WO2024196802A1 - Porte non à deux cellules comprenant des cellules immunosuppressives synthétiques - Google Patents

Porte non à deux cellules comprenant des cellules immunosuppressives synthétiques Download PDF

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WO2024196802A1
WO2024196802A1 PCT/US2024/020272 US2024020272W WO2024196802A1 WO 2024196802 A1 WO2024196802 A1 WO 2024196802A1 US 2024020272 W US2024020272 W US 2024020272W WO 2024196802 A1 WO2024196802 A1 WO 2024196802A1
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cell
antigen
cells
prior
immune
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Nishith REDDY
Milos Simic
Wendell A. Lim
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University of California Berkeley
University of California San Diego UCSD
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University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4225—Growth factors
    • A61K40/4226—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/49—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • a Sequence Listing is provided herewith as a Sequence Listing XML, “UCSF-726WO_SEQLIST”, created on March 15, 2024 and having a size of 7,400 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.
  • This disclosure provides a new “NOT” gate that relics on Boolean logic to improve therapeutic efficacy.
  • a cell therapy comprising: (i) a cytotoxic immune cell comprising an engineered immune receptor that recognizes an antigen on a target cell; and (ii) an immunosuppressive cell comprising a molecular' circuit comprising (a) a binding-triggered transcriptional switch (BTTS) that recognizes an antigen on a non-target cell and (b) a nucleic acid encoding an anti-inflammatory protein, wherein: binding of the engineered immune receptor to the antigen on the target cell in the absence of the immunosuppressive cell activates the cytotoxic immune cell and binding of the BTTS to the antigen on the surface of a non-target cell activates expression of the anti-inflammatory protein by the immunosuppressive cell and protects the non-target cell from the cytotoxic immune cell.
  • Methods of treatment are also provided.
  • a method of treating a subject is also provided.
  • the method may comprise administering to the subject the cell therapy, where the cells may be administered together or separately.
  • the engineered immune receptor may recognize an antigen that is expressed on cancerous cells; and the BTTS recognizes an antigen that is not on the cancerous cells (e.g., an antigen that is expressed in a non-target tissue).
  • the antigen recognized by the BTTS is tissue and/or organ specific.
  • the present therapy is believed to reduce “on-target/off-tumor” killing by the cytotoxic immune cells.
  • Fig 1. shows synNotch induced production of suppressive cytokine TGFb.
  • Figs. 2A and 2B show that suppressor cells that produce combination of TGFb (suppressive cytokine) and CD25 (IL2 sink) are very effective at suppressing CAR T killing in vitro.
  • TGFb suppressor cytokine
  • CD25 IL2 sink
  • Figs. 3A and 3B show that suppressor cells that produce combination of IL10 (suppressive cytokine) and CD25 (IL2 sink) are very effective at suppressing CAR T killing in vitro.
  • Figs. 4A and 4B shows that suppressor cells that produce combination of TGFb (suppressive cytokine) and CD25 (IL2 sink) are very effective at suppressing CAR T killing of tumors in vivo.
  • TGFb suppressor cytokine
  • CD25 IL2 sink
  • Fig. 5. shows that engineered T cells overexpressing CD25 increases consumption of IL2 and cell proliferation.
  • Fig. 6. shows that synNotch->ILl 0 synthetic suppressor cells can block autoimmune cell proliferation in brain and CNS in mouse neuroinflammation model.
  • Figs. 7A-7D show that synNotch circuits in CD4+ T cells can reconstitute Treg-like functions to drive local immune suppression.
  • Fig. 7A For many inflammatory disorders, one therapeutic strategy would be to locally suppress immune responses without systemic immune suppression.
  • the cell produces production of inhibitory cytokines and pro-inflammatory cytokine sinks (CD25 and IL-10/TGFbetal).
  • Fig. 7B SynNotch induced suppressive payloads are produced at high levels comparable to activated FoxP3+ regulatory T cells.
  • Fig. 7C Human CD4+ T cells engineered to express CD25 leads to increase consumption of IL2 (measured by ELISA) and increased expansion (measured by flow cytometry) in vitro compared to an untransduced T cell control.
  • Fig. 7D Synthetic suppressor cells with synNotch circuits that produce a combination of TGFpi and CD25 are more potent at suppression of CAR T cell expansion in vitro compared to each individual payload.
  • Figs. 8A-8C show that synthetic suppressor cells locally block inflammation without systemic immune suppression in vivo.
  • Fig. 8A Synthetic suppressor cells can act block off-target CAR T cell toxicity without systemic immune suppression.
  • Two tumor model K562 cells with one dual antigen tumor (Her2+ CD 19+) and one single antigen tumor (Her2+) is used to testantigen-specific suppression of CAR T cells by synthetic suppressor cells (T cells injected i.v.).
  • Human anti- Her2 CAR T cells can kill both tumors, while human synthetic suppressor cells expressing an anti-CD19 SynNotch will only induce SynNotch in the dual antigen tumor.
  • Fig. 8B Synthetic suppressors with anti-CD19 SynNotch circuits producing both CD25 and TGFpi are effective at suppressing anti-Her2 CAR T cell killing of the dual antigen tumor, but circuits producing either individual payloads was not sufficient for suppression.
  • Fig. 8C Flow analysis of isolated tumors shows reduced expansion of CAR T cells in dual antigen tumor.
  • Fig. 9 shows data obtained from replicates with different T cell donors.
  • Fig. 10 shows the present system is an improvement over other CAR NOT gates in vivo.
  • Fig. 11 shows some of the general principles of how tumor recognition can be enhanced using a two cell NOT gate.
  • Fig. 12 shows that suppressor T cells can protect bystander cells under immune attack.
  • Fig. 13 shows that suppressor T cells are not self-inactivating when active because synNotch bypasses native TCR signaling requirements.
  • Fig. 14 shows suppressor T cells can be programmed to induce non-native suppression programs.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect and/or a response related to the treatment.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or can be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • a “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (including biologic agents, such as cells), or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.
  • the individual is a human.
  • the individual is a non-human primate.
  • the individual is a rodent, e.g., a rat or a mouse.
  • the individual is a lagomorph, e.g., a rabbit.
  • binding-triggered transcriptional switch refers to any polypeptide or complex of the same that is capably of transducing a specific binding event on the outside of the cell (e.g., binding of an extracellular domain of the BTTS) to activation of a recombinant promoter within the nucleus of the cell.
  • Many BTTSs work by releasing a transcription factor that activates the promoter.
  • the BTTS is made up of one or more polypeptides that undergo proteolytic cleavage upon binding to the antigen to release a gene expression regulator that activates the recombinant promoter.
  • a BTTS may comprise (i) an extracellular domain comprising the antigen binding region of an antigen- specific antibody; (ii) a proteolytically cleavable sequence comprising one or more proteolytic cleavage sites; and (iii) an intracellular domain, wherein binding of the antigen binding region to the antigen induces cleavage of the sequence at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the intracellular domain activates transcription of an expression cassette.
  • a BTTS can be based on synNotch, A2, MESA, or force receptor, for example, although others are known or could be constructed.
  • a SNIPR Zhu et al 2022 Cell. 185: 1431-1443 and WO2021061856) may be used.
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.
  • Nb refers to the smallest antigen binding fragment or single variable domain (VHH) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids (Hamers-Casterman et al. (1993) Nature 363:446; Desmyter et al. (2015) Curr. Opin. Struct. Biol. 32:1). In the family of "camelids” immunoglobulins devoid of light polypeptide chains are found.
  • “Camelids” comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna ⁇ .
  • a single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody.
  • synthetic generally refer to artificially derived polypeptides or polypeptide encoding nucleic acids that are not naturally occurring.
  • Synthetic polypeptides and/or nucleic acids may be assembled de novo from basic subunits including, e.g., single amino acids, single nucleotides, etc., or may be derived from pre- existing polypeptides or polynucleotides, whether naturally or artificially derived, e.g., as through recombinant methods.
  • Chimeric and engineered polypeptides or polypeptide encoding nucleic acids will generally be constructed by the combination, joining or fusing of two or more different polypeptides or polypeptide encoding nucleic acids or polypeptide domains or polypeptide domain encoding nucleic acids.
  • Chimeric and engineered polypeptides or polypeptide encoding nucleic acids include where two or more polypeptide or nucleic acid “parts” that are joined are derived from different proteins (or nucleic acids that encode different proteins) as well as where the joined parts include different regions of the same protein (or nucleic acid encoding a protein) but the parts are joined in a way that does not occur naturally.
  • recombinant describes a nucleic acid molecule, e.g., a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature.
  • recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression from a recombinant polynucleotide.
  • recombinant as used with respect to a host cell or a virus means a host cell or virus into which a recombinant polynucleotide has been introduced.
  • Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).
  • material e.g., a cell, a nucleic acid, a protein, or a vector
  • a heterologous material e.g., a cell, a nucleic acid, a protein, or a vector
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • Operably linked nucleic acid sequences may but need not necessarily be adjacent.
  • a coding sequence operably linked to a promoter may be adjacent to the promoter.
  • a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences.
  • more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
  • a “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell.
  • heterologous means a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) nucleic acid or protein, respectively.
  • Heterologous nucleic acids or polypeptide may be derived from a different species as the organism or cell within which the nucleic acid or polypeptide is present or is expressed. Accordingly, a heterologous nucleic acids or polypeptide is generally of unlike evolutionary origin as compared to the cell or organism in which it resides.
  • cancer-associated refers to an antigen that is expressed in cancerous cells but not significantly non-cancerous cells of the same type. Some cancer-associated antigens are expressed on cancer cells and in normal tissues.
  • MSLN is considered a cancer- associated antigen since it is aberrantly expressed various cancer cells (e.g., lung cancers (adenocarcinoma and squamous carcinoma), ovary, peritoneum, endometrium, pancreas, stomach and colon, etc.) but it is also expressed on normal mesothelial cells in the pleura, pericardium, and peritoneum and in epithelial cells on the surface of the ovary, tunica vaginalis, rete testis, and fallopian tubes in trace amounts.
  • activates expression of in the context of activating the expression of a nucleic acid or protein, refers to activating the expression of the protein encoded by a nucleic acid. As would be understood, “activates expression of” includes transcription of the coding sequence to produce mRNA and translation of the mRNA to produce protein.
  • the present disclosure provides a two cell “NOT” gate therapeutic that dampens the ability of engineered cytotoxic immune cells to kill off-target cells. This principle is illustrated in Fig. 11.
  • the cell therapy may comprise: (i) a cytotoxic immune cell (e.g., a CD8 + T cell or an NK cell) comprising an engineered immune receptor (e.g., engineered T cell receptor (TCR) or chimeric antigen receptor (CAR)) that recognizes an antigen on a target cell and (ii) a immunosuppressive cell (e.g., a CD4 + T cell) comprising a molecular circuit comprising: (a) a binding-triggered transcriptional switch (BTTS) that recognizes an antigen on a non-target cell and one or any combination of: (b) a nucleic acid encoding a pro-inflammatory cytokine sink (e.g., CD25, at least the extracellular domain of IL-1R, IL-12R/CD25, IL-18R, TNFR1, TNFR2, IFNGR, GM-CSFR, a domain thereof that binds to its cognate ligand, or an antibody that is tethered to the
  • binding of the engineered immune receptor to the antigen on the target cell in the absence of the immunosuppressive cell activates the cytotoxic immune cell; and binding of the BTTS to the antigen on the surface of a non-target cell activates expression of one or any combination of (b)- (e) by the immunosuppressive cell and protects the non-target cell from the cytotoxic immune cell.
  • the immunosuppressive immune cell may be a T cell, a B cell, a macrophage, or a neutrophil.
  • the immunosuppressive immune cell may be a CD4 + T cell.
  • the engineered immune receptor may recognize a cancer antigen and the BTTS may recognize tissue and/or organ specific antigen that is at a different site to the antigen. For example, if the cancer antigen is expressed in brain tumor and in the liver, then the BTTS may recognize a liver-specific antigen.
  • binding of the BTTS to an antigen on a non-target cell activates expression of one or any combination of (b)-(e) by the immunosuppressive cell.
  • binding of the BTTS to a marker on the surface of a target cell may activate expression of (a), (b), (c), (d), (e), (a) and (b), (a) and (c), (a) and (d), (a) and (e), (b) and (c), (b) and (d), (b) and (e), (c) and (d), (c) and (e) or (d) and (e), etc., by the immunosuppressive cell.
  • binding of the BTTS to a marker on the surface of a target cell activates expression of the antiinflammatory cytokine of (b) and/or the pro-inflammatory cytokine sink of (c) by the engineered immune cell.
  • the circuit may comprise components (a) and (b), (a) and (c) or (a), (b) and (c).
  • binding of the BTTS to the antigen on the surface of a non-target cell activates expression of TGF01, CD25 and, optionally, IL-10.
  • BTTSs Binding-triggered, transcriptional switches
  • the BTTS is a cleavable fusion protein that contains: (a) an extracellular binding domain comprising a protein binding domain (e.g., scFv or nanobody) that binds to a cell surface marker on a cell, (b) an optional force sensing region, (c) a transmembrane domain, (d) one or more force-dependent cleavage sites that are cleaved, e.g., when the force sensing region is activated, and (e) an intracellular domain comprising a transcriptional activator, where binding of the binding domain to the marker on the surface of the other cell induces proteolytic cleavage of the one or more force-dependent cleavage sites to release the transcriptional activator.
  • a protein binding domain e.g., scFv or nanobody
  • the fusion protein is cleaved to release the intracellular domain when the extracellular domain of the fusion protein engages with a marker on another cell.
  • the fusion protein may contain a force sensing region (which is typically in the extracellular domain) and one or more force-dependent cleavage sites that are cleaved, e.g., when the force sensing region is activated.
  • the position of the force-dependent cleavage sites may vary and, in some embodiments the fusion protein may contain at least two cleavage sites. In some cases, one of the cleavage sites may be extracellular and the other may be in the transmembrane domain or within 10 amino acids of the transmembrane domain in the intracellular domain.
  • the force sensing region and/or the one or more forcedependent cleavage sites may be from a Delta/Serrate/Lag2 (DSL) superfamily protein, as reviewed by Pintar et al (Biology Direct 2007 2: 1-13).
  • DSL Delta/Serrate/Lag2
  • the force sensing region and/or the one or more force-dependent cleavage sites may be from Notch (see Morsut Cell.
  • vWF von Willebrand Factor
  • amyloid-beta CD16, CD44 , Delta, a cadherin , an ephrin-type receptor or ephrin ligand, a protocadherin, a filamin, a synthetic E cadherin, interleukin- 1 receptor type 2 (IL1R2), major prion protein (PrP), a neuregulin or an adhesion-GPCR.
  • IL1R2 interleukin- 1 receptor type 2
  • PrP major prion protein
  • neuregulin an adhesion-GPCR.
  • the one or more ligand-inducible proteolytic cleavage sites are selected from SI, S2, and S3 proteolytic cleavage sites.
  • the SI proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid.
  • the S2 proteolytic cleavage site is an ADAM-17-type protease cleavage site comprising an Ala- Vai dipeptide sequence.
  • the S3 proteolytic cleavage site is a y-secretase cleavage site comprising a Gly-Val dipeptide sequence.
  • the S3 proteolytic cleavage site is in the transmembrane domain.
  • the shear force generated by binding of the extracellular domain of this fusion protein to another cells unfolds the force sensing region (which, in the case of Notch contains EGF-like repeats whereas in other proteins is made up of other sequences such as the A2 domain in vWF (see, e.g., 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.
  • the fusion protein includes an SI ligand-inducible proteolytic cleavage site.
  • An SI ligand- inducible proteolytic cleavage site can be located between the HD-N segment and the HD-C segment.
  • the SI ligand-inducible proteolytic cleavage site is a furin-like protease cleavage site.
  • a furin-like protease cleavage site can have the canonical sequence Arg-X-(Arg/Lys)-Arg, where X is any amino acid; the protease cleaves immediately C-terminal to the canonical sequence.
  • an amino acid sequence comprising an S 1 ligand- inducible proteolytic cleavage site can have the amino acid sequence GRRRRELDPM (SEQ ID NO:1), where cleavage occurs between the “RE” sequence.
  • an amino acid sequence comprising an S 1 ligand-inducible proteolytic cleavage site can have the amino acid sequence RQRRELDPM (SEQ ID NO:2), where cleavage occurs between the “RE” sequence.
  • the fusion protein polypeptide includes an S2 ligand- inducible proteolytic cleavage site.
  • An S2 ligand-inducible proteolytic cleavage site can be located within the HD-C segment.
  • the S2 ligand-inducible proteolytic cleavage site is an ADAM-17-type protease cleavage site.
  • An ADAM-17-type protease cleavage site can comprise an Ala-Vai dipeptide sequence, where the enzyme cleaves between the Ala and the Vai.
  • amino acid sequence comprising an S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence KIEAVKSE (SEQ ID NOG), where cleavage occurs between the “AV” sequence.
  • amino acid sequence comprising an S2 ligandinducible proteolytic cleavage site can have the amino acid sequence KIEAVQSE (SEQ ID NO:4), where cleavage occurs between the “AV” sequence.
  • the fusion protein includes an S3 ligand-inducible proteolytic cleavage site.
  • An S3 ligand-inducible proteolytic cleavage site can be located within the TM domain.
  • the S3 ligand-inducible proteolytic cleavage site is a gamma- secretase (y-secretase) cleavage site.
  • a y-secretase cleavage site can comprise a Gly-Val dipeptide sequence, where the enzyme cleaves between the Gly and the Vai.
  • an S3 ligandinducible proteolytic cleavage site has the amino acid sequence VGCGVLLS (SEQ ID NO:5), where cleavage occurs between the “GV” sequence.
  • an S3 ligand-inducible proteolytic cleavage site comprises the amino acid sequence GCGVLLS (SEQ ID NO:6).
  • the fusion protein polypeptide lacks an SI ligand-inducible proteolytic cleavage site.
  • the BTTS lacks an S2 ligand-inducible proteolytic cleavage site.
  • the BTTS lacks an S3 ligand-inducible proteolytic cleavage site.
  • the BTTS lacks both an SI ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site.
  • the BTTS includes an S3 ligand-inducible proteolytic cleavage site; and lacks both an SI ligand- inducible proteolytic cleavage site and an S2 ligandinducible proteolytic cleavage site.
  • the fusion protein may have an vWF A2 sequence or a variation thereof, an ADAMTS13 cleavage site (which may be described by the consensus sequence HEXXHXXGXXHD; SEQ ID NO:7; Crawley, Blood. 2011 118:3212-21), and an S3 or y- secretase cleavage site, although many other arrangements exist.
  • the switch may contain components that arc borrowed from Notch. In other embodiments, the switch may not contain components that are from Notch.
  • BTTSs including but not limited to chimeric notch receptor polypeptides
  • BTTSs are primarily single polypeptide chains.
  • BTTSs including chimeric notch receptor polypeptides
  • constitutive joining of two portions of a split BTTS may be achieved by inserting a constitutive heterodimerization domain between the first and second portions of the split polypeptide such that upon heterodimerization the split portions are functionally joined.
  • MESA polypeptides comprises: a) a ligand binding domain; b) a transmembrane domain; c) a protease cleavage site; and d) a functional domain.
  • the functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor).
  • a MESA receptor comprises two polypeptide chains.
  • a MESA receptor comprises a single polypeptide chain.
  • Non-limiting examples of MESA polypeptides are described in, e.g., U.S. Patent Publication No. 2014/0234851; the disclosure of which is incorporated herein by reference in its entirety.
  • Useful BTTSs that may be employed in the subject methods include, but are not limited to, polypeptides employed in the TANGO assay.
  • the subject TANGO assay employs a TANGO polypeptide that is a heterodimer in which a first polypeptide comprises a tobacco etch virus (Tev) protease and a second polypeptide comprises a Tev proteolytic cleavage site (PCS) fused to a transcription factor.
  • Tev tobacco etch virus
  • PCS Tev proteolytic cleavage site
  • TANGO polypeptides are described in, e.g., Barnea et al. (Proc Natl Acad Sci USA. 2008 Jan. 8; 105( 1 ):64-9); the disclosure of which is incorporated herein by reference in its entirety.
  • a subject vWF cleavage domainbased BTTS will generally include: an extracellular domain comprising a first member of a binding pair; a von Willebrand Factor (vWF) cleavage domain comprising a proteolytic cleavage site; a cleavable transmembrane domain and an intracellular domain.
  • vWF von Willebrand Factor
  • 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 disclosure of which is incorporated herein by reference in its entirety.
  • the "SNIPR" switch is another example of a BTTS (Zhu et al 2022 Cell. 185: 1431-1443 and WO2021061856), although others exist and/or can be readily designed.
  • Expression of the BTTS in the cell may be constitutive or inducible, e.g., by binding of another BTTS to an antigen on another cell in the patient.
  • transcriptional activators that can be pail of the fusion protein are numerous and include artificial transcription factors (ATFs) such as, e.g., Zinc-finger-based artificial transcription factors (including e.g., 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.
  • ATFs artificial transcription factors
  • Zinc-finger-based artificial transcription factors including e.g., 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 Bio
  • the transcriptional activator may contain a GAL4 DNA binding domain, which binds to the Gal4 responsive UAS, which has been well characterized in the art.
  • suitable transcriptional activators include GAL4-VP16 and GAL4-VP64, although many others could be used.
  • the identity of the transcription activators may vary.
  • the transcription factor may have a DNA binding domain that binds to a corresponding promoter sequence and an activation domain.
  • the DNA binding domain transcription factor may be independently selected from Gal4-, LexA, Tet-, Lac-, dCas9-, zinc-finger- and TALE-based transcription factors.
  • TALE- and CRISPR/dCas9-based transcription factors are described in Lebar (Methods Mol Biol. 2018 1772: 191-203), among others.
  • the binding sites for such domains are well known or can be designed at will.
  • the transcription factors can have any suitable activation domain, e.g., VP16, VP64, Ela, Spl, VP16, CTF, GAL4 among many others.
  • the extracellular binding domain of the BTTS may bind to a tissue- or organ- specific cell-surface marker, a disease-specific cell-surface marker, or an off-target cell-surface marker, depending on how the cell is being used. For example, if one wanted to dampen the effects of the cytotoxic cells in the brain and/or spinal cord then the BTTS may have an extracellular domain that binds to a brain and/or CNS-specific cell-surface marker (e.g., MOG, CDH10, BCAN, CSPG5, PTPRZ1 or NRCAM) which are both preferentially expressed in the brain).
  • CNS-specific cell-surface marker e.g., MOG, CDH10, BCAN, CSPG5, PTPRZ1 or NRCAM
  • the BTTS may have an extracellular domain that binds to a pancreatic cell surface marker (e.g., GP2, CD 133, ion transport regulator 2 (FXYD2), tetraspanin 7 (TSPAN7), transmembrane protein 27 (TMEM27), discoidin domain receptor tyrosine kinase 1 (DDR1) and delta/notch- like EGF repeat containing (DNER), dispatched homologue 2 (DISP2), seizure related 6 homologue like (SEZ6L2), low density lipoprotein receptor- related protein 11 (LRP11), HEPACAM family member 2 (HEPACAM2), TSPAN7 and TMEM27, etc.) Tissue-specific cell-surface markers are available for the eye, retina, heart, skeletal muscle, smooth muscle, adrenal gland, parathyroid gland, thyroid gland, pituitary gland, lung, bone marrow, lymphoid
  • a pancreatic cell surface marker e.g., GP2, CD 133,
  • the extracellular binding domain may bind to a tissue or organ-specific cell surface marker in a transplanted organ (e.g., pancreas, liver, lung, or kidneys, etc.), thereby protecting it from attack from killer T cells.
  • a transplanted organ e.g., pancreas, liver, lung, or kidneys, etc.
  • the BTTS may bind to CD 19, for example.
  • the extracellular binding domain may bind to a marker in the off-target sites.
  • this marker may vary depending on the therapy being used.
  • many off-target markers are listed as “NOT” antigens in Dannenfelser (Cell Syst. 2020 11: 215-228) WO 2017/193059, WO 2020/097395 and PCT/US2021/045796).
  • the circuit may comprise a nucleic acid containing a promoter that is activated by the released transcriptional activator, and a coding sequence encoding an anti-inflammatory cytokine.
  • anti-inflammatory cytokine is intended to encompass natural molecules that have antiinflammatory activity (e.g., Il-lra, IL-4, IL-10, IL-11, IL-13, IL-35 and TGF-P), as well as nonnatural or “engineered” cytokines that have anti-inflammatory activity.
  • cytokines are secreted from the cell and their coding sequence will encode a secretion signal.
  • IL10 variants are described in Saxton et al (Science 2021 371: 6535); TGFb mimics are described in Johnston et al (Science Immun. 20205: 50); IL35 variants are described in Collison et al (Science 2021 371: 6535); and CD25-biased IL2 variants are described in Khoryati et al (Science Imm. 2020 5: 50), which publications are incorporated by reference for disclosure of the sequences.
  • pro-inflammatory cytokine sink is intended to refer to a protein that specifically binds to a pro-inflammatory cytokine (e.g., IL-2, CCL-21, IL-12, IL-7, IL-15 or IL- 21, etc.) and prevents it from binding with its cognate receptor on another immune cell.
  • the cytokine sink comprises at least the extracellular domain of a receptor for a pro-inflammatory cytokine, e.g., at least the extracellular domain of IL-1R, IL-2R/CD25, IL- 12R, IL-18R, TNFR1, TNFR2, IFNGR, GM-CSFR, etc., or a part thereof that binds to its cognate ligand.
  • the cytokine sink may have the extracellular domain of IL-1R (which binds to IL-1), IL-2R or CD25 (which binds to IL-2), IL-12R, IL-18R (which binds to IL- 18), TNFR1 and TNFR2 (which binds to TNF-a), IFNGR (which binds to IFNy) and GM- CSFR (which binds to GMCSF), or a subunit thereof that binds to its ligand.
  • This domain may be tethered to the cell via a transmembrane domain or it may be secreted.
  • a truncated or mutated form of the receptor may be used so that the receptor is incapable of signaling.
  • the full-length receptor may be used.
  • the cell may not have the internal machinery to transduce a signal from that receptor to the nucleus.
  • sink may contain the extracellular domain of CD25 (which is the receptor for IL-2), although others could be used too.
  • the cell may express CD25, which not only acts as a cytokine sink but it also causes the engineered CD4+ T cell to proliferate when it bind to its ligand. Stimulation of CD25 should survival/persistence of the T cells in the host.
  • an antibody e.g., a scFv that binds to the pro-inflammatory cytokine may be used.
  • the antibody may be tethered to the cell, e.g., via a transmembrane domain, or secreted.
  • the circuit may comprise a nucleic acid containing a promoter that is activated by the released transcriptional activator, and a coding sequence encoding a pro- inflammatory cytokine sink.
  • Ectonucleotidases are nucleotide metabolizing enzymes that are expressed on the plasma membrane and have externally oriented active sites. These enzymes metabolize nucleotides to nucleosides. Extracellular adenosine generated by the ectonucleotidases CD39 and CD73 is a newly recognized “immune checkpoint mediator” that is believed to interfere with anti-tumor immune responses. Expressing an ectonucleotidase such as CD39 or CD73 on a cell should dampen the immune response around that cell.
  • the circuit may comprise a nucleic acid containing a promoter that is activated by the released transcriptional activator, and a coding sequence encoding a ectonucleotidase .
  • expression of two or more of (a)-(e) may be induced by binding of the BTTS to the cell surface marker.
  • the different proteins may be on different constructs with the same promoter or their expression may be coordinated by an IRES.
  • IRES an IRES.
  • Other ways for co-expressing two proteins are known.
  • the two or more of (a)-(c) may be on the same vector or different vectors.
  • binding of BTTS to the non-target cell activates expression of one or more other proteins.
  • binding of the binding domain of the BTTS to the antigen on the surface of another cell induces proteolytic cleavage of the one or more forcedependent cleavage sites to release the transcriptional activator.
  • the released transcriptional activator then binds to a promoter that drives the expression of the one or more other proteins, thereby inducing expression of the one or more other proteins.
  • the general principles of a circuit are described in WO 2016/138034, U.S. Patent No. 9,670,281, U.S. Patent No.9,834,608, Roybal et al. Cell (2016) 167(2):419-432, Roybal et al. Cell (2016) 164(4):770-9, and Morsut et al. Cell (2016) 164(4):780-91, among others.
  • the present circuits make use of an engineered immune receptor that recognizes an antigen on a target cell, where binding of the engineered immune receptor to the antigen on the target cell (in the absence of the immunosuppressive cell) activates the cytotoxic immune cell and induces the cytotoxic immune cell to kill the target cell.
  • CARs and TCRs are examples of such immune receptors, although others are known.
  • chimeric antigen receptor and “CAR”, used interchangeably herein, refer to artificial multi-module molecules capable of triggering or inhibiting the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand/antigen binding domain), a transmembrane domain and one or more intracellular signaling domains.
  • the term CAR is not limited specifically to CAR molecules but also includes CAR variants.
  • CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules.
  • CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional heterodimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014/127261 Al and US Patent Application No. 2015/0368342 Al, the disclosures of which are incorporated herein by reference in their entirety).
  • CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR.
  • CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation.
  • CAR molecules and derivatives thereof i.e., CAR variants are described, e.g., in PCT Application No. US2014/016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ral72; 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.
  • Useful CARs also include the anti-CD19 — 4-1BB — CD3 ⁇ CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).
  • T cell receptor and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR complex is a disulfide-linked membrane- anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (P) chains expressed as part of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric p or y5 forms.
  • the complete endogenous TCR complex in heterodimeric P form includes eight chains, namely an alpha chain (referred to herein as TCRa or TCR alpha), beta chain (referred to herein as TCRP or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains.
  • TCRa or TCR alpha alpha chain
  • beta chain referred to herein as TCRP or TCR beta
  • delta chain gamma chain
  • two epsilon chains two zeta chains.
  • a TCR is generally referred to by reference to only the TCRa and TCRP chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and/or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate.
  • TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains.
  • engineered TCRs may include individual modified TCRa or modified TCRp chains as well as single chain TCRs that include modified and/or unmodified TCRa and TCR0 chains that are joined into a single polypeptide by way of a linking polypeptide.
  • the immune receptor may be bind to a cancer-associated antigen.
  • the antigen may be associated with hematological cancers (e.g., CD19, CD20, CD22, CD25, CD30 or CD33) or a solid tumor. Examples of cancer-associated antigens that are in solid tumors are listed in the table below.
  • this method may comprise administering a cell therapy described above to the subject.
  • primary immune cells may be purified from an individual, constructs encoding the above proteins may be introduced into the cells ex vivo, and the recombinant cells may be expanded and administered to the subject, e.g., by injection.
  • pre-made allogeneic cells (which may have abrogated MHC class I molecules) may be used instead.
  • the subject may have cancer.
  • the subject may be receiving a course of cytotoxic immune cells (e.g., CAR T or NK cells) that kill cancer cells in an antigen-specific manner.
  • the BTTS can be targeted to off- target sites (i.e., normal tissue that is not cancerous), thereby providing a way to protect those sites.
  • the BTTS may be an extracellular binding domain that binds to cells that are not part of the cancer.
  • Exemplary cell surface markers for off-target sites may be listed as "NOT" antigens in Dannenfelser (Cell Syst. 2020 11: 215-228) WO 2017/193059, WO 2020/097395 and PCT/US2021/045796), as described above.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneally ); s.c., subcutaneous(ly); and the like.
  • Engineered immune cells can produce immuno-suppressive payloads in response to a specific antigen.
  • Human CD4+ T cells can selectively induce immune inhibitory cytokine TGFpi in response to CD 19 antigen using SynNotch (measured by flow cytometry).
  • SynNotch measured by flow cytometry.
  • the results shown in Fig. 1 show that expression of the suppressive cytokine TGFb can be induced by synNotch binding to CD 19 on another cell.
  • Example 2 Suppressor cells that produce combination of TGFb (suppressive cytokine) and CD25 (IL2 sink) are very effective at suppressing CAR T killing in vitro.
  • T cells inducibly producing a combination of inhibitory cytokine TGF[ 1 and pro- inflammatory cytokine sink CD25 using synNotch show strong suppression of CAR T cell proliferation and killing in vitro.
  • In vitro immune suppression was assayed by co-culturing three cells: (1) human CD4+ T cells with anti-CD19 SynNotch inducing production of CD25, TGFfH, or both payloads, (2) K562 target cells expressing both a synNotch antigen, CD19, and a CAR antigen, Her2, and (3) human CD8+ T cells expressing an anti-Her2 4- IBB CAR. Cell counts were tracked over time using flow cytometry.
  • T cells inducibly producing a combination of inhibitory cytokine IL 10 and pro- inflammatory cytokine sink CD25 using synNotch show strong suppression of CAR T cell proliferation and killing in vitro.
  • In vitro immune suppression was assayed by co-culturing three cells: (1) human CD4+ T cells with anti-CD19 SynNotch inducing production of CD25, IL 10, or both payloads, (2) K562 target cells expressing both a synNotch antigen, CD19, and a CAR antigen, Her2, and (3) human CD8+ T cells expressing an anti-Her2 4- IBB CAR. Cell counts were tracked over time using flow cytometry.
  • Example 4 Suppressor cells that produce combination of TGFb (suppressive cytokine) and CD25 (IL2 sink) are very effective at suppressing CAR T killing of tumors in vivo
  • Synthetic immune-suppressive cells can locally suppress immune response.
  • K562 tumors, Her2+ and Her2+ CD19+ were subcutaneously injected in the flanks of N.S.G. mice. These mice were treated with either no T cells, anti-Her2 CAR T cells only, or anti-Her2 CAR T cells and synthetic suppressor cells (human CD4+ T cells with an anti-CD19 synNotch induciblely producing TGFpi and CD25) by i.v. injection after 7 days. Tumor volume was monitored by caliper measurement. Synthetic suppressor cells show strong local suppression of CAR T cell killing in the CD 19+ tumor without suppressing CAR T cell killing of the CD 19- tumor.
  • Engineered T cells overexpressing CD25 increases consumption of IL2 and cell proliferation.
  • Example 6 synNotch->IL10 synthetic suppressor cells can block autoimmune cell proliferation in brain and CNS in mouse neuroinflammation model.
  • IL10 expression can be activated by mouse brain specific antigen (CDH10).
  • SynNotch circuits in CD4+ T cells can reconstitute Treg-like functions to drive immune suppression.
  • Human CD4+ T cells with anti-CD19 synNotch circuits inducing different immune suppressive payloads were mixed with CD19+ or CD19- K562 target cells.
  • the level of CD25 on the engineered CD4+ T cells was measured by antibody staining after 72 hours of incubation with target cells.
  • the level of TGFb or IL10 cytokine secretion was measured by ELISA of the supernatant after 72 hours. This data is shown in Fig. 7B.
  • T cells Human CD4+ T cells were engineered to constitutively overexpress CD25. T cells were grown in media that contained added IL2. Cell counts were measured by flow cytometry of in vitro cell culture and IL2 levels were measured by ELISA of supernatant. This data is shown in Fig. c.
  • Human CD8+ T cells were engineered with an anti-Her2 CAR (killer T cells).
  • Human CD4+ T cells (suppressor T cells) were engineered with anti-CD19 synNotch circuits that either induces one or two pay loads. These T cells were mixed with K562 target cells that are engineered to express both Her2 and CD19. Cell counts were measured by flow cytometry. This data is shown in Fig. 7D.
  • BTTS circuits in CD4 + T cells induce antigen- specific production of immune suppressive payloads; suppressor T cells inducing immune suppressive payloads block CAR T cell expansion and killing in vitro; and suppressor T cells producing the combination of an inhibitory cytokine (TGFb or IL10) with CD25 leads to significantly stronger suppression of CAR T cell expansion and killing in vitro.
  • TGFb or IL10 an inhibitory cytokine
  • Synthetic suppressor cells act locally to block inflammation without systemic immune suppression in vivo
  • Two K562 tumors (Her2+ or Her2+ CD19+) were injected subcutaneously into two flanks of NSG mice. A week after tumor injection, anti-Her2 CAR T cells only or anti-Her2 CAR T cells and anti-CD19 synthetic suppressor T cells inducing different suppressive pay loads were injected i.v. Tumor volume was measured by calipers. This data is shown in Figs. 8A-8C.
  • present two cell system provides a robust two-cell NOT gate for CAR T cells in vivo; and the present two cell NOT gate is capable of blocking T cells from killing locally (dual antigen tumor) without systemic suppression (single antigen tumor) in vivo.
  • Fig. 9 shows replicates data using different T cell donors. The data is consistent between replicates.
  • Fig. 10 shows a comparison over an iCAR not gate.
  • the two cell NOT gate has improved performance over the iCAR NOT gate.
  • Suppressor T cells can protect bystander cells under immune attack
  • Fig. 12 shows that suppressor T cells protect bystander target cells that are in the neighborhood of cells with synNotch priming ligand.
  • target cells that have CD 19 synNotch priming ligand
  • the data shows that suppressor T cells can protect both priming cells and bystander cells from CAR T cell killing. This data shows that suppressor T cells can overcome heterogenous priming antigen expression.
  • suppressor T cells do not self-inactive when synNotch is active. Suppressor cells can continue to produce suppressive payloads like TGFb that inhibit TCR signaling during suppression. See Fig. 13. This data shows that suppressor T cell induce suppressive responses that are not dependent on TCR signaling.
  • Suppressor T cells can be programmed to induce non-native suppression programs
  • Suppressor T cells can produce a diverse set of suppressive signals and combinations, including non-native combinations like PD-L1 + CD25 which show synergistic suppression of CAR T cells.
  • the data shown in Fig 14 shows that synthetic suppressor T cells can induce custom programs that are not in endogenous suppressor cells such as Tregs.

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Abstract

L'invention concerne une thérapie cellulaire comprenant : (i) un lymphocyte T cytotoxique comprenant un récepteur immunitaire modifié qui reconnaît un antigène sur une cellule cible; et (ii) une cellule immunosuppressive comprenant un circuit moléculaire comprenant (a) un commutateur transcriptionnel déclenché par liaison (BTTS) qui reconnaît un antigène sur une cellule non cible et (b) une protéine anti-inflammatoire : la liaison du récepteur immunitaire modifié à l'antigène sur la cellule cible en l'absence de la cellule immunosuppressive activant le lymphocyte T cytotoxique et la liaison du BTTS à l'antigène sur la surface d'une cellule non cible activant l'expression de la protéine anti-inflammatoire par la cellule immunosuppressive et protégeant la cellule non cible du lymphocyte T cytotoxique. L'invention concerne également des méthodes de traitement.
PCT/US2024/020272 2023-03-21 2024-03-15 Porte non à deux cellules comprenant des cellules immunosuppressives synthétiques Ceased WO2024196802A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180355011A1 (en) * 2015-02-24 2018-12-13 The Regents Of The University Of California Binding-triggered transcriptional switches and methods of use thereof
US20220127373A1 (en) * 2018-11-08 2022-04-28 The Regents Of The University Of California Systems and methods for targeting cancer cells
WO2022118310A1 (fr) * 2020-12-01 2022-06-09 Lepton Pharmaceuticals Ltd. Méthodes d'amélioration de l'efficacité thérapeutique de cellules isolées pour la thérapie cellulaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180355011A1 (en) * 2015-02-24 2018-12-13 The Regents Of The University Of California Binding-triggered transcriptional switches and methods of use thereof
US20220127373A1 (en) * 2018-11-08 2022-04-28 The Regents Of The University Of California Systems and methods for targeting cancer cells
WO2022118310A1 (fr) * 2020-12-01 2022-06-09 Lepton Pharmaceuticals Ltd. Méthodes d'amélioration de l'efficacité thérapeutique de cellules isolées pour la thérapie cellulaire

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