host ratio was selected to achieve host-mediated graft killing of ≥60% of CD30.CAR EBVSTs on day 3. P values ​​were obtained by post hoc one-way ANOVA. *, P = 0.0193. (14C) Bar graph showing the percentage of tumor cells killed in round 2 in tricultures, normalized to tumor cells grown in monoculture. (14D) Schematic diagram of the in vivo allogeneic rejection model for 14E-14F, where 2.5 x 10 6 An engineered NALM6 (truncated positive for CD30 and HLA I and IIKO ) intravenously into NSG(MHC KO ) mice. After 18 days, 5x 10 6 eGFP-ffLuc-expressing transplanted CD30.CAR EBVST and 5x10 6 Alloreactive host T cells (allo-T) were co-injected intravenously. (14E) Bioluminescence image showing the level of transplanted CD30.CAR EBVSTs acquired by the IVIS Lumina S5 imaging system. (14F) Line graph showing the quantitative change in bioluminescence signal of transplanted CD30.CAR EBVSTs over time. (14G) Schematic diagram of the in vivo allogeneic rejection model of 14H-14K, in which 2.5 x 10 6 Engineered NALM6.eGFP-ffLuc were intravenously injected into NSG (MHC KO ) mice, and 15 days later, 5 x 10 6 transplanted CD30.CAR EBVST with 5x 10 6 Alloreactive host T cells (allo-T) were co-administered intravenously. (14H and 14I) Line graphs show flow cytometric analysis of blood samples at the indicated time points. The percentage of engrafted CD30.CAR EBVST cells (H) and the percentage of host allo-T cells (I) in the peripheral blood are shown. (14J) Bioluminescence images show the growth of engineered NALM6.eGFP-ffLuc tumors acquired on the IVIS Lumina S5 imaging system. (14K) Line graph shows quantification of the bioluminescent signal of tumor cells over time, normalized to the disease level at day 0. All in vivo data are presented as mean + SD. P values ​​were determined by one-way analysis of variance (K; day 11 after treatment) or two-way analysis of variance (F and H: P values ​​are shown for comparisons between CD30.CAR+Allo-T samples and SB9(CAS)-CD30.CAR+Allo-T samples) with Dunnett's multiple comparison correction, in which the sample means were compared with the mean of the CD30.CAR+Allo-T samples. Figures 15A to 15F Images and bar graphs show that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects against allogeneic rejection and enhances anti-tumor efficacy in vitro. (15A) The image shows FACS-sorted engineered NALM6 cells overexpressing truncated CD30 (tCD30) and expressed by HLA class I and II (HLA DKO) flow cytometric analysis of gene editing. (15B) The bar graph shows the percentage of host cells killing tumor cells when the engineered NALM6 tumor cells were co-cultured with host allo-T cells at a 1:1 ratio. (15C) The line graph shows the host-mediated transplant killing, Calculations show that when CD19.CAR ATC transplant cells, engineered NALM6 and host ATC are cultured at a ratio of 1:1:1, host ATC can recognize and kill transplant cells. (15D) The line graph shows the difference between CD30.CAR ATC transplant cells, host CD30 KO Host-mediated killing of CD30.CAR ATC-transplanted cells in a 1:4:1 tri-culture of ATC and engineered NALM6. This assay was performed in the absence of cytokines. (15E and 15F) Bar graphs show the number of tumor cells analyzed by flow cytometry after 3 days of tri-culture with CD19.CAR ATC (E) or CD30.CAR ATC (F).

[0596] Figures 16A to 16G Schematic diagrams, images, and line graphs show that allogeneic rejection of transplanted T cells can occur in vivo using alloreactive T cells. (16A) Schematic diagrams of the in vivo allogeneic rejection model of 16B and 16C, where 2.5x10 6 An engineered NALM6 (truncated positive for CD30 and HLA I and II KO ) intravenously into NSG(MHC KO ) mice. After 18 days, 5x 10 6 5 x 10 alloreactive host T cells (allo-T) 6 eGFP-ffLuc-expressing transplanted CD30.CAR (4-1BB interval) EBVSTs were injected intravenously. (16B) Bioluminescence image showing the level of transplanted CD30.CAR EBVSTs acquired by the IVISLumina S5 imaging system. (16C) Line graph showing the quantitative change in bioluminescence signal of transplanted CD30.CAR EBVSTs over time. (16D) Schematic diagram of the in vivo allogeneic rejection model of 16E-16G, where 2.5 x 10 6 Engineered NALM6.eGFP-ffLuc were intravenously injected into NSG (MHC KO ) mice, and after 15 days, the cells were either cultured with or without 5 x 10 6 5x10 alloreactive host T cells (allo-T) 6CD30.CAR EBVST cells were engrafted intravenously. (16E) Line graph shows flow cytometric analysis of blood samples at the indicated time points. The percentage of engrafted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images show the growth of engineered NALM6.eGFP-ffLuc tumors acquired by the IVIS Lumina S5 imaging system. (16G) Graph shows quantification of the bioluminescent signal of tumor cells over time, normalized to the disease level at day 0. Line graphs of all in vivo data are presented as mean + SD. P values ​​were determined using two-way ANOVA with Sidak's multiple comparison correction (E) or unpaired one-tailed t-test (C and G; day 11 after treatment).

[0597] Figures 17A to 17Q Schematic diagrams, line graphs, and bar graphs show that overexpression of SB9 (CAS) enhances the expansion of CD30.CAR (4-1BB spacer) and CD19.CAR ATCs after continuous co-culture with tumor cells. (17A) Schematic diagram shows the in vitro co-culture of CAR ATCs with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5, studying CD30.CAR and CD19.CAR ATCs, respectively. Every 2-3 days, cells were counted by flow cytometry and re-cocultured with new tumor cells at a fixed ratio. This process was repeated until complete tumor cell elimination by CAR T cells was impossible. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Line graphs show the cumulative fold change in the number of CD30.CAR ATCs over time in monoculture (B and F) and co-culture (C and G). (17D and 17H) Bar graphs show the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs show the expression of PD-1, Tim-3, and Lag-3 by CD30.CAR ATC at the start of coculture (day 0) and the last day of coculture (day 13). (17J, 17K, 17N, and 17O) Line graphs show the cumulative fold change in the number of CD19.CAR ATC over time in monoculture (J and N) and coculture (K and O). (17L and 17P) Bar graphs show the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs show the expression of PD-1, Tim-3, and Lag-3 by CD19.CAR ATC at the start of coculture (day 0) and the last day of coculture (day 8).

[0598] Figures 18A to 18DSchematic diagram and bar graph showing the cytokine secretion profiles of CD30.CAR EBVST (4-1BB spacer) expressing different forms of SB9 with and without CAR-mediated stimulation. (18A) Schematic diagram of the in vitro co-culture setup of 18B-18D, where effector CAR T cells were co-cultured with cloned engineered NALM6 (truncated positive CD30 and HLA I and II KO ) or KM-H2 tumor cells at a 1:1 ratio. Assays were performed in the absence of cytokines. (18B, 18C, and 18D) Bar graphs show cytokine concentrations measured using the 13-plex immunoassay kit (Merck) from supernatants of cells cultured for 24 hours monocultured (B) or cocultured with eng...

Claims

1. An immune cell, characterized in that The immune cell comprises a modification that increases the expression or activity of SERPINB9.

2. The immune cell according to claim 1, characterized in that The immune cell contains an exogenous nucleic acid encoding a SERPINB9 polypeptide.

3. The immune cell according to claim 2, characterized in that The exogenous nucleic acid encoding the SERPINB9 polypeptide is an expression vector, or is contained in an expression vector; optionally, the expression vector is a retroviral expression vector.

4. The immune cell according to claim 2 or 3, characterized in that The SERPINB9 polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, or a variant having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6 or 7.

5. The immune cell according to any one of claims 1 to 4, characterized in that The immune cells are effector immune cells; optionally, the effector immune cells are T cells or natural killer (NK) cells.

6. The immune cell according to any one of claims 1 to 5, characterized in that The immune cell comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

7. The immune cell according to claim 6, characterized in that The CAR comprises an antigen binding domain that binds to a cancer-associated antigen selected from the group consisting of CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA; optionally, the CAR comprises an antigen binding domain that binds to CD30.

8. The immune cell according to any one of claims 1 to 7, characterized in that The immune cells are virus-specific T cells or activated T cells (ATC).

9. The immune cell according to claim 8, characterized in that The virus-specific T cells are specific to a virus selected from the group consisting of Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV) or varicella-zoster virus (VZV); optionally, the virus is EBV.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the immune cell according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

11. The immune cell according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 10, for use in a method of treatment or prevention.

12. Use of the immune cell according to any one of claims 1 to 9, or the pharmaceutical composition according to claim 10, for the manufacture of a medicament for use in a medical or preventive method.

13. A method for treating or preventing a disease or condition in a subject, comprising administering to the subject an effective therapeutic or preventive dose of the immune cell according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10.

14. A method for reducing the activity of serine proteases or caspases in cells, comprising modifying the cells to increase the expression or activity of SERPINB9.

15. A method for increasing the resistance of a cell to serine protease or caspase activity, comprising modifying the cell to increase SERPINB9 expression or activity.

16. A method for increasing the resistance of cells to cell killing by granzyme B, comprising modifying the cells to increase the expression or activity of SERPINB9.

17. A method for increasing the resistance of cells to death receptor-mediated apoptosis, comprising modifying cells to increase SERPINB9 expression or activity.

18. The method according to any one of claims 14 to 17, characterized in that The method of modifying cells to increase SERPINB9 expression or activity comprises introducing a nucleic acid encoding a SERPINB9 polypeptide into the cells.

19. The method according to claim 18, characterized in that The nucleic acid encoding the SERPINB9 polypeptide is an expression vector, or is contained in an expression vector; optionally, the expression vector is a retroviral expression vector.

20. The method according to claim 18 or 19, characterized in that The SERPINB9 polypeptide comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, or a variant having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6 or 7.

21. The method according to any one of claims 14 to 20, characterized in that The cell is an effector immune cell; optionally, the effector immune cell is a T cell or a natural killer (NK) cell.

22. The method according to any one of claims 14 to 21, characterized in that The cells contain a nucleic acid encoding a chimeric antigen receptor (CAR).

23. The method of claim 22, wherein: The CAR comprises an antigen binding domain that binds a cancer-associated antigen selected from the group consisting of CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA; optionally, the CAR comprises an antigen binding domain that binds CD30.

24. The method according to any one of claims 14 to 23, characterized in that The cells are virus-specific T cells.

25. The method of claim 24, wherein: The virus-specific T cells are specific to a virus selected from the group consisting of Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV) or varicella-zoster virus (VZV); optionally, the virus is EBV.

26. An immune cell for treating or preventing cancer in a subject, characterized in that: The immune cell comprises a nucleic acid encoding a CAR, wherein the CAR comprises: (i) an antigen binding domain that binds CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); and The immune cell comprises a modification that increases the expression or activity of SERPINB9.

27. Use of immune cells in the manufacture of a drug for treating or preventing cancer in a subject, characterized in that: The immune cell comprises a nucleic acid encoding a CAR, wherein the CAR comprises: (i) an antigen binding domain that binds CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); and The immune cell comprises a modification that increases the expression or activity of SERPINB9.

28. A method for treating or preventing cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective dose of immune cells, wherein: The immune cell comprises a nucleic acid encoding a CAR, wherein the CAR comprises: (i) an antigen binding domain that binds CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM); and The immune cell comprises a modification that increases the expression or activity of SERPINB9.

29. The immune cell for use according to claim 26, the use according to claim 27, or the method according to claim 28, characterized in that: The immune cells are virus-specific T cells; optionally, the immune cells are Epstein-Barr virus (EBV)-specific T cells.

30. An immune cell for treating or preventing cancer in a subject, characterized in that: The immune cells are virus-specific T cells; optionally, the immune cells are Epstein-Barr virus (EBV)-specific T cells; and The immune cell comprises a modification that increases the expression or activity of SERPINB9.

31. Use of immune cells in the manufacture of a drug for treating or preventing cancer in a subject, characterized in that: The immune cells are virus-specific T cells; optionally, the immune cells are Epstein-Barr virus (EBV)-specific T cells; and The immune cell comprises a modification that increases the expression or activity of SERPINB9.

32. A method for treating or preventing cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective dose of immune cells, wherein: The immune cells are virus-specific T cells; optionally, the immune cells are Epstein-Barr virus (EBV)-specific T cells; and The immune cell comprises a modification that increases the expression or activity of SERPINB9.

33. The immune cell for use according to claim 30, the use according to claim 31, or the method according to claim 32, characterized in that: The immune cell comprises a nucleic acid encoding a CAR, wherein the CAR comprises: (i) an antigen binding domain that binds to CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM).

34. The immune cell, use or method according to any one of claims 26 to 33, characterized in that: With respect to the immune cells, the subject is allogeneic.

35. The immune cell, use or method according to any one of claims 26 to 34, characterized in that: The immune cell contains an exogenous nucleic acid encoding a SERPINB9 polypeptide.

36. The immune cell, use or method according to claim 35, characterized in that: The exogenous nucleic acid encoding the SERPINB9 polypeptide is an expression vector, or is contained in an expression vector; optionally, the expression vector is a retroviral expression vector.

37. The immune cell, use or method according to claim 35 or 36, characterized in that: The SERPINB9 polypeptide comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 1, 4, 5, 6 or 7, or a variant having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6 or 7.

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