Eukaryotic cell expressing exogenous inosine monophosphate dehydrogenase

Abstract

Provided herein are eukaryotic cells that express an exogenously introduced inosine 5 '-monophosphate dehydrogenase (IMPDH) that is resistant to purine biosynthesis inhibitors, uses of the enzyme in the production of therapeutic drugs and in the treatment of diseases or conditions, as well as related pharmaceutical compositions and methods of preparation thereof.

Description

Technical Field

[0001] This disclosure generally relates to eukaryotic cells expressing exogenously introduced inosine 5'-monophosphate dehydrogenase (IMPDH), and therapeutic methods comprising such cells and methods of using them. This disclosure also relates to the use of exogenously introduced IMPDH to construct engineered eukaryotic cells resistant to purine biosynthesis inhibitors. Background Technology

[0002] Adoptive transfer of engineered cells represents an emerging and innovative therapeutic strategy. While autologous cell therapy has been a breakthrough treatment for some cancers, several drawbacks limit its application to a wider range of patients. Allogeneic cell therapy may offer a more readily available treatment option, where cells (e.g., immune cells) are collected from a healthy donor, genetically modified, and then injected into the patient. However, allogeneic cell therapy is accompanied by certain complications, including, for example, rejection of the donor cells by the patient's own immune system. Although immunosuppressants have been proposed to mitigate the risk of such immune complications, these inhibitors often lead to other complications, including suppressing the efficacy of transferred engineered cells (e.g., CAR-T cells). Therefore, there remains a need in the art for improved constructs or engineered cells (e.g., CAR-T cells) for treating diseases or conditions that can optimize the balance between therapeutic efficacy and side effect control. Summary of the Invention

[0003] In one aspect, this article provides eukaryotic cells expressing exogenously introduced inosine 5'-monophosphate dehydrogenase (IMPDH); optionally, the eukaryotic cells also express a functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the exogenously introduced IMPDH is resistant to purine biosynthesis inhibitors.

[0004] In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH binding to the purine biosynthesis inhibitor is greater than that of wild-type human IMPDH binding to the purine biosynthesis inhibitor. In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH binding to the purine biosynthesis inhibitor is at least 20 times greater than that of wild-type human IMPDH binding to the purine biosynthesis inhibitor. In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH binding to the purine biosynthesis inhibitor is greater than 20 nM. In some embodiments, the half-maximum effective concentration (EC50) or half-maximum inhibitory concentration (IC50) of the purine biosynthesis inhibitor inhibiting exogenously introduced IMPDH is greater than the EC50 or IC50 inhibiting wild-type human IMPDH. In some embodiments, the half-maximum effective concentration (EC50) or half-maximum inhibitory concentration (IC50) of the purine biosynthesis inhibitor for exogenously introduced IMPDH is greater than 1 ng / ml. In some embodiments, the EC50 or IC50 of the exogenously introduced IMPDH is greater than 0.5 μg / ml for the purine biosynthesis inhibitor. In some embodiments, the exogenously introduced IMPDH catalyzes the conversion of IMP to XMP more efficiently than wild-type human IMPDH in the presence of the purine biosynthesis inhibitor. In some embodiments, the catalytic turnover number (Kcat) for the conversion of inosine monophosphate (IMP) to xanthoside monophosphate (XMP) by exogenously introduced IMPDH is greater than that of wild-type human IMPDH. In some embodiments, the catalytic turnover number (Kcat) for the conversion of inosine monophosphate (IMP) to xanthoside monophosphate (XMP) by exogenously introduced IMPDH is greater than 0.4 s. -1 .

[0005] In some embodiments of the various compositions and methods provided herein, the purine biosynthesis inhibitors include mycophenolic acid (MPA), ribavirin, mizoribine, tiazofurin, AVN-944 (VX-944), FF-10501, AS2643361, or BMS-986126, or their pharmaceutically acceptable esters, salts, or prodrugs. In some embodiments, the purine biosynthesis inhibitor is mycophenolic acid (MPA), its derivatives, analogs, or pharmaceutically acceptable salts, or a compound that can be converted into MPA intracellularly. In some embodiments, the purine biosynthesis inhibitor is mycophenolate, mycophenolate mofetil (MMF), sodium mycophenolate (MPS), calcium mycophenolate, potassium mycophenolate, or derivatives thereof. In some embodiments, the purine biosynthesis inhibitor is MMF.

[0006] IMPDH catalyzes the oxidation of inosine monophosphate (IMP) to xanthocyanin monophosphate (XMP) and the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. MPA inhibits IMPDH by binding to its active site (particularly the region interacting with NAD+). The exogenous IMPDHs presented herein may differ in their binding mode with the cofactor NAD+, which affects the mode of action of MPA. Therefore, exogenous IMPDHs (e.g., prokaryotic IMPDHs or variants thereof) have a higher affinity for NAD+ and may exhibit greater resistance to purine biosynthesis inhibitors such as MPA or its analogues or derivatives. In some embodiments, the exogenous IMPDHs have an affinity for nicotinamide adenine dinucleotide (NAD+) that is not less than or greater than that of wild-type human IMPDH. In some embodiments, the exogenous IMPDHs have a dissociation constant (Kd) for NAD+ that is not greater than or less than that of wild-type human IMPDH. In some embodiments, the Michaelis constant (Km) for NAD+ binding of exogenously introduced IMPDH is no greater than or less than that of wild-type human IMPDH. In some embodiments, exogenously introduced IMPDH allows for simultaneous binding of purine biosynthesis inhibitors and IMPDH substrates without mutual repulsion. In some embodiments, exogenously introduced IMPDH is able to catalyze the conversion of IMP to XMP in a normal manner, unaffected by purine biosynthesis inhibitors. Surprisingly, it has even been observed that eukaryotic cells expressing exogenously introduced IMPDH exhibit superior growth performance in the presence of purine biosynthesis inhibitors. In some embodiments, exogenously introduced IMPDH contains a cofactor binding site for substrate binding. In some embodiments, the substrate is inosine monophosphate (IMP). In some embodiments, the substrate is NAD+.

[0007] In some embodiments, the exogenously introduced IMPDH is a non-human IMPDH or a variant of a human IMPDH. In some embodiments, the exogenously introduced IMPDH is derived from a prokaryotic IMPDH, such as a bacterial IMPDH or a variant thereof. In some embodiments, the bacterial IMPDH is a Gram-positive bacterial IMPDH or a variant thereof. In some embodiments, the Gram-positive bacterial IMPDH is derived from Bacillus subtilis, Lactobacillus plantarum, or Staphylococcus aureus. In some embodiments, the bacterial IMPDH is a Gram-negative bacterial IMPDH or a variant thereof. In some embodiments, the Gram-negative bacterial IMPDH is derived from Escherichia coli or Mesoplasma florum. In some specific embodiments, the exogenously introduced IMPDH is Lactobacillus plantarum IMPDH, Escherichia coli IMPDH, Bacillus subtilis IMPDH, Staphylococcus aureus IMPDH, or Mesoplasma florum IMPDH, or a variant thereof.

[0008] In some embodiments, the exogenously introduced IMPDH comprises an amino acid sequence selected from SEQ ID NO:1-7, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1-7.

[0009] In some specific embodiments, *Lactobacillus plantarum* IMPDH contains the amino acid sequence of SEQ ID NO:1; and / or, *Escherichia coli* IMPDH contains the amino acid sequence of SEQ ID NO:2; and / or, *Bacillus subtilis* IMPDH contains the amino acid sequence of SEQ ID NO:3; and / or, *Staphylococcus aureus* IMPDH contains the amino acid sequence of SEQ ID NO:4; and / or, *Mesoplasma florum* IMPDH contains the amino acid sequence of SEQ ID NO:5.

[0010] In some embodiments, the IMPDH variant comprises an amino acid sequence that differs from naturally occurring IMPDH due to at least one amino acid alteration (e.g., substitution, addition, or deletion). The IMPDH variants described herein may have at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with naturally occurring IMPDH. In some embodiments, the IMPDH variant is a functional variant that has some biological characteristics modified or improved relative to the parent IMPDH, or will substantially retain some biological characteristics of the parent IMPDH, particularly resistance to purine biosynthesis inhibitors. In some embodiments, the IMPDH variant, relative to its naturally occurring IMPDH (parental IMPDH), includes mutations at the following sites: D13, D50, E54, D138, D200, A223, D243, D248, D338, E369, E373, E469, or combinations thereof. In some specific embodiments, the IMPDH variant comprises the amino acid sequence of SEQ ID NO: 6 or 7.

[0011] In some aspects of this disclosure, the prokaryotic IMPDH or variants thereof described herein are also contemplated and fall within the scope of this disclosure. In other aspects, the disclosure also contemplates the use of the prokaryotic IMPDH or variants thereof described herein for constructing engineered eukaryotic cells resistant to purine biosynthesis inhibitors. In some embodiments, the prokaryotic IMPDH or variants thereof are used for sorting or enriching eukaryotic cells engineered therefrom. By conferring resistance to purine biosynthesis inhibitors on eukaryotic cells, the screening, purification, sorting, or enrichment of engineered cells can be readily achieved in embodiments for producing cells (e.g., for treating diseases or conditions).

[0012] In some embodiments, the functional exogenous receptor is a T-cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), a T-cell antigen-conjugate (TAC) chimeric receptor, a chimeric switch receptor, a signal transduction receptor, an inducible regulation dimerization activation receptor (DARIC), a chimeric cytokine receptor, a co-stimulatory receptor, a dominant-negative receptor, or a component thereof. In some embodiments, the functional exogenous receptor is a CAR. In some embodiments, the extracellular antigen-binding domain is an antibody, a single-domain antibody (sdAb), a single-chain variable fragment (scFv), or a ligand. In some embodiments, the extracellular antigen-binding domain specifically recognizes tumor antigens, pathogen antigens, normal cell antigens, HLA antigens, or allogeneic antigens. In some embodiments, the transmembrane domain is derived from a molecule selected from CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α or CD28. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the co-stimulatory signaling domain includes a cytoplasmic domain of CD28 and / or a cytoplasmic domain of CD137. In some embodiments, the functional exogenous receptor further includes a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the functional exogenous receptor further includes a signal peptide located at the N-terminus of a polypeptide. In some implementations, the signal peptide is derived from CD8α.

[0013] In some embodiments, the functional exogenous receptor includes a single-domain antibody (sdAb) domain that binds GUCY2C in its extracellular domain. In some embodiments, the sdAb domain includes CDR1, CDR2, and CDR3, each containing the amino acid sequences of CDR1, CDR2, and CDR3 as shown in the sdAb domain containing any of the amino acid sequences in SEQ ID NO: 12-32. In some embodiments, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or combinations thereof. In some embodiments, the sdAb domain is a VHH domain.

[0014] In some embodiments of this disclosure, the engineered eukaryotic cells provided herein optionally, but not necessarily, contain or express a functional exogenous receptor. In some embodiments, the prokaryotic IMPDH introduced into the engineered eukaryotic cells can exert its biological activity in the absence of the functional exogenous receptor. In some embodiments, the functional exogenous receptor has immunogenic biological activity after introduction into the eukaryotic cells. In some embodiments, the functional exogenous receptor introduced into the engineered eukaryotic cells produces an additive or synergistic stimulatory effect with the introduced prokaryotic IMPDH.

[0015] In some embodiments, to facilitate the detection of IMPDH in transduced cells, exogenous IMPDH may be transduced into cells or co-expressed with additional detection markers. In some embodiments, detection markers include, but are not limited to, antibodies or ligands, resistance genes (e.g., antibiotic resistance genes such as puromycin resistance gene (Puro), G418 resistance gene (Neo), and blast fungicide resistance gene (BSD)) or selection markers (e.g., affinity tags such as histidine tags). Detection markers that are co-expressed with the protein being detected without affecting its expression and biological activity are well known in the art. In some embodiments, a functional exogenous receptor is introduced for the detection of engineered eukaryotic cells.

[0016] In some embodiments, the eukaryotic cells provided herein are derived from mammals. In some embodiments, the eukaryotic cells provided herein are derived from primates, rodents, etc. In some embodiments, the eukaryotic cells are derived from humans, rats, mice, guinea pigs, rabbits, sheep, goats, camels or alpacas, horses, donkeys, chimpanzees, or macaques. In some embodiments, the eukaryotic cells are derived from humans.

[0017] In some embodiments, the eukaryotic cells provided herein are primary cells. In this document, the term "primary cell" refers to cells isolated from an organism (e.g., human, animal, or plant tissues or fluids) that have not undergone prolonged in vitro culture and retain their original biological characteristics. In this document, "primary cell" is used in contrast to "cell line." The term "cell line" is typically established from primary cells through a process of "immortification"; immortalization usually involves mutations or alterations to genes related to cell cycle regulation, thereby creating a stable cell line. It has been observed that the exogenously introduced IMPDH provided herein can confer enhanced resistance to purine biosynthesis inhibitors in primary cells compared to cell lines, and even provide improved growth performance. Therefore, in some specific embodiments, the eukaryotic cells provided herein are primary cells. In some particular embodiments, the eukaryotic cells may be cell lines.

[0018] In some embodiments, the eukaryotic cells provided herein are immune cells. In some embodiments, the eukaryotic cells provided herein are lymphocytes, phagocytes, or dendritic cells.

[0019] In some implementations, the eukaryotic cells described herein are T cells, γδT cells, regulatory T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, monocytes, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, or normal tissue cells. In some specific implementations, the eukaryotic cells are T cells.

[0020] On the other hand, this document provides a polypeptide or polypeptide combination comprising IMPDH and a functional exogenous receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the IMPDH is resistant to purine biosynthesis inhibitors. In some embodiments, the IMPDH and the functional exogenous receptor in the polypeptide combination may be unlinked. In some embodiments, the IMPDH and the functional exogenous receptor in the polypeptide combination may be linked or fused, i.e., as a single polypeptide comprising an IMPDH region and a functional exogenous receptor region. In some embodiments, the amino acid sequence of the prokaryotic IMPDH is located at the N-terminus or C-terminus of the amino acid sequence of the functional exogenous receptor. In some embodiments, the IMPDH and the functional exogenous receptor are linked by a cleavable peptide linker. In some embodiments, the IMPDH is an exogenously introduced IMPDH provided herein. In some embodiments, the functional exogenous receptor is a functional exogenous receptor provided herein. In some embodiments, the cleavable peptide linker is a 2A self-cleaving peptide, optionally selected from F2A, E2A, P2A, T2A, and variants thereof. In some embodiments, the 2A self-cleaving peptide is a T2A fragment containing the amino acid sequence of SEQ ID NO:11.

[0021] On the other hand, this document provides isolated nucleic acids comprising nucleic acid sequences encoding polypeptides or combinations of polypeptides provided herein. In some embodiments, this document provides isolated nucleic acids comprising a first region encoding an exogenously introduced IMPDH provided herein, and a second region encoding a functional exogenous receptor provided herein. On another aspect, this document provides a cluster of nucleic acids comprising a first nucleic acid encoding an exogenously introduced IMPDH provided herein, and a second nucleic acid encoding a functional exogenous receptor provided herein. On yet another aspect, this document provides vectors or clusters of vectors comprising isolated nucleic acids or clusters of nucleic acids provided herein.

[0022] In another aspect, this document provides a method for preparing eukaryotic cells, comprising introducing isolated nucleic acids, or nucleic acid clusters, or vectors, or vector groups provided herein into eukaryotic cells. Eukaryotic cells produced according to this method are included in this disclosure. In yet another aspect, this document also provides a eukaryotic cell comprising isolated nucleic acids, or nucleic acid clusters, or vectors, or vector groups provided herein; or prepared by the method for preparing eukaryotic cells provided herein.

[0023] On the other hand, this document provides pharmaceutical compositions comprising eukaryotic cells, polypeptides or combinations of polypeptides, isolated nucleic acids, nucleic acid clusters or carriers or groups of carriers provided herein; and pharmaceutically acceptable carriers. In some embodiments, the pharmaceutical compositions provided herein further comprise purine biosynthesis inhibitors. In some embodiments, the pharmaceutical compositions provided herein further comprise agents that inhibit antigen-presenting cell (APC) function.

[0024] In another respect, this article provides methods for treating a disease or condition in a subject, including administering to the subject an effective amount of the pharmaceutical composition provided herein.

[0025] In another aspect, this document provides a method for preparing cell therapies for treating diseases or conditions, comprising preparing eukaryotic cells expressing exogenously introduced IMPDH and functional exogenous receptors; culturing the eukaryotic cells prepared in vitro in a culture medium containing a purine biosynthesis inhibitor; and collecting the cells after in vitro culture. In some embodiments, the eukaryotic cells are prepared according to the methods provided herein. In some embodiments, the method further includes administering the collected cells to a subject suffering from a disease or condition and administering the purine biosynthesis inhibitor to the subject. In some embodiments, the method further includes administering an agent that inhibits the function of antigen-presenting cells (APCs).

[0026] In some embodiments, the treatment methods or cell therapies provided herein are allogeneic cell therapies. In other embodiments, the treatment methods or cell therapies provided herein are autologous cell therapies.

[0027] In some implementations, the disease or condition is an autoimmune disease, pathogen infection, solid tumor, or hematologic malignancy.

[0028] In another aspect, this document provides methods for reducing and / or preventing host-resistant graft responses, comprising preparing eukaryotic cells expressing exogenously introduced IMPDH, said IMPDH being resistant to purine biosynthesis inhibitors. In some embodiments, the eukaryotic cells are the eukaryotic cells provided herein. In some embodiments, the method further includes administering the eukaryotic cells to a subject suffering from a disease or condition and administering a purine biosynthesis inhibitor to the subject. In some embodiments, the method further includes administering an agent that inhibits the function of antigen-presenting cells (APCs). In some embodiments, the graft is an allogeneic graft.

[0029] In some embodiments of the various compositions or methods provided herein, the agent that inhibits APC function is selected from abatacept, tofacitinib, secukinumab, ipilimumab, cyclosporine A, fingolimod, methotrexate, and their equivalents.

[0030] On the other hand, this document provides a single-domain antibody (sdAb) against GUCY2C, said sdAb comprising or consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of any of SEQ ID NO:12-32. In some embodiments, the anti-GUCY2C sdAb is a camel sdAb or a humanized sdAb. In some embodiments, the sdAb is a VHH antibody. In some embodiments, the anti-GUCY2C sdAb is genetically fused or chemically conjugated with other agents. It should be understood that the primary goal of humanization is to reduce the immunogenicity of antibodies from other species, but such sequence alterations may affect or reduce the biological activity of the parent antibody. It has been observed that the exemplary humanized sdAbs provided in this paper unexpectedly exhibit enhanced biological activity, especially the functional exogenous receptors constructed from humanized sdAbs and the engineered cells, which demonstrate enhanced cytotoxicity and killing efficiency against target cells.

[0031] In another embodiment, this document provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular antigen-binding domain comprising a single-domain antibody (sdAb) domain binding to GUCY2C, wherein the sdAb domain comprises the amino acid sequence of any one of SEQ ID NO:12-32; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the transmembrane domain is derived from molecules selected from CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α, and optionally comprises the amino acid sequence of SEQ ID NO:57. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ, and optionally comprises the amino acid sequence of SEQ ID NO:60. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. In some embodiments, the co-stimulatory signal transduction domain comprises a cytoplasmic domain of CD28, and optionally, the co-stimulatory signal transduction domain comprises the amino acid sequence of SEQ ID NO:59. In some embodiments, the co-stimulatory signal transduction domain comprises a cytoplasmic domain of CD137 (4-1BB), and optionally, the co-stimulatory signal transduction domain comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, the CAR of this disclosure further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α, and optionally, the hinge domain comprises the amino acid sequence of SEQ ID NO:56. In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α, and optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO:55. In some embodiments, the CAR provided herein comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of any one of SEQ ID NO:33-54.

[0032] In another aspect, this document provides an isolated nucleic acid comprising a nucleic acid sequence encoding the sdAb or CAR provided herein. In another aspect, this document provides a vector comprising the isolated nucleic acid provided herein. In another aspect, this document provides eukaryotic cells comprising the sdAb, CAR, isolated nucleic acid, or vector provided herein. In another aspect, this document provides a pharmaceutical composition comprising the sdAb, CAR, isolated nucleic acid, vector, or eukaryotic cell provided herein. In another aspect, this document provides a method of treating a disease or condition, including administering the sdAb, CAR, isolated nucleic acid, vector, eukaryotic cell, or pharmaceutical composition provided herein. Attached Figure Description

[0033] Figure 1 The in vitro killing efficiency of exemplary CAR-T cells constructed from camel-derived VHH, humanized VHH, and VHH with FR region mutations is shown.

[0034] Figure 2A-2C The study shows tumor growth in a T84 tumor NCG mouse model after injection of exemplary CAR-T cells.

[0035] Figures 3A-3F This study shows the changes in cell survival and CAR+% of eukaryotic primary immune cells (such as T cells, NK cells, and NKT cells) expressing exogenous prokaryotic IMPDH and its mutants in the presence of purine synthesis inhibitors (such as IMPDH inhibitors (MPA or MMF)). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; PWDF-104 refers to cells transduced with *E. coli* IMPDH; and PWDF-104m refers to cells transduced with the *E. coli* IMPDH A223T point mutant.

[0036] Figures 4A-4BThe study showed the survival of eukaryotic primary immune cells (such as T cells) expressing IMPDH from different species in the presence of purine synthesis inhibitors (such as IMPDH inhibitors, such as MMF). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; the PWDF-98 group refers to cells transduced with Lactobacillus plantarum IMPDH; the PWDF-100 group refers to cells transduced with Mesoplasma florum IMPDH; the PWDF-102 group refers to cells transduced with a truncated variant of Escherichia coli IMPDH; the PWDF-104 group refers to cells transduced with Escherichia coli IMPDH; the PWDF-105 group refers to cells transduced with Bacillus subtilis IMPDH; the PWDF-106 group refers to cells transduced with Staphylococcus aureus IMPDH; and the PWDF-103 group refers to cells transduced with human IMPDH2. T333I / S351Y double mutant; PWDF-109 group refers to cell transduction of rat IMPDH2 (Rattus norvegicus IMPDH2); PWDF-110 group refers to cell transduction of Penicillium brevicompactum IMPDH.

[0037] Figure 5 This study shows the enrichment of CAR-T cells expressing exogenous prokaryotic (e.g., *E. coli*) IMPDH in the presence of purine synthesis inhibitors (e.g., IMPDH inhibitors, such as MMF). Mock-T refers to T cells that do not transduce the viral vector; the control group (PWDF-80) refers to cells without exogenous IMPDH transduction; PWDF-104 refers to cells transduced with *E. coli* IMPDH; and PWDF-104m refers to cells transduced with the *E. coli* IMPDH A223T point mutant.

[0038] Figures 6A-6BThis study demonstrates the ability of donor cells expressing exogenous prokaryotic (e.g., *E. coli*) IMPDH to tolerate host versus graft reaction (HVGR) in the presence of purine synthesis inhibitors (e.g., IMPDH inhibitors, such as MMF) by assessing mixed lymphocyte reaction (MLR). Mock-T refers to T cells without transduction of the viral vector; the control group (PWDF-80) refers to cells without exogenous IMPDH transduction; PWDF-104 refers to cells transduced with *E. coli* IMPDH; and PWDF-104m refers to cells transduced with the *E. coli* IMPDH A223T point mutant.

[0039] Figure 7 This study demonstrates the in vitro killing efficiency of CAR-T cells expressing exogenous prokaryotic IMPDH (such as *E. coli* or its variants) after a single round of stimulation with T84 tumor cells in the presence of purine synthesis inhibitors (such as IMPDH inhibitors, such as MMF). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; PWDF-104 refers to cells transduced with *E. coli* IMPDH; and PWDF-104m refers to cells transduced with the *E. coli* IMPDH A223T point mutant.

[0040] Figure 8 This study demonstrates the in vitro killing efficiency of CAR-T cells expressing exogenous prokaryotic IMPDH (such as *E. coli* or its variants) after six rounds of stimulation with T84 tumor cells in the presence of purine synthesis inhibitors (such as IMPDH inhibitors, such as MMF). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; PWDF-104 refers to cells transduced with *E. coli* IMPDH; and PWDF-104m refers to cells transduced with the *E. coli* IMPDH A223T point mutant.

[0041] Figures 9A-9EThis study demonstrates the changes in survival and CAR+% of eukaryotic primary immune cells (e.g., T cells) expressing exogenous prokaryotic IMPDH in the presence of different concentrations of purine synthesis inhibitors (e.g., IMPDH inhibitors, such as MPA). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; the PWDF-98 group refers to cells transduced with *Lactobacillus plantarum* IMPDH; the PWDF-103 group refers to cells transduced with the human IMPDH2 T333I / S351Y double mutant; the PWDF-104 group refers to cells transduced with *Escherichia coli* IMPDH; the PWDF-105 group refers to cells transduced with *Bacillus subtilis* IMPDH; and the PWDF-110 group refers to cells transduced with *Penicillium brevicompactum* IMPDH.

[0042] Figures 10A-10B This study demonstrates the changes in survival and CAR+% of eukaryotic primary immune cells (e.g., T cells) expressing exogenous prokaryotic IMPDH in the presence of different drug forms of purine synthesis inhibitors (e.g., IMPDH inhibitors such as MPA, MMF, and MPAS). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; the PWDF-104 group refers to cells transduced with E. coli IMPDH.

[0043] Figure 11A-11B This study demonstrated the differences in resistance to purine synthesis inhibitors (such as IMPDH inhibitors, like MPA) among different prokaryotic IMPDHs in eukaryotic primary immune cells (e.g., T cells), and statistically analyzed the half-maximal inhibitory concentration (IC50). The control group (PWDF-80) was defined as cells without exogenous IMPDH transduction; the PWDF-98 group was defined as cells transduced with *Lactobacillus plantarum* IMPDH; the PWDF-103 group was defined as cells transduced with the human IMPDH2 T333I / S351Y double mutant; the PWDF-104 group was defined as cells transduced with *Escherichia coli* IMPDH; and the PWDF-105 group was defined as cells transduced with *Bacillus subtilis* IMPDH.

[0044] Figure 12A-12DThis study demonstrates the differences in resistance to purine synthesis inhibitors (such as IMPDH inhibitors, like MPA) among different prokaryotic IMPDHs in primary cells (e.g., T cells) and cell lines (e.g., Jurkat cells), and statistically analyzes the half-maximal inhibitory concentration (IC50). The control group (PWDF-80) refers to cells without exogenous IMPDH transduction; the PWDF-98 group refers to cells transduced with *Lactobacillus plantarum* IMPDH; the PWDF-103 group refers to cells transduced with the human IMPDH2 T333I / S351Y double mutant; the PWDF-104 group refers to cells transduced with *Escherichia coli* IMPDH; and the PWDF-105 group refers to cells transduced with *Bacillus subtilis* IMPDH.

[0045] Figure 13 This study shows the changes in the number of eukaryotic primary T cells and Jurkat cell lines expressing prokaryotic IMPDH under conditions with or without purine synthesis inhibitors (such as IMPDH inhibitors, like MPA). The PWDF-104 group refers to cells transduced with E. coli IMPDH.

[0046] Figure 14 This study shows the changes in the number of TCR-T cells expressing prokaryotic IMPDH under conditions of purine synthesis inhibitors (such as IMPDH inhibitors, like MPA). The control group (WDHG007) refers to cells without exogenous IMPDH transduction; WDHG007-247 refers to cells transduced with E. coli IMPDH.

[0047] Figures 15A-15B The expression of the human IMPDH2 T333I / S351Y double mutant in cells is shown. Mock-T refers to T cells that are not transduced with the viral vector; the PWDF-103 group refers to T cells transduced with the human IMPDH2 T333I / S351Y double mutant.

[0048] Figures 16A-16BThe immunogenicity of IMPDH from different species was demonstrated, and the levels of the cytokine IFN-gamma (IFN-γ) were detected. Mock-T refers to T cells that did not transduce the viral vector; the control group (PWDF-103) refers to cells transduced with the human IMPDH2 T333I / S351Y double mutant; PWDF-104 refers to cells transduced with Escherichia coli IMPDH; PWDF-105 refers to cells transduced with Bacillus subtilis IMPDH; PWDF-106 refers to cells transduced with Staphylococcus aureus IMPDH; PWDF-109 refers to cells transduced with rat IMPDH2 (Rattus norvegicus IMPDH2); and PWDF-110 refers to cells transduced with Penicillium brevicompactum IMPDH. Detailed Implementation

[0049] This disclosure is based in part on surprising discoveries of improved function and properties in therapies involving engineered eukaryotic cells expressing certain exogenously introduced inosine 5'-monophosphate dehydrogenase (IMPDH).

[0050] While allogeneic cell therapy may offer patients more accessible treatment options and other benefits, a challenge it faces is the attack of the patient's own immune system on the engineered donor cells. Therefore, various immunosuppressants have been explored in this field to reduce the attack by the host immune system.

[0051] However, due to the broad effects of immunosuppressants on the immune system, their use often leads to a wide range of side effects. The most concerning effect is reduced therapeutic efficacy of transferred engineered immune cells. For example, by broadly suppressing T cell activity, such immunosuppressants can limit the function and proliferation of engineered T cells injected into a patient. If engineered T cells cannot proliferate or function well, this can reduce the overall therapeutic effect. Finding the right balance in the art regarding the dosage and duration of immunosuppressants to maximize therapeutic benefit while minimizing risk is extremely challenging.

[0052] This disclosure provides a novel strategy (and compositions of substances or uses thereof) that has been shown herein to be unexpectedly effective in controlling the negative effects of immunosuppressants on transfer-engineered cells while maintaining their cytotoxicity or efficacy against a target disease or condition. More specifically, this disclosure innovatively utilizes certain types of IMPDH resistant to purine biosynthesis inhibitors and provides eukaryotic cells expressing such exogenous IMPDH.

[0053] IMPDH is a key regulatory enzyme in purine nucleotide biosynthesis and plays a crucial role in maintaining normal cell function and growth, as well as in maintaining cell proliferation and immune responses. For example, normal activation and function of B and T lymphocytes depend on IMPDH and exhibit upregulated IMPDH expression. The exemplary exogenous IMPDH provided herein is resistant to MPA (including its analogues or derivatives). MPA and its analogues or derivatives exert their immunosuppressive effects at least in part by inhibiting IMPDH. This disclosure strategically targets this pathway and uses exogenous IMPDH resistant to purine biosynthesis inhibitors (e.g., MPA) to construct therapeutic eukaryotic cells. As shown in the Examples section below, engineered eukaryotic cells expressing this exogenous IMPDH exhibit resistance to MPA (and its analogues or derivatives) and are therefore able to proliferate and expand adequately upon treatment with mycophenolate mofetil (MMF). Importantly, the expression of exogenous IMPDH does not affect the cytotoxicity of these engineered eukaryotic cells. In fact, as shown in the figures, these cells exhibit long-term cytotoxicity, making them a viable approach for developing therapeutically effective cell therapies.

[0054] Furthermore, by expressing exogenous IMPDH resistant to purine biosynthesis inhibitors, engineered eukaryotic cells can be easily screened and prepared for infusion into patients. Patients can then later receive such purine biosynthesis inhibitors to control the host immune system's attack on the engineered eukaryotic cells without diminishing the therapeutic effect. Further details regarding the currently available material compositions and methods of use are provided in the following sections.

[0055] 5.1 Definition

[0056] The techniques and procedures described or referenced herein include conventional methods that are generally well understood and / or commonly used by those skilled in the art, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd ed. 2001); *Current Protocols in Molecular Biology* (Ausubel et al. eds., 2003); *Therapeutic Monoclonal Antibodies: From Bench to Clinic* (An ed. 2009); *Monoclonal Antibodies: Methods and Protocols* (Albitar ed. 2010); and the widely used methods described in *Antibody Engineering Vols 1 and 2* (Kontermann and Dübel eds., 2nd ed. 2010). Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following terminology will be used, and where appropriate, terms expressed in the singular will also include the plural, and vice versa. If any description of the terms conflicts with any document incorporated herein by reference, the description of the terms below shall prevail.

[0057] The terms “antibody,” “immunoglobulin,” or “Ig” are used interchangeably herein and in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions having multiple or single epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), single-chain antibodies formed from at least two intact antibodies, single-domain antibodies (e.g., VHH), and fragments thereof. Antibodies can be human, humanized, chimeric, and / or affinity-mature, as well as antibodies from other species, such as mice, rabbits, camels, etc. The term "antibody" is intended to include polypeptide products of B-cell immunoglobulin-like peptides capable of binding specific molecular antigens, and consisting of two pairs of identical polypeptide chains, each pair having a heavy chain (approximately 50-70 kDa) and a light chain (approximately 25 kDa), each amino-terminal portion of each chain comprising a variable region of approximately 100 to approximately 130 or more amino acids, and each carboxyl-terminal portion of each chain comprising a constant region. See, for example, Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3ded. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinant antibodies, single-domain antibodies or humanized variants thereof, including those from camel species (e.g., llamas or alpacas), intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above antibodies, where a functional fragment is a portion of the antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which that fragment originates. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. Specifically, the antibodies described herein include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing antigen-binding sites that bind antigens (e.g., one or more CDRs of an antibody).Such antibody fragments can be found in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; Day, Advanced Immunochemistry (2d ed. 1990). The antibodies described herein can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Antibodies can be agonistic or antagonistic. Antibodies may be neither agonistic nor antagonistic.

[0058] An "antigen" is a structure that an antibody can selectively bind to. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In some embodiments, the antigen is cell-related, for example, present on or within cells.

[0059] A "complete" antibody is an antibody that contains an antigen-binding site, as well as a CL and at least heavy chain constant regions CH1, CH2, and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. In some embodiments, a complete antibody has one or more effector functions.

[0060] "Single-chain Fv" can also be abbreviated as "sFv" or "scFv," and is an antibody fragment containing VH and VL antibody domains linked together to form a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the sFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0061] As used herein, "sdAb" or "monolithic antibody" refers to a single monomeric variable antibody domain capable of binding an antigen. Single-domain antibodies include the VHH domain described herein. Examples of single-domain antibodies include, but are not limited to, antibodies naturally lacking a light chain, such as antibodies from camelid species (e.g., llamas), single-domain antibodies derived from conventional four-chain antibodies, engineered antibodies, and single-domain scaffolds other than antibody-derived domain scaffolds. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. For example, as described herein, single-domain antibodies can be derived from antibodies produced in camelid species, such as camels, llamas, dromedary camels, alpacas, and guanacos. Other species besides camelids may produce heavy-chain antibodies naturally lacking a light chain, and VHHs derived from these other species are also included within the scope of this disclosure. In some embodiments, the single-domain antibodies (e.g., VHHs) provided herein have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As described herein, single-domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., a reagent). Single-domain antibodies can also be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0062] As used in this article, the term "ligand" refers to a molecule that specifically binds to biological macromolecules (such as proteins, nucleic acids, or other biopolymers). These ligands can include small molecule compounds, peptides, antibodies, metal ions, or other bioactive molecules, and are commonly used to regulate biological processes, as drugs, or as molecular probes. Ligands exert their effects by binding to their targets, characterized by binding affinity, specificity of the binding site, and kinetic properties of the interaction. The relationship between the ligand and the binding target is, for example, a function of charge, hydrophobicity, and molecular structure.

[0063] The term "binding" refers to intermolecular interactions, including, for example, the formation of complexes. Interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. Complexes can also comprise two or more molecules bound together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interaction between a single antigen-binding site on an antibody and a single epitope of a target molecule (such as an antigen) is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k0) of the binding molecule (e.g., antibody) from a monovalent antigen is also considered. off ) and binding rate (k on The ratio of (k) off / k on ) is the dissociation constant K D It is inversely proportional to affinity. K D The lower the value, the higher the antibody affinity. K DThe value varies depending on the different complexes of the antibody and antigen, and depends on k. on and k off The dissociation constant K of the antibody provided in this article D The affinity can be determined using any of the methods provided herein or any other methods known to those skilled in the art. The affinity at a single binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants (such as a multivalent antigen) comes into contact with an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the likelihood of a reaction at a second site. The strength of this multiple interaction between a multivalent antibody and an antigen is called affinity.

[0064] Regarding the binding molecules described herein, terms such as "binding to," "specifically binding to," and similar terms may be used interchangeably within this text, referring to binding molecules that specifically bind to the antigen-binding domain of an antigen (such as a polypeptide). For example, this can be achieved through immunoassay, Alternatively, other techniques known to those skilled in the art may be used to identify binding molecules or antigen-binding domains that bind or specifically bind to antigens. In some embodiments, binding or specific binding of an antigen occurs when a binding molecule or antigen-binding domain binds to an antigen with a higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective reaction will be at least twice the background signal or noise, and possibly more than 10 times the background signal. See, for example, the discussion of binding specificity in Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In some embodiments, the binding of a binding molecule or antigen-binding domain to a “non-target” protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, for example, by FACS analysis or RIA determination. Binding molecules or antigen-binding domains that bind antigens include binding molecules or antigen-binding domains capable of binding antigens with sufficient affinity such that the binding molecule can be used as, for example, a therapeutic agent and / or diagnostic agent targeting the antigen. In some embodiments, the binding molecule or antigen-binding domain of the antigen has a dissociation constant (KB) less than or equal to 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. DIn some implementations, the binding molecule or antigen-binding domain binds to a conserved antigenic epitope in antigens from different species.

[0065] In some embodiments, the binding molecule or antigen-binding domain may contain a “chimeric” sequence in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody species or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody species or subclass. The binding molecule or antigen-binding domain may also contain a fragment of such antibody, provided it exhibits the desired biological activity (see U.S. Patent No. 4,816,567; Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). The chimeric sequence may include a humanized sequence.

[0066] In some embodiments, the binding molecule or antigen-binding domain may comprise a portion of a “humanized” form of a non-human (e.g., camelid, murine, or non-human primate) antibody containing a sequence from a human immunoglobulin (e.g., a receptor antibody), wherein native CDR residues are replaced by corresponding CDR residues from a non-human species (e.g., camelids, mice, rats, rabbits, or non-human primates) (e.g., donor antibodies) having the desired specificity, affinity, and capability. In some cases, one or more FR region residues of a human immunoglobulin sequence are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the receptor or donor antibody. These modifications are used to further refine the antibody's performance. The heavy or light chain of the humanized antibody may contain substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to the CDRs of non-human immunoglobulins, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. In some embodiments, the humanized antibody will contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); US Patent Nos.: 6,800,738; 6,719,971; 6,639,055; 6,407,213; 6,054,297.

[0067] In some embodiments, the binding molecule or antigen-binding domain may contain portions of a “fully human antibody” or “human antibody,” terms which are used interchangeably herein and refer to an antibody containing a human variable region and, for example, a human constant region. The binding molecule may contain a single-domain antibody sequence. In a particular embodiment, the term refers to an antibody containing both a human variable region and a constant region. In some embodiments, a “fully human antibody” may also include an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a natural somatic variant of a human immunoglobulin nucleic acid sequence. The term “fully human antibody” includes antibodies having variable and constant regions corresponding to human immunoglobulin sequences, as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A “human antibody” is an antibody having an amino acid sequence corresponding to an antibody produced by humans and / or prepared using any technique for preparing human antibodies. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.Mol.Biol.227:381(1991); Marks et al., J.Mol.Biol.222:581(1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68(2006)). Other available methods for preparing human monoclonal antibodies can be found in Cole et al., Monoclonal Antibodies and Cancer Therapy 77(1985); Boerner et al., J.Immunol.147(1):86-95(1991); and van Dijk and van de Winkel, Curr. Opin. Pharmacol.5:368-74(2001).Human antibodies can be prepared by administering an antigen to a transgenic animal modified to produce the antibody in response to an antigen attack, but whose endogenous loci have been invalidated, such as in mice (for XENOMOUSE™ technology, see, for example, Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995); Brüggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997); US Patent Nos. 6,075,181 and 6,150,584). For human antibodies produced by human B-cell hybridoma technology, see, for example, Liet al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0068] In some implementations, the binding molecule or antigen-binding domain may comprise a portion of a “recombinant human antibody,” a term that includes human antibodies prepared, expressed, created, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from transgenic or transchromosomal animals (e.g., mice or cattle) possessing human immunoglobulin genes (see, for example, Taylor, L.D. et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other method involving splicing a human immunoglobulin gene sequence to another DNA sequence. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242). However, in some implementations, such recombinant human antibodies undergo in vitro mutagenesis (or, when using transgenic animals with human Ig sequences, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences derived from and associated with human VH and VL sequences, but these sequences are not naturally present in the antibody gene pool in the human body.

[0069] In some embodiments, the binding molecule or antigen-binding domain may comprise a portion of a “monoclonal antibody,” whereby the term refers herein to an antibody derived from a highly homogeneous group of antibodies, such that individual antibodies in that group are identical except for the possible presence of a small number of naturally occurring mutations or known post-translational modifications (e.g., amino acid isomerization, deamidation, methionine oxidation, or aspartic acid or glutamine deamidation), and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, “monoclonal antibody” herein refers to an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to any particular method of antibody preparation. For example, monoclonal antibodies available in this disclosure can be prepared by a hybridoma method originally described by Kohler et al. (Kohler et al., Nature 256:495 (1975)), or using recombinant DNA methods from bacterial or eukaryotic or plant cells (e.g., see U.S. Patent 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using methods described, for example, those described by Clackson et al. (Nature 352:624-28 (1991)) and Marks et al. (J.Mol.Biol.222:581-97 (1991)). Other methods for preparing clonal cell lines and monoclonal antibodies expressed therefrom are known in the field; see, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0070] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). For IgG antibodies, the four-chain unit is typically approximately 150,000 Daltons. Each L chain is linked to the H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form the interface between the variable domains of the light and heavy chains. The pairing of VH and VL together forms a single antigen-binding site. For information on the structure and properties of different types of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); Immunobiology (Janeway et al. eds., 5th ed. 2001).

[0071] "Fab" or "Fab region" refers to the antibody region that binds to the antigen. Conventional IgG typically contains two Fab regions, located on the two arms of a Y-shaped IgG structure. Each Fab region typically consists of a variable domain and a constant domain of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, respectively, while the variable and constant regions of the light chain are the VL and CL regions, respectively. Within the Fab region, the four portions VH, CH1, VL, and CL can be arranged in various ways to achieve the antigen-binding capacity according to this disclosure. For example, the VH and CH1 regions can be located on one polypeptide chain, while the VL and CL regions can be located on another polypeptide chain, similar to the Fab region of conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be located on the same polypeptide chain and arranged in different orders as described in the more detailed section below.

[0072] The term "variable region," "variable domain," "V region," or "V domain" refers to the portion of an antibody's light or heavy chain typically located at the amino terminus. These regions are approximately 120 to 130 amino acids long in the heavy chain and approximately 100 to 110 amino acids long in the light chain. These regions are responsible for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be called "VH," and the variable region of the light chain may be called "VL." The term "variable" refers to the extensive sequence differences between certain segments of the variable region across antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, this variability is not uniformly distributed within the 110 amino acid range of the variable region. Instead, the V region consists of short segments with high variability (very high variability) (called hypervariable regions) and segments separated by them with less variation (i.e., relatively invariant) (called frame regions, FRs). Frame regions are approximately 15 to 30 amino acids long, and each hypervariable region is approximately 9 to 12 amino acids long. The variable regions of both the heavy and light chains each contain four Fc regions, primarily in a β-sheet configuration, interconnected by three hypervariable regions. These hypervariable regions form linking loops and, in some cases, constitute part of the β-sheet configuration. The hypervariable regions in each chain are closely linked together by Fc regions and, together with the hypervariable regions of the other chain, form the antibody's antigen-binding site (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). Constant regions do not directly participate in antibody-antigen binding but have various effector functions, such as involvement in antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region sequences vary considerably between different antibodies. In a specific embodiment, the variable region is a human variable region.

[0073] The term "heavy chain" used when referring to antibodies refers to a polypeptide chain of approximately 50-70 kDa, in which the amino-terminal portion contains a variable region of approximately 120 to 130 or more amino acid residues, while the carboxyl-terminal portion contains a constant region. The constant region can be one of five different types (similar to serotypes), classified as α, δ, ε, γ, and μ based on the amino acid sequence of the heavy chain's constant region. These different heavy chains differ in size: α, δ, and γ contain approximately 450 amino acid residues, while μ and ε contain approximately 550 amino acid residues. When combined with a light chain, these different heavy chain types produce five known antibody classes (similar to serotypes): IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.

[0074] When referring to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, where the amino-terminal portion contains a variable region of approximately 100 to 110 or more amino acids, while the carboxyl-terminal portion contains a constant region. The average length of a light chain is 211 to 217 amino acids. There are two distinct types of light chains, classified as κ or λ based on the amino acid sequence of the constant region.

[0075] The terms “hypervariant region,” “HVR,” “complementarity-determining region,” and “CDR” used in this article are used interchangeably. “CDR” refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VL β-sheet framework of an antibody. CDR1, CDR2, and CDR3 in the VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. Similarly, CDR1, CDR2, and CDR3 in the VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Therefore, CDRs are variable region sequences scattered throughout the frame region sequence.

[0076] CDR regions are well known to those skilled in the art, and their definition follows well-known numbering systems. For example, the Kabat Complementary Determinant Region (CDR) is based on sequence variability and is the most commonly used (see, for example, Kabat et al., supra; Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia, on the other hand, is based on the position of the structural ring (see, for example, Chothia and Lesk, J.Mol.Biol.196:901-17 (1987)). When using the Kabat numbering system, the end of the Chothia CDR-H1 ring varies between H32 and H34 depending on the length of the ring (this is because the Kabat numbering scheme inserts bit numbers at H35A and H35B; if neither 35A nor 35B exists, the ring ends at bit 32; if only 35A exists, the ring ends at bit 33; if both 35A and 35B exist, the ring ends at bit 34). The AbM hypervariable region represents a trade-off between the Kabat CDR and Chothia structural rings and is used in Oxford Molecular's AbM antibody modeling software (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dübeleds., 2nd ded. 2010). The "Contact" hypervariable region is based on the analysis of the available complex crystal structures. Another widely developed and adopted universal numbering system is ImMunoGeneTics (IMGT) Information. (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77(2003)). IMGT is an integrated information system dedicated to immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complex (MHC) in humans and other vertebrates. In this paper, CDRs are named according to the amino acid sequence and position within the light or heavy chain. Since the “position” of CDRs in the variable domain structure of immunoglobulins is conserved across species and exists in so-called loop structures, CDRs and framework residues can be easily identified by using a numbering system that compares variable domain sequences based on structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into receptor frameworks that are typically derived from human antibodies. Honegger and Plückthun (Honegger and Plückthun, J. Mol. Biol. 309:657-70(2001)) developed another numbering system (AHon). Numbering systems, including, for example, the correspondence between the Kabat numbering system and the IMGT unique numbering system, are well known to those skilled in the art (see, for example, Kabat, ibid.; Chothia and Lesk, ibid.; Martin, ibid.; Lefranc et al., ibid.). Residues from each of these hypervariable regions or CDRs are illustrated in the table below.

[0077] Exemplary CDRs based on different numbering systems

[0078]

[0079]

[0080] The boundaries of a given CDR may vary depending on the identification scheme. Therefore, unless otherwise stated, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., a variable region), and individual CDRs (e.g., CDR-H1, CDR-H2) of an antibody or its region, should be understood to encompass complementarity-determining regions defined by any known scheme described above. In some cases, the scheme used to identify one or more specific CDRs is specified, for example, according to the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR is given. It should be noted that CDR regions can also be defined according to a combination of multiple numbering systems, such as a combination of the Kabat and Chothia numbering systems, or a combination of the Kabat and IMGT numbering systems. Therefore, terms such as “CDR as shown in a specific VH or VHH” include, but are not limited to, any CDR1 defined by the exemplary CDR numbering systems described above. When a variable region (e.g., VHH, VH, or VL) is given, those skilled in the art will understand that CDRs within that region can be defined by different numbering systems or combinations thereof.

[0081] The high-variable region may include the following "extended high-variable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL; and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

[0082] The term "constant region" or "constant domain" refers to the carboxyl-terminal portion of the light and heavy chains, which does not directly participate in antibody-antigen binding but exhibits various effector functions, such as interaction with Fc receptors. The term also refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence and contains antigen-binding sites compared to other parts (variable regions) of the immunoglobulin. The constant region can contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0083] The term "frame" or "FR" refers to the variable domain residues that flank a CDR. FR residues are found in, for example, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies (e.g., single-domain antibodies), biantibodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.

[0084] In this document, the term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, including, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at Cys226 or from Pro230 to its C-terminus. The C-terminal lysine of the Fc region (residue K447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, the composition of a complete antibody may include a group of antibodies with all K447 residues removed, a group of antibodies with K447 residues not removed, and a group of antibodies having a mixture of antibodies containing and without K447 residues. The "functional Fc region" has the "effect function" of the native sequence Fc region. Exemplary "effect functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require an Fc region combined with a binding region or binding domain (e.g., antibody variable region or domain) and can be evaluated using various assays known to those skilled in the art. A “variant Fc region” comprises an amino acid sequence that differs from the native sequence Fc region due to at least one amino acid modification (e.g., substitution, addition, or deletion). In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the parental peptide Fc region, for example, about one to about ten amino acid substitutions, or about one to about five amino acid substitutions, in the native sequence Fc region or the parental peptide Fc region. The variant Fc region described herein may have at least about 80% homology with the native sequence Fc region and / or with the parental peptide Fc region, or at least about 90% homology with it, for example, at least about 95% homology with it.

[0085] The "percentage of amino acid sequence identity (%)" and "homology" for peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, provided that the sequences are aligned and vacancies are introduced (if necessary) to achieve maximum sequence identity, and no conserved substitutions are considered part of the sequence identity. Sequence alignment used to determine the percentage of amino acid sequence identity can be performed using various methods known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, or MEGALIGN. TM Publicly available computer software such as DNASTAR is available. Those skilled in the art can determine the appropriate parameters for determining alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0086] As used herein, the term "functional exogenous receptor" refers to an exogenous receptor (e.g., a TCR, such as a recombinant or engineered TCR, cTCR, a TAC-like chimeric receptor, or CAR) that retains its biological activity upon introduction into immune effector cells (e.g., T cells). Biological activity includes, but is not limited to, the ability of the exogenous receptor to specifically bind molecules and to properly transmit downstream signals, such as inducing cell proliferation, producing cytokines, and performing regulatory or cytolytic effector functions.

[0087] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells (e.g., T cells). Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some implementations, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain specific to one or more antigens (e.g., tumor antigens) for T cells and / or other receptors. "CAR-T cell" refers to a T cell expressing a CAR.

[0088] As used herein, the term "recombinant or engineered TCR" is included within the category of functional exogenous receptors provided herein and refers to peptides expressed in immune cells. The functions of a recombinant or engineered TCR may include, for example, redirecting immune cells to immune activity against specific cell types, such as cancer cells and infected cells with specific surface markers. It may be expressed as an alternative to or concurrently with an endogenous TCR. In some embodiments, such recombinant TCRs are single-stranded TCRs containing open reading frames in which variable Vα and Vβ domains pair with protein linkers. This involves the molecular cloning of TCR genes known to be specific for selected antigens. These strands are then introduced into T cells, typically via retroviral vectors. Thus, expression of the cloned TCRα and TCRβ genes confers transduced T cells with functional specificity determined by the pairing of these new genes. Components of a recombinant or engineered TCR refer to any functional subunits of a TCR, such as recombinant TCRα and TCRβ, encoded by exogenous polynucleotide sequences introduced into the cell.

[0089] In some embodiments, the functional exogenous receptor provided herein is a chimeric TCR (cTCR) that combines antigen binding and T cell activation functions. For example, a cTCR may include: (a) an extracellular ligand-binding domain comprising an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3); (b) optionally, a linker; (c) optionally, an extracellular domain or a portion thereof of a first TCR subunit (e.g., CD3ε); (d) a transmembrane domain comprising a transmembrane domain of a second TCR subunit (e.g., CD3ε); and (e) an intracellular signaling domain comprising an intracellular signaling domain of a third TCR subunit (e.g., CD3ε); wherein the first, second, and third TCR subunits are all selected from TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ. In some embodiments, the first, second, and third TCR subunits are identical (e.g., all are CD3ε). In some embodiments, the first, second, and third TCR subunits are different. In some embodiments, the cTCR also includes a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the cTCR also includes a signal peptide located at the N-terminus of the cTCR, such as a signal peptide derived from CD8α.

[0090] In some embodiments, the functional exogenous receptor is a T-cell antigen-coupled agent (TAC), for example comprising: (a) an extracellular ligand-binding domain containing an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3); (b) optionally, a first adapter; (c) an extracellular TCR-binding domain that specifically recognizes an extracellular domain of a TCR subunit (e.g., CD3ε); (d) optionally, a second adapter; (e) optionally, a first TCR helper receptor (e.g., (f) an extracellular domain or a portion thereof of a second TCR co-receptor (e.g., CD4); and (g) optionally, an intracellular signaling domain comprising an intracellular signaling domain of a third TCR co-receptor (e.g., CD4); wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ; and wherein the first, second, and third TCR co-receptors are all selected from CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are the same. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC further includes a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the TAC further includes a signal peptide located at the N-terminus of the TAC, such as a signal peptide derived from CD8α. In some embodiments, the extracellular ligand-binding domain is located at the N-terminus of the extracellular TCR-binding domain. In some embodiments, the extracellular ligand-binding domain is located at the C-terminus of the extracellular TCR-binding domain.

[0091] In some embodiments, the functional exogenous receptor is a TAC-like chimeric receptor, for example comprising: (a) an extracellular ligand-binding domain containing an antigen-binding fragment (e.g., sdAb, scFv) that specifically recognizes one or more epitopes of a tumor antigen (e.g., GPC3); (b) optionally, a first adapter; (c) an extracellular TCR-binding domain that specifically recognizes the extracellular domain of a first TCR subunit (e.g., TCRα); (d) optionally, a second adapter; (e) optionally, the extracellular domain of a second TCR subunit (e.g., CD3ε) or a portion thereof; (f) a transmembrane domain containing the transmembrane domain of a third TCR subunit (e.g., CD3ε); and (g) optionally, an intracellular signaling domain containing the intracellular signaling domain of a fourth TCR subunit (e.g., CD3ε); wherein the first, second, third, and fourth TCR subunits are all selected from TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, and CD3δ. In some embodiments, the second, third, and fourth TCR subunits are identical. In some embodiments, the first, second, third, and fourth TCR subunits are identical. In some embodiments, the first, second, third, and fourth TCR subunits are different. In some embodiments, the second, third, and fourth TCR subunits are identical but different from the first TCR subunit. In some embodiments, the extracellular ligand-binding domain is located at the N-terminus of the extracellular TCR-binding domain. In some embodiments, the extracellular ligand-binding domain is located at the C-terminus of the extracellular TCR-binding domain. In some embodiments, the TAC-like chimeric receptor further includes a hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the TAC-like chimeric receptor further includes a signal peptide located at the N-terminus of the TAC-like chimeric receptor, such as a signal peptide derived from CD8α.

[0092] In some embodiments of this disclosure, the engineered eukaryotic cells provided herein optionally, but not necessarily, contain or express a functional exogenous receptor. In some embodiments, the prokaryotic IMPDH introduced into the engineered eukaryotic cells can exert its biological activity in the absence of a functional exogenous receptor. In some embodiments, the functional exogenous receptor has immunogenic biological activity after introduction into the eukaryotic cells. In some embodiments, the functional exogenous receptor introduced into the engineered eukaryotic cells produces an additive or synergistic stimulatory effect with the introduced prokaryotic IMPDH. In some embodiments, to facilitate the detection of IMPDH in transduced cells, the exogenous IMPDH can be transduced into the cells or co-expressed with additional detection markers. In some embodiments, detection markers include, but are not limited to, antibodies or ligands, resistance genes (e.g., antibiotic resistance genes such as puromycin resistance gene (Puro), G418 resistance gene (Neo), and blast fungicide resistance gene (BSD)) or selection markers (e.g., affinity tags such as histidine tags). Detection markers that are co-expressed with the protein being detected without affecting its expression and biological activity are well known in the art. In some implementations, a functional exogenous receptor is introduced for the detection of engineered eukaryotic cells.

[0093] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be dispersed with non-amino acids. These terms also cover polymers of amino acids that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. This definition also includes, for example, polypeptides containing one or more amino acid analogs, including but not limited to non-natural amino acids, and other modifications known in the art. It should be understood that because the polypeptides of this disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, a “polypeptide” may appear as a single chain or as two or more associated chains.

[0094] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogues, or any substrate incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. As used herein, “oligonucleotide” refers to a short, generally single-stranded synthetic polynucleotide whose length is generally, but not necessarily, about 200 nucleotides. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides. Cells that produce the binding molecules of this disclosure may include parental hybridoma cells, as well as bacterial and eukaryotic host cells incorporating nucleic acids encoding antibodies. Unless otherwise stated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is referred to as the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition from 5' to 3' of the newly formed RNA transcript is called the transcription direction; the 5' sequence region on the DNA strand that has the same sequence as the RNA transcript and is located at the 5' end of the RNA transcript is called the "upstream sequence"; the 3' sequence region on the DNA strand that has the same sequence as the RNA transcript and is located at the 3' end of the RNA transcript is called the "downstream sequence".

[0095] "Isolated nucleic acid" is a nucleic acid, such as RNA, DNA, or a mixture of nucleic acids, that is substantially separate from other genomic DNA sequences naturally associated with its natural sequence, as well as proteins or complexes (e.g., ribosomes and polymerases). An "isolated" nucleic acid molecule is a nucleic acid molecule that is separate from other nucleic acid molecules present in the natural source of that nucleic acid molecule. Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding a single-domain antibody or the antibody described herein are isolated or purified. The term includes nucleic acid sequences removed from their naturally occurring environment and includes recombinant or cloned DNA isolates as well as chemically synthesized analogs or analogs biosynthesized from a heterologous system. A substantially pure molecule may include the isolated form of that molecule. Specifically, an "isolated" nucleic acid molecule encoding the CAR described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminating nucleic acid molecule typically associated with its production environment.

[0096] The term "control sequence" refers to the DNA sequence required to express the coding sequence of an operational link in a particular host organism. Suitable control sequences for example, in prokaryotes, include promoters, and optionally also operon sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0097] As used herein, the term "operational link" and similar phrases (e.g., gene fusion), when used in relation to nucleic acids or amino acids, refer to an operational link between nucleic acid sequences or amino acid sequences that are functionally related to each other. For example, the promoter, enhancer element, open reading frame, 5' and 3' UTR, and terminator sequence of an operational link lead to the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, the nucleic acid element of an operational link leads to the transcription of the open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operationally linked peptide is a peptide in which functional domains are positioned at appropriate distances from each other to confer the intended function on each domain.

[0098] The term "vector" refers to a substance used to deliver or include a nucleic acid sequence for introducing that nucleic acid sequence into a host cell, including, for example, nucleic acid sequences encoding binding molecules (e.g., antibodies) as described herein. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selective sequences or markers for stable integration into the host cell chromosome. Additionally, a vector may include one or more selective marker genes and appropriate expression control sequences. Selective marker genes may include, for example, providing resistance to antibiotics or toxins, supplementing nutrient deficiencies, or supplying critical nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well-known in the art. When two or more nucleic acid molecules (e.g., antibody heavy and light chains or antibody VH and VL) are to be co-expressed, the two nucleic acid molecules may be inserted, for example, into a single expression vector or into separate expression vectors. For single-vector expression, the encoding nucleic acid may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the nucleic acid molecule into the host cell can be confirmed using methods well-known in the art. Such methods include, for example, nucleic acid analysis, such as RNA blotting or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that nucleic acid molecules are expressed in amounts sufficient to produce the desired product, and will also understand that expression levels can be optimized using methods well-known in the art to achieve adequate expression.

[0099] As used in this article, the term "host" refers to an animal, such as a mammal (e.g., a human).

[0100] As used herein, the term "host cell" refers to a specific subject cell that can be transfected with nucleic acid molecules and its offspring or potential offspring. Due to mutations or environmental influences that may occur in subsequent generations, or the integration of nucleic acid molecules into the host cell genome, the offspring of such cells may differ from the parent cells transfected with nucleic acid molecules.

[0101] As used in this article, the term "self" means that the individual from which any material is the same individual as the individual into which the material is later reintroduced.

[0102] "Allogeneic" refers to grafts that originate from different individuals of the same species.

[0103] As used herein, the terms “transfection” or “transformation” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. Cells that are “transfected” or “transformed” or “transduced” are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny.

[0104] As used herein, the term “pharmaceutically acceptable” means approved by the relevant regulatory agency or listed in the Chinese Pharmacopoeia, United States Pharmacopoeia, European Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and more specifically in humans.

[0105] "Excipient" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, humectants, lubricants, fragrances, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or medium.

[0106] In some implementations, the excipients are pharmaceutically acceptable. Examples of pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (e.g., fewer than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN. TM Polyethylene glycol (PEG) and PLURONICS TMOther examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed. 1990).

[0107] In one embodiment, each component is "pharmaceutically acceptable" in the sense of compatibility with other components of the pharmaceutical formulation and is suitable for use in contact with human or animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, in proportion to a reasonable benefit / risk ratio. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipients are non-toxic to the cells or mammals exposed to them at the doses and concentrations used. In some implementations, the pharmaceutically acceptable excipient is a pH-buffered aqueous solution.

[0108] In some embodiments, the excipients are sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the composition (e.g., a pharmaceutical composition) is administered intravenously, an exemplary excipient is water. Saline solutions, as well as glucose and glycerol solutions, can also be used as liquid excipients, especially for injectable solutions. Excipients may also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also contain trace amounts of wetting agents or emulsifiers, or pH buffers. The composition may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral compositions, including formulations, may include standard excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate.

[0109] The composition, including the pharmaceutical compound, may contain, for example, a conjugated molecule (e.g., an antibody) in an isolated or purified form, and an appropriate amount of excipient.

[0110] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount of immune effector cells or a composition comprising an agent and the provided engineered immune effector cells or pharmaceutical composition sufficient to produce the desired results.

[0111] The terms “subject” and “patient” are used interchangeably herein. As used herein, in some embodiments, the subject is a mammal, such as a non-primate or a primate (e.g., a human). In a particular embodiment, the subject is a human. In one embodiment, the subject is a mammal diagnosed with a disease or condition, such as a human. In another embodiment, the subject is a mammal at risk of developing a disease or condition, such as a human.

[0112] "Administer" means the act of injecting or otherwise physically delivering a substance that is present outside the body into a patient's body, such as via mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art.

[0113] As used herein, the terms “treat”, “treatment”, and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of a disease or condition resulting from the application of one or more therapies. Treatment can be determined by assessing whether there is a reduction, alleviation, and / or mitigation of one or more symptoms associated with an underlying condition that results in an observed improvement in the patient, although the patient may still have the underlying condition. The term “treatment” includes managing and improving a disease. The terms “manage”, “managing”, and “management” refer to the beneficial effects that a subject derives from a therapy, not necessarily a cure for the disease.

[0114] The term “prevent, preventing, and prevention” refers to reducing the likelihood of the onset (or recurrence) of a disease, condition, illness, or related symptoms (such as cancer).

[0115] As used herein, “delayed” cancer development refers to postponing, hindering, slowing, delaying, stabilizing, and / or postponing the development of the disease. The length of this delay can vary depending on medical history and / or the individual being treated. It will be apparent to those skilled in the art that a sufficient or significant delay can effectively include prevention, as the individual will not develop the disease. Methods for “delayed” cancer development involve reducing the likelihood of disease development and / or reducing the severity of the disease over a given time period, compared to not using the methods described. Such comparisons are typically based on clinical studies using a statistically significant number of individuals. Cancer development can be detected using standard methods, including but not limited to computed tomography (CAT), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Cancer development can also refer to cancer progression, which may initially be undetectable, including occurrence, recurrence, and onset.

[0116] The terms “about” and “approximately” mean within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a given value or range.

[0117] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used in this disclosure and claims include the plural forms.

[0118] It should be understood that when the term "comprising" is used to describe an implementation herein, other similar implementations described using "consisting of" and / or "substantially consisting of" are also provided. It should also be understood that when the term "substantially consisting of" is used to describe an implementation herein, other similar implementations described using "consisting of" are also provided.

[0119] The term "between" in phrases such as "between A and B" or "between A and B" refers to a range that includes both A and B.

[0120] The term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); B (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following implementations: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0121] 5.2 Engineered eukaryotic cells

[0122] In one aspect, this disclosure provides a eukaryotic cell comprising an exogenously introduced IMPDH that is resistant to inhibitors of purine biosynthesis. In some embodiments, the eukaryotic cell provided by this disclosure also expresses a functional exogenous receptor, such as (but not limited to) a T-cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), a T-cell antigen-coupled device (TAC) chimeric receptor, a chimeric switch receptor, a signal transduction receptor, an inducible regulation dimerization activation receptor (DARIC), a chimeric cytokine receptor, a co-stimulatory receptor, a dominant-negative receptor, or a component thereof. The IMPDH and functional exogenous receptors provided herein and expressed in the eukaryotic cell described herein will be described in more detail below.

[0123] eukaryotic cells

[0124] In some embodiments, the eukaryotic cells described herein are derived from mammals. In some embodiments, the eukaryotic cells provided herein are derived from primates, rodents, etc. In some embodiments, the eukaryotic cells are derived from humans, rats, mice, guinea pigs, rabbits, sheep, goats, camels or alpacas, horses, donkeys, chimpanzees, or macaques. In some embodiments, the eukaryotic cells are derived from humans.

[0125] In some embodiments, the eukaryotic cells provided herein are primary cells. In this document, the term "primary cell" refers to cells isolated from an organism (e.g., human, animal, or plant tissues or fluids) that have not undergone prolonged in vitro culture and retain their original biological characteristics. In this document, "primary cell" is used in contrast to "cell line." The term "cell line" is typically established from primary cells through an "immortification" process; immortalization usually involves mutations or alterations to genes related to cell cycle regulation, thereby creating a stable cell line. It has been observed that the exogenously introduced IMPDH provided herein can confer enhanced resistance to purine biosynthesis inhibitors in primary cells compared to cell lines, and even provide improved growth performance. Therefore, in some specific embodiments, the eukaryotic cells described herein are primary cells. In some particular embodiments, the eukaryotic cells may be cell lines.

[0126] In some embodiments, the eukaryotic cells provided herein are immune cells. In some embodiments, the eukaryotic cells provided herein are lymphocytes, phagocytes, or dendritic cells. In some embodiments, the eukaryotic cells provided herein are T cells, γδT cells, regulatory T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, monocytes, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, or normal tissue cells. In some specific embodiments, the eukaryotic cells are T cells.

[0127] Purine biosynthesis inhibitor resistance IMPDH

[0128] In some embodiments of the various compositions and methods provided herein, the purine biosynthesis inhibitors include mycophenolic acid (MPA), ribavirin, mizoribine, tiazofurin, AVN-944 (VX-944), FF-10501, AS2643361, or BMS-986126, or their pharmaceutically acceptable esters, salts, or prodrugs. In some embodiments, the purine biosynthesis inhibitor is mycophenolic acid (MPA), its derivatives, analogs, or pharmaceutically acceptable salts, or a compound that can be converted into MPA intracellularly. In some embodiments, the purine biosynthesis inhibitor is mycophenolate, mycophenolate mofetil (MMF), sodium mycophenolate (MPS), calcium mycophenolate, potassium mycophenolate, or derivatives thereof. In some embodiments, the purine biosynthesis inhibitor is MMF. An exemplary purine biosynthesis inhibitor according to this disclosure is mycophenolic acid (MPA) or its derivatives or analogs. Numerous MPA derivatives and analogs have been reported, including but not limited to U.S. Patent Nos. 4,686,234; 4,725,622; 4,727,069; 4,748,173; 4,753,935; 4,786,637; 4,808,592; 4,861,776; 4,868,153; 4,948,793; 4,952,579; 4,959,387; 4,922,467; and 5,247. MPA, including MPA derivatives with the 4-hydroxy group replaced by an amino substituent, 5,083; 5,380,879 (including MPA derivatives with the 6-methoxy group replaced by other substituents); and PCT international patent applications PCT / US92 / 09932 (WO94 / 12184) and PCT / US93 / 06410 (WO94 / 01105), the entire contents of which are incorporated herein by reference. MPA and its analogues or derivatives have been shown to be effective in preventing acute rejection in organ transplant recipients. MPA is the active ingredient in a variety of immunosuppressive drugs primarily used to prevent organ transplant rejection. These drugs include, for example, mycophenolate mofetil (MMF), such as CellCept. MMF is a prodrug that is converted to MPA in vivo and is commonly used to prevent rejection in kidney, heart, and liver transplants. Sodium mycophenolate, such as Myfortic, is a formulation that delivers mycophenolic acid in the form of enteric-coated tablets designed to reduce gastrointestinal side effects and improve patient tolerance.

[0129] In some embodiments, the exogenously introduced IMPDH described in this disclosure has a low binding affinity to purine biosynthesis inhibitors (e.g., MPA), and is therefore more resistant to the inhibitory effects of purine biosynthesis inhibitors (e.g., MPA).

[0130] In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH binding to a purine biosynthesis inhibitor is greater than that of wild-type human IMPDH binding to a purine biosynthesis inhibitor. In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH binding to a purine biosynthesis inhibitor is at least 20 times greater than that of wild-type human IMPDH binding to a purine biosynthesis inhibitor. More specifically, in some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH provided herein binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 20 nM. In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH provided herein binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 30 nM, for example greater than 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, or greater.

[0131] As illustrated in the examples below, prokaryotic IMPDHs with an inhibition constant (Ki) greater than 400 nM when binding to a purine biosynthesis inhibitor (e.g., MPA) exhibit sufficient resistance and are suitable for use in the eukaryotic cells described herein. Therefore, in some embodiments, the exogenously introduced IMPDH provided herein exhibits an inhibition constant (Ki) greater than 400 nM when binding to a purine biosynthesis inhibitor (e.g., MPA). In other embodiments, the exogenously introduced IMPDH provided herein exhibits an inhibition constant (Ki) greater than 500 nM when binding to a purine biosynthesis inhibitor (e.g., MPA). In other embodiments, the exogenously introduced IMPDH provided herein exhibits an inhibition constant (Ki) greater than 600 nM when binding to a purine biosynthesis inhibitor (e.g., MPA). In other embodiments, the exogenously introduced IMPDH provided herein exhibits an inhibition constant (Ki) greater than 700 nM when binding to a purine biosynthesis inhibitor (e.g., MPA). In other embodiments, the exogenously introduced IMPDH provided herein exhibits an inhibition constant (Ki) greater than 800 nM when binding to a purine biosynthesis inhibitor (e.g., MPA). In other embodiments, the inhibition constant (Ki) of the exogenously introduced IMPDH provided herein binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 900 nM. In other embodiments, the inhibition constant (Ki) of the exogenously introduced IMPDH provided herein binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 1 μM, for example, greater than 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, or 19 μM. In other embodiments, the inhibition constant (Ki) of the exogenously introduced IMPDH provided herein binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 20 μM. In some embodiments, the inhibition constant (Ki) of exogenously introduced IMPDH binding to a purine biosynthesis inhibitor (e.g., MPA) provided herein is at least 20 times greater than that of wild-type human IMPDH, for example, at least 25, 30, 35, or 40 times greater.

[0132] Alternatively or concurrently, the purine biosynthesis inhibitors described herein (e.g., MMF) exhibit lower antagonistic efficacy against exogenously introduced IMPDH as provided herein, manifested by a higher half-maximal effective concentration (EC50) or half-inhibitory concentration (IC50). In this document, EC50 or IC50 refers to the drug concentration of the purine biosynthesis inhibitor (e.g., MMF) at half the maximum cell number. In some embodiments, the EC50 or IC50 of the purine biosynthesis inhibitor against exogenously introduced IMPDH is greater than that against wild-type human IMPDH. In some embodiments, the EC50 or IC50 value of the purine biosynthesis inhibitor described herein (e.g., MMF) against exogenously introduced IMPDH as provided herein is greater than that against wild-type human IMPDH. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) inhibits exogenously introduced IMPDH as described herein by approximately two times, for example, three times, four times, or more, the EC50 or IC50 value compared to the EC50 or IC50 value inhibiting wild-type human IMPDH. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 1 ng / mL for exogenously introduced IMPDH as described herein. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 2 ng / mL for exogenously introduced IMPDH as described herein. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 3 ng / mL for exogenously introduced IMPDH as described herein. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 4 ng / mL for exogenously introduced IMPDH as described herein. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 5 ng / mL for exogenously introduced IMPDH as described herein, for example, greater than 6 ng / mL, 7 ng / mL, 8 ng / mL, or 9 ng / mL. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 10 ng / mL for exogenously introduced IMPDH as described herein. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, or greater for exogenously introduced IMPDH as described herein.

[0133] In certain embodiments of the various compositions or uses according to this disclosure, the eukaryotic cells provided herein are mammalian cells, wherein the introduced IMPDH is derived from prokaryotic cells, such as bacterial cells. In some embodiments, bacterial IMPDH catalyzes the conversion of IMP to XMP more efficiently than wild-type human IMPDH in the presence of a purine biosynthesis inhibitor. In some embodiments, bacterial IMPDH has a greater catalytic turnover number (Kcat) for the conversion of inosine monophosphate (IMP) to xanthoside monophosphate (XMP) than wild-type human IMPDH. A significant difference between bacterial IMPDH and mammalian IMPDH is that the catalytic turnover rate of bacterial IMPDH is much faster than that of mammalian IMPDH, which is reflected in a higher catalytic turnover number (Kcat). Specifically, the Kcat of mammalian IMPDH is approximately 0.4 s. -1 The Kcat of bacterial IMPDH may be 10 s. -1 Inhibitors of purine biosynthesis, such as MPA, exhibit strong inhibitory effects on slower mammalian IMPDH but poor inhibitory effects on rapid bacterial enzymes, suggesting a potential mechanistic link between drug affinity and catalytic activity. Therefore, in other embodiments, the Kcat of exogenously introduced IMPDH provided herein is greater than 0.4 s. -1 greater than 0.5s -1 greater than 1.0s -1 greater than 2.0s -1 greater than 3.0s -1 greater than 4.0s -1 greater than 5.0s -1 greater than 6.0s -1 greater than 7.0s -1 Greater than 8.0s -1 greater than 9.0s -1 In some implementations, the Kcat for externally introduced IMPDH provided herein is approximately 10.0 seconds. -1 .

[0134] Human IMPDH has been shown to catalyze the oxidation of inosine monophosphate (IMP) to xanthoside monophosphate (XMP) while simultaneously reducing nicotinamide adenine dinucleotide (NAD+) to NADH. MPA inhibits human IMPDH by binding to its active site, particularly in the region that interacts with NAD+. Exogenous IMPDHs (e.g., prokaryotic IMPDHs) provided herein may exhibit differences in their binding mode with the cofactor NAD+, affecting the mode of action of MPA on this enzyme in prokaryotic cells. Therefore, exogenous IMPDHs (e.g., bacterial IMPDHs or variants thereof) may have a high affinity for NAD+ and may be resistant to purine biosynthesis inhibitors such as MPA or its analogues or derivatives. Therefore, in some embodiments of the various compositions or uses according to this disclosure, the exogenous IMPDHs have an affinity for NAD+ that is no less than or greater than that of eukaryotic IMPDHs, such as wild-type human IMPDHs. In some embodiments, the dissociation constant (Kd) of exogenously introduced IMPDH with NAD+ is not greater than or less than that of wild-type human IMPDH. Alternatively, in some embodiments, the Michaelis constant (Km) of exogenously introduced IMPDH with NAD+ is not greater than or less than that of wild-type human IMPDH. Or, in some embodiments, the spatial configuration of the active and / or binding sites of the exogenously introduced IMPDH provided herein allows for the simultaneous binding of purine biosynthesis inhibitors (e.g., MPA) and substrates (e.g., IMP and / or NAD+) without mutual repulsion. In some embodiments, the exogenously introduced IMPDH is able to catalyze the conversion of IMP to XMP in a normal manner, unaffected by the purine biosynthesis inhibitor. Surprisingly, it has even been observed that eukaryotic cells expressing exogenously introduced IMPDH exhibit superior growth performance in the presence of purine biosynthesis inhibitors. In some embodiments, the exogenously introduced IMPDH provided herein contains a cofactor binding site for binding substrates (e.g., IMP and / or NAD+), and this site is not inhibited by purine biosynthesis inhibitors (e.g., MPA).

[0135] In some embodiments, the exogenously introduced IMPDH provided herein is a non-human IMPDH or a variant of a human IMPDH. In some embodiments, the exogenously introduced IMPDH is derived from a prokaryotic IMPDH. In some embodiments, the prokaryotic IMPDH is derived from a bacterial IMPDH or a variant thereof, such as a Gram-positive bacterial IMPDH or a variant thereof, or a Gram-negative bacterial IMPDH or a variant thereof. Exemplary Gram-positive bacterial IMPDHs include, but are not limited to, Bacillus subtilis IMPDH, Lactobacillus plantarum IMPDH, Staphylococcus aureus IMPDH, or variants thereof. Exemplary Gram-negative bacterial IMPDHs include, but are not limited to, Escherichia coli IMPDH, Mesoplasma florum IMPDH, or variants thereof. In some preferred embodiments, the exogenously introduced IMPDH is Lactobacillus plantarum IMPDH, Escherichia coli IMPDH, Bacillus subtilis IMPDH, Staphylococcus aureus IMPDH, or Mesoplasma florum IMPDH, or variants thereof.

[0136] In some embodiments, the exogenously introduced IMPDH comprises an amino acid sequence selected from SEQ ID NO:1-7, or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1-7.

[0137] In some more specific embodiments, the exogenously introduced IMPDH provided herein is derived from *Lactobacillus plantarum* IMPDH, which contains the amino acid sequence of SEQ ID NO:1. In some embodiments, the exogenously introduced IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors described herein (e.g., MPA or its analogues or derivatives).

[0138] In some more specific embodiments, the exogenous IMPDH provided herein is derived from *E. coli* IMPDH containing the amino acid sequence of SEQ ID NO:2. In some embodiments, the exogenous IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:2, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors described herein (e.g., MPA or its analogues or derivatives).

[0139] In some more specific embodiments, the exogenously introduced IMPDH provided herein is derived from Bacillus subtilis IMPDH, which contains the amino acid sequence of SEQ ID NO:3. In some embodiments, the exogenously introduced IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:3, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors (e.g., MPA or its analogues or derivatives) provided herein.

[0140] In some more specific embodiments, the exogenously introduced IMPDH provided herein is derived from Staphylococcus aureus IMPDH, which contains the amino acid sequence of SEQ ID NO:4. In some embodiments, the exogenously introduced IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors (e.g., MPA or its analogues or derivatives) provided herein.

[0141] In some more specific embodiments, the exogenously introduced IMPDH provided herein is derived from the floral intermediate protoplasm IMPDH, which contains the amino acid sequence of SEQ ID NO:5. In some embodiments, the exogenously introduced IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors (e.g., MPA or its analogues or derivatives) provided herein.

[0142] In some embodiments, the IMPDH variant comprises an amino acid sequence that differs from its naturally occurring IMPDH (parental IMPDH) due to at least one amino acid alteration (e.g., substitution, addition, or deletion). The IMPDH variants described herein may have at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with naturally occurring IMPDH. In some embodiments, the IMPDH variant is a functional variant that has some biological characteristics modified or improved relative to the parental IMPDH, or will substantially retain some biological characteristics of the parental IMPDH, particularly resistance to purine biosynthesis inhibitors. In some embodiments, the IMPDH variant, relative to its naturally occurring IMPDH (parental IMPDH), includes mutations at the following sites: D13, D50, E54, D138, D200, A223, D243, D248, D338, E369, E373, E469, or combinations thereof.

[0143] In some more specific embodiments, the exogenously introduced IMPDH provided herein is a truncated variant of *E. coli* IMPDH containing the amino acid sequence of SEQ ID NO:6. In some embodiments, the exogenously introduced IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:6, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors (e.g., MPA or its analogues or derivatives) provided herein.

[0144] In some more specific embodiments, the exogenously introduced IMPDH provided herein is an *E. coli* IMPDH mutant containing the amino acid sequence of SEQ ID NO:7. In some embodiments, the exogenously introduced IMPDH provided herein contains an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:7, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors (e.g., MPA or its analogues or derivatives) provided herein.

[0145] The percentage of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be determined using mathematical algorithms. A non-limiting preferred example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264 2268 (1990), and revised versions Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873 5877 (1993)). This algorithm was incorporated into the NBLAST and XBLAST procedures of Altschul et al. (Altschul et al., J. Mol. Biol. 215:403 (1990)). BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, such as score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set, such as score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparison purposes, Gapped BLAST as described by Altschul et al. (Altschul et al., Nucleic Acids Res. 25:3389 3402 (1997)) can be used. Alternatively, an iterative search can be performed using PSI BLAST to detect the proximity of molecules. When using BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI), website: web,ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller (Myers and Miller, CABIOS 4:11-17 (1998)). This algorithm has been incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used.

[0146] In some aspects of this disclosure, the prokaryotic IMPDH described herein or variants thereof are contemplated and fall within the scope of this disclosure. In other aspects, the disclosure also contemplates the use of the prokaryotic IMPDH described herein or variants thereof for constructing engineered eukaryotic cells resistant to purine biosynthesis inhibitors. In some embodiments, the prokaryotic IMPDH or variants thereof are used for sorting or enriching eukaryotic cells engineered therefrom. By conferring resistance to purine biosynthesis inhibitors on eukaryotic cells, the screening, purification, sorting, or enrichment of engineered cells can be readily achieved in embodiments for producing cells (e.g., for treating diseases or conditions). In some embodiments, the disclosure also contemplates modifications or variations to the amino acid sequence of the IMPDH described herein. Variations may be substitutions, deletions, or insertions of one or more codons encoding a polypeptide, resulting in an altered amino acid sequence compared to the original polypeptide.

[0147] Amino acid substitution can be the result of replacing one amino acid with another amino acid that has a similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conserved amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that induce amino acid substitutions. Insertions or deletions may optionally range from about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions relative to the original molecule include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids. In one particular embodiment, the substitution is a conserved amino acid substitution performed at one or more predicted non-essential amino acid residues. Permissible variations can be determined by systematically inserting, deleting, or substituting amino acids in the sequence and testing the variants against the activity exhibited by the parent antibody.

[0148] Polypeptides derived through conserved amino acid substitution are included in this disclosure. In conserved amino acid substitution, an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. As described above, families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced into all or part of the coding sequence, for example, through saturation mutagenesis, and the resulting mutants can be screened for bioactivity to identify those that retain activity. After mutagenesis, the encoded protein can be expressed and its activity can be determined. Conserved substitutions (e.g., within amino acid groups with similar properties and / or side chains) can be made to maintain or not significantly alter the properties.

[0149] Amino acids can be grouped according to the similarity of their side chain characteristics (see, for example, Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Nonpolar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped according to common side-chain properties: (1) hydrophobic: leucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. For example, any cysteine ​​residue that does not participate in maintaining the correct conformation of the polypeptide can be replaced by another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Non-conservative substitution involves replacing members of one of these categories with members of another category.

[0150] Amino acid sequence insertions include peptides with fusion lengths ranging from one residue to one hundred or more residues at the amino and / or carboxyl ends, as well as the insertion of single or multiple amino acid residues within the sequence. These variations can be achieved using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scan mutagenesis, and PCR mutagenesis. Site-directed mutagenesis (see, for example, Carter, Biochem J. 237:1-7 (1986); Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, for example, Wells et al., Gene 34:315-23 (1985)) or other known techniques can be applied to cloned DNA to produce polypeptide variant DNA.

[0151] Functional exogenous receptors

[0152] In some embodiments of this disclosure, the engineered eukaryotic cells provided herein optionally, but not necessarily, contain or express a functional exogenous receptor. In some embodiments, the prokaryotic IMPDH introduced into the engineered eukaryotic cells can exert its biological activity in the absence of a functional exogenous receptor. In some embodiments, the functional exogenous receptor has immunogenic biological activity after introduction into the eukaryotic cells. In some embodiments, the functional exogenous receptor introduced into the engineered eukaryotic cells produces an additive or synergistic stimulatory effect with the introduced prokaryotic IMPDH. In some embodiments, the functional exogenous receptor is introduced for the detection of engineered eukaryotic cells. In some embodiments, the eukaryotic cells provided herein also express a functional exogenous receptor, such as, but not limited to, T-cell receptors (TCRs), chimeric antigen receptors (CARs), chimeric TCRs (cTCRs), T-cell antigen-coupled device (TAC) chimeric receptors, chimeric switch receptors, signal transduction receptors, induced regulation dimerization activation receptors (DARICs), chimeric cytokine receptors, co-stimulatory receptors, dominant-negative receptors, or components thereof. Any functional exogenous receptor is included in this disclosure. Chimeric antigen receptors (CARs) will be described in more detail below, provided only as exemplary functional exogenous receptors for the purposes of this document, but not limiting the scope of this disclosure.

[0153] The CAR in the immune effector cells described herein comprises a polypeptide including: (a) an extracellular antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular signal transduction domain, wherein each domain and additional regions will be described in more detail below.

[0154] Extracellular antigen-binding domain

[0155] The extracellular antigen-binding domain of the CAR described herein includes one or more antigen-binding domains. In some embodiments, the extracellular antigen-binding domain of the CAR described herein is monospecific. In other embodiments, the extracellular antigen-binding domain of the CAR described herein is multispecific. In other embodiments, the extracellular antigen-binding domain of the CAR described herein is monovalent. In other embodiments, the extracellular antigen-binding domain of the CAR described herein is multivalent. In some embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains fused together directly by peptide bonds or by peptide linkers.

[0156] In some implementations, the extracellular antigen-binding domain includes a ligand or a fragment thereof.

[0157] In some embodiments, the extracellular antigen-binding domain comprises an antibody or a fragment thereof. For example, the binding domain may be derived from monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibodies with multi-epitope or single-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, provided they exhibit the desired biological activity), antibodies formed from at least two intact antibodies, single-chain antibodies, single-domain antibodies, and fragments thereof (e.g., VHH or domain antibodies). Antibodies may be human, humanized, chimeric, and affinity-mature, or may be derived from other species, such as mice, rabbits, camels, etc. In some embodiments, the antibody comprises a polypeptide product of a B-cell immunoglobulin-like polypeptide capable of binding a specific molecular antigen and consisting of two pairs of identical polypeptide chains, each pair having a heavy chain (approximately 50-70 kDa) and a light chain (approximately 25 kDa), each amino-terminal portion of each chain comprising a variable region of approximately 100 to approximately 130 or more amino acids, and each carboxyl-terminal portion of each chain comprising a constant region. See, for example, Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinant antibodies, single-domain antibodies or their humanized variants derived from camel species (e.g., llamas or alpacas), intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above antibodies. A functional fragment is a portion of the antibody's heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which that fragment originates. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. Specifically, the antibodies described herein include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, such as antigen-binding domains or molecules containing antigen-binding sites that bind antigens (e.g., one or more CDRs of an antibody).Such antibody fragments can be found in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; Day, Advanced Immunochemistry (2d ed. 1990). The antibodies described herein can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Antibodies can be agonistic or antagonistic. Antibodies may be neither agonistic nor antagonistic.

[0158] In one specific embodiment, the extracellular antigen-binding domain of the CAR described herein comprises a single-chain Fv (sFv or scFv). scFv is an antibody fragment comprising VH and VL antibody domains linked together as a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0159] In another specific embodiment, the extracellular antigen-binding domain of the CAR described herein comprises one or more single-domain antibodies (sdAbs). sdAbs may be derived from the same or different sources and may have the same or different sizes. Exemplary sdAbs include, but are not limited to, the heavy chain variable domain of pure heavy chain antibodies (e.g., VHH or VNAR), naturally occurring light chain-deficient binding molecules, single-domain antibodies derived from conventional four-chain antibodies (such as VH or VL), and humanized pure heavy chain antibodies. Human single-domain antibodies produced from transgenic mice or rats expressing human heavy chain fragments and single-domain scaffolds other than antibody-derived domain scaffolds are also included. Any single-domain antibody known in the art or developed according to this disclosure, including the single-domain antibodies described above in this disclosure, can be used to construct the CAR described herein. Single-domain antibodies may be derived from any species, including but not limited to mice, rats, humans, camels, alpacas, lampreys, fish, sharks, goats, rabbits, and cattle. The single-domain antibodies contemplated in this disclosure also include naturally occurring single-domain antibody molecules from species other than camelids and sharks.

[0160] In some embodiments, the sdAb is derived from naturally occurring single-domain antigen-binding molecules, i.e., heavy-chain antibodies lacking light chains (also known as "pure heavy-chain antibodies"). Such single-domain molecules are disclosed, for example, in WO 94 / 04678 and Hamers-Casterman, C. et al., Nature 363:446-448 (1993). For clarity, variable domains derived from naturally occurring heavy-chain molecules lacking light chains are referred to herein as VHHs to distinguish them from the conventional VHs of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, llamas, vicuñas, dromedaries, alpacas, and guanacos). Other species besides camelids may also produce naturally occurring heavy-chain molecules lacking light chains, and such VHHs are within the scope of this disclosure. Furthermore, humanized forms of VHHs, as well as other modifications and variants, are contemplated and fall within the scope of this disclosure. It should be understood that the primary goal of humanization is to reduce the immunogenicity of antibodies from other species, but such sequence alterations may affect or reduce the biological activity of parental antibodies. It has been observed that the exemplary humanized VHHs provided herein unexpectedly exhibit enhanced biological activity, particularly the enhanced cytotoxicity and killing efficiency of the functional exogenous receptors constructed from humanized VHHs and engineered cells against target cells. In some embodiments, sdAbs are derived from the variable regions of immunoglobulins found in cartilaginous fish. For example, sdAbs can be derived from an immunoglobulin isotype called a novel antigen receptor (NAR) found in shark serum. Methods for generating single-domain molecules derived from the variable regions of NARs (“IgNAR”) are available in WO 03 / 014161 and Streltsov, Protein Sci. 14:2901-2909 (2005).

[0161] In some embodiments, naturally occurring VHH domains targeting a specific antigen or target can be obtained from a (raw or immune) library of camel VHH sequences. This approach may or may not involve screening such a library using one or more screening techniques known in the art with the antigen or target (or at least a portion, fragment, antigenic determinant, or epitope thereof). Such libraries and techniques are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694. Alternatively, modified synthetic or semi-synthetic libraries derived from (raw or immune) VHH libraries can be used, such as VHH libraries obtained from (raw or immune) VHH libraries through techniques such as random mutation and / or CDR rearrangement, as described in WO 00 / 43507.

[0162] In some embodiments, the sdAb is recombinant, CDR-transplanted, humanized, camelized, deimmunized, and / or in vitro generated (e.g., by phage display screening). In some embodiments, the amino acid sequence of the frame region can be altered by “camelization” of specific amino acid residues in the frame region. Camelization refers to replacing or substituting one or more amino acid residues in the amino acid sequence of the (naturally occurring) VH domain from a conventional 4-chain antibody with one or more amino acid residues appearing at corresponding positions in the VHH domain of a heavy chain antibody. This can be performed in a manner known in the art and will be apparent to those skilled in the art. Such “camelization” substitutions are preferably inserted at amino acid sites that form and / or are present at the VH-VL interface and / or at so-called cameloid marker residues, as defined herein (see, for example, WO 94 / 04678; Davies and Riechmann FEBS Letters 339:285-290 (1994); Davies and Riechmann, Protein Engineering 9(6):531-537 (1996); Riechmann, J. Mol. Biol. 259:957-969 (1996); Riechmann and Muyldermans, J. Immunol. Meth. 231:25-38 (1999).

[0163] In some implementations, sdAb is a human single-domain antibody produced by transgenic mice or rats expressing the human heavy chain segment. See, for example, US20090307787, US Patent No. 8,754,287, US20150289489, US20100122358, and WO2004049794.

[0164] In some implementations, single-domain antibodies are generated from conventional tetrachain antibodies. See, for example, EP 0368684; Ward et al., Nature, 341(6242):544-6 (1989); Holt et al., Trends Biotechnol., 21(11):484-490 (2003); WO 06 / 030220; WO 06 / 003388.

[0165] In some implementations, the extracellular antigen-binding domain comprises a humanized antibody or a fragment thereof. The humanized antibody may contain human frame region and human constant region sequences.

[0166] Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to CDR transplantation (European Patent No. EP 239,400; International Publication No. WO 1 / 09967; US Patent Nos. 5,225,539, 5,530,101 and 5,585,089); matte or surface remodeling (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, PNAS 91:969-973); chain tampering (US Patent No. 5,565,332); and other disclosed techniques, such as US Patent No. 6,407,213, US Patent No. 5,766,886, WO 9317105; Tan et al., J. Immunol. 169:1119 25 (2002); Caldas et al., Protein Eng. 13 (5): 353-60 (2000); Morea et al., Methods 20 (3): 267 79 (2000); Baca et al. al., J.Biol.Chem.272(16):10678-84(1997); Roguska et al., Protein Eng.9(10):895 904(1996); Couto et al., Cancer Res.55(23Supp):5973s-5977s(1995); Couto et al., Cancer Res.55(8):1717-22(1995); Sandhu JS, Gene 150(2):409-10(1994); Pedersen et al., J.Mol.Biol.235(3):959-73(1994). See also US Patent Publication No. US2005 / 0042664 A1 (February 24, 2005), the entire contents of which are incorporated herein by reference.

[0167] Various methods for humanizing nonhuman antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are generally referred to as “input” residues and are typically derived from an “input” variable domain. Humanization can be performed by replacing the corresponding sequence of a human antibody with a hypervariable region sequence, for example, with reference to the methods of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36.

[0168] In some cases, humanized antibodies are constructed via CDR transplantation, where the amino acid sequence of six CDRs from a parental non-human (e.g., mouse) antibody is transplanted into the human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in the CDR actually contact the antigen, and termed these residues "specificity-determining residues" or SDRs (Padlan et al., 1995, FASEB J.9:133-39). In SDR transplantation techniques, only SDR residues are transplanted into the human antibody framework (see, for example, Kashmiri et al., 2005, Methods 36:25-34).

[0169] The selection of human (heavy and light chain) variable domains used to prepare humanized antibodies can be important for reducing antigenicity. For example, according to the so-called "best fit" method, sequences of variable domains of non-human (e.g., mouse) antibodies are screened against an entire library of known human variable domain sequences. Human sequences that are closest to the sequences of non-human (e.g., mouse) antibodies can be selected as the human frame for the humanized antibody (Sims et al., 1993, J. Immunol. 151:2296-308; Chothia et al., 1987, J. Mol. Biol. 196:901-17). Another approach uses a specific frame derived from a common sequence of all human antibodies from a specific light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the shared sequences of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). In another approach, germline genes are used as the source of the framework region.

[0170] In an alternative paradigm called hyperhumanization based on CDR comparison, FR homology is irrelevant. This approach involves comparing non-human sequences with functional human genotypes. Genes encoding typical structures identical to or closely related to mouse sequences are then selected. Next, among genes sharing typical structures with non-human antibodies, those with the highest homology within the CDR are selected as FR donors. Finally, the non-human CDR is transplanted onto these FRs (see, for example, Tan et al., 2002, J. Immunol. 169:1119-25).

[0171] Generally, it is desirable to humanize antibodies while retaining their affinity for antigens and other advantageous biological properties. To achieve this, humanized antibodies are prepared according to a method that analyzes parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are widely available and well-known to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences can be used. These programs include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and SwissPDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). ​​Examining these displays allows analysis of the possible functional roles of residues in the candidate immunoglobulin sequences, such as analyzing residues that affect the ability of the candidate immunoglobulin to bind its antigens. In this way, FR residues can be selected and combined from the acceptor and input sequences to achieve desired antibody characteristics, such as increased affinity for target antigens. Generally, hypervariable residues are directly and most significantly involved in influencing antigen binding.

[0172] Another approach to antibody humanization is based on an indicator of antibody humanity called Human String Content (HSC). This method compares mouse sequences to human germline gene lines and counts the differences as HSC. The target sequence is then humanized by maximizing its HSC, rather than using a global identity metric, to generate multiple distinct humanized variants (Lazar et al., 2007, Mol. Immunol. 44: 1986-98).

[0173] In addition to the methods described above, empirical methods can also be used to generate and select humanized antibodies. These methods include those based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosome, and yeast display libraries, as well as through bacterial colony screening (see, for example, Hoogenboom, 2005, Nat. Biotechnol. 23:1105-16; Dufner et al., 2006, Trends Biotechnol. 24:523-29; Feldhaus et al., 2003, Nat. Biotechnol. 21:163-70; Schlapschy et al., 2004, Protein Eng. Des. Sel. 17:847-60).

[0174] It should be understood that the primary goal of humanization is to reduce the immunogenicity of antibodies from other species, but such sequence alterations may affect or reduce the biological activity of parental antibodies. It has been observed that the exemplary humanized antibodies presented herein unexpectedly exhibit enhanced biological activity, particularly the enhanced cytotoxicity and killing efficiency of the functional exogenous receptors constructed from humanized antibodies and the engineered cells against target cells.

[0175] In the FR library approach, a set of residue variants is introduced at specific positions in the FR, and the library is then screened to select the FR that best supports the transplantation of the CDR. The residues to be replaced may include some or all of the “vernier” residues of a more limited set of target residues identified as potentially contributing to the CDR structure (see, for example, Foote and Winter, 1992, J.Mol.Biol.224:487-99) or Baca et al. (Baca et al. 1997, J.Biol.Chem.272:10678-84).

[0176] In FR shuffling, the entire FR is combined with a nonhuman CDR, rather than creating a combined library of selected residue variants (see, for example, Dall'Acqua et al., 2005, Methods 36:43-60). Library screening can be performed in two steps: first VL, then VH humanization. Alternatively, a one-step FR shuffling approach can be used. This approach has proven to be more efficient than the two-step screening method because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermostability (see, for example, Damschroder et al., 2007, Mol. Immunol. 44:3049-60).

[0177] The "humanizing" approach is based on the experimental identification of the minimum specificity determinant (MSD) and involves sequentially replacing non-human fragments into a human FR library and assessing binding affinity. This method begins with the CDR3 region of the non-human VH and VL chains and progressively replaces other regions of the non-human antibody with human FRs, including the CDR1 and CDR2 of VH and VL. This approach typically preserves and identifies epitopes of multiple antibody subclasses with different human V-segment CDRs. Humanizing can isolate antibodies with 91% to 96% homology to human germline antibodies (see, for example, Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

[0178] The "human-engineered" approach involves altering non-human antibodies or antibody fragments (e.g., mouse antibodies or fragments, or chimeric antibodies or fragments) by making specific changes to the amino acid sequence of the antibody, thereby producing modified antibodies with reduced immunogenicity in humans while still retaining the desired binding properties of the original non-human antibody. Generally, this technique involves classifying amino acid residues of non-human (e.g., mouse) antibodies into "low-risk," "intermediate-risk," or "high-risk" residues. Classification is performed using a global risk / reward calculation that evaluates the predicted benefit (e.g., immunogenicity in humans) of making a specific substitution versus the risk that the substitution would affect the folding of the resulting antibody. Specific human amino acid residues to be substituted at a given position (e.g., low-risk or intermediate-risk) in the non-human (e.g., mouse) antibody sequence can be selected by aligning the amino acid sequence from the variable region of the non-human antibody with the corresponding region of a specific or shared human amino acid sequence. Based on the alignment, amino acid residues at low-risk or intermediate-risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence. For details on the technology for preparing human engineered proteins, please refer to: Studnicka et al., 1994, Protein Engineering 7:805-14; US Patent Nos. 5,766,886; 5,770,196, 5,821,123, 5,869,619; PCT Publication WO 93 / 11794.

[0179] For example, Composite Human Antibody can be used. TMThe technology (Antitope Ltd., Cambridge, United Kingdom) produces complex human antibodies. To produce complex human antibodies, the variable region sequences are designed in a manner that avoids T-cell epitopes, using fragments of multiple human antibody variable region sequences, thereby minimizing the immunogenicity of the resulting antibody. These antibodies may include human constant region sequences, such as the human light chain and / or heavy chain constant regions.

[0180] Deimmunizing antibodies are antibodies that have had T-cell epitopes removed. Methods for preparing deimmunizing antibodies have been described (see, for example, Jones et al., Methods Mol Biol. 2009; 525:405-23, xiv, and De Groot et al., Cell. Immunol. 244:148-153 (2006)). Deimmunizing antibodies contain a variable region with reduced T-cell epitopes and a human constant region. In short, the VH and VL regions of the antibody are cloned, and T-cell epitopes are then identified by testing overlapping peptides derived from the antibody's VH and VL regions in a T-cell proliferation assay. T-cell epitopes are identified using computational methods to identify peptides that bind to human class II MHC. Mutations are introduced into the VH and VL regions to eliminate binding to human class II MHC. The mutated VH and VL regions are then used to generate deimmunizing antibodies.

[0181] In some embodiments, the extracellular antigen-binding domain comprises multiple binding domains. In some embodiments, the extracellular antigen-binding domain comprises a multispecific antibody or a fragment thereof, such as an extracellular antigen-binding domain comprising multiple tandem binding domains (e.g., multiple scFvs). In other embodiments, the extracellular antigen-binding domain comprises a multivalent antibody or a fragment thereof. The term "specificity" refers to the selective recognition of a specific antigenic epitope by an antigen-binding protein. As used herein, the term "multispecific" means that an antigen-binding protein has two or more antigen-binding sites, wherein at least two sites bind different antigens. As used herein, the term "valence" refers to the presence of a specified number of binding sites in an antigen-binding protein. A full-length antibody has two binding sites and is divalent. Therefore, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" indicate that an antigen-binding protein has two, three, four, five, and six binding sites, respectively.

[0182] Multispecific antibodies, such as bispecific antibodies, are antibodies that possess binding specificity against at least two different antigens. Methods for preparing multispecific antibodies are known in the art, for example, by co-expressing two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (see, for example, Milstein and Cuello, 1983, Nature 305:537-40). For more detailed information on the generation of multispecific antibodies (e.g., bispecific antibodies), see, for example, "Bispecific Antibodies" (Kontermann ed., 2011).

[0183] These antibodies can be multivalent antibodies having two or more antigen-binding sites (e.g., tetravalent antibodies) and can be readily prepared by recombinant expression of nucleic acids encoding antibody polypeptide chains. In some embodiments, multivalent antibodies comprise (or consist of) three to eight antigen-binding sites. In one embodiment, multivalent antibodies comprise (or consist of) four antigen-binding sites. Multivalent antibodies contain at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain contains two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, respectively, and n is 0 or 1. For example, the polypeptide chain may comprise: a VH-CH1-flexible linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibodies described herein may also contain at least two (e.g., four) light chain variable domain polypeptides. The multivalent antibodies described herein may, for example, comprise about two to about eight light chain variable domain peptides. The light chain variable domain peptides envisioned herein include light chain variable domains, and optionally also include CL domains.

[0184] In cases where multiple binding domains exist within the extracellular antigen-binding domain of the CAR described herein, these domains can be interconnected via peptide linkers. In some embodiments, the domains are directly interconnected without any peptide linkers. Peptide linkers can be identical or different. Depending on the structural and / or functional characteristics of each domain, each peptide linker can have the same or different lengths and / or sequences. Each peptide linker can be independently selected and optimized. The length, flexibility, and / or other characteristics of the peptide linkers used in the CAR may influence the properties, including but not limited to affinity, specificity, or affinity for one or more specific antigens or epitopes. In some embodiments, the peptide linkers contain flexible residues (e.g., glycine and serine) that allow adjacent domains to move freely relative to each other. For example, a glycine-serine dinucleotide may be a suitable peptide linker.

[0185] Peptide linkers may have naturally occurring or non-naturally occurring sequences. For example, sequences derived from the hinge region of a pure heavy chain antibody can be used as linkers. See, for example, WO1996 / 34103. In some embodiments, peptide linkers are flexible linkers. Exemplary flexible linkers include, but are not limited to, glycine polymers ((G)n), glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. The CARs described herein may include other connectors known in the art, see, for example, WO2016014789, WO2015158671, WO2016102965, US20150299317, WO2018067992, US7741465, Colcher et al., J.Nat.Cancer Inst.82:1191-1197 (1990), Bird et al., Science 242:423-426 (1988), the contents of which are incorporated herein by reference.

[0186] In some embodiments, the extracellular antigen-binding domain provided in the CAR described herein recognizes antigens as target cell surface markers associated with a specific disease state. In some embodiments, the antigen is a tumor antigen. Tumors express many proteins that can serve as target antigens for immune responses, particularly T cell-mediated immune responses. The antigen targeted by the CAR can be an antigen on a single diseased cell or an antigen expressed on different cells that contribute to the disease. The antigen targeted by the CAR may be directly or indirectly involved in the disease.

[0187] In some implementations, the antigens on the target cells are antigens on the surface of cancer cells. In some implementations, the antigens are tumor-specific antigens, tumor-associated antigens, or neoantigens.

[0188] In some implementations, the target cells are cancer cells, such as those of adrenal cancer, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic disease, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer. In some implementation schemes, cancers include adrenal cancer, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic disease, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer.

[0189] Tumor antigens are proteins produced by tumor cells that can trigger immune responses, especially T-cell-mediated immune responses. Exemplary tumor antigens include, but are not limited to: glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muta-hsp70-2, M-CSF, kallikrein (Prostase), prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, Prostein, PSMA, HER2 / neu, survival proteins and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoprotein B2, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0190] In some implementations, the cancer antigen is carcinoembryonic antigen (CEA), immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, EpCAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survival protein, BAGE family antigen, CAGE family antigen, GAGE ​​family antigen, MAGE family antigen, SAGE family antigen, XAGE family antigen, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A, MART-1, Gp100, pmel17, tyrosinase, TRP-1, TRP-2, tissue polypeptide-specific antigen (TPS), MC1R, prostate-specific antigen, β-catenin, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII, or MUC1.

[0191] In some implementations, tumor antigens contain one or more antigenic oncotopes associated with malignancy. Malignant tumors express a variety of proteins that can serve as targets for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the category of transformation-related molecules, such as the oncogene HER2 / Neu / ErbB-2. Another class of target antigens is onco-fetal antigen, such as carcinoembryonic antigen (CEA).

[0192] In some implementations, tumor antigens are tumor-specific antigens (TSA) or tumor-associated antigens (TAA). TSAs are antigens specific to tumor cells and are not present on other cells in the body. TAA-associated antigens, however, are not unique to tumor cells and can also be expressed in normal cells under conditions where immune tolerance to the antigen cannot be induced. Antigens on tumor cells may be expressed under conditions that enable the immune system to respond to them. TAAs may be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond; or they may be antigens that are normally present at very low levels in normal cells but are expressed at significantly increased levels in tumor cells.

[0193] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7.

[0194] Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0195] Other non-limiting exemplary targets for chimeric antigen receptors (CARs) provided herein include GPC2, CD276, delta-like protein ligand 3 (DLL3), NY-ESO-1, melanoma-associated antigen 4; survival protein, synovial sarcoma X breakpoint protein 2, CD3, epidermal growth factor receptor (EGFR), ERBB2 tyrosine kinase receptor, HER2, CEA, CD66, CD66e, ROR1, NTRK1 tyrosine kinase receptor, GPC3, mesothelin, glutamate carboxypeptidase II, PMSA, PD-L1, folate receptor α, PSCA, mucin 1, HLA antigens (such as HLA class I antigen A-2α, HLA class I antigen A-11α, and HLA class II antigens), c-Met, hepatocyte growth factor receptor, K-Ras GTPase (KRAS), IL-15 receptor, Kit tyrosine kinase, PDGF receptor β, RET tyrosine kinase receptor; Raf1 protein kinase, Raf B protein kinase, thymidine synthase, topoisomerase II, Brachyury protein, Flt3 tyrosine kinase, VEGF, VEGF receptors (VEGF-1 receptor, VEGF-2 receptor and VEGF-3 receptor), estrogen receptor, neoantigen, human papillomavirus E6, heat shock protein.

[0196] In other specific embodiments, at least one target antigen of the CDR described herein is Muc1, GAP, CD33, CEA, PSCA, Her2, mesothelin, CD19, CD20, CD22, BCMA, VEGFR2, FAP, EpCam, GPC3, CD133, IL13Ra, EGFRIII, EphA2, CD70, CD123, ROR1, PSMA, CD5, or GD2.

[0197] In some embodiments, the chimeric antigen receptor (CAR) described herein is capable of binding to B-cell antigens. In some embodiments, the B-cell antigens are CD1a, CD1b, CD1c, CD1d, CD2, CD5, CD6, CD9, CD11a, CD11b, CD11c, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD29, CD30, CD31, CD32a, CD32b, CD35, CD37, CD38, CD39, CD40, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49b, CD49c, and CD49d. , CD50, CD52, CD53, CD54, CD55, CD58, CD60a, CD62L, CD63, CD68, CD69, CD70, CD72, CD73, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82 , CD83, CD84, CD85E, CD85I, CD85J, CD86, CD92, CD95, CD97, CD98, CD99, CD100, CD102, CD108, CD119, CD120a, CD120b, CD121b, CD122, CD124, CD125 , CD126, CD130, CD132, CD137, CD138, CD139, CD147, CD148, CD150, CD152, CD162, CD164, CD166, CD167a, CD170, CD171, CD175, CD175s, CD180, CD1 84. CD185, CD192, CD196, CD197, CD200, CD205, CD201a, CDw210b, CD212, CD213a1, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, C D224, CD225, CD226, CD227, CD229, CD230, CD232, CD252, CD252, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD 267, CD268, CD269, CD270, CD272, CD274, CD275, CD277, CD279, CD283, CD289, CD290, CD295, CD298, CD300, CD300c, CD305, CD306, CD307a, CD307b,CD307c, CD307d, CD307e, CD314, CD215, CD316, CD317, CD319, CD321, CD327, CD328, CD329, CD338, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD360, CD361, CD362, or CD363 antigens.

[0198] In one embodiment, the chimeric antigen receptor (CAR) described herein targets a pathogen. In some embodiments, the target cell is a cell containing the pathogen.

[0199] In some implementations, the pathogen can cause infectious diseases selected from the following: acute flaccid myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, Haemophilus influenzae type b (Hib or H-influenza), Hantavirus pulmonary syndrome (HPS), hemolytic uremic syndrome (HUS), hepatitis A (Hep A), hepatitis B (Hep B), hepatitis C (Hep C), hepatitis D (Hep D), hepatitis E (Hep E), herpes, and herpes zoster (Herpes Zoster).Shingles, Histoplasma capsulatum infection, Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Legionella infection, Shigella gastroenteritis, Smallpox, Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus food poisoning (Enterotoxin B poisoning), Vancomycin-resistant Staphylococcus aureus (VISA), Vancomycin-resistant Staphylococcus aureus (VRSA), Invasive Group A Streptococcal disease, Group B Streptococcal disease B) Streptococcal toxic shock syndrome (STSS), syphilis (primary, secondary, early latent period, late latent period, congenital), tetanus infection, trichomoniasis, trichinosis, pulmonary tuberculosis (TB), latent pulmonary tuberculosis (LTBI), tularemia, typhoid fever group D, vaginitis, varicella (Varicella, Chickenpox), campylobacter infection, carbapenem-resistant infection, chancroid, chikungunya virus infection, chlamydia, ciguatoxin poisoning, and more. Clostridium difficile infection, Clostridium perfringens infection, Coccidioidomycosis (fungal infection), Coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jakob disease / infectious spongiform encephalopathy, Cryptosporidiosis, Cyclosporidiosis, Dengue fever types 1, 2, 3 or 4, Diphtheria, Escherichia coli infection / Shiga toxin-producing type (STEC), Eastern equine encephalitis, Hemorrhagic fever (Ebola), Ehrlich disease, Encephalitis, Vector-borne or parainfectious encephalopathy, Non-polio enterovirus, D68 enterovirus Virus (EV-D68), Giardiasis, Meredithromycin, Gonococcal Infection, Venereal Granuloma, Vibrio cholerae (cholera), Vibrio infection (Vibrio), Ebola Virus Hemorrhagic Fever, Lhasa Virus Hemorrhagic Fever, Marburg Virus Hemorrhagic Fever, West Nile Virus, Yellow Fever, Yersinia Infection, Zika Virus Infection, Leprosy (Hansen's Disease), Leptospirosis, Listeria Infection (Listeria), Lyme Disease, Lymphogranuloma Venereum (LGV), Malaria, Measles, Meredithromycin, Viral Meningitis Meningococcal disease (bacterial meningitis), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), mumps, norovirus, head lice, pelvic inflammatory disease (poliomyelitis), Bouvasin disease, psittacosis, pubic lice, pustular skin diseases (smallpox, monkeypox, cowpox), Queensland fever (Q fever), rabies, rickettsial disease (Rocky Mountain spotted fever), rubella (German measles), Salmonella gastroenteritis (Salmonella), scabies, mackerel disease, septicemia, severe acute respiratory syndrome (SARS).

[0200] In some embodiments, the pathogen is bacteria. In some embodiments, the bacteria belong to the genera *Bacillus*, *Bartonella*, *Bordezoella*, *Bordezoella burgdorferi*, *Brucella*, *Campylobacter*, *Chlamydia*, *Chlamydophila*, *Clostridium*, *Corynebacterium*, *Enterococcus*, *Escherichia coli*, *Francis*, *Haemophilus*, *Helicobacter*, *Legionella*, *Leptospira*, *Listeria*, *Mycobacterium*, *Mesoplasma*, *Neisseria*, *Pseudomonas*, *Rickettsia*, *Salmonella*, *Shigella*, *Staphylococcus*, *Streptococcus*, *Treponema pallidum*, *Ureaplasma*, *Vibrio*, or *Yersinia*. In some embodiments, the bacteria belong to the genus *Bacillus*. In some embodiments, the bacteria belong to the genus *Bartonella*. In some embodiments, the bacteria belong to the genus *Bordezoella*. In some embodiments, the bacteria belong to the genus *Bordezoella*. In some embodiments, the bacteria belong to the genus *Bordezoella*. In some embodiments, the bacteria belong to the genus *Brucella*. In some embodiments, the bacteria belong to the genus *Campylobacter*. In some embodiments, the bacteria belong to the genus *Chlamydia*. In some embodiments, the bacteria belong to the genus *Chlamydia*. In some embodiments, the bacteria belong to the genus *Clostridium*. In some embodiments, the bacteria belong to the genus *Corynebacterium*. In some embodiments, the bacteria belong to the genus *Enterococcus*. In some embodiments, the bacteria belong to the genus *Intermediate*. In some embodiments, the bacteria belong to the genus *Neisseria*. In some embodiments, the bacteria belong to the genus *Pseudomonas*. In some embodiments, the bacteria belong to the genus *Rickettsia*. In some embodiments, the bacteria belong to the genus *Salmonella*. In some embodiments, the bacteria belong to the genus *Shigella*. In some embodiments, the bacteria belong to the genus *Staphylococcus*. In some embodiments, the bacteria belong to the genus *Streptococcus*. In some embodiments, the bacteria belong to the genus *Treponema*. In some embodiments, the bacteria belong to the genus *Ureaplasma*. In some embodiments, the bacteria belong to the genus *Vibrio*. In some embodiments, the bacteria belong to the genus *Yersinia*. In some embodiments, the bacteria belong to the genus *Escherichia*. In some embodiments, the bacteria belong to the genus *Francis*. In some embodiments, the bacteria belong to the genus *Haemophilus*. In some embodiments, the bacteria belong to the genus *Helicobacter*. In some embodiments, the bacteria belong to the genus Legionella. In some embodiments, the bacteria belong to the genus Leptospira. In some embodiments, the bacteria belong to the genus Listeria. In some embodiments, the bacteria belong to the genus Mycobacterium.

[0201] In some embodiments, the pathogen is a parasite. In some embodiments, the parasite is a protozoan, worm, or ectoparasite. In some embodiments, the protozoan is an amoeba, Giardia lamblia, Leishmania, baculopoda, Plasmodium, or Cryptosporidium. In some embodiments, the worm is a fluke, tapeworm, acanthocephalan, or nematode. In some embodiments, the ectoparasite is an arthropod.

[0202] In some implementations, the pathogen is a virus. In some implementations, the virus belongs to the Adenoviridae, Arenaviridae, Astroviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepatoviridae, Hepatitisviridae, Orthomyxoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Polyomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Clonorviridae families.

[0203] In some implementations, the virus is adenovirus, coronavirus, Coxsackievirus, EB virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, or varicella-zoster virus.

[0204] Transmembrane domain

[0205] The CAR disclosed herein includes a transmembrane domain that can be directly or indirectly fused to an extracellular antigen-binding domain. The transmembrane domain can be of natural or synthetic origin. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in the cell membrane (preferably the eukaryotic cell membrane). The transmembrane domain suitable for the CAR described herein can be obtained from naturally occurring proteins. Alternatively, it can be a synthetic, non-naturally occurring protein fragment, such as a hydrophobic protein fragment that is thermodynamically stable in the cell membrane.

[0206] Transmembrane domains can be classified based on their three-dimensional structure. For example, transmembrane domains can form α-helices, complexes of multiple α-helices, β-barrels, or other stable structures capable of crossing the cellular phospholipid bilayer. Furthermore, transmembrane domains can also be classified according to the topological characteristics of their transmembrane structure, including the number of times the transmembrane domain crosses the cell membrane and the orientation of the protein. For example, single-transmembrane proteins cross the cell membrane once, while multi-transmembrane proteins cross the cell membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Based on the topological structure of their terminals and transmembrane segments relative to the cell interior and exterior, membrane proteins can be defined as type I, type II, or type III. Type I membrane proteins have only a single transmembrane region, and their orientation is such that the N-terminus of the protein is located on the extracellular side of the cellular lipid bilayer, while the C-terminus is located on the cytoplasmic side. Type II membrane proteins also have only a single transmembrane region, but their orientation is such that the C-terminus of the protein is located on the extracellular side of the cellular lipid bilayer, while the N-terminus is located on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subdivided into subclasses based on the number of transmembrane segments and the positions of the N-terminus and C-terminus.

[0207] In some embodiments, the transmembrane domain of the CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, the transmembrane domain of a multiple-pass membrane protein may also be used in the CAR described herein. Multiple-pass membrane proteins may contain complex (at least 2, 3, 4, 5, 6, 7 or more) α-helices or β-sheets. In some embodiments, the N-terminus and C-terminus of the multiple-pass membrane protein are located on opposite sides of the lipid bilayer; for example, the N-terminus of the protein is located on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein is located on the extracellular side.

[0208] The transmembrane region used in the CAR described herein may also comprise at least a portion of a synthetic, non-naturally occurring protein fragment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring α-helix or β-sheet. In some embodiments, the protein fragment comprises at least about 20 amino acids, such as at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the related art, such as U.S. Patent No. 7,052,906 and PCT Publication WO 2000 / 032776, the contents of which are incorporated herein by reference.

[0209] The transmembrane domains described herein may include a transmembrane region and a cytoplasmic region located on one side of the C-terminus of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain may contain three or more amino acids, facilitating the localization of the transmembrane domain within the lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain contains positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain contains the amino acids arginine, serine, and lysine.

[0210] In some embodiments, the transmembrane region of the transmembrane domain contains hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the CAR provided herein contains an artificially hydrophobic sequence. For example, a triplet sequence of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region primarily contains hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region contains a polyleucine-alanine sequence. The hydrophilicity (hydrophobic or hydrophilic characteristic) of a protein or protein fragment can be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0211] In some implementations, the transmembrane domain of the CAR includes transmembrane domains selected from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1a, CD18), I COS(CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD1 9. IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl Transmembrane domains of ld, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (TACTILE), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0212] In some specific embodiments, the transmembrane domain is derived from CD8α. In other specific embodiments, the transmembrane domain is derived from CD28α.

[0213] Intracellular signal transduction domains

[0214] The intracellular signaling domains in the CARs described herein are responsible for activating at least one normal effector function in CAR-expressing immune effector cells. The term "effector function" refers to a specific function of a cell. For example, the effector function of a T cell might be cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the term "cytoplasmic signaling domain" refers to a portion of a protein that transduces effector signals and directs the cell to perform a specific function. While the entire cytoplasmic signaling domain can often be used, in many cases it is not necessary to use the entire strand. A truncated portion of the cytoplasmic signaling domain can be used in place of the complete strand, as long as it can transduce effector signals. Therefore, the term "cytoplasmic signaling domain" is intended to include any truncated portion of a cytoplasmic signaling domain capable of transducing effector signals.

[0215] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the CAR comprises an intracellular signaling domain that is essentially composed of primary intracellular signaling domains of immune effector cells. A “primary intracellular signaling domain” refers to a cytoplasmic signaling sequence that can induce immune effector function upon stimulation. In some embodiments, the primary intracellular signaling domain comprises a signaling motif called an immune receptor tyrosine activation motif (ITAM). As used herein, “ITAM” is a conserved protein motif commonly found in the tail portion of signaling molecules expressed by many immune cells. This motif may comprise two repeating amino acid sequences YxxL / I, separated by 6-8 amino acids, where x is each any amino acid independently, forming the conserved motif YxxL / Ix. (6-8) YxxL / I. The ITAM within the signaling molecule is crucial for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. The ITAM can also function as a binding site for other proteins involved in signaling pathways. Exemplary primary cytoplasmic signaling sequences containing the ITAM include sequences derived from CD3z, FcRγ (FCER1G), FcRβ (Fc Epsilon Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0216] Co-stimulatory signal transduction domain

[0217] In some implementations, the CAR includes at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to a portion of a protein that mediates intracellular signal transduction to induce an immune response (e.g., effector function). Many immune effector cells require auxiliary stimuli in addition to stimulation by specific antigen signals to promote cell proliferation, differentiation, and survival, as well as to activate effector functions.

[0218] The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain derived from a costimulatory protein, capable of transducing signals and regulating responses mediated by immune cells (such as T cells, NK cells, macrophages, neutrophils, or eosinophils). The "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to a homologous binding chaperone molecule on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand, thereby mediated by the immune cell to conduct costimulatory responses, such as, but not limited to, proliferation and survival.

[0219] In some embodiments, the intracellular signaling domain includes a co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain includes two or more (e.g., about 2, 3, 4, or more) co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain includes two or more identical co-stimulatory signaling domains. In some embodiments, the intracellular signaling domain includes two or more co-stimulatory signaling domains from different co-stimulatory proteins, such as any two or more co-stimulatory proteins described herein. In some embodiments, the intracellular signaling domain includes a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z) and one or more co-stimulatory signaling domains. In some embodiments, one or more co-stimulatory signaling domains and the primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z) are fused together via an optional peptide linker. The primary intracellular signaling domain and the one or more co-stimulatory signaling domains can be arranged in any suitable manner. In some implementations, one or more co-stimulatory signaling domains are located between a transmembrane domain and a primary intracellular signaling domain (e.g., the cytoplasmic signaling domain of CD3z). Multiple co-stimulatory signaling domains can provide additive or synergistic stimulation effects.

[0220] Activation of costimulatory signaling domains in host cells (e.g., immune cells) may induce the cell to increase or decrease cytokine production and secretion, phagocytosis, proliferation, differentiation, survival, and cytotoxicity. The costimulatory signaling domain of any costimulatory molecule is suitable for the CAR described herein. The type of costimulatory signaling domain can be selected based on factors such as the type of immune effector cells to which the effector molecule will be expressed (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC) effect).Examples of co-stimulatory signaling domains that can be used in the CARs described herein include cytoplasmic signaling domains of co-stimulatory proteins, including but not limited to members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF). R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α and TNF RII / TNFRSF1B); SLAM family members (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150); and any other co-stimulatory molecules such as CD2, CD7, CD53, CD82 / Kai -1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, Class I HLA, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.In some embodiments, one or more co-stimulatory signal transduction domains are selected from: CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0221] In some embodiments, the costimulatory signaling domain is a variant of any costimulatory signaling domain described herein, enabling these costimulatory signaling domains to modulate the immune response of immune cells. In some embodiments, the costimulatory signaling domain contains up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residues different from a wild-type control. Such costimulatory signaling domains containing one or more amino acid variations are referred to as variants. Mutations in amino acid residues of a costimulatory signaling domain may result in increased signal transduction and enhanced stimulation of the immune response relative to a costimulatory signaling domain without the mutation. Mutations in amino acid residues of a costimulatory signaling domain may result in decreased signal transduction and reduced stimulation of the immune response relative to a costimulatory signaling domain without the mutation.

[0222] signal peptide

[0223] In some embodiments, the CARs provided herein may contain a signal peptide (also known as a signal sequence) located at the N-terminus of a polypeptide. Generally, a signal peptide is a peptide sequence that targets a polypeptide to a specific site within the cell. In some embodiments, the signal peptide directs effector molecules to the cell's secretory pathway and allows the effector molecules to integrate and anchor into the lipid bilayer. It will be apparent to those skilled in the art that signal peptides suitable for the CARs described herein include signal sequences of native proteins or synthetic, non-natural signal sequences. In some embodiments, the signal peptide is derived from molecules selected from CD8α, GM-CSF receptor α, and the IgG1 heavy chain.

[0224] Hinge area

[0225] In some embodiments, the functional exogenous receptors provided herein include a hinge domain located between an extracellular antigen-binding domain and a transmembrane domain. A hinge domain is an amino acid segment typically present between two domains of a protein and enables the protein to be flexible and allows one or both domains to move relative to each other. Any amino acid sequence that provides this flexibility and mobility of the extracellular antigen-binding domain relative to the transmembrane domain of an effector molecule can be used.

[0226] Hinge domains of antibodies (such as IgG, IgA, IgM, IgE, or IgD antibodies) are also applicable to the chimeric receptor systems described herein. In some embodiments, the hinge domain is a hinge domain connecting the constant domains CH1 and CH2 of the antibody. In some embodiments, the hinge domain is the hinge domain of the antibody and includes the hinge domain of the antibody as well as one or more constant regions of the antibody. In some embodiments, the hinge domain includes the hinge domain of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain includes the hinge domain of the antibody as well as the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region includes the hinge region as well as the CH2 and CH3 constant regions of the IgG1 antibody. In some embodiments, the hinge region includes the hinge region as well as the CH3 constant region of the IgG1 antibody.

[0227] The hinge domain of the chimeric receptor described herein may also use a non-naturally occurring peptide. In some embodiments, the hinge domain located between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of the Fc receptor is a peptide linker, such as a (GxS)n linker, where x and n can each be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.

[0228] The hinge domain may contain about 10 to 100 amino acids, such as about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the length of the hinge domain is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0229] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. The hinge domain of any protein known in the art that contains a hinge domain can be used in the chimeric receptor described herein. In some embodiments, the hinge domain is part of the hinge domain of a naturally occurring protein and imparts flexibility to the chimeric receptor.

[0230] In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, a hinge domain segment of CD8α containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids. In other embodiments, the hinge domain is derived from CD28α. In some embodiments, the hinge domain is a portion of the hinge domain of CD28α, for example, a hinge domain segment of CD28α containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids.

[0231] Exemplary chimeric antigen receptor (CAR)

[0232] The CARs that may be used in this disclosure include, but are not limited to, Kymria. TM (Tisagenlecleucel), Yescart TM(Axicabtagene ciloleucel), ALEXIS AIDT-2EOC (Kiromic Biopharma Inc.), CIK-CAR.PSMA (Formula Pharmaceuticals), ADI-002 (Adicet Bio), TSC-200 (TScan Therapeutics), TSC-100 (TScan Therapeutics), RB-H21 (Refuge Biotechnologies), ADP-A2AFP (Adaptimmune Therapeutics plc), CT-0729 (Carisma Therapeutics), CT-1119 (Carisma Therapeutics), CCT-301-59 (EXUMA Biotech), BOXR-889 (Unum Therapeutics), meso-CAR-T+PD-78 (MirImmune), MAGE-A10C796T (Adaptimmune Therapeutics plc), NKG2D-DARIC T cells (Bluebird) Bio, AGENT-NY-ESO-1X (AgenTus Therapeutics), MB-105 (City of Hope Medical Center), P-MUC1C-101 (Poseida Therapeutics), TT-16 (Baylor College of Medicine), ACTR-087 (Unum Therapeutics), EGFR-806 (Seattle Children's Hospital), TAC01-ROR1 (Triumvira Immunologics), CCT-301-38 (EXUMA Biotech), MB-103 (Mustang Bio), TAB-28z (OncoTab), ET-1402 (Eureka Therapeutics), ITI-1000 (Duke University), CIDeCAR (Bellicum Pharmaceuticals), MT-201 (Myeloid Therapeutics), CT-0508 (Carisma Therapeutics), ADP-A2M4 (Adaptimmune)Therapeutics, AMG-119 (Amgen), iCasp9M28z cells (Sloan Kettering Cancer Center), P-PSMA-101 (Poseida Therapeutics), ACE-1702 (Acepodia), AIC-100 (Cornell University), PRGN-3005 (Precigen), UniCAR-T-PSMA (GEMoaB Monoclonals GmbH), IMA-204 (University of Texas MD Anderson Cancer Center), DSG3-CAART (University of Pennsylvania), LXF-821 (University of Pennsylvania), MOv19-BBzCAR T cells (University of Pennsylvania), Tn MUC-1CAR-T (University of Pennsylvania), huMesoCART (University of Pennsylvania), IMA-203 (Immatics NV), IMA-202 (Immatics NV). NV), XYP-317 (Xyphos), CYAD-101 (Celyad Oncology), huMNC2-CAR44 T cells (Minerva Biotechnologies), HER2Bi-armed ATC (Roger Williams Medical Center), CYAD-200 series (Celdara Medical LLC), Lisocabtagene Maraleucel (liso-cel), FT596 (Fate Therapeutics), KTE-C19 (Kite Pharma), KTE-X19 (Kite Pharma), KITE-718 (Kite Pharma), KITE-439 (Kite Pharma), JCAR-024 (Fred Hutchinson Cancer Research Center), JCAR-023 (Juno Therapeutics), BPX-201 (Bellicum Pharmaceuticals), BPX-601 (Bellicum Pharmaceuticals), BPX-603 (Bellicum Pharmaceuticals, MCY-M11 (MaxCyte), KUR-503 (Baylor College of Medicine), ICS-200 (University of Alabama at Birmingham), ADP-A2M4CD8 (Adaptimmune Therapeutics), GLYCAR (Baylor College of Medicine), ALLO-501A (Allogene), CTX110 (CRISPR Therapeutics), ATA3219 (Atara)Biotherapeutics, Inc., RD13-01 (BiohengTherapeutics, Inc.)

[0233] In some embodiments, the CAR provided herein comprises an amino acid sequence having a certain percentage of identity with any of the exemplary CARs described above and in Section 6 below. In some embodiments, the CAR provided herein comprises or is composed of an extracellular domain having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of the CARs described above and in Section 6 below.

[0234] In some embodiments, amino acid sequence modifications of the CAR are contemplated herein. For example, it may be necessary to optimize the binding affinity and / or other biological properties of the extracellular domain, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, in addition to the extracellular domains described herein, it is contemplated that variants of the domains described herein can be prepared. For example, single-chain antibody (scFv) variants can be prepared by introducing appropriate nucleotide changes into the coding DNA or by synthesizing the desired antibody or peptide. Those skilled in the art will understand that amino acid changes may alter the post-translational process of the antibody.

[0235] Mutations can be substitutions, deletions, or insertions of one or more codons encoding the polypeptide, resulting in a change in the amino acid sequence compared to the original antibody or polypeptide. Target sites for substitution mutations include complementarity-determining regions (CDRs) and frame regions (FRs).

[0236] Amino acid substitutions can be achieved by replacing one amino acid with another that has similar structure and / or chemical properties, such as replacing leucine with serine, i.e., a conserved amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, to result in amino acid substitutions. Insertions or deletions may optionally range from about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a particular embodiment, the substitution is a conserved amino acid substitution performed at one or more predicted non-essential amino acid residues. Permissible variations can be determined by systematically inserting, deleting, or substituting amino acids in the sequence and testing the activity exhibited by the resulting variants relative to the parent polypeptide.

[0237] Peptides resulting from conserved amino acid substitutions are included in this disclosure. Conserved substitutions can be made (e.g., within amino acid groups having similar properties and / or side chains) to maintain or not significantly alter the properties.

[0238] One type of substitution variant involves replacing one or more hypervariable residues of the parent antibody (e.g., humanized or human antibody) or a fragment thereof in the extracellular antigen-binding domain of the CAR described herein. Typically, the resulting variant selected for further research will have some biological properties modified (improved) relative to the parent antibody (e.g., increased affinity, decreased immunogenicity), or will substantially retain some of the biological properties of the parent antibody. A typical substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques as described herein. In short, one or more CDR residues are mutated, and these variant antibodies are displayed on phages and screened for specific biological activities (e.g., binding affinity). It should be understood that the primary goal of humanization is to reduce the immunogenicity of antibodies from other species, but such sequence alterations may affect or reduce the biological activity of the parent antibody. It has been observed that the exemplary humanized antibodies provided herein unexpectedly exhibit enhanced biological activity, particularly functional exogenous receptors constructed from humanized antibodies and engineered cells exhibiting enhanced cytotoxicity and killing efficiency against target cells.

[0239] Alterations (e.g., substitutions) can be made in the CDR to improve antibody affinity. Such alterations can be made on CDR “hotspots” (residues encoded by codons that mutate frequently during somatic maturation, see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or the SDR (a-CDR), followed by binding affinity testing on the resulting variant antibody or fragment thereof. Affinity maturation methods involving the construction of a secondary library and subsequent selection are described, for example, by Hoogenboom et al. (Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In affinity maturation implementations, diversity can be introduced into the selected variant gene to be maturated using various methods (e.g., error-prone PCR, strand truncation, or oligonucleotide site-directed mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves CDR-directed methods, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. The CDR residues involved in antigen binding can be specifically identified using methods such as alanine scan mutagenesis or modeling.

[0240] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as these changes do not significantly reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions as described herein) may be made in CDRs that do not significantly reduce binding affinity. In some embodiments, each CDR either remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0241] An efficient method for identifying residues or regions in an antibody that can be mutated is called "alanine scanning mutagenesis," see Cunningham and Wells, Science, 244:1081-1085 (1989). Alternatively, or furthermore, the contact points between the antibody and antigen can be determined by the crystal structure of the antigen-antibody complex. These contact residues and their adjacent residues can be targeted or eliminated as replacement candidates. Variants can be screened to determine if they possess the desired properties.

[0242] Amino acid sequence insertions include peptides with a length ranging from one residue to one hundred or more residues fused to the amino and / or carboxyl ends, as well as insertions of single or multiple amino acid residues within the sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusing the N-terminus or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme-catalyzed prodrug therapy, ADEPT) or a peptide that increases the serum half-life of the antibody.

[0243] These mutations can be achieved using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scan mutagenesis, and PCR mutagenesis. Site-directed mutagenesis (see, for example, Carter, Biochem J.237:1-7 (1986); Zoller et al., Nucl. Acids Res.10:6487-500 (1982)), cassette mutagenesis (see, for example, Wells et al., Gene 34:315-23 (1985)) or other known techniques can be applied to cloned DNA to produce antibody variant DNA.

[0244] This disclosure also includes immune effector cells containing two or more functional exogenous receptors, such as dual chimeric antigen receptors (dual CARs) capable of binding to two different targets.

[0245] Antibodies that bind to GUCY2C

[0246] On the other hand, this document provides a single-domain antibody (sdAb) against GUCY2C, said sdAb comprising or consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of any of SEQ ID NO:12-32. In some embodiments, the anti-GUCY2C sdAb is a camel sdAb or a humanized sdAb. In some embodiments, the sdAb is a VHH antibody. In some embodiments, the anti-GUCY2C sdAb is genetically fused or chemically conjugated with other agents. It should be understood that the primary goal of humanization is to reduce immunogenicity, but such sequence alterations may affect or reduce the biological activity of the parent antibody. It has been observed that the exemplary humanized sdAbs provided in this paper unexpectedly exhibit enhanced biological activity, especially the functional exogenous chiropractic bodies and engineered cells constructed from humanized sdAbs, which demonstrate enhanced cytotoxicity and killing efficiency against target cells.

[0247] Anti-GUCY2C CAR

[0248] On the other hand, this document provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular antigen-binding domain including an extracellular antigen-binding domain comprising a single-domain antibody (sdAb) binding domain that binds to GUCY2C, (b) a transmembrane domain, and (c) an intracellular signal transduction domain. In some embodiments, the sdAb domain comprises CDR1, CDR2, and CDR3, which respectively comprise the amino acid sequences of CDR1, CDR2, and CDR3 as shown in the sdAb domain comprising any of SEQ ID NO:12-32.

[0249] The CDR can be determined according to any CDR encoding scheme known in the art, such as Kabat, AbM, Chothia, Contact, IMGT, or combinations thereof. In some embodiments, the CDR is determined according to the Kabat encoding scheme. In some embodiments, the CDR is determined according to the AbM encoding scheme. In some embodiments, the CDR is determined according to the Chothia encoding scheme. In some embodiments, the CDR is determined according to the Contact encoding scheme. In some embodiments, the CDR is determined according to the IMGT encoding scheme. In some specific embodiments, the sdAb structure field is a VHH structure field.

[0250] The anti-GUCY2C CAR provided in this article may also include one or more of the following: transmembrane domain, intracellular signal transduction domain, hinge domain, and / or signal peptide, which have been described in detail above.

[0251] More specifically, in some embodiments, the transmembrane domain is derived from a molecule selected from CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α, and optionally, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:57. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ, and optionally, the primary intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:60. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83 ligand, and combinations thereof. In some embodiments, the co-stimulatory signal transduction domain comprises a cytoplasmic domain of CD28, and optionally, the co-stimulatory signal transduction domain comprises the amino acid sequence of SEQ ID NO:59. In some embodiments, the co-stimulatory signal transduction domain comprises a cytoplasmic domain of CD137 (4-1BB), and optionally, the co-stimulatory signal transduction domain comprises the amino acid sequence of SEQ ID NO:58. In some embodiments, the CAR described herein further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α, and optionally, the hinge domain comprises the amino acid sequence of SEQ ID NO:56. In some embodiments, the CAR further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α, and optionally, the signal peptide comprises the amino acid sequence of SEQ ID NO:55. In some embodiments, the CAR provided herein comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence of any of SEQ ID NO:33-54.

[0252] In some embodiments, the anti-GUCY2C CAR provided herein is a functional exogenous receptor present in eukaryotic cells or peptides containing the aforementioned IMPDH. In other embodiments, the anti-GUCY2C CAR is expressed in eukaryotic cells not containing the aforementioned IMPDH.

[0253] In another embodiment, this document provides an isolated nucleic acid comprising a nucleic acid sequence encoding the anti-GUCY2C sdAb or anti-GUCY2CCAR provided herein. In another embodiment, this document provides a vector comprising the isolated nucleic acid provided herein. In another embodiment, this document provides a eukaryotic cell comprising the anti-GUCY2C sdAb or anti-GUCY2C CAR provided herein, the isolated nucleic acid, or the vector. In another embodiment, this document provides a pharmaceutical composition comprising the anti-GUCY2C sdAb, anti-GUCY2C CAR, isolated nucleic acid, vector, or eukaryotic cell provided herein. In another embodiment, this document provides a method of treating a disease or condition, comprising administering the anti-GUCY2C sdAb, anti-GUCY2C CAR, isolated nucleic acid, vector, eukaryotic cell, or pharmaceutical composition provided herein. The following sections provide a more detailed description of eukaryotic cells expressing the anti-GUCY2C sdAb and anti-GUCY2C CAR described herein, and their uses.

[0254] eukaryotic cells

[0255] The exemplary (but not limiting) eukaryotic cells provided herein are derived from mammals. In some embodiments, the eukaryotic cells provided herein are derived from primates, rodents, etc. In some embodiments, the eukaryotic cells are derived from humans, rats, mice, guinea pigs, rabbits, sheep, goats, camels or alpacas, horses, donkeys, chimpanzees, or macaques. In some embodiments, the eukaryotic cells are derived from humans.

[0256] In some implementations, the eukaryotic cells provided herein are primary cells.

[0257] Primary cells

[0258] "Primary cells" refer to cells isolated from an organism (e.g., tissues or fluids of humans, animals, or plants) that have not undergone long-term in vitro culture and retain their original biological characteristics. In this document, "primary cells" is used in contrast to "cell lines." The term "cell line" is typically established from primary cells through a process of "immortification"; immortalization usually involves mutations or alterations in genes related to cell cycle regulation to create a stable cell line. It has been observed that the exogenously introduced IMPDH provided herein can confer enhanced resistance to purine biosynthesis inhibitors in primary cells compared to cell lines, and even provide improved growth performance. Therefore, in some specific embodiments, the eukaryotic cells provided herein are primary cells. In some specific embodiments, the eukaryotic cells may be cell lines.

[0259] In some embodiments, the eukaryotic cells described in this disclosure include normal tissue cells and immune cells, such as lymphocytes, phagocytes, and dendritic cells. For example, the eukaryotic cells provided herein may be T cells, such as γδT cells and regulatory T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, monocytes, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. Any engineered eukaryotic cells carrying exogenously introduced IMPDH and resistant to purine biosynthesis inhibitors are within the scope of this disclosure. Immune effector cells are described in detail below only as one example of the eukaryotic cells provided herein and are not intended to limit the scope of this disclosure.

[0260] In some embodiments, the eukaryotic cells provided herein are immune cells. In some embodiments, the eukaryotic cells provided herein are lymphocytes, phagocytes, or dendritic cells. In some embodiments, the eukaryotic cells provided herein are T cells, γδT cells, regulatory T cells, natural killer (NK) cells, NKT cells, B cells, macrophages, monocytes, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, or normal tissue cells. In some specific embodiments, the eukaryotic cells are T cells.

[0261] Immune effector cells

[0262] "Immune effector cells" refer to immune cells capable of performing immune effector functions. In some implementations, immune effector cells express at least FcγRIII and perform antibody-dependent cell-mediated cytotoxicity (ADCC) effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0263] In some embodiments, the immune effector cells are T cells. In some embodiments, the T cells are CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8+, or combinations thereof. In some embodiments, the T cells produce IL-2, TNF, and / or TNF upon expressing a functional exogenous receptor (such as CAR) and binding to target cells. In some embodiments, CD8+ T cells lyse antigen-specific target cells upon expressing a functional exogenous receptor (such as CAR) and binding to target cells.

[0264] In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells may be established cell lines, such as NK-92 cells.

[0265] In some implementations, immune effector cells differentiate from stem cells, such as hematopoietic stem cells, pluripotent stem cells, induced pluripotent stem cells (iPS), or embryonic stem cells.

[0266] Engineered immune effector cells are prepared by introducing the peptides provided herein into immune effector cells (such as T cells). In some embodiments, the peptides can be introduced into immune effector cells by transfecting any of the isolated nucleic acids or any of the vectors described above.

[0267] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. Vectors can be transferred to immune effector cells by physical, chemical, or biological methods.

[0268] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are known in the art. For example, see Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cells via electroporation.

[0269] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most common method for inserting genes into mammalian (e.g., human) cells.

[0270] Chemical methods for introducing carriers into immune effector cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal dispersion system used in vitro as a delivery carrier is a liposome (e.g., an artificial membrane vesicle).

[0271] In some embodiments, the RNA molecule encoding any of the polypeptides described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into immune effector cells by known methods (e.g., mRNA electroporation). See, for example, Rabinovich et al., Human Gene Therapy 17:1027-1035 (2006).

[0272] In some embodiments, after the introduction of the vector or isolated nucleic acid, the transduced or transfected immune effector cells can be cultured in vitro to proliferate. In some embodiments, the transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select engineered mammalian cells. For example, the transduced or transfected immune effector cells can be cultured for a period of time in a medium containing a purine biosynthesis inhibitor (such as MMF) and then collected.

[0273] Other methods may also be employed, such as using reporter genes to identify potentially transfected cells and assess the function of regulatory sequences. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue, and the expression of its encoded polypeptide is indicated by some easily detectable property, such as enzyme activity. The expression of the reporter gene is measured after DNA has been introduced into the recipient cells for an appropriate period. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available.

[0274] Methods for confirming the presence of nucleic acids encoding peptides in engineered immune effector cells include, for example, molecular biological detection methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical detection methods, such as detecting the presence of a specific peptide by immunological methods (e.g., ELISA and Western blotting).

[0275] T cell origin

[0276] The immune effector cells particularly useful in this disclosure are T cells. In some embodiments, the T cell source is obtained from a subject prior to expansion and genetic modification of the T cells. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines existing in the art, such as Jurkat cells, can be used. In some embodiments, any number of methods known to those skilled in the art (e.g., Ficoll) can be used. TMApheresis is a method of obtaining T cells from a unit of blood collected from a subject. In some embodiments, cells in an individual's circulating blood can be obtained via apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, these cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is calcium-free and may be magnesium-free, or may be free of most (if not all) divalent cations. Performing the initial activation step under calcium-free conditions can enhance activation. As will be readily understood by those skilled in the art, the washing step can be performed by methods known in the art, such as using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, BaxterCytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as calcium-free buffers. 2+ Mg 2+ PBS, PlasmaLyte A, or other physiological saline with or without buffer can be used. Alternatively, unwanted components in the apheresis sample can be removed, and the cells can be directly resuspended in the culture medium.

[0277] In some implementations, T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example, through PERCOLL TM Centrifugation along a gradient or by counterflow centrifugal elutriation can be performed. Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can also be further isolated using positive or negative selection techniques. For example, in some embodiments, magnetic beads conjugated to anti-CD3 / anti-CD28 (i.e., 3×28) antibodies (such as...) can be used. M-450 CD3 / CD28 T cells are incubated for a sufficient time to positively select the desired T cells for isolation. In some embodiments, the time is approximately 30 minutes. In some embodiments, the time is from 30 minutes to 36 hours or longer, and all integer values ​​within this range. In some embodiments, the time is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time is 10 to 24 hours. In some embodiments, the incubation time is 24 hours. For isolating T cells from leukemia patients, using a longer incubation time (such as 24 hours) can increase cell yield. In any case where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals, a longer incubation time can be used to isolate T cells.

[0278] Furthermore, using a longer incubation time can improve the efficiency of capturing CD8+ T cells. Therefore, in some embodiments, simply shortening or lengthening the binding time of T cells to CD3 / CD28 magnetic beads, or by increasing or decreasing the ratio of magnetic beads to T cells, can preferentially select or exclude T cell subsets at the start of culture or at other points in the process. Additionally, by increasing or decreasing the proportion of anti-CD3 and / or anti-CD28 antibodies on the magnetic beads, T cell subsets can also be preferentially selected or excluded at the start of culture or at other desired points in the process. Those skilled in the art will recognize that multiple screenings can be used. In some embodiments, a screening procedure may need to be performed, and “non-screened” cells may be used for activation and expansion processes. “Non-screened” cells may also be subject to further screening procedures.

[0279] T cell population enrichment can be achieved through antibody combinations targeting surface markers specific to negatively selected cells. One approach is cell sorting and / or screening via negative magnetic immunoadsorption or flow cytometry, using a mixture of monoclonal antibodies targeting the surface markers of the negatively selected cells. For example, to enrich CD4+ cells through negative selection, a mixture of monoclonal antibodies containing antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8 is typically used. In some embodiments, it may be necessary to enrich or positively select regulatory T cells that typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some embodiments, T regulatory cells can be reduced by conjugating anti-C25 antibodies to magnetic beads or other similar selection methods.

[0280] For separating a desired cell population using either positive or negative selection, the cell concentration and surface area (e.g., particles such as magnetic beads) can vary. In some embodiments, to ensure maximum cell contact with the magnetic beads, it may be necessary to significantly reduce the total volume of cells mixed with the beads (i.e., increase the cell concentration). For example, in one embodiment, a concentration of 2 billion cells per milliliter is used. In another embodiment, a concentration of 1 billion cells per milliliter is used. In a further embodiment, a concentration of more than 100 million cells per milliliter is used. In a further embodiment, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per milliliter are used. In another embodiment, concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per milliliter are used. In a further embodiment, a concentration of 125 million or 150 million cells per milliliter can be used. Using high concentrations may increase cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations can more effectively capture cells that may weakly express the target antigen (such as CD28-negative T cells), or obtain cells from samples containing large numbers of tumor cells (such as leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are worth acquiring. In some implementations, using high cell concentrations can more effectively screen for CD8+ T cells that typically have weak CD28 expression.

[0281] In some implementations, lower cell concentrations may be required. Significantly diluting the mixture of T cells and the surface (e.g., particles, such as magnetic beads) minimizes particle-cell interactions. This allows screening for cells expressing high levels of the desired antigen to bind to the particles. For example, at diluted concentrations, CD4+ T cells express higher levels of CD28 than CD8+ T cells and are more effective at capture. In some implementations, a cell concentration of 5 × 10⁻⁶ cells is used. 6 / ml. In some implementations, the concentration used can be approximately 1×10⁻⁶. 5 / ml to 1×10 6 / ml, including any integer value in between.

[0282] In some implementations, cells can be incubated for a certain period of time on a rotator at different speeds and temperatures (e.g., 2-10°C or room temperature).

[0283] T cells used for stimulation can also be frozen after the washing step. Without being theoretically limited, freezing and subsequent thawing steps may provide a more homogeneous product because granulocytes and some degree of monocytes can be removed from the cell population. After the washing step to remove plasma and platelets, the cells can be suspended in a cryoprotectant. Many cryoprotectants and related parameters are available in the prior art and are applicable here. One method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40, 5% glucose, 20% human serum albumin, and 7.5% DMSO, or a solution containing 31.25% PlasmaLyte-A, 31.25% glucose, 5% sodium chloride, 10% dextran 40, 5% glucose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell cryoprotectants (e.g., containing Hespan and PlasmaLyte A). The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the gas phase of a liquid nitrogen tank. Other controlled freezing methods can also be used, as well as instantaneous uncontrolled freezing at -20°C or in liquid nitrogen.

[0284] In some implementations, the cryopreserved cells are thawed and washed as described herein, left to stand at room temperature for one hour, and then activated.

[0285] This disclosure also envisions collecting blood samples or apheresis products from subjects some time before the amplified cells described herein are needed. Therefore, the cell source for amplification can be collected at any necessary point in time, and the desired cells (e.g., T cells) can be isolated and frozen for use in T-cell therapy for any disease or condition that may benefit from T-cell therapy (as described herein). In one embodiment, blood samples or apheresis products are collected from generally healthy subjects. In some embodiments, blood samples or apheresis products are collected from generally healthy subjects who are at risk of disease but do not yet have the disease, and the desired cells are isolated and frozen for later use. In some embodiments, T cells can be amplified, frozen, and used later. In some embodiments, samples can be collected from patients shortly after a specific disease is diagnosed (as described herein) but before any treatment is initiated. In a further implementation, cells are isolated from the subject's blood sample or apheresis product prior to any relevant treatment (including but not limited to the use of drug therapy (such as natamizumab, efarizidumab), antiviral agents, chemotherapy, radiotherapy, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablation agents (such as CAMPATH, anti-CD3 antibody, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228) and radiotherapy). These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or the p70S6 kinase (rapamycin), which plays an important role in growth factor-induced signaling (Liu et al., Cell 66:807-815 (1991); Henderson et al., Immun 73:316-321 (1991); Bierer et al., Curr. Opin. Immun. 5:763-773 (1993)). In a further embodiment, cells are isolated from the patient and frozen for future use in conjunction with bone marrow or stem cell transplantation or T-cell ablation therapy (using chemotherapy drugs such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH) (e.g., before, during, or after). In another embodiment, cells may be isolated prior to B-cell ablation therapy (e.g., using drugs targeting CD20, such as rituximab) and cryopreserved for subsequent treatment.

[0286] In some implementations, T cells are obtained directly from the patient after treatment. In this regard, the quality of the T cells obtained after certain cancer treatments (especially those using drugs that damage the immune system) may be optimal, or their in vitro expansion capacity may be enhanced, during the short recovery period typically following treatment. Similarly, these cells may be in a state more conducive to enhanced in vivo colonization and expansion after in vitro manipulation using the methods described herein. Therefore, this document envisions collecting blood cells, including T cells, dendritic cells, or other hematopoietic lineage cells, during this recovery phase. Furthermore, in some implementations, mobilization (e.g., using GM-CSF) and modulation protocols may be employed to create conditions in the subject that favor the reproliferation, recycling, regeneration, and / or expansion of specific cell types, particularly within a specific time window after treatment. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0287] Activation and expansion of T cells

[0288] In some embodiments, T cells can be activated and expanded using methods described, for example, those in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 20060121005, before or after genetic modification of T cells with a functional exogenous receptor (e.g., CAR) as described herein.

[0289] Typically, T cells can proliferate through surface contact with reagents bearing signals associated with the CD3 / TCR complex and ligands that stimulate co-stimulatory molecules on the T cell surface. Specifically, T cell populations can be stimulated as described herein, for example, by contact with surface-immobilized anti-CD3 antibodies or their antigen-binding fragments, or anti-CD2 antibodies; or by contact with a combination of protein kinase C activators (such as lichenin) and calcium ionophores. For co-stimulation of T cell surface helper molecules, ligands that bind to helper molecules can be used. For example, under conditions suitable for stimulating T cell proliferation, T cell populations can be contacted with anti-CD3 and anti-CD28 antibodies. Anti-CD3 and anti-CD28 antibodies can be used to stimulate the proliferation of CD4+ or CD8+ T cells. Examples of anti-CD3 antibodies include UCHT1, OKT3, and HIT3a (BioLegend, San Diego, USA), and other methods known in the art may also be used (Graves J, et al., J. Immunol. 146:2102 (1991); Li B, et al., Immunology 116:487 (2005); Rivollier A, et al., Blood 104:4029 (2004)). Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besançon, France), and other methods known in the art may also be used (Berg et al., Transplant Proc. 30(8): 3975-3977 (1998); Haanen et al., J. Exp. Med. 190(9): 1319-1328 (1999); Garland et al., J. Immunol Meth. 227(1-2): 53-63 (1999)).

[0290] In some embodiments, the primary and co-stimulatory signals of T cells can be provided by different methods. For example, the reagent providing each signal can be in solution form or bound to a surface. When bound to a surface, the reagent can bind to the same surface (i.e., the "cis" configuration) or to a different surface (i.e., the "trans" configuration). Alternatively, one reagent can bind to a surface while the other is in solution form. In one embodiment, the reagent providing the co-stimulatory signal binds to the cell surface, while the reagent providing the primary stimulatory signal is in solution form or bound to a surface. In some embodiments, both reagents can be in solution form. In another embodiment, the reagent can be in a soluble form and then cross-linked to a surface (e.g., cells expressing Fc receptors, antibodies, or other binding agents that can bind to these reagents). In this regard, see, for example, U.S. Patent Application Publications 20040101519 and 20060034810, which describe artificial antigen-presenting cells (aAPCs) that can be used to activate and expand T cells in some embodiments of this disclosure.

[0291] In some embodiments, T cells bind to reagent-coated microbeads, then the microbeads are separated from the cells, and the cells are then cultured. In another embodiment, the reagent-coated microbeads and cells are cultured together without separation prior to culture. In a further embodiment, the microbeads and cells are first concentrated by applying a force (e.g., magnetic force) to increase the binding of cell surface markers, thereby inducing cell stimulation.

[0292] For example, cell surface proteins can be linked by contacting T cells with magnetic beads coated with anti-CD3 and anti-CD28 (3×28 magnetic beads). In one embodiment, cells (e.g., 10 4 Up to 4×10 8Target cells (T cells) and microparticles (e.g., anti-CD3 / CD28 MACSiBead particles at a recommended titer of 1:100) are bound in a buffer (preferably free of divalent cations, such as calcium- and magnesium-free PBS). Those skilled in the art will readily understand that any cell concentration can be used. For example, target cells may be very rare in a sample, comprising only 0.01% of the sample, or the entire sample (i.e., 100%) may consist of target cells. Therefore, any number of cells is within the scope of this disclosure. In some embodiments, it may be necessary to significantly reduce the volume of the particle and cell mixture (i.e., increase the cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a cell concentration of approximately 2 billion cells per milliliter is used. In another embodiment, a concentration greater than 100 million cells per milliliter is used. In further embodiments, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per milliliter are used. In another embodiment, a concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per milliliter is used. In a further embodiment, a concentration of 125 million or 150 million cells per milliliter can be used. Using high concentrations may increase cell yield, cell activation, and cell expansion. Furthermore, using high cell concentrations can more effectively capture cells that may weakly express the target antigen (such as CD28-negative T cells). Such cell populations may have therapeutic value and are worth acquiring in some embodiments. For example, using high cell concentrations can more effectively screen for CD8+ T cells that typically have weak CD28 expression.

[0293] In some embodiments, the mixture may be cultured for several hours (about 3 hours) to 14 days or any integer number of hours in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment, the microbeads and T cells are cultured together for about 8 days. In another embodiment, the microbeads and T cells are cultured together for 2 to 3 days. Several stimulation cycles may also be required to extend the culture time of the T cells to 60 days or longer. Suitable conditions for T cell culture include appropriate culture media (e.g., basal medium, RPMI 1640 medium, or X-vivo 15 medium (Lonza)) containing components necessary for cell proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, interferon-γ (IFN-γ), IL-4, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, IL-12, IL-15, transforming growth factor-β (TGFβ), and tumor necrosis factor-α (TNF-α), or any other additives known to those skilled in the art for cell growth. Other additives required for cell growth include, but are not limited to, surfactants, human plasma proteins, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, optimizers containing added amino acids, sodium pyruvate, and vitamins, either serum-free or with the addition of adequate serum (or plasma) or a predetermined set of hormones, and / or sufficient amounts of cytokines to promote T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in cell cultures injected into subjects. Target cells are maintained under conditions necessary to support their growth, such as a suitable temperature (e.g., 37°C) and a gaseous environment (e.g., air plus 5% CO2). T cells treated with different stimulation times may exhibit different characteristics. For example, in typical blood or apheresis peripheral blood mononuclear cell products, the number of helper T cells (TH, CD4+) is greater than that of cytotoxic or suppressor T cells (TC, CD8). In vitro expansion of T cells by stimulating CD3 and CD28 receptors produces a T cell population primarily composed of TH cells by approximately day 8-9, followed by a gradual increase in TC cells after approximately day 8-9. Therefore, depending on the therapeutic objective, infusing the subject with a T cell population primarily containing TH cells may be advantageous. Similarly, if an antigen-specific TC cell subset has been isolated, expanding this subset may be beneficial.

[0294] Furthermore, while other phenotypic markers besides CD4 and CD8 vary considerably, most are reproducible during cell expansion. This reproducibility allows for the customization of activated T-cell products for specific purposes.

[0295] In some implementations, as described in Section 5.8 below, the engineered eukaryotic cells (such as engineered T cells) described herein can be sorted and screened by culturing them in a culture medium containing an inhibitor of purine biosynthesis.

[0296] 5.3 Peptides containing functional exogenous receptors and exogenous IMPDH

[0297] On the other hand, this document provides a polypeptide or polypeptide combination comprising an IMPDH resistant to purine biosynthesis inhibitors provided herein and at least one functional exogenous receptor (e.g., CAR, TCR, and TAC). In some embodiments, the IMPDH and functional exogenous receptor in the polypeptide combination may be unlinked. In some embodiments, the IMPDH and functional exogenous receptor in the polypeptide combination may be linked or fused, i.e., as a single polypeptide comprising an IMPDH region and a functional exogenous receptor region. In some embodiments, in the polypeptide or polypeptide combination, the amino acid sequence of the prokaryotic IMPDH is located at the N-terminus or C-terminus of the amino acid sequence of the functional exogenous receptor. In some embodiments, the IMPDH and functional exogenous receptor are linked together via a cleavable peptide linker. The IMPDH and functional exogenous receptor in the polypeptide described herein are each selected from the IMPDH and functional exogenous receptor described in Section 5.2 above. For example, in some embodiments, the IMPDH provided herein has a low binding affinity for purine biosynthesis inhibitors (e.g., MPA). In some embodiments, the inhibition constant (Ki) of this IMPDH binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 20 nM. In some embodiments, the inhibition constant (Ki) of this IMPDH binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 30 nM, for example, greater than 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, or greater. In other embodiments, the inhibition constant (Ki) of IMPDH binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 400 nM, for example, greater than 500 nM, 600 nM, 700 nM, 800 nM, or 900 nM. In other embodiments, the inhibition constant (Ki) of IMPDH binding to a purine biosynthesis inhibitor (e.g., MPA) is greater than 1 μM, for example, greater than 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 19 μM, or 20 μM. In still other embodiments, the inhibition constant (Ki) of IMPDH provided by the peptides described herein binding to a purine biosynthesis inhibitor (e.g., MPA) is at least 20 times greater than that of wild-type human IMPDH, for example, greater than at least 25 times, 30 times, 35 times, or 40 times. In other embodiments, the purine biosynthesis inhibitors (e.g., MMF) provided herein have low antagonistic efficacy against the IMPDH provided by the peptides described herein. In some embodiments, the purine biosynthesis inhibitors (e.g., MMF) provided herein inhibit IMPDH by a greater EC50 or IC50 value than those inhibiting wild-type human IMPDH.In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) inhibits the EC50 or IC50 values ​​of the IMPDH described herein by approximately two times, for example, three times, four times, or more, than the EC50 or IC50 values ​​inhibiting wild-type human IMPDH. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) has an EC50 or IC50 value greater than 1 ng / mL for the IMPDH described herein. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) has an EC50 or IC50 value greater than 2 ng / mL for the IMPDH described herein, for example, greater than 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL. In some embodiments, the purine biosynthesis inhibitor (e.g., MMF) exhibits an EC50 or IC50 greater than 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, or greater against the IMPDH provided herein. In some embodiments, the Kcat value for the conversion of IMP to XMP by the IMPDH provided herein is higher than the Kcat value of wild-type human IMPDH. In some embodiments, the Kcat value of the IMPDH provided herein is greater than 0.4 s. -1 In some implementations, the Kcat of the IMPDH provided herein is greater than 0.5s. -1 In some implementations, the Kcat of the IMPDH provided herein is greater than 1.0s. -1 In some implementations, the Kcat of the IMPDH provided herein is greater than 2.0s. -1 For example, greater than 3.0s -1 4.0s -1 5.0s -1 6.0s -1 7.0s -1 8.0s -1 Or 9.0s -1 In some implementations, the Kcat for the IMPDH provided herein is approximately 10.0 s. -1In some embodiments, the binding affinity of IMPDH to NAD+ is not less than or greater than that of eukaryotic IMPDH (e.g., wild-type human IMPDH). In some embodiments, the dissociation constant (Kd) of IMPDH to NAD+ is not more than or less than that of wild-type human IMPDH. In some embodiments, the Michaelis constant (Km) of IMPDH to NAD+ is not more than or less than that of wild-type human IMPDH. In some embodiments, the IMPDH provided herein allows for the simultaneous binding of purine biosynthesis inhibitors (e.g., MPA) and substrates (e.g., IMP and / or NAD+) without mutual repulsion, for example, the active site and / or binding site have spatial configurations that confer multiple binding capabilities. In some embodiments, exogenously introduced IMPDH is capable of catalyzing the conversion of IMP to XMP in a normal manner without interference from purine biosynthesis inhibitors; for example, in the presence of purine biosynthesis inhibitors, exogenously introduced IMPDH can catalyze the conversion of IMP to XMP more efficiently. Surprisingly, it has even been observed that eukaryotic cells expressing exogenously introduced IMPDH exhibit superior growth performance in the presence of purine biosynthesis inhibitors. In some embodiments, the IMPDH provided herein includes a cofactor binding site for binding substrates (e.g., IMP and / or NAD+), which is not inhibited by purine biosynthesis inhibitors (e.g., MPA). In some embodiments, the IMPDH provided herein is a non-human IMPDH or a variant of human IMPDH. In some embodiments, the IMPDH is derived from prokaryotic IMPDH. In some embodiments, the prokaryotic IMPDH is derived from bacterial IMPDH, such as Gram-positive bacterial IMPDH or Gram-negative bacterial IMPDH. Exemplary (but not limiting) Gram-positive bacterial IMPDHs include Bacillus subtilis IMPDH, Lactobacillus plantarum IMPDH, Staphylococcus aureus IMPDH, or variants thereof. Exemplary (but not limiting) Gram-negative bacterial IMPDHs include Escherichia coli IMPDH, Mesoplasma florum IMPDH, or variants thereof.

[0298] In some more specific embodiments, the IMPDH provided herein comprises the amino acid sequence of any one of SEQ ID NO: 1 to 7. In some embodiments, the exogenously introduced IMPDH provided herein comprises an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of any one of SEQ ID NO: 1 to 7, wherein the IMPDH retains resistance to the purine biosynthesis inhibitors (e.g., MPA or its analogues or derivatives) provided herein.

[0299] In some embodiments, the IMPDH variant comprises an amino acid sequence that differs from naturally occurring IMPDH due to at least one amino acid alteration (e.g., substitution, addition, or deletion). The IMPDH variants described herein may have at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with naturally occurring IMPDH. In some embodiments, the IMPDH variant is a functional variant that has some biological characteristics modified or improved relative to the parent IMPDH, or will substantially retain some biological characteristics of the parent IMPDH, particularly resistance to purine biosynthesis inhibitors. In some embodiments, the IMPDH variant, relative to its naturally occurring IMPDH (parental IMPDH), includes mutations at the following sites: D13, D50, E54, D138, D200, A223, D243, D248, D338, E369, E373, E469, or combinations thereof. In some specific embodiments, the IMPDH variant comprises the amino acid sequence of SEQ ID NO: 6 or 7.

[0300] In some embodiments of the various peptides provided herein, the IMPDH and the functional exogenous receptor are linked together via a cleavable peptide. In some embodiments, the peptide linker is a self-cleaving peptide, such as a 2A self-cleaving peptide, so that the IMPDH is cleaved into a separate peptide within the cell.

[0301] Members of the 2A peptide family are named after the virus in which they were first described. For example, F2A was the first 2A peptide described, derived from foot-and-mouth disease virus. These 18-22 amino acid-long, self-cleaving 2A peptides mediate "ribosomal jumping" between proline and glycine residues and inhibit peptide bond formation without affecting downstream translation. These peptides allow multiple proteins to be encoded as polyproteins, which are broken down into constituent proteins during translation. Self-splicing peptides are found in members of the Picornaviridae family, including the genus *Aphthous stomatitis virus*, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), *TaV*, and porcine cheshvirus 1 (PTV-1) (see Donnelly et al., J. Gen. Virol., 82:1027-101 (2001); Ryan et al., J. Gen. Virol., 72:2727-2732 (2001)); and the genus *Cardiovirus*, such as Theylvirus (e.g., Theyl rat encephalomyelitis virus) and encephalocarditis virus. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are sometimes referred to as “F2A,” “E2A,” “P2A,” and “T2A,” respectively, and are included in this disclosure, see, for example, Donnelly et al., J. Gen. Virol., 78:13-21 (1997); Ryan and Drew, EMBO J., 13:928-933 (1994); Szymczaketal., Nature Biotech., 5:589-594 (2004); Hasegawa et al., Stem Cells, 25(7):1707-12 (2007). In other embodiments, intron-mediated protein splicing is employed herein (see, for example, Shah and Muir, Chem Sci., 5(1):446–461 (2014); Topilina and Mills, Mobile DNA, 5(5)(2014)). Other methods known in the art may also be used in the constructs described in this article.

[0302] In some embodiments, the 2A self-cleaving peptide is selected from F2A, E2A, P2A, T2A, or variants thereof. In one specific embodiment, the self-cleaving peptide is a T2A fragment comprising the amino acid sequence of SEQ ID NO:11.

[0303] In another aspect, this article provides nucleic acids encoding the aforementioned polypeptides or combinations of polypeptides, which contain the functional exogenous receptors (e.g., CARs) and exogenous IMPDHs provided herein, which are described in more detail below.

[0304] 5.4 Polynucleotides

[0305] On the other hand, this disclosure provides polynucleotides encoding polypeptides or combinations of polypeptides provided herein, including those described in sections 5.2 and 5.3 above. More specifically, in some embodiments, this document provides a polynucleotide encoding a polypeptide or combination of polypeptides comprising a functional exogenous receptor (e.g., CAR) provided herein and an exogenous IMPDH provided herein, wherein the functional exogenous receptor and the IMPDH are fused to each other via a peptide linker (e.g., a 2A self-cleaving linker).

[0306] On the other hand, this document provides a polynucleotide comprising a region encoding a functional exogenous receptor provided herein and a region encoding an exogenous IMPDH provided herein, as described in Section 5.2 above. In some embodiments, both regions are controlled by the same promoter. For example, in some embodiments, this document uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, these regions are controlled by independent promoters.

[0307] In another aspect, this document provides a composition comprising a first polynucleotide encoding a functional exogenous receptor and a second polynucleotide encoding the exogenous IMPDH described in Section 5.2 above.

[0308] The polynucleotides described in this disclosure can be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; it can be double-stranded or single-stranded, and if single-stranded, it can be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0309] This disclosure also relates to variants of the aforementioned polynucleotides, wherein the variants encode fragments, analogs, and / or derivatives of, for example, the polypeptides or combinations of polypeptides described in this disclosure. In some embodiments, this disclosure provides a polynucleotide comprising a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (in some embodiments, at least about 96%, 97%, 98%, or 99% identity) with a polynucleotide sequence encoding a polypeptide or combination of polypeptides described in this disclosure. The term “polynucleotide having a nucleotide sequence having at least, for example, 95% identity with a reference nucleotide sequence” as used herein means that the nucleotide sequence of the polynucleotide is identical to the reference nucleotide sequence, but the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having at least 95% identity with a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. Variations in these reference sequences can occur at the 5' or 3' ends of the reference nucleotide sequence, or anywhere between these ends, either scattered individually among the nucleotides of the reference sequence or present in one or more consecutive groups within the reference sequence.

[0310] These polynucleotide variants may contain variations in coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain changes that produce silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide or combination of polypeptides. In some embodiments, the polynucleotide variants contain silent substitutions that do not cause changes in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be generated for a variety of reasons, such as optimizing codon expression for a specific host (i.e., changing codons in human mRNA to codons preferred by a bacterial host (such as E. coli)). In some embodiments, the sequence of the polynucleotide variant contains at least one silent mutation in a non-coding or coding region.

[0311] In some embodiments, polynucleotide variants are generated to modulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded polypeptide. In some embodiments, the expression of the encoded polypeptide by the polynucleotide variant is increased compared to the parental polynucleotide sequence. In some embodiments, the expression of the encoded polypeptide by the polynucleotide variant is decreased compared to the parental polynucleotide sequence.

[0312] 5.5 Carrier

[0313] This document also provides vectors containing the polynucleotide or nucleic acid molecules described herein. In one embodiment, the nucleic acid molecule may be integrated into a recombinant expression vector.

[0314] This disclosure provides vectors for cloning and expressing any of the polypeptides described herein. In some embodiments, the vectors are suitable for replication and integration in eukaryotic cells (e.g., mammalian cells). In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, vaccinia virus vectors, herpes simplex virus vectors, and derivatives thereof. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0315] Various virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. These heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using methods known in the art. The recombinant virus can then be isolated and delivered in vitro or ex vivo to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying sequences encoding immunomodulators (such as immune checkpoint inhibitors) and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged according to methods known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (such as primary human T cells) using methods known in the art. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their spread in daughter cells. Lentiviral vectors also have low immunogenicity and are capable of transducing non-proliferating cells.

[0316] In some embodiments, the vector contains a nucleic acid sequence encoding any of the polypeptides described herein. The nucleic acid can be cloned into the vector using any molecular cloning method known in the art (e.g., using restriction endonuclease sites and one or more selective markers). In some embodiments, the nucleic acid is operatively linked to a promoter. Various promoters for gene expression in mammalian cells have been explored, and any promoter known in the art can be used in this disclosure. Promoters can be broadly classified into constitutive promoters or regulatory promoters, such as inducible promoters.

[0317] In some embodiments, the nucleic acid sequence encoding the polypeptide is operatively linked to a constitutive promoter. A constitutive promoter enables the sustained expression of a heterologous gene (also known as a transgene) in a host cell. Exemplary constitutive promoters contemplated in this disclosure include, but are not limited to, the mouse stem cell virus (MSCV) promoter, the cytomegalovirus (CMV) promoter, the human elongation factor-1α (hEF1α), the ubiquitin C promoter (UbiC), the glycerol phosphokinase promoter (PGK), the simian virus 40 early promoter (SV40), and the chicken β-actin promoter (CAGG) coupled to the CMV early enhancer. The efficiency of these constitutive promoters in driving transgene expression has been extensively compared in numerous studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF1α promoter not only induced the highest transgene expression levels but also achieved optimal maintenance in CD4 and CD8 human T cells (Molecular Therapy, 17(8):1453-1464(2009)). In some embodiments, the nucleic acid encoding the CAR is operatively linked to the hEF1α promoter. In some embodiments, the nucleic acid encoding the CAR is operatively linked to the MSCV promoter.

[0318] In some implementations, the nucleic acid encoding the polypeptide is operatively linked to an inducible promoter. Inducible promoters fall under the category of regulatory promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of engineered immune effector cells, the physiological state of engineered immune effector cells, an inducing agent (i.e., an inducing reagent), or a combination thereof.

[0319] In some embodiments, the induction conditions do not induce the expression of endogenous genes in engineered mammalian cells or in a subject receiving the pharmaceutical composition. In some embodiments, the induction conditions are selected from: inducers, radiation (such as ionizing radiation, light), temperature (such as heat), redox state, tumor environment, and the activation state of engineered mammalian cells.

[0320] In some implementations, the vector also contains a selective marker gene or a reporter gene to screen for cells expressing the polypeptide from a host cell population transfected with the lentiviral vector. Both the selective marker and the reporter gene can have appropriate regulatory sequences flanking them, enabling expression in host cells. For example, the vector may contain transcription and translation terminators, a start sequence, and a promoter for regulating nucleic acid sequence expression.

[0321] In some specific embodiments, the vectors provided herein contain the MND promoter (a promoter with the negative control region deleted and the dl587rev primer binding site replaced). In some specific embodiments, the vectors provided herein contain the MSCV promoter. In other specific embodiments, the vectors provided herein contain EF1α.

[0322] 5.6 Preparation method

[0323] In another aspect, this disclosure provides a method for preparing engineered eukaryotic cells (e.g., immune effector cells, such as CAR-T cells, TCR-T cells, or TAC-T cells), the method comprising introducing one or more polynucleotides or vectors provided herein (e.g., as described in Sections 5.4 and 5.5 above) into eukaryotic cells (e.g., immune effector cells, such as T cells).

[0324] More specifically, in some embodiments, the eukaryotic cells described herein can be produced by introducing one or more nucleic acids encoding the polypeptide described in Section 5.3 or one or more nucleic acids described in Section 5.4 into T cells.

[0325] The functional exogenous receptors (e.g., CARs) and IMPDHs provided herein can be introduced into eukaryotic cells (e.g., T cells) as separate peptides. For example, the nucleic acids encoding the functional exogenous receptor and the nucleic acids encoding IMPDH provided herein can be introduced into eukaryotic cells (e.g., T cells) separately. Alternatively, the nucleic acids encoding the functional exogenous receptor and the nucleic acids encoding IMPDH provided herein can be introduced together as a single peptide into eukaryotic cells (e.g., T cells) via a single nucleic acid fragment that is cleaved during intracellular translation. The self-cleaving peptide linker has been described in more detail above. In some embodiments, the 2A self-cleaving peptide is selected from F2A, E2A, P2A, T2A, or variants thereof. In a specific embodiment, the self-cleaving peptide is a T2A fragment containing the amino acid sequence of SEQ ID NO:11.

[0326] Furthermore, the eukaryotic cells (e.g., T cells) described herein can be generated from polynucleotides comprising multiple regions, such as regions encoding functional exogenous receptors and regions encoding the IMPDH described herein. Different regions can be controlled by the same promoter. For example, in some embodiments, the internal ribosome entry site (IRES) is used herein to express multiple genes via a single promoter. In other embodiments, different regions are controlled by independent promoters.

[0327] In another aspect, this article provides an engineered eukaryotic cell, such as an immune effector cell (e.g., CAR-T cell), prepared according to the methods provided herein.

[0328] 5.7 Pharmaceutical Compositions

[0329] In one aspect, this disclosure further provides pharmaceutical compositions comprising the engineered cells described herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the engineered T cells described herein and a pharmaceutically acceptable excipient.

[0330] In specific implementations, the term "excipient" may also refer to diluents, adjuvants (e.g., Freund's adjuvant (complete or incomplete), carriers, or mediators). Pharmaceutical excipients can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetically derived oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Aqueous solutions of saline and glucose and glycerol can also be used as liquid excipients. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Examples of suitable pharmaceutical excipients can be found in Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). These compositions will contain a preventative or therapeutically effective amount of the active ingredient described herein, for example in a purified form, along with appropriate excipients to provide a suitable form of administration to the patient. The formulation should be suitable for the route of administration.

[0331] In some implementations, the choice of excipients depends in part on the specific cells and / or administration method. Therefore, a variety of suitable formulations exist.

[0332] Generally, acceptable carriers, excipients, or stabilizers are non-toxic to subjects receiving the doses and concentrations used, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium sulfite); preservatives, isotonic agents, stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents (e.g., EDTA) and / or nonionic surfactants.

[0333] Buffers can be used to control the pH of a solution, keeping it within a range that optimizes therapeutic efficacy, especially where stability depends on pH. Suitable buffers applicable to this disclosure include organic and inorganic acids and their salts. Examples include citric acid, phosphoric acid, succinic acid, tartaric acid, fumaric acid, gluconic acid, oxalic acid, lactic acid, and acetic acid. Furthermore, buffers may also contain histidine and trimethylamine salts, such as tris(hydroxymethyl)aminomethane (Tris).

[0334] Preservatives can be added to inhibit microbial growth. Suitable preservatives applicable to this disclosure include octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzyl ammonium halides (e.g., chlorides, bromides, iodides), benzyl ammonium chloride; thimerosal, phenol, butanol, or benzyl alcohol; alkyl p-hydroxybenzoates (e.g., methyl or propyl p-hydroxybenzoates); catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0335] Tensile agents, sometimes called "stabilizers," may be present to adjust or maintain the surface tension of liquids in a composition. They are often referred to as "stabilizers" when used with large, charged molecules such as proteins and antibodies because they are able to interact with the charged groups on the side chains of amino acids, thereby reducing the likelihood of intermolecular and intramolecular interactions. Exemplary tensile agents include polyhydroxy sugar alcohols, trihydroxy or higher molecular weight sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol.

[0336] Other exemplary excipients include: (1) thickeners, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. These excipients include: the polyhydroxy sugar alcohols listed above; amino acids (such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.); organic sugars or sugar alcohols (such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbitol, xylose, ribose, ribitol, inositol isobutyrate, inositol (myonisitose, myonisitol), galactose, galactitol, glycerol). Cyclohydric alcohols (e.g., inositol, polyethylene glycol); sulfur-containing reducing agents (e.g., urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate); low molecular weight proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose); and polysaccharides (e.g., dextrin or dextran).

[0337] Nonionic surfactants or detergents (also known as "wetting agents") may be present to help dissolve the therapeutic agent and protect the therapeutic protein from aggregation caused by agitation. This allows the formulation to be exposed to surface shear stress without causing denaturation of the active therapeutic protein or antibody. Suitable nonionic surfactants include, for example, polysorbates (20, 40, 60, 65, 80, etc.), poloxamer (184, 188, etc.). Polyol ( polyol), Polyoxyethylene sorbitan monooleate ( (etc.), polidocanol 400, polyoxyethylene stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Available anionic detergents include sodium dodecyl sulfate, sodium dioctylsulfonate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzyl chloride.

[0338] The route of administration follows known and acceptable methods, such as single or multiple administration or prolonged infusion, in an appropriate manner, such as injection or infusion via subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, or intra-articular routes, local administration, inhalation, or sustained-release or delayed-release methods.

[0339] In another embodiment, the pharmaceutical composition may be provided in the form of a controlled-release or sustained-release system. In one embodiment, a pump may be used to achieve the controlled-release or sustained-release effect (see, for example, Sefton, Crit. Ref. Biomed. Eng. 14:201-40 (1987); Buchwald et al., Surgery 88:507-16 (1980); Saudek et al., N. Engl. J. Med. 321:569-74 (1989)). In another embodiment, polymeric materials can be used to achieve the controlled or sustained release effect of a preventive or therapeutic agent (e.g., the fusion protein described herein) or a composition provided herein (see, for example, Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Balleds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126 (1983); Levy et al., Science 228:190-92 (1985); During et al., Ann. Neurol. 25:351-56 (1989); Howard et al. al., J. Neurosurg. 71:105-12 (1989); US Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326; PCT Publications WO 99 / 15154, WO 99 / 20253. Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), polyethylene-vinyl acetate copolymer, poly(methacrylic acid), polyglycolic acid (PLG), polyanhydride, poly(N-vinylpyrrolidone), polyvinyl alcohol, polyacrylamide, polyethylene glycol, polylactic acid (PLA), poly(lactic-co-glycolic acid) copolymer (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leaching impurities, storage-stable, sterile, and biodegradable.In another embodiment, the controlled-release or sustained-release system can be placed near a specific target tissue, such as the nasal cavity or lungs, thereby requiring only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled-release systems are also described, for example, Langer, Science 249:1527-33 (1990). Any technique known to those skilled in the art can be used to prepare sustained-release formulations comprising one or more agents described herein (see, for example, U.S. Patent No. 4,526,938; PCT Publications WO91 / 05548, WO 96 / 20698; Ning et al., Radiotherapy & Oncology 39:179-89 (1996); Songet et al., PDA J. of Pharma. Sci. & Tech. 50:372-97 (1995); Cleek et al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-54 (1997); Lam et al., Proc. Int'l. Symp. Control. Rel. Bioact. Mater. 24:759-60 (1997)).

[0340] The pharmaceutical compositions described herein may also contain one or more active compounds or agents to meet the needs of the specific condition being treated. Alternatively, or additionally, the compositions may contain cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, or growth inhibitors. These molecules are appropriately combined in amounts effective for the intended purpose.

[0341] The active ingredient can also be encapsulated in microcapsules, colloidal drug delivery systems, or crude emulsions. Microcapsules are prepared, for example, using coagulation or interfacial polymerization techniques, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. Colloidal drug delivery systems include, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. These technologies are described in Remington's Pharmaceutical Sciences (18th edition).

[0342] Various compositions and delivery systems are known and can be used in the therapeutic agents provided herein, including but not limited to liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing the single-domain antibodies or therapeutic molecules described herein, and the construction of nucleic acids as part of retroviruses or other vectors.

[0343] In some embodiments, the pharmaceutical compositions provided herein comprise binding molecules and / or cells in an amount effective in treating or preventing a disease or condition (e.g., a therapeutically effective amount or a preventatively effective amount). In some embodiments, the therapeutic or preventative effect is monitored by periodically evaluating the treated subject. For repeated dosing over several days or longer, treatment will be repeated as needed until the desired suppression of disease symptoms is achieved. However, other dosing regimens may be useful and may be determined as appropriate.

[0344] 5.8 Methods and Applications

[0345] On the other hand, this document provides methods for using the engineered eukaryotic cells described herein and their uses. The methods and uses provided herein include therapeutic methods and uses, such as those involving administering these cells or compositions containing the cells to a subject suffering from a disease or condition. In some embodiments, an effective amount of cells is administered to treat the disease or condition. Uses include the use of cells in these methods and treatments, as well as in the preparation of medicaments for performing these therapeutic methods. In some embodiments, these methods are performed by administering these cells or compositions containing them to a subject suffering from or suspected of suffering from a disease or condition. In some embodiments, these methods thereby treat the subject's disease or condition.

[0346] The eukaryotic cells according to this disclosure also have a further advantage, enabling a convenient and efficient method for the automated selection of engineered cells, for example, for therapeutic purposes. More specifically, eukaryotic cells transduced with nucleic acids encoding exogenous IMPDH resistant to purine biosynthesis inhibitors can be cultured for a period of time in a medium containing purine biosynthesis inhibitors. Eukaryotic cells successfully transduced and expressing this IMPDH can survive the culture process and can be automatically sorted out and then prepared for infusion to patients. This will greatly simplify the sorting process required before infusion to patients.

[0347] Therefore, in another aspect, this document provides a method for preparing cell therapies for treating diseases or conditions, comprising preparing eukaryotic cells expressing exogenously introduced IMPDH and optionally a functional exogenous receptor, for example, prepared according to the method described in Section 5.6 above; culturing the eukaryotic cells in vitro for a period of time in a medium containing a purine biosynthesis inhibitor; and collecting the cells after in vitro culture. These selected eukaryotic cells can then be administered, together with the purine biosynthesis inhibitor, to patients suffering from the disease or condition. In some embodiments, the method further includes administering an agent that inhibits the function of antigen-presenting cells (APCs), such as abatacept, tofacitinib, secukinumab, ipilimumab, cyclosporine A, fingolimod, methotrexate, or equivalents thereof.

[0348] In addition, this document provides methods for reducing and / or preventing host-graft reactions associated with cell therapy. In some embodiments, this document provides methods for reducing and / or preventing host-graft reactions, which include preparing eukaryotic cells expressing exogenously introduced IMPDH resistant to purine biosynthesis inhibitors.

[0349] In some implementations, the treatments provided herein may result in complete or partial improvement or reduction of the disease or condition, symptoms, adverse reactions, or outcomes. The intended effects of the treatment include, but are not limited to: prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or relief of the disease state, and relief or improvement of prognosis. These terms include, but are not limited to, complete cure of the disease or complete elimination of all symptoms or effects on all symptoms or outcomes.

[0350] As used herein, in some embodiments, the treatments provided herein can delay the development of a disease or condition, such as postponing, hindering, slowing, inhibiting, stabilizing, suppressing, and / or delaying the development of a disease (e.g., cancer). The duration of this delay will vary depending on the medical history of the disease and / or the individual receiving treatment. It will be apparent to those skilled in the art that a sufficient or significant delay can effectively encompass prevention, i.e., the individual will not develop the disease or condition. For example, advanced cancer (e.g., metastasis) can be delayed. In other embodiments, the methods or uses provided herein are capable of preventing a disease or condition.

[0351] In some embodiments, the cell therapies described herein are used to treat solid tumor cancers. In other embodiments, the cell therapies described herein are used to treat hematologic malignancies. In other embodiments, the disease or condition is an autoimmune or inflammatory disease. In other embodiments, the disease or condition is an infectious disease (e.g., a pathogen infection).

[0352] In some implementations, the disease or condition is a disease of abnormal cell growth and / or dysregulation of apoptosis. Examples of such diseases include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, ovarian cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, bone cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal (stomach, colon and / or duodenum) cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, testicular cancer, hepatocellular carcinoma (liver and / or bile duct) cancer, primary or secondary central nervous system tumors, primary... Secondary or secondary brain tumors, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, T-cell or B-cell lymphoid malignancies, melanoma, multiple myeloma, oral cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, kidney and / or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brainstem glioma, pituitary adenoma, adrenocortical carcinoma, gallbladder cancer, spleen cancer, bile duct cancer, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of the above diseases.

[0353] In some implementation schemes, the disease or condition is selected from: bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, T-cell or B-cell-derived lymphoid malignancies, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, and spleen cancer.

[0354] In some implementations, the disease or condition is a blood cancer, such as leukemia, lymphoma, or myeloma. In some implementations, the cancer is selected from: Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), cutaneous B-cell lymphoma, activated B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular central lymphoma, transformed lymphoma, moderately differentiated lymphocytic lymphoma, moderately differentiated lymphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centricell lymphoma, diffuse small cleavage cell lymphoma (DSCCL), peripheral T-cell lymphoma (PTCL), cutaneous T-cell lymphoma, and marginal zone lymphoma. Low-grade follicular lymphoma, multiple myeloma (MM), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), myelodysplastic syndrome (MDS), acute T-cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia, acute myeloid leukemia, acute megakaryocytic leukemia, precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt leukemia (Burkit lymphoma), acute biphenotypic leukemia, chronic myeloid lymphoma, chronic myeloid leukemia (CML), and chronic monocytic leukemia. In one specific implementation, the disease or condition is myelodysplastic syndrome (MDS). In another specific implementation, the disease or condition is acute myeloid leukemia (AML). In another specific implementation, the disease or condition is chronic lymphocytic leukemia (CLL). In another specific implementation, the disease or condition is multiple myeloma (MM).

[0355] In other implementations, the disease or condition is a solid tumor cancer. In some implementations, the solid tumor cancer is selected from: malignant tumors, adenocarcinoma, adrenocortical carcinoma, colonic adenocarcinoma, colorectal adenocarcinoma, colorectal cancer, ductal cell carcinoma, lung cancer, thyroid cancer, nasopharyngeal carcinoma, melanoma, non-melanoma skin cancer, liver cancer, and lung cancer.

[0356] In some implementation schemes, cancers include adrenal cancer, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gestational trophoblastic disease, head and neck cancer, Hodgkin's lymphoma, intestinal cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma (MM), neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spinal cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer.

[0357] In some embodiments, adrenal carcinoma is adrenocortical carcinoma (ACC), adrenal cortex cancer, pheochromocytoma, or neuroblastoma. In some embodiments, anal cancer is squamous cell carcinoma, anal-origin carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In some embodiments, appendiceal cancer is neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal-type adenocarcinoma, or signet ring cell adenocarcinoma. In some embodiments, bile duct cancer is extrahepatic bile duct cancer, adenocarcinoma, hilar bile duct cancer, perihepatic bile duct cancer, distal bile duct cancer, or intrahepatic bile duct cancer. In some embodiments, bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, or sarcoma. In some implementations, bone cancer is primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, soft tissue osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In some implementations, brain cancer is astrocytoma, brainstem glioma, glioblastoma, meningioma, ependymoma, oligodendroglioma, mixed glioma, pituitary cancer, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, medulloblastoma, or primary central nervous system (CNS) lymphoma. In some implementations, breast cancer is adenocarcinoma of the breast, invasive breast cancer, non-invasive breast cancer, breast sarcoma, metaplastic carcinoma, adenoid cystic carcinoma, phyllodes tumor, angiosarcoma, HER2-positive breast cancer, triple-negative breast cancer, or inflammatory breast cancer. In some implementations, cervical cancer is squamous cell carcinoma or adenocarcinoma. In some embodiments, colon cancer is colonic adenocarcinoma, primary colonic lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, signet ring cell adenocarcinoma, gastrointestinal carcinoid tumor, or melanoma. In some embodiments, esophageal cancer is adenocarcinoma or squamous cell carcinoma. In some embodiments, gallbladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In some embodiments, gestational trophoblastic disease (GTD) is hydatidiform mole, gestational trophoblastic tumor (GTN), choriocarcinoma, placental site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In some embodiments, head and neck cancer is laryngeal cancer, nasopharyngeal cancer, laryngopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, salivary gland cancer, oral cavity cancer, oropharyngeal cancer, or tonsil cancer. In some implementations, Hodgkin lymphoma is defined as classical Hodgkin lymphoma, nodular sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-dominant Hodgkin lymphoma (NLPHL). In some implementations, intestinal cancer is defined as small intestine cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma.In some implementations, renal cell carcinoma is renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urothelial carcinoma, renal pelvis carcinoma, or renal sarcoma. In some implementations, leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), or myelodysplastic syndrome (MDS). In a specific implementation, leukemia is AML. In some implementations, hepatocellular carcinoma is hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastases. In some implementations, lung cancer is defined as small cell lung cancer, small cell carcinoma, mixed small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large cell undifferentiated carcinoma, pulmonary nodules, metastatic lung cancer, adenosquamous carcinoma, large cell neuroendocrine carcinoma, salivary gland type lung cancer, pulmonary carcinoid, mesothelioma, pulmonary sarcomatoid carcinoma, or malignant granular cell lung cancer. In some implementations, melanoma is defined as superficial diffuse melanoma, nodular melanoma, acral lentigines melanoma, malignant lentigines melanoma, amelanotic melanoma, fibroproliferative melanoma, ocular melanoma, or metastatic melanoma. In some implementations, mesothelioma is defined as pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some implementations, multiple myeloma is defined as active myeloma or smoldering myeloma. In some implementations, neuroendocrine tumors are defined as gastrointestinal neuroendocrine tumors, pancreatic neuroendocrine tumors, or pulmonary neuroendocrine tumors.In some implementations, non-Hodgkin's lymphoma includes anaplastic large cell lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, follicular lymphoma, cutaneous T-cell lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), precursor T-cell lymphoblastic leukemia / lymphoma, acute lymphoblastic leukemia (ALL), adult T-cell lymphoma / leukemia (ATLL), hairy cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, and primary central nervous system (CNS) lymphoma. Mantle cell lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, marginal zone B-cell lymphoma of lymph nodes, marginal zone B-cell lymphoma of spleen, lymphoplasmacytic lymphoma, B-cell non-Hodgkin lymphoma, T-cell non-Hodgkin lymphoma, natural killer cell lymphoma, cutaneous T-cell lymphoma, mycosis fungoides (Alibert-Bazin syndrome), Cezari syndrome, primary cutaneous anaplastic large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), systemic ALCL, enteropathy-type T-cell lymphoma (EATL), or hepatosplenic γ / δ T-cell lymphoma. In some implementation schemes, oral cancer includes squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granuloma, fibroma, granular cell tumor, keratoacanthoma, leiomyoma, osteochondroma, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminata, verrucous xanthoma, pyogenic granuloma, rhabdomyosarcoma, odontogenic tumor, leukoplakia, erythroplakia, squamous cell carcinoma of the lip, basal cell carcinoma of the lip, oral cancer, gingival cancer, or tongue cancer. In some implementations, ovarian cancer is defined as ovarian epithelial carcinoma, mucinous epithelial ovarian carcinoma, endometrioid epithelial ovarian carcinoma, clear cell epithelial ovarian carcinoma, undifferentiated epithelial ovarian carcinoma, low-grade malignant potential ovarian tumor, primary peritoneal carcinoma, fallopian tube cancer, germ cell tumor, teratoma, dysgerminoma, ovarian germ cell carcinoma, endodermal sinus tumor, sex cord-stromal tumor, sex cord-gonadal-stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa-theca cell tumor, Sertoli-Leydig cell tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenocarcinoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Klukenberg's tumor, or ovarian cyst. In some implementations, pancreatic cancer is defined as pancreatic exocrine gland carcinoma, pancreatic endocrine gland carcinoma, pancreatic adenocarcinoma, islet cell tumor, or neuroendocrine tumor. In some implementations, prostate cancer is defined as prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor.In some implementations, sinus cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic cell carcinoma, acinar cell carcinoma, undifferentiated sinus carcinoma, nasal cavity carcinoma, paranasal sinus carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma, or nasopharyngeal carcinoma. In some implementations, skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel cell carcinoma, Kaposi's sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoacanthoma. In some implementations, soft tissue cancer is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid / round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In some implementations, spinal cancer is a metastatic tumor of the spine. In some implementations, gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, type I ECL cell carcinoid, type II ECL cell carcinoid, or type III ECL cell carcinoid. In some implementations, testicular cancer is seminoma, non-seminomatous tumor, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, gonadal stromal tumor, testicular stromal cell tumor, or Sertoli cell tumor. In some implementations, laryngeal cancer is squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal tumor, pharyngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, lymphoepithelioma, spindle cell carcinoma, verrucous carcinoma, undifferentiated carcinoma, or lymph node carcinoma. In some implementations, thyroid cancer is papillary carcinoma, follicular carcinoma, eosinophilic cell carcinoma, medullary thyroid carcinoma, or undifferentiated carcinoma. In some embodiments, uterine cancer is endometrial cancer, endometrial adenocarcinoma, endometrioid adenocarcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma, or undifferentiated sarcoma. In some embodiments, vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, vulvar cancer is squamous cell carcinoma or adenocarcinoma.

[0358] In some implementations, the disease or symptom is caused by a pathogen. In some implementations, the pathogen may cause an infectious disease selected from the following: acute flaccid myelitis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, Haemophilus influenzae type b (Hib or H-influenza), Hantavirus pulmonary syndrome (HPS), hemolytic uremic syndrome (HUS), hepatitis A (Hep A), hepatitis B (Hep B), hepatitis C (Hep C), hepatitis D (Hep D), hepatitis E (Hep E), herpes, and herpes zoster (Herpes zoster).Shingles, Histoplasma capsulatum infection, Human Immunodeficiency Virus / AIDS (HIV / AIDS), Human Papillomavirus (HPV), Influenza (Flu), Legionella infection, Shigella gastroenteritis, Smallpox, Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus food poisoning (Enterotoxin B poisoning), Vancomycin-resistant Staphylococcus aureus (VISA), Vancomycin-resistant Staphylococcus aureus (VRSA), Invasive Group A Streptococcal disease, Group B Streptococcal disease B) Streptococcal toxic shock syndrome (STSS), syphilis (primary, secondary, early latent period, late latent period, congenital), tetanus infection, trichomoniasis, trichinosis, pulmonary tuberculosis (TB), latent pulmonary tuberculosis (LTBI), tularemia, typhoid fever group D, vaginitis, varicella (Varicella, Chickenpox), campylobacteriosis, carbapenem-resistant infection, chancroid, chikungunya virus infection, chlamydia, ciguatera toxin poisoning, Clostridium difficile infection, Clostridium perfringens infection. Coccidioidomycosis (fungal infection), coronavirus infection, Covid-19 (SARS-CoV-2), Creutzfeldt-Jakob disease / infectious spongiform encephalopathy, cryptosporidiosis, cyclosporidiosis, dengue fever types 1, 2, 3 or 4, diphtheria, Escherichia coli infection / Shiga toxin-producing type (STEC), eastern equine encephalitis, hemorrhagic fever (Ebola), erythrozoonosis, encephalitis, vector-borne or parainfectious encephalopathy, non-polio enterovirus, D68 enterovirus (EV-D68), giardiasis, glanders, gonorrhea Bacterial infections, granulomatous venereum, cholerae vibrio (cholera), vibriosis (Vibrio), Ebola virus hemorrhagic fever, Lhasa virus hemorrhagic fever, Marburg virus hemorrhagic fever, West Nile virus, yellow fever, Yersinia infection, Zika virus infection, leprosy (Hansen's disease), leptospirosis, listeriosis (Listeria), Lyme disease, lymphogranuloma venereum (LGV), malaria, measles, melioidosis, viral meningitis, meningococcal disease (bacterial meningitis), Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Mumps, norovirus, head lice, pelvic inflammatory disease (PID), pertussis, plague (Black Death, septicemia, pneumonic plague), pneumococcal disease, poliomyelitis, Boui...

Claims

1. An engineered eukaryotic cell, said engineered eukaryotic cell comprising: (i) the prokaryotic IMPDH; and (ii) Functional exogenous receptors.

2. The engineered eukaryotic cell according to claim 1, wherein the prokaryotic IMPDH is resistant to purine biosynthesis inhibitors.

3. The engineered eukaryotic cell according to claim 1 or 2, wherein the inhibition constant (Ki) of the prokaryotic IMPDH binding to the purine biosynthesis inhibitor is greater than the inhibition constant (Ki) of the wild-type human IMPDH binding to the purine biosynthesis inhibitor; optionally, wherein the inhibition constant (Ki) of the prokaryotic IMPDH binding to the purine biosynthesis inhibitor is at least 20 times greater than the inhibition constant (Ki) of the wild-type human IMPDH binding to the purine biosynthesis inhibitor; more optionally, wherein the inhibition constant (Ki) of the prokaryotic IMPDH binding to the purine biosynthesis inhibitor is greater than 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM or greater.

4. The engineered eukaryotic cells according to claim 1 or 2, wherein the half-maximal effective concentration (EC50) or half-maximal inhibitory concentration (IC50) of the purine biosynthesis inhibitor inhibiting the prokaryotic IMPDH is greater than the EC50 or IC50 inhibiting wild-type human IMPDH; optionally, wherein the EC50 or IC50 value of the purine biosynthesis inhibitor inhibiting the prokaryotic IMPDH is two, three, four, or more times higher than the EC50 or IC50 value inhibiting wild-type human IMPDH; more preferably, wherein the EC50 or IC50 of the purine biosynthesis inhibitor on the prokaryotic IMPDH is greater than 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, or greater.

5. The engineered eukaryotic cells according to any one of claims 2 to 4, wherein the purine biosynthesis inhibitor comprises mycophenolic acid (MPA), ribavirin, mizoribine, tiazofurin, AVN-944 (VX-944), FF-10501, AS2643361 or BMS-986126, or their pharmaceutically acceptable esters, salts or prodrugs.

6. The engineered eukaryotic cell according to any one of claims 2 to 4, wherein the purine biosynthesis inhibitor is mycophenolic acid (MPA), its derivatives, analogs, or pharmaceutically acceptable salts, or compounds that can be converted into MPA intracellularly; optionally, the purine biosynthesis inhibitor is mycophenolate ester, mycophenolate mofetil (MMF), sodium mycophenolate (MPS), calcium mycophenolate, potassium mycophenolate, or derivatives thereof.

7. The engineered eukaryotic cell according to any one of claims 1 to 6, wherein the prokaryotic IMPDH is bacterial IMPDH or a variant thereof.

8. The engineered eukaryotic cell according to any one of claims 1 to 7, wherein the prokaryotic IMPDH is a Gram-positive bacterium IMPDH or a variant thereof, or a Gram-negative bacterium IMPDH or a variant thereof.

9. The engineered eukaryotic cell according to claim 8, wherein the Gram-positive bacterium IMPDH is derived from Bacillus subtilis, Lactobacillus plantarum, or Staphylococcus aureus; and / or, the Gram-negative bacterium IMPDH is derived from Escherichia coli or Flora intercalation.

10. The engineered eukaryotic cell according to any one of claims 1 to 9, wherein the prokaryotic IMPDH is Lactobacillus plantarum IMPDH, Escherichia coli IMPDH, Bacillus subtilis IMPDH, Staphylococcus aureus IMPDH, or Flora intercalation IMPDH, or variants thereof.

11. The engineered eukaryotic cell according to claim 9 or 10, wherein: (i) Lactobacillus plantarum IMPDH contains the amino acid sequence shown in SEQ ID NO:1; (ii) Escherichia coli IMPDH contains the amino acid sequence shown in SEQ ID NO:2; (iii) Bacillus subtilis IMPDH contains the amino acid sequence shown in SEQ ID NO:3; (iv) Staphylococcus aureus IMPDH contains the amino acid sequence shown in SEQ ID NO:4; and / or (v) The flower intermediate protoplasm IMPDH contains the amino acid sequence shown in SEQ ID NO:

5.

12. The engineered eukaryotic cell according to any one of claims 7 to 11, wherein the IMPDH variant comprises an amino acid sequence that differs from the parental IMPDH due to at least one amino acid alteration (e.g., substitution, addition, or deletion); optionally, the amino acid sequence of the IMPDH variant has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the parental IMPDH.

13. The engineered eukaryotic cell according to any one of claims 7 to 12, wherein the IMPDH variant is a functional variant that substantially retains the resistance of the parental IMPDH to purine biosynthesis inhibitors relative to the parental IMPDH.

14. The engineered eukaryotic cell according to any one of claims 7 to 13, wherein the IMPDH variant comprises a mutation at the following sites relative to the parental IMPDH: D13, D50, E54, D138, D200, A223, D243, D248, D338, E369, E373, E469 or a combination thereof; optionally, wherein the IMPDH variant comprises an amino acid sequence as shown in SEQ ID NO: 6 or 7.

15. The engineered eukaryotic cell according to any one of claims 1 to 14, wherein the eukaryotic cell is derived from a mammal; optionally, wherein the eukaryotic cell is derived from a human.

16. The engineered eukaryotic cell according to any one of claims 1 to 15, wherein the eukaryotic cell is a primary cell; optionally, wherein the eukaryotic cell is a human primary cell.

17. The engineered eukaryotic cell according to any one of claims 1 to 15, wherein the eukaryotic cell is a T cell, γδT cell, regulatory T cell, natural killer (NK) cell, NKT cell, B cell, macrophage, monocyte, peripheral blood mononuclear cell (PBMC), hematopoietic stem cell, pluripotent stem cell, embryonic stem cell or normal tissue cell.

18. The engineered eukaryotic cell according to any one of claims 1 to 15, wherein the eukaryotic cell is an immune cell; optionally, wherein the immune cell is a lymphocyte, a phagocyte, or a dendritic cell.

19. The engineered eukaryotic cell according to any one of claims 1 to 18, wherein the functional exogenous receptor comprises at least one of: (a) at least one extracellular antigen-binding domain; (b) at least one transmembrane domain; and (c) at least one intracellular signal transduction domain.

20. The engineered eukaryotic cell according to any one of claims 1 to 19, wherein the functional exogenous receptor comprises a T cell receptor (TCR), a chimeric antigen receptor (CAR), a chimeric TCR (cTCR), a T-cell antigen-coupled device (TAC) chimeric receptor, a chimeric switch receptor, a signal transduction receptor, an inducible regulation dimerization activation receptor (DARIC), a chimeric cytokine receptor, a co-stimulatory receptor, a dominant or negative receptor, or a component thereof.

21. The engineered eukaryotic cell according to claim 19 or 20, wherein the extracellular antigen-binding domain is an antibody, a single-domain antibody (sdAb), a single-chain variable fragment (scFv), or a ligand.

22. The engineered eukaryotic cell according to any one of claims 19 to 21, wherein the extracellular antigen-binding domain comprises a single-domain antibody (sdAb) domain that binds GUCY2C; optionally, the sdAb domain comprises CDR1, CDR2, and CDR3, which respectively comprise, for example, SEQ ID NO:26, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, or SEQ ID NO:

26. The amino acid sequences of CDR1, CDR2, and CDR3 shown in the sdAb domain of any of NO:32; optionally, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof.

23. The engineered eukaryotic cell of claim 19, wherein the transmembrane domain is derived from a molecule selected from CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1; optionally, the transmembrane domain is derived from CD8α; more optionally, the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO:

57.

24. The engineered eukaryotic cell of claim 19, wherein the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune effector cell; optionally, the primary intracellular signal transduction domain is derived from CD3ζ; more optionally, the primary intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO:

60.

25. The engineered eukaryotic cell according to claim 19 or 24, wherein the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain; optionally, wherein the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof; Optionally, the co-stimulatory signal transduction domain is derived from CD28; more optionally, the co-stimulatory signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO:59; or Optionally, the costimulatory signal transduction domain is derived from CD137(4-1BB); more preferably, the costimulatory signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO:

58.

26. The engineered eukaryotic cell according to any one of claims 19 to 25, wherein the functional exogenous receptor further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain; optionally, wherein the hinge domain is derived from CD8α; more optionally, the hinge domain comprises an amino acid sequence as shown in SEQ ID NO:

56.

27. The engineered eukaryotic cell according to any one of claims 19 to 26, wherein the functional exogenous receptor further comprises a signal peptide located at the N-terminus of the functional exogenous receptor; optionally, wherein the signal peptide is derived from CD8α; more optionally, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO:

55.

28. An engineered eukaryotic cell comprising the prokaryotic organism IMPDH.

29. The engineered eukaryotic cell according to claim 28, wherein the prokaryotic IMPDH is the prokaryotic IMPDH as defined in any one of claims 2 to 14.

30. The engineered eukaryotic cell according to claim 28 or 29, wherein the eukaryotic cell is a eukaryotic cell as defined in any one of claims 15 to 18.

31. A variant of a prokaryotic IMPDH, wherein the variant of the IMPDH is an IMPDH variant as defined in any one of claims 12 to 14.

32. Use of prokaryotic IMPDH or variants thereof for the construction of engineered eukaryotic cells resistant to inhibitors of purine biosynthesis.

33. The use according to claim 32, wherein the prokaryotic IMPDH is a prokaryotic IMPDH as defined in any one of claims 2 to 14; optionally, wherein the variant of the IMPDH is an IMPDH variant as defined in any one of claims 12 to 14.

34. The use according to claim 32 or 33, wherein the eukaryotic cell is a eukaryotic cell as defined in any one of claims 15 to 18.

35. A polypeptide or combination of polypeptides comprising a prokaryotic IMPDH and a functional exogenous receptor.

36. The polypeptide or combination of polypeptides according to claim 35, wherein the prokaryotic IMPDH and the functional exogenous receptor are not connected.

37. The polypeptide or combination of polypeptides according to claim 35, wherein the prokaryotic IMPDH and the functional exogenous receptor are linked; optionally, in the polypeptide or combination of polypeptides, the amino acid sequence of the prokaryotic IMPDH is located at the N-terminus or C-terminus of the amino acid sequence of the functional exogenous receptor.

38. The polypeptide or combination of polypeptides according to claim 37, wherein the prokaryotic IMPDH and the functional exogenous receptor are linked by a cleavable peptide linker; optionally, wherein the cleavable peptide linker is a 2A self-cleaving peptide, optionally selected from F2A, E2A, P2A, T2A and variants thereof. More optionally, the polypeptide or polypeptide combination comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of any of SEQ ID NO:61-70.

39. The polypeptide or combination of polypeptides according to any one of claims 35 to 38, wherein the prokaryotic IMPDH is the prokaryotic IMPDH as defined in any one of claims 2 to 14.

40. The polypeptide or combination of polypeptides according to any one of claims 35 to 39, wherein the functional exogenous receptor is a functional exogenous receptor as defined in any one of claims 19 to 27.

41. An isolated nucleic acid or group of nucleic acids comprising a nucleotide sequence encoding a variant of the prokaryotic IMPDH of claim 31; or comprising a nucleotide sequence encoding a polypeptide or combination of polypeptides of any one of claims 35 to 40.

42. The nucleic acid or group of nucleic acids according to claim 41, comprising a first region encoding the prokaryotic IMPDH; and a second region encoding the functional exogenous receptor; and / or comprising a first nucleic acid encoding the prokaryotic IMPDH and a second nucleic acid encoding the functional exogenous receptor.

43. A vector or group of vectors comprising the nucleic acid or group of nucleic acids as described in claim 41 or 42.

44. A method for preparing engineered eukaryotic cells, comprising introducing a nucleic acid or nucleic acid group as described in claim 41 or 42, or a vector or vector group as described in claim 43, into eukaryotic cells; optionally, wherein the eukaryotic cells are eukaryotic cells as defined in any one of claims 15 to 18.

45. An engineered eukaryotic cell comprising the nucleic acid or nucleic acid group as described in claim 41 or 42, or the vector or vector group as described in claim 43; or prepared by the method of claim 44.

46. ​​A pharmaceutical composition comprising: engineered eukaryotic cells according to any one of claims 1 to 30 and 45; a variant of the prokaryotic IMPDH according to claim 31; a polypeptide or combination of polypeptides according to any one of claims 35 to 40; a nucleic acid or group of nucleic acids according to claim 41 or 42; or a carrier or group of carriers according to claim 43; optionally, further comprising a pharmaceutically acceptable carrier or excipient; or optionally, further comprising a purine biosynthesis inhibitor.

47. The engineered eukaryotic cell according to any one of claims 1 to 30 and 45; a variant of the prokaryotic IMPDH according to claim 31; The polypeptide or combination of polypeptides according to any one of claims 35 to 40; the nucleic acid or group of nucleic acids according to claim 41 or 42; the vector or group of vectors according to claim 43; Or the use of the pharmaceutical composition of claim 46 in the preparation of cells or pharmaceuticals for treating diseases or conditions or reducing and / or preventing host resistance to grafts.

48. The use according to claim 47, wherein the disease or condition is an autoimmune disease, a pathogen infection, or cancer; optionally, the cancer includes solid tumors or hematologic malignancies.

49. A single-domain antibody (sdAb) against GUCY2C, said sdAb comprising CDR1, CDR2, and CDR3, which respectively comprise, as shown in SEQ ID NO:26, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, or SEQ ID NO:

26. The amino acid sequences of CDR1, CDR2, and CDR3 shown in the sdAb of any of NO:32; optionally, CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or a combination thereof.

50. The anti-GUCY2C sdAb according to claim 49, wherein the sdAb is a camel sdAb or a humanized sdAb.

51. The anti-GUCY2C sdAb according to claim 49 or 50, wherein the sdAb is a VHH antibody.

52. The anti-GUCY2C sdAb according to any one of claims 49 to 51, wherein the sdAb comprises or is composed of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:26, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, or SEQ ID NO:

32.

53. The anti-GUCY2C sdAb according to any one of claims 49 to 52, wherein the sdAb is genetically fused with or chemically conjugated with an additional agent.

54. A chimeric antigen receptor (CAR) comprising: (a) an extracellular antigen-binding domain comprising the anti-GUCY2C sdAb of any one of claims 49 to 52; (b) a transmembrane domain; and (c) an intracellular signal transduction domain.

55. The CAR of claim 54, wherein the transmembrane domain is derived from a molecule selected from CD8α, CD4, CD28, CD137, CD80, CD86, CD152 and PD1; optionally, the transmembrane domain is derived from CD8α; more optionally, the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO:

57.

56. The CAR according to claim 54 or 55, wherein the intracellular signal transduction domain comprises a primary intracellular signal transduction domain of an immune effector cell; optionally, wherein the primary intracellular signal transduction domain is derived from CD3ζ; more optionally, the primary intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO:

60.

57. The CAR according to any one of claims 54 to 56, wherein the intracellular signal transduction domain further comprises a co-stimulatory signal transduction domain; optionally, wherein the co-stimulatory signal transduction domain is derived from a co-stimulatory molecule selected from ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof; Optionally, the co-stimulatory signal transduction domain is derived from CD28; more optionally, the co-stimulatory signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO:59; or Optionally, the costimulatory signal transduction domain is derived from CD137(4-1BB); more preferably, the costimulatory signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO:

58.

58. The CAR according to any one of claims 54 to 57, wherein the CAR further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain; optionally, wherein the hinge domain is derived from CD8α; more optionally, the hinge domain comprises an amino acid sequence as shown in SEQ ID NO:

56.

59. The CAR according to any one of claims 54 to 58, wherein the CAR further comprises a signal peptide located at the N-terminus of the CAR; optionally, wherein the signal peptide is derived from CD8α; more optionally, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO:

55.

60. The CAR according to any one of claims 54 to 59, wherein the CAR comprises or is composed of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in any one of SEQ ID NO:54, SEQ ID NO:47, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, or SEQ ID NO:

53.

61. An isolated nucleic acid comprising a nucleotide sequence encoding a single-domain antibody (sdAb) against GUCY2C as described in any one of claims 49 to 52; or comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) as defined in any one of claims 54 to 60.

62. A vector comprising the nucleic acid of claim 61.

63. An engineered eukaryotic cell comprising the single-domain antibody (sdAb) against GUCY2C as described in any one of claims 49 to 52; a chimeric antigen receptor (CAR) as defined in any one of claims 54 to 60; the nucleic acid as described in claim 61; or the vector as described in claim 62.

64. A pharmaceutical composition comprising a single-domain antibody (sdAb) against GUCY2C as described in any one of claims 49 to 52; a chimeric antigen receptor (CAR) as defined in any one of claims 54 to 60; a nucleic acid as described in claim 61; a vector as described in claim 62; or engineered eukaryotic cells as described in claim 63; optionally, further comprising a pharmaceutically acceptable vector or excipient.

65. The use of the single-domain antibody (sdAb) against GUCY2C as described in any one of claims 49 to 52; the chimeric antigen receptor (CAR) as defined in any one of claims 54 to 60; the nucleic acid as described in claim 61; the vector as described in claim 62; the engineered eukaryotic cell as described in claim 63; or the pharmaceutical composition as described in claim 64, for the preparation of a medicament for treating a disease or condition of a subject.

66. The use according to claim 65, wherein the disease or condition is an autoimmune disease, a pathogen infection, or cancer; optionally, the cancer includes solid tumors or hematologic malignancies.

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