Viral vectors for glioblastoma cell therapy

Abstract

The present disclosure provides, inter alia, a recombinant nucleic acid comprising, in order from 5'to 3 ': (a) a first promoter, (b) a first coding sequence wherein the first coding sequence encodes an anti-EphA2 / IL13R [alpha] 2 tandem CAR and is operably linked to the first promoter, (c) a second promoter; and (d) a second coding sequence encoding a binding trigger transcriptional switch (BTTS), which is activated by binding to EGFRvIII or a brain specific marker, and which is operably linked to the second promoter. In these embodiments, transfection of a viral vector into a cytotoxic immune cell results in expression of BTTS on the surface of the cytotoxic immune cell, and the expressed BTTS binds to an antigen on the surface of the target cell or a protein in the extracellular matrix, thereby activating the cytotoxic immune cell to express a CAR. Methods of treating glioblastoma are also provided.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 464,497, filed May 5, 2023, which is incorporated herein by reference.

[0003] [Government Rights]

[0004] This invention was completed with government support under license numbers U19 CA264338 and R35 NS105068 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention.

[0005] [Reference Electronic Sequence List]

[0006] The entire contents of the electronic sequence list (UCSF-736WO_Seq_List.xml; size: 10,343 bytes; creation date: May 1, 2024) are incorporated herein by reference. [Background Technology]

[0007] Despite the immense promise of CAR T-cell therapy, some major challenges facing this novel cancer treatment include accurately distinguishing between cancerous and healthy tissue and addressing the immunosuppressive tumor microenvironment (TME) to enhance T-cell function. Next-generation therapeutic cell design, such as multi-antigen “initiate and kill” therapeutic T-cell circuits, shows great promise in addressing these challenges, but all require the ability to deliver multiple and larger transgenes to support the engineering of these novel therapeutic cells. A significant subsequent challenge is translating these more complex research-grade multigene designs and successfully adapting them to clinical-grade vectors optimized to the stringent standards required for human use for efficient, large-scale production.

[0008] This disclosure is considered to have resolved the problem. [Summary of the Invention]

[0009] This disclosure specifically provides a recombinant nucleic acid design optimized for the preparation of therapeutic T cells with a multi-antigen “initiate and kill” therapeutic circuit for the treatment of glioblastoma. The recombinant nucleic acid encodes two main components: (1) an inducible transgene encoding a therapeutic payload (such as an EphA2 / IL13Rα2 tandem (i.e., dual-affinity) CAR), and (2) a constitutive transgene encoding a trigger transcription switch (BTTS) designed to sense glioblastoma-specific antigens (such as EGFRvIII) or brain-specific tissue markers (e.g., BCAN, etc.) and upregulate the expression of the inducible transgene upon activation. The recombinant nucleic acid can be configured to place the two transgenes in tandem and in the positive direction of the viral backbone, with the inducible transgene positioned upstream of the constitutive transgene.

[0010] In some embodiments, the recombinant nucleic acid may sequentially comprise from 5' to 3': (a) a first promoter, (b) a first coding sequence, wherein the first coding sequence encodes an anti-EphA2 / IL13Rα2 tandem CAR and is operatively linked to the first promoter, (c) a second promoter; and (d) a second coding sequence, wherein the second coding sequence encodes a binding trigger transcription switch (BTTS), which is activated by binding to EGFRvIII or a brain-specific marker, and is operatively linked to the second promoter. In these embodiments, transfection of the recombinant nucleic acid into cytotoxic immune cells results in the expression of BTTS on the surface of the cytotoxic immune cells, and the expressed BTTS binds to antigens on the surface of target cells or proteins in the extracellular matrix, thereby activating CAR expression in the cytotoxic immune cells.

[0011] In some implementations, the single-vector encodes all components of the "initiating and killing" BTTS multiantigen therapeutic circuit. This design reduces the preparation costs of both the virus and cells, improving feasibility for clinical application. Furthermore, the vector is compatible with current cGMP viral production methods, clinical preparation of therapeutic T cells, and use in human patients. The design encodes protein binders optimized for bioactivity and viral production, has no potentially immunogenic protein tags, and is compatible with advanced third-generation lentiviral systems to improve efficacy and biosafety. Additionally, the dual-transgenic configuration enhances the inducibility of the BTTS therapeutic circuit while minimizing basal, ligand-independent circuit activation, thereby improving safety by minimizing the likelihood of adverse reactions on the target tumor. Finally, compared to multi-vector designs, the single-vector design requires fewer viral integration events, improving patient safety by reducing the risk of insertional mutagenesis and genotoxicity.

[0012] It also provides vectors and cells containing recombinant nucleic acids, as well as methods for using cells to treat glioblastoma.

[0013] Examples of such recombinant nucleic acids, cells, and methods are described in further detail below. [Attached Image Description]

[0014] Figures 1A-1C Overview of E-SYNC therapeutic T cells and design of p885 E-SYNC clinical single lentiviral transfer vector from original dual-vector research-grade design. Figure 1A A schematic diagram of the "Initiate and Kill" E-SYNC T cell circuit. Figure 1B , 1C: A schematic diagram of the construct used.

[0015] Figures 2A-2CExamples of single lentiviral vector configurations for BTTS-driven CAR loops are shown. Several different lentiviral vector configurations are illustrated, including transgenic tandem (…). Figure 2A and 1B ) or opposite ( Figure 2C )place.

[0016] Figures 3A-3B E-SYNC T cells prepared using the p885 single vector showed EGFRvIII-driven CAR expression and killed GBM tumor cells in vitro in a manner similar to the control dual-vector cells. Figure 3A P885 T cells can activate CAR expression in response to EGFRvIII exposure, reaching levels similar to dual-carrier reference cells, while maintaining low basal CAR expression in the absence of stimulation. Figure 3B Measurements were performed using CD8 transducers from P168 / 322, P830, or P885. + The killing assay of T cells' ability to clear GBM cells showed that the single-vector design could be matched with the dual-vector reference.

[0017] Figure 4 E-SYNC T cells prepared using the p885 clinical vector effectively eliminated EGFRvIII in mice. + Xenografted brain tumors derived from GBM patients.

[0018] Figure 5A and 5B E-SYNC T cells transduced with p885 single vector can differentiate between brain tumors and ventral tumors in vivo based on tumor EGFRvIII expression. (Model example follows.) Figure 5A As shown. The results are as follows. Figure 5B As shown.

[0019] Figures 6A-6C An overview of the BCAN-initiated SynNotch-CAR circuit (B-SYNC), the composition of the pB-SYNC1 single lentiviral transfer vector, and the tumor cell killing performance of B-SYNC T cells. Figure 6A An overview of the design of the pB-SYNC1 vector for producing BCAN-initiated SynNotch-CAR (B-SYNC) T cells. Figure 6B A schematic diagram of B-SYNC therapeutic T cells. Figure 6C In this in vitro co-culture killing assay of GBM6 PDX, T cells transduced with pB-SYNC1 single vector showed better performance than dual-vector control cells.

[0020] Figure 7B-SYNC T cells prepared using the pB-SYNC1 clinical vector effectively eliminated xenografted tumors derived from GBM patients in mice and improved survival rates.

[0021] Figure 8A and 8B p885 ( Figure 8A ) and pB-SYNC1 ( Figure 8B The plasmid map of the vector.

[0022] Figures 9A-9C : A single-vector design variant with BTTS-controlled multi-protein payload expression. Figure 9A Serial design of single CAR payload. Figure 9B . Serial carrier design. Figure 9C Another alternative design with a bidirectional promoter.

[0023]

definition

[0024] As used herein, the terms “treatment,” “treating,” “treat,” etc., refer to achieving the desired pharmacological and / or physiological effects and / or treatment-related responses. Effects may be preventative, completely or partially preventing the disease or its symptoms, and / or therapeutic, partially or completely curing the disease and / or adverse reactions caused by the disease. As used herein, “treatment” encompasses any treatment of diseases in mammals (particularly humans), including: (a) preventing the development of the disease in subjects who are susceptible to it but have not yet been diagnosed with it; (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing its remission.

[0025] "Therapeutic effective amount" or "effective amount" refers to the amount of a drug (including biological agents, such as cells), or a combination of two drugs, that is sufficient to produce such a therapeutic effect when administered to a mammal or other subject for the treatment of a disease. Therapeutic effective amount will vary depending on the drug, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0026] In this document, the terms “individual,” “subject,” “host,” and “patient” used interchangeably refer to mammals, including but not limited to rodents (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., horses, cattle, sheep, pigs, goats), and lagomorphs. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, such as a rat or mouse. In some cases, the individual is a lagomorph, such as a rabbit.

[0027] As used herein, the term “binding-triggered transcription switch” or “BTTS” refers to any polypeptide or complex capable of transducing a specific binding event outside the cell (e.g., binding of the extracellular domain of a BTTS) to activation of a recombinant promoter within the cell nucleus. Many BTTSs function by releasing transcription factors that activate promoters. In these embodiments, a BTTS consists of one or more polypeptides that undergo proteolytic cleavage upon binding to an antigen to release gene expression regulators that activate recombinant promoters. For example, a BTTS may comprise (i) an extracellular domain containing an antigen-specific antibody-binding region; (ii) a proteolytically cleavable sequence containing one or more proteolytic cleavage sites; and (iii) an intracellular domain wherein binding of the antigen-binding region to the antigen induces cleavage at one or more proteolytic cleavage sites, thereby releasing the intracellular domain, and wherein the intracellular domain activates transcription of the expression cassette. For example, BTTS can be based on Notch, A2, MESA or a mechanoceptor, although other receptors are known (e.g., see Zhu et al., 2022 Cell. 185:1431-1443, US20200331985 and WO2021061856).

[0028] The terms “chimeric antigen receptor” and “CAR” used interchangeably in this document refer to artificially engineered multi-modal molecules capable of triggering or inhibiting the activation of immune cells, which typically (but not exclusively) contain extracellular domains (e.g., ligand / antigen-binding domains), transmembrane domains, and one or more intracellular signaling domains. The term CAR is not limited to CAR molecules in particular, but also includes CAR variants. CAR variants include split CARs, in which the extracellular portion (e.g., ligand-binding portion) and the intracellular portion (e.g., intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants also include ON-switch CARs, which are conditionally activatable CARs, for example, comprising split CARs, in which the conditional heterodimerization of the two parts of the split CAR is pharmacologically controlled (e.g., as described in PCT Publication No. WO 2014 / 127261 A1 and U.S. Patent Application No. 2015 / 0368342 A1, the entire disclosures of which are incorporated herein by reference). CAR variants also include bispecific CARs, which include a secondary CAR-binding domain that can amplify or inhibit primary CAR activity. CAR variants also include inhibitory chimeric antigen receptors (iCARs), for example, which can be used as components of bispecific CAR systems, wherein binding to the secondary CAR-binding domain leads to inhibition of primary CAR activation. For example, in PCT application number US2014 / 016527; Fedorov et al., Sci Transl Med (2013); 5(215): 215ra172; Glienke et al., Front Pharmacol (2015) 6: 21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al., Cancer J (2014) 20(2): 141-4; Pegram et al., Cancer J (2014) 20(2): 127-33; Cheadle et al., Immunol Rev (2014) 257(1): 91-106; Barrett et al., Annu Rev Med (2014) 65: 333-47; Sadelain et al., Cancer Discov (2013) 3(4): 388-98; Cartellieri et al., J Biomed CAR molecules and their derivatives (i.e., CAR variants) are described in Biotechnol (2010) 956304; the entire contents of that publication are incorporated herein by reference.

[0029] As used herein, the terms “synthetic,” “chimeric,” and “engineered” generally refer to artificially derived peptides or peptides encoding non-naturally occurring nucleic acids. Synthetic peptides and / or nucleic acids can be assembled de novo from basic subunits (including, for example, single amino acids, single nucleotides, etc.) or can be derived from pre-existing peptides or polynucleotides, whether of natural or artificial origin, for example, through recombinant methods. Chimeric and engineered peptides or peptides encoding nucleic acids are generally constructed by combining, linking, or fusing two or more different peptides or peptide domains encoding nucleic acids. Chimeric and engineered peptides or peptides encoding nucleic acids include cases where the two or more linked peptides or nucleic acids are “partially” derived from different proteins (or nucleic acids encoding different proteins), and where the linked portions comprise different regions of the same protein (or nucleic acid encoding a protein), but these portions are linked in a non-natural manner.

[0030] As used herein, the term "recombinant" describes a nucleic acid molecule, such as a genome, cDNA, virus, or a semi-synthetic and / or synthetically derived polynucleotide, which, due to its origin or manipulation, is unrelated to all or part of its associated polynucleotide sequence in nature. When referring to proteins or peptides, the term recombinant means a peptide produced by the expression of a recombinant polynucleotide. When referring to host cells or viruses, the term recombinant means a host cell or virus in which a recombinant polynucleotide has been introduced. When referring to materials (e.g., cells, nucleic acids, proteins, or vectors), recombinant is also used herein to refer to the modification of materials by introducing a heterologous material (e.g., cells, nucleic acids, proteins, or vectors).

[0031] The term "operably linked" refers to juxtaposition, where the components described so far are in a relationship that allows them to function in their intended manner. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is operably linked to the coding sequence. Operatically linked nucleic acid sequences may not necessarily be adjacent. For example, in some cases, the coding sequence operably linked to the promoter may be adjacent to the promoter. In some cases, the coding sequence operably linked to the promoter may be separated by one or more insert sequences (including coding and non-coding sequences). Furthermore, in some cases, more than two sequences may be operably linked, including but not limited to, for example, two or more coding sequences operably linked to a single promoter.

[0032] The terms “polynucleotide” and “nucleic acid” used interchangeably in this document refer to polymers of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0033] The terms “polypeptide,” “peptide,” and “protein,” used interchangeably in this document, refer to a polymer of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone. This term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusion proteins with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; immunolabeled proteins, etc.

[0034] A "vector" or "expression vector" is a replicon, such as a plasmid, bacteriophage, virus, or kinetid, that can attach another DNA fragment, called an "insert," to enable the replication of the attached fragment within the cell.

[0035] As used herein, the term "heterologous" refers to a nucleotide or polypeptide sequence that is not found in natural (e.g., naturally occurring) nucleic acids or proteins. Heterologous nucleic acids or polypeptides may originate from a species different from the organism or cell in which they are present or expressed. Therefore, heterologous nucleic acids or polypeptides typically have a different evolutionary origin compared to the cell or organism in which they are present.

[0036] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, as these embodiments may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0037] When a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value (the tenths of the lower limit unit) between the upper and lower limits of the range, as well as any other stated or intermediate value within the range, is covered in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered in this invention, subject to any explicitly excluded limits within the range. When the range includes one or both limits, the range excluding one or both of these included limits is also included in this invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein are used in the practice or testing of this invention, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0039] It must be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes multiple such cells, reference to “the cell” includes one or more cells, and references to their equivalents known to those skilled in the art, etc. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this description is intended to provide a priori basis for the use of exclusive terms such as “solely,” “only,” or the use of negative restrictions in the description of claim elements.

[0040] It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to the invention are specifically included in the invention and disclosed herein, as if each combination were individually and explicitly disclosed herein. Furthermore, all sub-combinations of the various embodiments and their elements are specifically included in the invention and disclosed herein, as if each such sub-combination were individually and explicitly disclosed herein.

[0041] The publications discussed herein provide only their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to a prior invention prior to that disclosure date. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

Detailed Implementation Methods

[0042] As described above, this disclosure provides a recombinant nucleic acid comprising, from 5' to 3', sequentially: (a) a first promoter; (b) a first coding sequence, wherein the first coding sequence encodes an anti-EphA2 / IL13Rα2 tandem CAR and is operatively linked to the first promoter; (c) a second promoter; and (d) a second coding sequence, wherein the second coding sequence encodes a binding trigger transcription switch (BTTS), which is activated by binding to EGFRvIII or a brain-specific marker, and is operatively linked to the second promoter. Transfection of the recombinant nucleic acid (e.g., via a viral vector) into cytotoxic immune cells results in the expression of BTTS on the surface of the cytotoxic immune cells, and the expressed BTTS binds to antigens on the surface of target cells or proteins in the extracellular matrix, thereby activating CAR expression in the cytotoxic immune cells. In some embodiments, BTTS may be activated by binding to EGFRvIII (see, for example, Johnson et al., Sci. Transl. Med 2015 7:275ra22). An example of such a construct is as follows: Figure 1C As shown. In other implementations, BTTS may be activated by binding with MOG, CDH10, CSPG5, PTPRZ1, BCAN, Neurexin 1b, and NrCAM. For example, BTTS is activated by binding with BCAN (see, for example, WO2007056536A1). An example of such an architecture is shown below. Figure 7 As shown in Figure A.

[0043] The publications of WO2019195586, US20200331985, and Choe et al. (Sci. Transl. Med. 2021 13: eabe7378) are expressly incorporated herein by reference for all purposes.

[0044] Chimeric antigen receptor (CAR)

[0045] As described above, the CAR encoded by the first coding sequence can be a tandem CAR (also known as a "tan CAR" or "tanCAR"), which is a bispecific CAR that includes binding domains for EphA2 and IL13Rα2. These domains can be activated by binding to either hepatin A receptor 2 (EphA2) or interleukin-13 receptor α2 (IL13Rα2) and are expressed on the surface of many GBM cells (see, for example, Bielamowicz et al., Neuro Oncol 2018 20, 506-518; Hegde et al., Mol. Ther. 2013 21, 2087-2101; and Wykosky et al., Mol. Cancer Res 2005 3, 541-551). In other words, the extracellular domain of the CAR has a first binding domain that binds to EphA2 and a second binding domain that binds to IL13Rα2, wherein the CAR is activated by binding to either EphA2 or IL13Rα2. The CAR used in this article may have an extracellular region containing an α-EphA2 single-chain antibody and an IL13 mutant protein (a variant of the IL13 ligand that binds to IL13Rα2 with a higher affinity than IL13Rα1) (see, for example, Choe et al., Sci. Transl. Med. 202113: eabe7378, Chow et al., Mol. Ther. J. Am. Soc. Gene Ther 2013 21, 629-637, Krenciute et al., Mol. Ther. J. Am. Soc. Gene Ther 2016 24, 354-363 and Kahlon et al., Cancer Res 64, 9160-9166 2004). Non-limiting examples of tandem CARs include those described in U.S. Patent Nos. 9,447,194; 10,155,038; 10,189,903; and 10,239,948; U.S. Patent Application Publication No. 20130280220; and PCT Application Publication No. WO / 2013 / 123061; the entire disclosure of which is incorporated herein by reference.

[0046] CARs activate immune cells upon binding to their homologous antigens. CARs can be engineered in several ways (see, for example, Guedan et al., Methods and Clinical Development 2019 12:145-156) and may include extracellular domains containing antigen-binding domains (such as scFv, nanobodies, or ligands), hinges, transmembrane regions (possibly derived from CD4, CD8α, or CD28), co-stimulatory signaling domains (possibly derived from the CD28 family (e.g., CD28 and ICOS) or the tumor necrosis factor receptor (TNFR) gene family (e.g., 4-1BB, OX40, or CD27), and ITAM domains, such as signaling domains derived from the human CD3 complex ζ chain (CD3zeta). In practice, any of these domains may be a variant of the wild-type sequence. In practice, any of these sequences may be a variant of the wild-type sequence, for example, a sequence that has at least 90%, 95, or 98% identity with the sequence described in WO2014127261.

[0047] In any implementation, the CAR may contain at least one immune receptor tyrosine-based activation motif (ITAM). In these implementations, the ITAM (immunoreceptor tyrosine-based activation motif) can be described using the formula YX1X2L / I, where X1 and X2 are independent amino acids. In some cases, the intracellular signaling domain of the subject-engineered immune receptor contains 1, 2, 3, 4, or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular signaling domain, where the first and second ITAM motifs are separated by 6 to 8 amino acids, for example, (YX1X2L / I)(X3). n(YX1X2L / I), where n is an integer from 6 to 8, and each of the 6 to 8 X3 can be any amino acid. In some cases, the intracellular signaling domain of the subject-engineered immune receptor contains three ITAM motifs. Suitable ITAMs can be derived from peptides containing ITAM motifs. For example, a suitable intracellular signaling domain can be an ITAM-motif-containing domain from any protein containing an ITAM motif. Therefore, a suitable intracellular signaling domain does not need to contain the entire sequence of the protein from which it originates. Examples of suitable ITAM-motif-containing peptides include, but are not limited to: DAP12; FCER1G (Fcε receptor Iγ chain); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD3Z (CD3ζ); and CD79A (antigen receptor complex-associated protein α chain), although in practice functional variants of these domains (e.g., domains with at least 90% or 95% sequence identity with the wild-type domain) can be used in many cases.

[0048] In some embodiments, the CAR comprises EphA2 scFv (Goldgur et al., Growth Factors 2014 32, 214-222) and IL13 mutant protein [E13K,K105R] (Krebs et al., Cytotherapy 2014 16, 1121-1131), or IL13 mutant protein [E13K,K105R]-G4Sx4-EphA2 scFv (Goldgur et al., Growth Factors 2014 32, 214-222; Krebs et al., Cytotherapy 2014 16, 1121-1131), which are linked to the hinge region of the human CD8α chain and the transmembrane and cytoplasmic regions of human 4-1BB, as well as the CD3z signaling domain.

[0049] In any implementation, the IL13Rα2 binding domain may have the following sequence:

[0050] LTCLGGFASP GPVPPSTALR KLIEELVNIT QNQKAPLCNG SMVWSINLTA GMYCAALESLINVSGCSAIE KTQRMLSGFC PHKVSAGQFS SLHVRDTKIE VAQFVKDLLL HLRKLFREGR FN (SEQ IDNO: 1),

[0051] Or a sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0052] In any implementation, the EphA2 binding domain may have the following sequence:

[0053] QVQLLESGGG LVQPGGSLRL SCAASGFTFS SYTMSWVRQA PGQALEWMGT ISSGGTYTYYPDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREA IFTYWGRGTL VTSSGGGGSG GGGSGGGGSDIQLTQSPSSL SASVGDRVTI TCKASQDINN YLSWYQQKPG QAPRLLIYRA NRLVDGVPDR FSGSGYGTDFTLTINNIESE DAAYYFCLKY DVFPYTFGQG TKVEIKS (SEQ ID NO: 2)

[0054] Or a sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0055] In any implementation, the EGFRvIII binding domain may have the following sequence:

[0056] DIQMTQSPSS LSASVGDRVT ITCRASQGIR NNLAWYQQKP GKAPKRLIYA ASNLQSGVPSRFTGSGSGTE FTLIVSGSSLQP EDFATYYCLQ HHSYPLTSGG GTKVEIKGST SGSGKPGSGE GSEVQVLESGGGLVQPGGSL RLSCAASGFT FSSYAMSWVR QAPGKGLEWV SAISGSGGST NYADSVKGRF TISRDNSKNTLYLQMNSLRA EDTAVYYCAG SSGWSEYWGQ GTLVTVSS (SEQ ID NO: 3)

[0057] Or a sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0058] As mentioned above, CAR expression may be controlled by BTTS.

[0059] [Binding to Triggered Transcription Switch (BTTS)]

[0060] BTTS is a cleavable fusion protein comprising: (a) an extracellular binding domain containing a protein-binding domain (e.g., scFv or nanobody) that binds to EGFRvIII; (b) a transmembrane domain; and (c) an intracellular domain containing a transcription activator, wherein the binding domain binds to a marker on another cell surface or in the extracellular matrix, thereby inducing proteolytic cleavage of BTTS to release the transcription activator. In some embodiments, BTTS may additionally contain an extracellular force-sensing region between regions (a) and (b), and (d) one or more force-dependent cleavage sites in (c) that are cleaved when the force-sensing region is activated.

[0061] In this switch, when the extracellular domain of the fusion protein binds to a marker on another cell, the fusion protein is cleaved to release its intracellular domain. Therefore, in many cases, the fusion protein may contain a sensing region (typically in the extracellular domain) and one or more cleavage sites that are cleaved when the sensing region is activated. The location of the cleavage sites may vary, and in some embodiments, the fusion protein may contain at least two cleavage sites. In some cases, one cleavage site may be extracellular, and another may be within the transmembrane domain or within 10 amino acids of the transmembrane domain in the intracellular domain. In any embodiment, the sensing region and / or one or more cleavage sites may be derived from the δ / Serrate / Lag2 (DSL) superfamily of proteins, as reviewed by Pintar et al. (Biology Direct 2007 2: 1-13). For example, the sensing region and / or one or more cleavage sites may originate from Notch (see Morsut Cell. 2016164:780-91), von Willebrand factor (vWF), amyloid β, CD16, CD44, δ, cadherin, hepatocyte ligand or hepatocyte ligand, protocadherin, filamin, synthetic E cadherin, interleukin-1 receptor type 2 (IL1R2), major prion protein (PrP), neuregulin, or adhesion-GPCR. Several other examples of this protein type are known and are listed in the Pintar section above. Many members of this family appear to share similar structures: regions that unfold and open protease cleavage sites (e.g., EGF-like repeats; see Cordle et al., Nat. Struct. Mol. Biol. 2008 15: 849-857), transmembrane segments, and relatively short (approximately 100–150 amino acids) intracellular domains. These sequences allow transcriptional activators to be released from the membrane via binding triggers in their native environment and can be readily applied to this paper.

[0062] In some cases, one or more ligand-induced proteolytic cleavage sites are selected from S1, S2, and S3 proteolytic cleavage sites. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site containing the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. In some cases, the S2 proteolytic cleavage site is an ADAM-17 type protease cleavage site containing the Ala-Val dipeptide sequence. In some cases, the S3 proteolytic cleavage site is a γ-secretase cleavage site containing the Gly-Val dipeptide sequence. The S3 proteolytic cleavage site is located within a transmembrane domain. In some cases, shear forces generated by the binding of the extracellular domain of the fusion protein to another cell or extracellular matrix cause the force-sensitive region to unfold (in Notch's case, this region contains EGF-like repeats, while in other proteins it consists of other sequences, such as the A2 domain in vWF (see, for example, J Thromb Haemost. 2009 7:2096-105, Lippok Biophys J. 2016 110:545-54, Lynch Blood. 2014 123:2585-92, Crawley, Blood. 2011 118:3212-21 and Xy J Biol Chem. 2013 288:6317-24) or a modified A2 domain, for example, with R1597W, E1638K, and I1628T substitutions. The structures of such proteins are described in particular, for example, Morsut et al., Cell. 2016. 164:780-91, WO2016138034 and WO2019099689.

[0063] In some cases, the fusion protein includes an S1 ligand-induced proteolytic cleavage site. This site can be located between the HD-N and HD-C fragments. In other cases, the S1 ligand-induced proteolytic cleavage site is a furin-like protease cleavage site. A furin-like protease cleavage site can have the typical sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid; the protease cleaves immediately at the C-terminus of the classic sequence. For example, in some cases, the amino acid sequence containing the S1 ligand-induced proteolytic cleavage site can have the amino acid sequence GRRRRELDPM (SEQ ID NO: 4), where cleavage occurs between the “RE” sequences. As another example, the amino acid sequence containing the S1 ligand-induced proteolytic cleavage site can have the amino acid sequence RQRRELDPM (SEQ ID NO: 5), where cleavage occurs between the “RE” sequences.

[0064] In some cases, the fusion protein polypeptide includes an S2 ligand-inducible proteolytic cleavage site. The S2 ligand-inducible proteolytic cleavage site may be located within the HD-C fragment. In some cases, the S2 ligand-inducible proteolytic cleavage site is an ADAM-17 protease cleavage site. The ADAM-17 protease cleavage site may contain an Ala-Val dipeptide sequence, wherein the enzyme cleaves between Ala and Val. For example, in some cases, the amino acid sequence containing the S2 ligand-inducible proteolytic cleavage site may have the amino acid sequence KIEAVKSE (SEQ ID NO: 6), wherein the cleavage occurs between the "AV" sequences. As another example, the amino acid sequence containing the S2 ligand-inducible proteolytic cleavage site may have the amino acid sequence KIEAVQSE (SEQ ID NO: 7), wherein the cleavage occurs between the "AV" sequences.

[0065] In some cases, the fusion protein includes an S3 ligand-induced proteolytic cleavage site. The S3 ligand-induced proteolytic cleavage site may be located within a TM domain. In some cases, the S3 ligand-induced proteolytic cleavage site is a γ-secretase cleavage site. The γ-secretase cleavage site may contain a Gly-Val dipeptide sequence, wherein the enzyme cleaves between Gly and Val. For example, in some cases, the S3 ligand-induced proteolytic cleavage site has the amino acid sequence VGCGVLLS (SEQ ID NO: 8), wherein cleavage occurs between the “GV” sequences. In some cases, the S3 ligand-induced proteolytic cleavage site contains the amino acid sequence GCGVLLS (SEQ ID NO: 9).

[0066] In some cases, the fusion peptide lacks an S1 ligand-induced proteolytic cleavage site. In some cases, BTTS lacks an S2 ligand-induced proteolytic cleavage site. In some cases, BTTS lacks an S3 ligand-induced proteolytic cleavage site. In some cases, BTTS lacks both S1 and S2 ligand-induced proteolytic cleavage sites. In some cases, BTTS includes an S3 ligand-induced proteolytic cleavage site and lacks both S1 and S2 ligand-induced proteolytic cleavage sites.

[0067] In some embodiments, the fusion protein may have a vWF A2 sequence or a variant thereof, an ADAMTS13 cleavage site (which may be described by the concordant sequence HEXXHXXGXXHD (SEQ ID NO: 10); Crawley, Blood. 2011 118:3212-21), and an S3 or γ-secretase cleavage site, although many other arrangements exist. In some embodiments, the switch may contain components borrowed from Notch. In other embodiments, the switch may not contain components from Notch.

[0068] For simplicity, BTTSs (including but not limited to chimeric notch receptor peptides) are primarily single polypeptide chains. However, BTTSs (including chimeric notch receptor peptides) may be split or cleaved into two or more separate polypeptide chains, with the linkage of these chains forming a functional BTTS. For example, a chimeric notch receptor peptide may be constitutively or conditionally controlled. For instance, a constitutive heterodimerization domain can be inserted between the first and second parts of a split polypeptide to achieve a constitutive link between the two parts of a split BTTS, such that the split parts are functionally linked after heterodimerization.

[0069] Useful BTTS that may be employed in the methods described herein include, but are not limited to, modular extracellular sensor architecture (MESA) peptides. MESA peptides comprise: (a) a ligand-binding domain; (b) a transmembrane domain; (c) a protease cleavage site; and (d) a functional domain. The functional domain may be a transcriptional regulator (e.g., a transcriptional activator or repressor). In some cases, MESA receptors comprise two polypeptide chains. In some cases, MESA receptors comprise a single polypeptide chain. Non-limiting examples of MESA peptides are described, for example, in U.S. Patent Publication No. 2014 / 0234851; the entire disclosure of which is incorporated herein by reference.

[0070] Useful BTTS that may be employed in the methods described herein include, but are not limited to, the peptides used in the TANGO assay. The TANGO assay used in this study employs a TANGO peptide, which is a heterodimer in which the first peptide contains a tobacco etch virus (Tev) protease, and the second peptide contains a Tev proteolytic cleavage site (PCS) fused to a transcription factor. When the two peptides approach each other, this approach is mediated by native protein-protein interactions, and Tev cleaves the PCS to release the transcription factor. Non-limiting examples of TANGO peptides are described, for example, by Barnea et al. (Proc Natl Acad Sci USA. January 8, 2008; 105(1): 64-9); the entire disclosure of which is incorporated herein by reference.

[0071] Useful BTTSs that may be employed in the methods described herein include, but are not limited to, BTTSs based on the von Willebrand factor (vWF) cleavage domain, such as, but not limited to, BTTSs containing, for example, an unmodified or modified vWF A2 domain. VWF cleavage domain-based BTTSs described herein generally include: extracellular domains containing the first member of the binding pair; von Willebrand factor (vWF) cleavage domains containing proteolytic cleavage sites; cleavable transmembrane domains; and intracellular domains. Non-limiting examples of vWF cleavage domains and vWF cleavage domain-based BTTSs are described in Langridge & Struhl (Cell (2017) 171(6): 1383-1396), the entire contents of which are incorporated herein by reference.

[0072] Useful BTTS that may be employed in the methods described herein include, but are not limited to, chimeric Notch receptor peptides, such as, but not limited to, for example, synNotch peptides, non-limiting examples of which are described in PCT Publication No. WO 2016 / 138034, U.S. Patent No. 9,670,281, U.S. Patent No. 9,834,608, Royal et al., Cell (2016) 167(2): 419-432, Royal et al., Cell (2016) 164(4): 770-9, and Morsut et al., Cell (2016) 164(4): 780-91; the entire contents of which are incorporated herein by reference. The “SNIPR” switch is another example of a BTTS (see Zhu et al., 2022 Cell. 185: 1431-1443 and WO2021061856), although other BTTS exist (e.g., US20200331985) and / or can be readily designed.

[0073] BTTS expression in cells can be constitutive or inducible, for example, by the binding of another BTTS to an antigen on another cell. Therefore, in some embodiments, the second promoter may be constitutive in the cell. For example, the second promoter may be a CMV, EF-1, hPGK, or RPBSA promoter, although many other options are available.

[0074] In any implementation, BTTS may include:

[0075] (i) Extracellular binding domains that bind to EGFRvIII or brain-specific markers,

[0076] (ii) Optional force-sensing area,

[0077] (iii) Transmembrane domains,

[0078] (iv) One or more force-dependent cut sites that are cut when the force-sensing region is activated, and

[0079] (v) Intracellular domains containing transcription activators,

[0080] The extracellular binding domain binds to EGFRvIII or brain-specific markers, inducing proteolytic cleavage at one or more force-dependent cleavage sites to release transcriptional activators.

[0081] The released transcription activator induces transcription of the first coding sequence, which in turn leads to CAR expression.

[0082] Numerous examples of transcription activators that can be part of a fusion protein exist, including artificial transcription factors (ATFs), such as, for example, zinc finger-based artificial transcription factors (including, for example, those described in Sera T. Adv Drug Deliv Rev. 2009 61(7-8):513-26; Collins et al., Curr Opin Biotechnol. 2003 14(4):371-8; Onori et al., BMC Mol Biol. 2013 14:3). In some cases, transcription activators may contain a GAL4 DNA-binding domain that binds to a Gal4-responsive UAS, which is well characterized in the art. Examples of suitable transcription activators include GAL4-VP16 and GAL4-VP64, although many other factors may be used. As understood, the identity of transcription activators may vary. In some embodiments, transcription factors may have both a DNA-binding domain and an activation domain that bind to the corresponding promoter sequence. In many embodiments, the DNA-binding domain transcription factor may be independently selected from transcription factors based on Gal4, LexA, Tet, Lac, dCas9, zinc finger, and TALE. TALE- and CRISPR / dCas9-based transcription factors are particularly described in Lebar (Methods Mol Biol. 2018 1772:191-203). The binding sites of such domains are well-known or can be arbitrarily designed. The transcription activator can have any suitable activation domain, particularly, for example, VP16, VP64, Ela, Sp1, VP16, CTF, and GAL4. The first promoter will contain one or more copies of the binding site of the transcription activator, for example, GAL4 UAS.

[0083] As can be seen from the figure, in any implementation, the nucleic acid is a double-stranded nucleic acid, wherein CAR and BTTS are encoded by the top strand of the recombinant nucleic acid.

[0084] [Carriers and Particles]

[0085] In any implementation, the recombinant nucleic acid can be in a vector, such as a viral vector, for example, adenovirus vector, retrovirus vector, lentivirus vector, adeno-associated virus vector, herpes simplex virus vector, etc. Replication-deficient viruses can also be advantageous. Some vectors are incorporated into the nuclear genome of the host cell, while others are not. The use of viral vectors (particularly retroviruses and lentiviruses) for expressing CARs in immune cells is known (see, in particular, Lanne et al., Viruses. 2021 13:1528 and Moco et al., Methods Mol Biol 2020 2086:69-76).

[0086] Viral particles containing a viral vector are also provided. In these embodiments, the particles can be prepared by encapsulating the vector within the viral particles. Many methods for producing viral particles are known in the art (see, in particular, TiscornaNat Protoc. 2006 1:241-5).

[0087] [Cells and Treatment Methods]

[0088] Suitable host cells include cytotoxic cells, including CD8 cells. + T cells, natural killer (NK) cells, and neutrophils are cells that mediate cytotoxic responses. Recombinant nucleic acids may or may not be integrated into the cell's nuclear genome. In some cases, the introduction and / or genetic modification of the subject's nucleic acid is performed in vitro. For example, T lymphocytes, stem cells, or NK cells may be obtained from the individual; and the cells obtained from the individual are modified to express components of the circuit disclosed herein. Thus, the modified cells can be redirected to one or more selected antigens, defined by one or more antigen-binding domains present on the introduced circuit component. In some cases, the modified cells are regulated in vitro. In other cases, the cells are introduced (e.g., from the individual from whom the cells are obtained) and / or are already present in the individual; and the cells are regulated in vivo, for example, by administering nucleic acids or vectors into the individual. Alternatively, allogeneic immune cells may be used.

[0089] As described above, cells can be used to treat subjects with GBM, which may be epidermal growth factor receptor variant III (EGFRvIII) positive or negative. In some embodiments, glioblastoma is an EGFRvIII-positive glioblastoma, and the method involves administering cells containing recombinant nucleic acids to the subject (e.g., a human subject). This method is particularly useful in treating subjects with GBM, including cases where the subject's GBM exhibits EGFRvIII heterogeneity. Such treatments may include at least one EGFRvIII inhibitor targeting heterogeneous EGFRvIII-positive tumors. - Cell types (or their subpopulations) achieve the desired effect. As used herein, the terms "heterogeneous positive EGFRvIII" and "EGFRvIII" are used interchangeably. + GBM or EGFRvIII + "Tumor" generally refers to GBM tumors containing at least some cells expressing EGFRvIII. Such tumors may include EGFRvIII-positive cells. - Cells, or may evolve to contain EGFR during tumor progression. - . cells.

[0090] Therefore, in the treatment methods described herein, nucleic acids encoding circuits or their components may be administered in vitro, ex vivo, or in vivo. In some cases, cells may be collected from a subject and transfected with nucleic acids, and the transfected cells may be administered to the subject, with or without further processing, including but not limited to, in vitro amplification. In some cases, nucleic acids (with or without a delivery vector) may be administered directly to the subject.

[0091] In some cases, the methods of this disclosure can be used to target, treat, or eliminate minimal residual disease (MRD) remaining in a subject after prior GBM treatment. Whether or not the MRD has been determined to be refractory to prior treatment, the immediate approach can be used to target, treat, and / or eliminate GBM MRD. In some cases, the methods of this disclosure can be used to target, treat, and / or eliminate the subject's MRD after determining that the MRD is refractory to prior treatment or one or more available treatment options other than the treatment employed in the circuit described herein.

[0092] Regarding GBM as a whole, the desired effect of the treatment can result in a reduction in the number of cells in GBM, a reduction in the size of GBM tumors, a reduction in the overall proliferation of GBM, a reduction in the overall growth rate of GBM tumors, etc. For example, an effective treatment in some cases is one in which, when administered to an individual in need, the number of cancer cells and / or the size of the tumor in the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or more than 75% compared to the number of cancer cells and / or the size of the tumor in the individual without treatment. The reduction in the number of cancer cells or the size of the tumor mass can be defined relative to the heterogeneous tumor as a whole or relative to the target cells of GBM.

[0093] In some embodiments, an effective treatment is defined as a reduction, when administered alone (e.g., in monotherapy) or in combination with one or more other therapeutic agents (e.g., in combination therapy), in one or more doses, of the tumor growth rate, GBM cell number, and tumor mass compared to the absence of treatment, of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more. The reduction in tumor growth rate, GBM cell number, or tumor mass can be defined relative to the entire heterogeneous tumor or relative to the target cells of the GBM.

[0094] As will be readily understood, the treatments described herein may be combined with one or more conventional therapies in some cases. For example, in the case of GBM oncology, the methods described herein may be combined with conventional GBM treatments, including but not limited to, for example, conventional chemotherapy, conventional radiotherapy, conventional immunotherapy, surgery, etc. Also as stated above, in some cases, the treatments described herein may be used after conventional treatments, for example, to treat heterogeneous GBM refractory to conventional therapy, to treat heterogeneous GBM relapsed after conventional therapy, to treat MRD in subjects after conventional therapy, etc.

[0095] In some cases, the methods described herein may be used before or after routine treatment. For example, the methods described herein may be used as adjunctive therapy, such as when improvement is observed in a subject after routine treatment, or when a subject does not respond to routine treatment. In some cases, the methods described herein may be used before additional treatment, such as preparing a subject for additional treatment, such as routine treatment as described herein.

[0096] Standard treatment for GBM includes surgery (e.g., surgical removal of cancerous tissue), radiation therapy, chemotherapy, antibody therapy, biological response modifier therapy, and certain combinations thereof.

[0097] Radiation therapy includes, but is not limited to, X-rays or gamma rays delivered from external sources (such as beams) or via implanted small radiation sources.

[0098] Antibodies suitable for or under investigation for the treatment of GBM include, but are not limited to, naked antibodies, such as trastuzumab (Herceptin) and bevacizumab (Avastin). TM ), cetuximab (Erbitux) TM Panitumumab (Vectibix) TM Ipilimumab (Yervoy) TM ), rituximab (Rituxan), alemtuzumab (Lemtrada) TM Oregovomab (OvaRex) TM Lambrolizumab (also known as pembrolizumab, MK-3475, or Keytruda) TM ranibizumab (Lucentis) TM ), and conjugated antibodies, such as conjugated antibodies of the antibodies listed above.

[0099] Conventional cancer treatment also includes targeted cancer therapy, including but not limited to bevacizumab (Avastin), which targets the VEGF ligand (approved for glioblastoma).

[0100] In some cases, the methods disclosed herein may be used without any other conventional treatment, including, for example, where the methods described herein are the only method for treating a subject. For instance, in oncology, the methods described herein may be the only method for treating a subject with GBM (including, for example, primary GBM, recurrent GBM, etc.).

[0101] This indicates when to determine the timing of combination therapy (e.g., involving the administration of one or more agents that improve one or more side effects of the treatment described herein, or involving the administration of one or more agents that enhance the treatment described herein), and that the specific administration of such combination therapy is within the skill level of the relevant physician. In some cases, the dosage regimen and treatment plan for combination therapy may be determined through clinical trials.

[0102] T cell population

[0103] A T-cell population is also provided. In some embodiments, these cells may be present in vitro and may be progenitor cells of primary T cells that have been genetically modified to have recombinant nucleic acids. In some embodiments, as discussed above, the cells may contain nucleic acids encoding immune receptors. As mentioned above, in some embodiments, the T cells may be genetically modified to be allogeneic in a human host. In these embodiments, the cells may be frozen. The population may contain any number of CAR-T cells (e.g., 100,000 to 1 billion (1 Bn) cells). However, in some embodiments, the population may contain 100,000 to 50 million (50M) CAR-T cells.

[0104] In some embodiments, the harvested cells may be cryopreserved, where the term "cryopreservation" refers to preserving or maintaining cells by cooling to sub-zero temperatures, such as 77 K or -196 °C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including biochemical reactions that would lead to cell death, is effectively stopped. Useful methods for cryopreserving and thawing cryopreserved cells, and related methods and reagents, include, but are not limited to, those described, for example, in U.S. Patent Nos. 10,370,638; 10,159,244; 9,078,430; 7,604,929; 6,136,525; and 5,795,711, the entire disclosure of which is incorporated herein by reference. Conversely, when referring to cells, the term "fresh" as used herein may mean cells that have not been cryopreserved, for example, cells that may be obtained directly and / or used (e.g., transplantation, culture, etc.) after being collected from a subject or their organs.

[0105] The therapeutic cell populations and their therapeutic or pharmaceutical compositions obtained by the methods described herein can be present in any suitable container (e.g., culture vessel, test tube, flask, vial, cryotube, cryopreservation bag, etc.) and may be delivered using any suitable delivery method and / or device (e.g., for administration to a subject). Such cell populations and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some cases, the therapeutic cell populations and their pharmaceutical compositions may be prepared in a “ready-to-use” form, including, for example, the therapeutic cells being present in a suitable diluent and / or at the desired delivery concentration (e.g., in unit dose form) or at a concentration that can be easily diluted to the desired delivery concentration (e.g., with a suitable diluent or medium). The therapeutic cell populations and their pharmaceutical compositions may be prepared in a delivery device or apparatus compatible with the desired delivery mechanism or route, such as, but not limited to, syringes, infusion bags, etc.

[0106] In some cases, this disclosure provides one or more cell therapy doses, for example, each dose contained in a suitable container. Cell therapy doses may be generated by a variety of methods. Dividing the expanded therapeutic cell population into cell therapy doses may be done in a variety of ways.

[0107] In some embodiments, the composition may include therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffer solution, etc. Such compositions may contain one or more additives, such as salts (e.g., NaCl, MgCl2, KCl, MgSO4), buffers (Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholine)ethanesulfonic acid (MES), sodium 2-(N-morpholine)ethanesulfonate (MES), 3-(N-morpholine)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), solubilizers, detergents (e.g., nonionic detergents, such as Tween-20), nuclease inhibitors, glycerol, chelating agents, etc.

[0108] The population may include a therapeutically effective amount of cells. "Therapeutically effective amount" means the number of cells sufficient to produce the desired result, for example, the amount sufficient to affect beneficial or desired therapeutic (including preventative) outcomes, such as reducing symptoms of a disease or condition, for example, associated with the target cells or their population. The effective amount may be administered in one or more applications.

[0109] The "therapeutic effective amount" of such cells may vary depending on factors such as the subject's disease state, age, sex, and weight, as well as the cells' ability to elicit the desired response in the subject. Therapeutic effective amount is also the amount by which any toxic or harmful effects of the cells are outweighed by the beneficial effects of treatment. The term "therapeutic effective amount" includes the amount effective for "treating" a subject (e.g., a patient). When referring to a therapeutic amount, the precise amount of composition considered for administration in a particular embodiment may be determined by a physician based on the instructions and taking into account age, weight, tumor size, degree of infection or metastasis, and individual differences in the patient's (subject's) condition. In some embodiments, the therapeutic effective amount of T cells may be 100,000 to 50 million (50M) T cells. However, in other embodiments, the therapeutic effective amount of T cells may be 50 million (50M) to 500 million (500M) cells.

[0110] The cells of this disclosure can be incorporated into various formulations for therapeutic application. More specifically, the cells of this disclosure can be formulated for application in combination with suitable excipients, diluents, and / or the like.

[0111] Cellular preparations suitable for administration to patients (e.g., suitable for human administration) are typically sterile and may further be free of detectable pyrogens or other contaminants that are contraindicated for administration to patients according to the chosen route of administration.

[0112] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intraventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. In some embodiments, the carrier-containing cells may be locally administered to a brain tumor, for example, intracranial or intratumoral (e.g., by direct injection into the brain cavity or tumor).

[0113] Aqueous formulations of cells may be prepared in pH buffer solutions, for example, with a pH range of about 4.0 to about 7.0, or about 5.0 to about 6.0, or optionally about 5.5. Examples of buffers suitable for this pH range include phosphate, histidine, citrate, succinate, acetate buffers, and other organic acid buffers. Buffer concentrations may be about 1 mM to about 100 mM, or about 5 mM to about 50 mM, depending on, for example, the osmotic pressure required by the buffer and the formulation.

[0114] The formulation may include an osmoregulator to adjust the osmotic pressure of the formulation. Exemplary osmoregulators include any components and combinations thereof, such as sodium chloride, potassium chloride, glycerol, amino acids, and sugars. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" means a solution that has the same osmotic pressure as some other solution, such as physiological saline or serum. The amount of osmoregulator used may be from about 5 mM to about 350 mM, for example, from 100 mM to 350 mM.

[0115] In some embodiments, the composition comprises the cells of this disclosure, and one or more of the reagents identified above (e.g., surfactants, buffers, stabilizers, osmotic agents), and is substantially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride, and combinations thereof. In other embodiments, the formulation contains a preservative, for example, in a concentration range of about 0.001 to about 2% (w / v).

[0116] [Cell Preparation Methods]

[0117] Methods for cell preparation are also provided. In some embodiments, the method may include in vitro amplification of T cells containing recombinant nucleic acids to produce amplified T cells, and harvesting the amplified T cells to produce a T cell population, wherein the term "harvest" is intended to refer to the step of removing the cells from the amplification container / bioreactor. In some embodiments, if desired, the cells may be concentrated, for example, by centrifugation, suitable cell separation techniques (e.g., magnetic beads), and / or similar methods.

[0118] Cells can be prepared by any method, for example, by introducing recombinant nucleic acids into T cells. A general procedure for preparing CAR-T cells typically involves isolating peripheral blood mononuclear cells (PBMCs). Next, PBMCs or T cells further enriched from PBMCs are stimulated with antibody-coated beads (e.g., Dynabeads) or plate-binding antibodies to induce T cell activation, and then genetically modified using lentiviral vectors, gamma-retroviral vectors, or other delivery methods to express the cell surface CAR molecule. Subsequently, these engineered T cells are expanded in culture medium for several days in the presence of one or more cytokines (e.g., IL-2, IL-15, IL-7, or any combination thereof (e.g., IL-2 and IL-15 or IL-7 and IL-15)) to reach the cell numbers required for experimental testing or clinical treatment.

[0119] In some implementations, T cells may be harvested within 3 to 8 days after the start of cell expansion (e.g., on day 3, 4, 5, 6, 7, or 7), earlier than the standard procedure (which typically requires 10 to 14 days of cell expansion). In these implementations, the harvested T cell population may contain 100,000 to 50 million (50M) CAR-T cells, but more or fewer cells may be used. In other implementations, T cells may be harvested within 8 to 16 days after the start of cell expansion, in which case the population may contain 50 million (50M) to 1 billion (1 Bn) cells.

[0120] Cell populations prepared according to this method are also provided.

[0121]

Example

[0122] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors consider their invention, nor to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is the weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular (ly); ip, intraperitoneal; sc, subcutaneous, etc.

[0123] [Example 1: Overview of E-SYNC therapeutic T cells and generation of p885 E-SYNC clinical single lentiviral transfer vector from a primitive dual-vector research-grade design]

[0124] Figure 1A This diagram illustrates the “initiation and killing” E-SYNC T cell circuitry. E-SYNC is initiated upon exposure to cells presenting GBM-specific EGFRvIII markers, which triggers activation of the synNotch receptor (orange), thereby activating the expression of anti-EphA2 / IL13Rα2 tandem CARs. Once initiated, E-SYNC T cells are able to kill GBM cells that naturally overexpress these markers.

[0125] Figure 1B The EGFRvIII-initiated synNotch-CAR GBM therapeutic loop demonstrated by Choe et al. (Sci. Transl. Med. 2021 13: eabe7378) utilizes a dual-lentiviral vector system consisting of a vector expressing synNotch (p168) and a payload vector encoding a CAR under a synNotch-inducible promoter (p322). Although effective in all experiments described in the publication, this dual-vector system may be potentially unsuitable for preparing T cells for clinical applications due to its cost-inefficient dual-vector design and the use of epitope tags (purple) and fluorescent markers.

[0126] Figure 1C A schematic diagram of the clinical E-SYNC single-vector design (p885) is shown. An inducible CAR transgene (blue) is placed tandemly upstream of the constitutive synNotch transgene. All redundant DNA elements (tags, markers) are removed, and partial coding sequences are optimized to improve expression and viral titer. The entire vector payload is subcloned into a third-generation lentiviral backbone compatible with cGMP viral preparation.

[0127] [Example 2: Example of a single lentivirus vector configuration for a BTTS-driven CAR loop]

[0128] Several different lentiviral vector configurations were explored, with transgenes placed in tandem (A and B) or anisotropically (C). These different configurations were evaluated as follows: (i) strongly BTTS-driven CAR expression (e.g., synNotch) and (ii) low basal CAR expression in the absence of BTTS stimulation. Observations are summarized below. Given the significant risk of adverse reactions associated with basal CAR expression detectable in therapeutic cells, only design A was considered for single-vector development for both E-SYNC and B-SYNC.

[0129] [Example 3: Similar to the control dual-vector cells, E-SYNC T cells prepared using p885 single vector showed EGFRvIII-driven CAR expression and killed GBM tumor cells in vitro]

[0130] Figure 3A The study showed that P885 T cells can activate CAR expression in response to EGFRvIII exposure, reaching levels similar to dual-carrier control cells, while maintaining low basal CAR expression in the absence of stimulation. CD8 + T cells were transduced using a dual-vector design (P168 and P322) and two finalized single-vector designs (P830 and P885). Differences in binder composition are listed in the table on the left. The ability of T cells to induce CAR expression was tested. 50k T cells were co-cultured with K562 cells at a 1:1 ratio for 48 h, and then CAR levels were stained (middle two figures) or synNotch (right figure). Basal CAR expression was assessed using parental K562 cells, while EGFRvIII was used. + Activation of synNotch-CAR was measured in K562 cells.

[0131] Figure 3B The measurement of CD8 transducers using P168 / 322, P830, or P885 is shown. + The killing assay of T cells' ability to eliminate GBM cells showed that the single-vector design could be matched with a dual-vector reference. U87 MG (EGFRvIII negative) or GBM6 PDX (EGFRvIII negative) were used. + Target cells were co-cultured with 50k T cells for 48 h (E:T=1). Target cell viability was analyzed by flow cytometry and normalized relative to control wells containing untransduced T cells. Data represent two technical replicates.

[0132] Example 4: E-SYNC T cells prepared using p885 clinical vector effectively eliminated EGFRvIII in mice. + Xenografted brain tumors derived from GBM patients

[0133] Similar to the dual-vector control cells, T cells transduced with a single P885 candidate vector were able to eliminate GBM6 PDX tumors. GBM6 patient-derived xenograft tumor cells were orthotopically implanted into the brains of NCG mice, followed by CD4+ transduction with a P168 / P322 dual-vector and a P885 clinical single-vector candidate. + and CD8 +Intravenous administration of T cells or untransduced T cells (timelines and doses are listed below). Tumor size was measured every 7 days following tumor induction using an IVIS spectral imager via bioluminescent in vivo imaging with effLuc markers. Tumor size trajectories for individual mice in each group are plotted below.

[0134] [Example 5: E-SYNC T cells transduced with p885 single vector can differentiate between brain tumors and ventral tumors in vivo based on tumor EGFRvIII expression]

[0135] T cells prepared using a single P885 candidate vector to eliminate EGFRvIII + Brain tumor, but not controlling EGFRvIII in the flank - Negative tumors indicate that the spatial control of CAR activity in vivo is similar to that of the published dual-vector reference design. Orthotopic implantation of activator U87 MG tumors (EGFRvIII) into the brains of NCG mice. + and EGFRvIII - A 1:1 mixture of cells) was implanted in the flank of a non-initiated U87 tumor (EGFRvIII). - U87 MG cells). Mice were then transduced with CD4+ cells transduced using a dual-vector of P168 / P322 and a single-vector of P885 (a clinical candidate cell). + and CD8 + Intravenous administration of T cells or untransduced T cells (timelines and doses are listed below). Tumor size was measured every 7 days following tumor induction using an IVIS spectral imager via bioluminescent in vivo imaging with effLuc markers. Tumor size trajectories for individual mice in each group are plotted below.

[0136] [Example 6: Overview of the BCAN-initiated SynNotch-CAR circuit (B-SYNC), composition of the pB-SYNC1 single lentiviral transfer vector, and tumor cell killing performance of B-SYNC T cells]

[0137] Figure 6A An overview of the pB-SYNC1 vector design for producing BCAN-initiated SynNotch-CAR (B-SYNC) T cells is provided. The design is based on the p885 vector, with the difference being the expression of an anti-BCAN binder as part of the synNotch receptor.

[0138] Figure 6BThis is a schematic diagram of B-SYNC therapeutic T cells. Unlike E-SYNC, which requires the presence of GBM-specific EGFRvIII antigen, B-SYNC cells are activated by exposure to the brain-specific ligand Brevican (BCAN). This extends the final expression of the dual-affinity CAR to the entire central nervous system, thereby enabling the treatment of a wider range of EGFRvIII. - Negative GBM patients.

[0139] Figure 6C The study demonstrated that T cells transduced with pB-SYNC1 single-vector showed superior performance compared to dual-vector control cells in an in vitro co-culture killing assay of this GBM6 PDX. The assay was prepared using T cells transduced with clinical pB-SYNC1 single-vector, dual-vector control, constitutive dual-affinity CAR, or untransduced control. T cells and GBM6 were then compared with parental or BCAN cells. + K562 cells were mixed in a ternary co-culture at a 1:1:1 ratio to provide circuit initiation. After 72 h of culture, the remaining target GBM6 cells were analyzed by flow cytometry and normalized relative to wells containing untransduced T cells. The mean of two replicates is shown, with error bars representing standard deviation.

[0140] [Example 7: B-SYNC T cells prepared using pB-SYNC1 clinical vector effectively eliminated xenograft tumors derived from GBM patients in mice and improved survival rate]

[0141] Similar to dual-vector reference T cells, T cells transduced with a single clinical vector candidate of pB-SYNC1 were able to eliminate GBM6 brain tumors in mice. GBM6 patient-derived xenograft tumor cells were orthotopically implanted into the brains of NCG mice, followed by CD4+ transduction with a vector expressing constitutive CAR using a single clinical vector candidate of pB-SYNC1 and a reference dual-vector. + and CD8 + Intravenous administration of T cells or untransduced T cells (timelines and doses are listed below). Tumor size was measured every 7 days following tumor induction using an IVIS spectral imager via bioluminescent in vivo imaging with effLuc markers. The mean tumor size trajectory for each group of individual mice is plotted below. Kaplan-Meier survival curves for different mouse groups are shown on the right.

[0142]

Example 8: Single-carrier design

[0143] Figure 8A and 8B The plasmid maps of the p885 and pB-SYNC1 vectors as described above are shown.

[0144] Figure 9AThe initial tandem was shown, which is suitable for a first-generation therapeutic BTTS-controlled CAR expression circuit, but may be limited by a single protein payload. To overcome the challenges of the tumor microenvironment and enhance T cell function in vivo, expressing additional protein payloads along with the CAR may be advantageous.

[0145] Figure 9B The study demonstrates that tandem vector designs can accommodate more than one regulatory protein by using 2A peptides or internal ribosome entry sites (IRES), thereby creating polycistronic transgenes that can produce multiple proteins under BTTS control.

[0146] Figure 9C An alternative design is shown, utilizing a bidirectional BTTS-regulated synthetic promoter composed of a BTTS transcription factor binding array, flanked by two minimal promoters in opposite directions. This synthetic bidirectional promoter can induce the expression of two separate transgenes under the control of a single BTTS. This strategy can also be combined with bicistronic elements (…). Figure 9B This allows for the expression of more than two protein payloads under BTTS control.

[0147] Although the invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes and equivalent substitutions can be made without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to bring particular circumstances, materials, compositions, methods, or one or more method steps into accordance with the purpose, spirit, and scope of the invention. All such modifications are intended to fall within the scope of the appended claims.

Claims

1. Recombinant nucleic acid, which consists of the following from 5' to 3': (a) The first promoter; (b) A first coding sequence, wherein the first coding sequence encodes an anti-EphA2 / IL13Rα2 tandem CAR and is operatively connected to the first promoter; (c) The second promoter; and (d) A second coding sequence, wherein the second coding sequence encodes a binding trigger transcription switch (BTTS) that is activated by binding to EGFRvIII or a brain-specific marker and is operatively linked to the second promoter. in: Transfection of recombinant nucleic acids into cytotoxic immune cells resulted in the expression of BTTS on the surface of the cytotoxic immune cells, and The expressed BTTS binds to antigens on the surface of target cells or proteins in the extracellular matrix, thereby activating the expression of CAR in the cytotoxic immune cells.

2. The recombinant nucleic acid according to claim 1, wherein the BTTS is activated by binding to EGFRvIII.

3. The recombinant nucleic acid according to claim 1, wherein the BTTS is activated by binding to MOG, CDH10, CSPG5, PTPRZ1, BCAN, Neurexin 1b or NrCAM.

4. The recombinant nucleic acid according to claim 3, wherein the BTTS is activated by binding to BCAN.

5. The recombinant nucleic acid according to any one of claims 1 to 4, wherein the BTTS comprises: (i) Extracellular binding domains that bind to EGFRvIII or the aforementioned brain-specific markers, (ii) Transmembrane domains, (iii) One or more force-dependent cleavage sites that are cleaved when the extracellular binding domain binds to EGFRvIII or the brain-specific marker, and (iv) Intracellular domains containing transcription activators, The binding of the extracellular binding domain to EGFRvIII or the brain-specific marker induces proteolytic cleavage of one or more force-dependent cleavage sites to release the transcriptional activator. The released transcription activator induces transcription of the first coding sequence.

6. The recombinant nucleic acid according to any one of the prior claims, wherein the second promoter is constitutively present in the cytotoxic immune cells.

7. The recombinant nucleic acid according to any one of the prior claims, wherein the nucleic acid is double-stranded, and wherein the CAR and BTTS are encoded by the top strand of the recombinant nucleic acid.

8. A vector comprising the recombinant nucleic acid according to any one of claims 1 to 7.

9. The vector according to claim 7, wherein the vector is a viral vector.

10. The vector according to claim 9, wherein the viral vector is a lentiviral vector.

11. The vector according to claim 9, wherein the viral vector is a retroviral vector.

12. Viral particles comprising a vector according to any one of claims 8 to 11.

13. A cell comprising the recombinant nucleic acid according to any one of claims 1 to 7.

14. The cell of claim 13, wherein the recombinant nucleic acid is integrated into the nuclear genome of the cell.

15. The cell of claim 13, wherein the recombinant nucleic acid is not integrated into the nuclear genome of the cell.

16. The cell according to any one of claims 13 to 14, wherein the cell is a cytotoxic immune cell.

17. The cell of claim 16, wherein the cell is a cytotoxic T cell.

18. A method for treating a subject with epidermal growth factor receptor variant III (EGFRvIII) positive glioblastoma, comprising administering to the subject the cells according to any one of claims 13 to 17.

19. The method of claim 18, wherein the subject is a human.

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