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WO2024232911A1 - Compositions de car-t commutables contenant des motifs barnase-barstar - Google Patents

Compositions de car-t commutables contenant des motifs barnase-barstar Download PDF

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WO2024232911A1
WO2024232911A1 PCT/US2023/066644 US2023066644W WO2024232911A1 WO 2024232911 A1 WO2024232911 A1 WO 2024232911A1 US 2023066644 W US2023066644 W US 2023066644W WO 2024232911 A1 WO2024232911 A1 WO 2024232911A1
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car
moiety
barstar
cell
barnase
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Sergey DEEV
Alexander Gabibov
Alexey Stepanov
Richard A. Lerner
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Scripps Research Institute
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Scripps Research Institute
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • 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]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
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    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4203—Receptors for growth factors
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/27—Endoribonucleases producing 3'-phosphomonoesters (3.1.27)
    • C12Y301/27002—Bacillus subtilis ribonuclease (3.1.27.2)
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
    • A61K2239/15—Non-antibody based
    • A—HUMAN NECESSITIES
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    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A61K2239/24—Dimerizable CARs; CARs with adapter
    • A—HUMAN NECESSITIES
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C12N2510/00—Genetically modified cells

Definitions

  • CAR T cell therapy has proven its efficacy in therapy -resistant chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • CRS cytokine release syndrome
  • Another concern in CAR-based therapy is the choice of target.
  • CD 19 is a suitable target for CAR T cells because it is expressed only by B cells populations.
  • targeted antigens are also expressed at low levels but broadly across various normal tissues.
  • the on-target off-tissue activity of CAR T cells has resulted in severe toxicities in the translation of CAR-T therapy to solid tumors in the clinic. Therefore, developing approaches to control CAR T cell activity after infusion is a high priority.
  • a promising solution to this problem is the design of switchable CARs.
  • artificial molecular switchers "bridge" the tumor cell with CAR T cells. By varying the switcher concentration, it became possible to control cytotoxicity and the sensitivity of the CAR T cells to antigen density.
  • the predictable pharmacokinetics and pharmacodynamics of switcher allow for controlling CAR T cell effector functionality and persistency.
  • bi-functional moieties could be used to control CAR T activity in vivo.
  • the invention provides chimeric antigen receptors (CARs) that contain in the extracellular domain a bamase moiety or a barstar moiety.
  • CARs chimeric antigen receptors
  • the invention provides CAR-T cells that contain such CARs.
  • the invention provides switch molecules for targeting inert CAR-T cells.
  • the switch molecules contain a barnase moiety or a barstar moiety that is fused to a targeting moiety that specifically recognizes a surface molecule of a tumor cell.
  • the invention provides chimeric antigen receptor T (CAR-T) cell combinations (platforms or systems) for targeting a tumor cell.
  • the combinations contain (a) a CAR-T cell comprising in the extracellular domain of its CAR a barstar moiety, and a switch molecule comprising a cognate barnase moiety that is fused to a targeting moiety, or (b) a CAR-T cell comprising in the extracellular domain of its CAR a barnase moiety, and a switch molecule comprising a cognate barstar moiety that is fused to a targeting moiety; wherein the targeting moiety specifically recognizes a surface molecule of the tumor cell.
  • the employed barstar moiety and the barnase moiety are from Bacillus amyloliquefaciens.
  • the employed barnase moiety contains a full length barnase protein
  • the barstar moiety contains a full length barstar protein or variant thereof.
  • the employed barnase moiety contains the sequence shown in SEQ ID NO: 1, a conservatively modified variant thereof, or a sequence substantially identical thereto
  • the employed barstar moiety contains the sequence shown in any one of SEQ ID NOs:2-6, a conservatively modified variant thereof, or a sequence substantially identical thereto.
  • the switch molecule contains a barnase moiety that is covalently linked to the targeting moiety via a spacer motif.
  • the CAR molecules contains a barstar moiety that is inserted at its N-terminus via a linker that is at least about 12 amino acid residues in length.
  • the employed linker contains tandem repeats of GGGGS (SEQ ID NO:21), GGGS (SEQ ID NO: 10) or GGS.
  • the employed targeting moiety contains a designed ankyrin repeat protein (DARPin), an antibody moiety or a small molecule.
  • the targeting moiety is a designed ankyrin repeat protein (DARPin), and the DARPin is fused at its C- terminus via a linker to a barnase moiety.
  • the employed DARPin is specific for a tumor antigen.
  • the tumor antigen is HER2 or EpCAM.
  • the employed DARPin is DARPin G3 (SEQ ID NO:7), DARPin 9.29 (SEQ ID NO:8), or DARPin ECI (SEQ ID NO:9).
  • the invention provides methods for targeting a CAR-T cell to a tumor cell present in a population of heterogeneous cells.
  • the methods entail contacting the population of heterogeneous cells with a CAR-T combination that contains (a) a CAR-T cell having in the extracellular domain of its CAR a barstar moiety, and a switch molecule having a cognate barnase moiety that is fused to a targeting moiety, or (b) a CAR-T cell having in the extracellular domain of its CAR a barnase moiety, and a switch molecule having a cognate barstar moiety that is fused to a targeting moiety.
  • the targeting moiety is the switch molecule specifically recognizes a surface molecule of the tumor cell.
  • the employed barstar moiety and barnase moiety in the CAR-T combination are from Bacillus amyloliquefaciens.
  • the employed barnase moiety contains a full length barnase
  • the barstar moiety contains a full length barstar protein or a barstar variant.
  • the employed targeting moiety contains a designed ankyrin repeat protein (DARPin).
  • the targeted tumor cell is a breast cancer cell.
  • the invention provides methods for treating a cancer in a subject.
  • the methods involve administering to the subject a switchable CAR-T combination that contains (a) a CAR-T cell having in the extracellular domain of its CAR a barstar moiety, and a switch molecule having a cognate barnase moiety that is fused to a targeting moiety, or (b) a CAR-T cell having in the extracellular domain of its CAR a barnase moiety, and a switch molecule having a cognate barstar moiety that is fused to a targeting moiety.
  • the targeting moiety in the switch molecule specifically recognizes a cell surface molecule of the cancer.
  • the employed barstar moiety and barnase moiety in the CAR-T combination are from Bacillus amyloliquefaciens.
  • the employed targeting moiety contains a designed ankyrin repeat protein (DARPin) that is specific for the cancer.
  • DARPin ankyrin repeat protein
  • the cancer to be treated is breast cancer.
  • FIG. 1 Cell death analysis of the 9.29-Bn treated BT-474 cells with Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit.
  • the Annexin V Alexa Fluor 488 (AV) fluorescence was analyzed in the FL1 channel (excitation laser 488 nm, emission filter 530/30 nm), the propidium iodide (PI) fluorescence was analyzed in the FL3 channel (excitation laser 488 nm, emission filter 670LP nm).
  • Quadrants bottom left - live cells (AV-, PI-), bottom right - early apoptosis (AV-, PI+), top right - late apoptosis/necrosis (AV+, PI+), top left - cell debris (AV-, PI+).
  • FIG. 3 Clonogenic assay. BT-474 cells were incubated with Trastuzumab, Barnase, and 9.29-Bn and evaluated with crystal violet staining after three weeks of cultivation.
  • Figure 4 The sequences of the barstar mutants and spacers (SEQ ID NOs:25-30, respectively) used for generation of the barnase binding domain of BsCAR. Variant: 1 - wild type, 2 - cysteine alanine mutant (C40 and C82), 3 - wild type with an extended flexible GS hinge (or linker) sequence, 4 - cysteine alanine mutant (C40 and C82) with an extended flexible GS hinge, 5 - mutant of 187 (I87E) with the extended flexible hinge, 6 - double mutant (C40, C82, and I87E) with the extended flexible hinge.
  • Variant 1 - wild type, 2 - cysteine alanine mutant (C40 and C82), 3 - wild type with an extended flexible GS hinge (or linker) sequence, 4 - cysteine alanine mutant (C40 and C82) with an extended flexible GS hinge, 5 - mutant of 187 (I87E) with the extended flexible hinge, 6 - double mutant (
  • FIG. 5 The luciferase reporter assay.
  • A The diagram illustrating BsCAR Jurkat reporter luciferase assay. The reporter luciferase gene is under the control of the NF AT promoter.
  • ECl-Bn induces BsCAR T cells to kill EpCAM-positive tumor cells.
  • the data were analyzed by two-way ANOVA with Sidak’s multiple comparisons and represented as mean ⁇ SD. Statistical significance: **p ⁇ 0.01, ****p ⁇ 0.0001.
  • FIG. 7 The monotherapy with the BsCAR T cells or 9.29-Bn does not suppress tumor growth in vivo.
  • A Tumor growth kinetics and
  • Figure 8. 9.29-Bn drives BsCAR T cells into the tumor. Single-cell suspensions were stained with anti-IgG4-APC antibodies and FITC labeled barnase (Bn-FITC) to detect BsCAR and analyzed by flow cytometry.
  • Bn-FITC FITC labeled barnase
  • FIG. 9 BsCAR T cells targeted by DARPin-Bn suppress HER2 -positive tumors in vivo.
  • CAR T cell therapy has become a critical milestone in modem oncotherapy.
  • CAR T cells therapy has become a critical milestone in modem oncotherapy.
  • the problem of safety and efficacy of CAR T cells therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects.
  • Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for precise control of CAR T cell cytotoxicity.
  • the RNA together with Ribonucleases (RNases) was shown to possess a broad therapeutic potential.
  • RNases Ribonucleases
  • the barnase-barstar toxin-antitoxin system is orchestrated by the RNase toxin bamase and inactivated by the cognate barstar antitoxin, representing an outstanding example of molecular switching based on the extraordinary affinity of the bamase/barstar complex (KD-W 14 ).
  • the present invention is derived in part from studies undertaken by the inventors to guide barstar-modified CAR (BsCAR) T cells to tumor cells by barnase-based molecular switches.
  • BsCAR barstar-modified CAR
  • RNase toxin bamase as a targeting module
  • an additional benefit of the system is the cytotoxic effects by barnase itself.
  • barnase was fused with designed ankyrin repeat proteins (DARPins) that are specific to tumor antigens HER2 (human epidermal growth factor receptor 2) and EpCAM.
  • DARPin-Bn DARPin-barnase
  • BsCAR barstar CAR
  • DARPin-barnase switches enable targeting of different tumor antigens with a single BsCAR.
  • a gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads.
  • the switchable BsCAR T cell therapy was able to eradicate the HER2 -positive ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.
  • the present invention provides novel switchable CAR-T compositions and their use in cancer therapies as described in detail below.
  • the present invention can be performed using standard procedures, as described, for example in Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1st edition (1997) (ISBN-13: 978-0121821906); U.S. Pat. Nos.
  • Anticalin® proteins are based on human lipocalins, a family of proteins that normally transport small molecules such as steroids and lipids within the body. They are a promising new class of next generation therapeutics. Anticalin proteins are smaller than conventional protein or antibody therapeutics and offer the potential for greater penetration into target tissues and delivery through non-injectable routes such as inhalation. Inhaled delivery of Anticalin proteins directly to the lungs, for example, could potentially improve patient convenience and tolerability, and enable use of lower doses compared to systemically administered antibodies. See, e.g., Rothe and Skerra, BioDrugs. 2018; 32: 233-243.
  • binding target such as the binding of an immunoglobulin or small molecule agent to a target molecule or antigen, e.g., an epitope on a particular polypeptide, peptide, or other target (e.g. a glycoprotein target), and means binding that is measurably different from a non-specific interaction (e.g., a non-specific interaction can be binding to bovine serum albumin or casein).
  • Specific binding can be measured, for example, by determining binding of a binding moiety (e.g., a small molecule agent), or an immunoglobulin, to a target molecule compared to binding to a control molecule.
  • a binding moiety e.g., a small molecule agent
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.
  • the term “specific binding” or “specifically binds to” or is “specific for” a particular target molecule or an epitope on a particular target molecule can be exhibited, for example, by a molecule having a I ⁇ d for the target of at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater.
  • the term “specific binding” refers to binding where a binding moiety binds to a particular target molecule or epitope on the target molecule without substantially binding to any other molecule or
  • conjugation refers to any and all forms of covalent or non-covalent linkage, and include, without limitation, direct genetic or chemical fusion, coupling through a linker or a cross-linking agent, and non-covalent association.
  • fusion is used herein to refer to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their coding nucleotide sequences.
  • fusion explicitly encompasses internal fusions, i.e., insertion of sequences of different origin within a polypeptide chain, in addition to fusion to one of its termini.
  • fusion is used herein to refer to the combination of amino acid sequences of different origin.
  • epitope includes any molecular determinant capable of specific binding to an immunoglobulin.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain aspects, can have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an immunoglobulin.
  • a “binding region” is a region on a binding target bound by a binding molecule.
  • target or “binding target” is used in the broadest sense and specifically includes polypeptides, without limitation, nucleic acids, carbohydrates, lipids, cells, and other molecules with or without biological function as they exist in nature.
  • antigen refers to an entity or fragment thereof, which can bind to an immunoglobulin or trigger a cellular immune response.
  • An immunogen refers to an antigen, which can elicit an immune response in an organism, particularly an animal, more particularly a mammal including a human.
  • antigen includes regions known as antigenic determinants or epitopes, as defined above.
  • a nucleic acid is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • Percent (%) amino acid sequence identity with respect to a peptide or polypeptide sequence, i.e., the h38C2 antibody polypeptide sequences or GCN4 derived peptides identified herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • Two sequences are "substantially identical” if they have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the well-known sequence comparison algorithms or by manual alignment and visual inspection.
  • Treating” or “treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a targeted pathologic condition or disorder.
  • Those in need of treatment include those already with the disorder, as well as those prone to have the disorder, or those in whom the disorder is to be prevented.
  • a subject or mammal is successfully “treated” for cancer, if, after receiving a therapeutic amount of a subject immunoconjugate according to the methods of the present invention, the subject shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slowing to some extent and preferably stopping) of cancer cell infiltration into peripheral organs, including the spread of cancer into soft tissue and bone; inhibition (i.e., slowing to some extent and preferably stopping) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent of one or more of the symptoms associated with the specific cancer; reduced morbidity and/or mortality, and improvement in quality of life issues.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • betabranched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the term “contacting” has its normal meaning and refers to combining two or more agents (e.g., polypeptides or phage), combining agents and cells, or combining two populations of different cells.
  • Contacting can occur in vitro, e.g., mixing an antibody and a cell or mixing a population of antibodies with a population of cells in a test tube or growth medium.
  • Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by co-expression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
  • Contacting can also occur in vivo inside a subject, e.g., by administering an agent to a subject for delivering the agent to a target cell.
  • subject refers to human and non-human animals (especially nonhuman mammals).
  • subject is used herein, for example, in connection with therapeutic and diagnostic methods, to refer to human or non-human animals.
  • specific examples of non-human subjects include, e.g., cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.
  • Artificial T cell receptors also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs) or T-bodies
  • CARs chimeric antigen receptors
  • T-bodies are engineered receptors, which graft an arbitrary specificity onto an immune effector cell.
  • these receptors are used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral or lentiviral vectors or by transposons.
  • CAR- engineered T cells are genetically engineered T cells armed with chimeric receptors whose extracellular recognition unit is comprised of an antibody- derived recognition domain and whose intracellular region is derived from one or more lymphocyte stimulating moieties.
  • the structure of the prototypic CAR is modular, designed to accommodate various functional domains and thereby to enable choice of specificity and controlled activation of T cells.
  • the preferred antibody-derived recognition unit is a single chain variable fragment (scFv) that combines the specificity and binding residues of both the heavy and light chain variable regions of a monoclonal antibody.
  • the most common lymphocyte activation moieties include a T-cell costimulatory (e.g.
  • CD28 and/or 4-1BB domain in tandem with a T-cell triggering (e.g. CD3zeta) moiety.
  • T-cell triggering e.g. CD3zeta
  • the engineered cell is re-directed with a pre-defined specificity to any desired target antigen, in a non-HLA restricted manner.
  • CAR constructs are introduced ex vivo into T cells from peripheral lymphocytes of a given patient using retroviral or lentiviral vectors or transposons. Following infusion of the resulting CAR-engineered T cells back into the patient, they traffic, reach their target site, and upon interaction with their target cell or tissue, they undergo activation and perform their predefined effector function.
  • Therapeutic targets for the CAR approach include cancer and HIV-infected cells, or autoimmune effector cells.
  • a switchable CAR-T system (“CAR-T platform” or “CAR-T combination”) refers to a pharmaceutical combination that contains a CAR-T switch molecule and a complementary CAR-T cell.
  • the CAR-T switch molecule (“CAR-T switch”) contains a targeting moiety (e.g., a DARPin, an antibody or antigen-binding fragment thereof) that is capable of specifically binding to a target molecule on the surface of a target cell (e.g., a tumor cell).
  • the CAR-T switch is also able to bind to the CAR of the complementary CAR-T cell.
  • the extracellular domain of the CAR of the CAR-T cell contains a binding moiety (e.g., a barstar moiety or an antibody moiety) that specifically recognizes a cognate binding partner (e.g., a bamase moiety, an antigen, a peptide, or a small molecule) in the CAR-T switch.
  • a binding moiety e.g., a barstar moiety or an antibody moiety
  • a cognate binding partner e.g., a bamase moiety, an antigen, a peptide, or a small molecule
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment.
  • Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors”.
  • the invention provides switchable CAR-T systems (also termed switchable CAT- T platforms or switchable CAR-T combinations) that are based on the barnase-barstar protein-protein binding.
  • the systems contain a switchable molecule that contains a barnase moiety that is conjugated to (e.g., covalently fused to ) a targeting moiety, and a corresponding CAR-T cell having in the extracellular domain of its CAR a barstar moiety that specifically binds to the bamase moiety in the switch molecule.
  • the systems can have a switchable molecule that contains a barstar moiety conjugated to a targeting moiety, and a corresponding CAR-T cell having in the extracellular domain of its CAR a barnase moiety that specifically binds to the barstar moiety in the switch molecule.
  • barnase moiety refers to a bamase protein or fragment thereof that is capable of forming a strong non-covalent binding with a cognate barstar molecule.
  • barstar moiety refers to a barstar protein or fragment thereof that is capable of forming a strong non-covalent binding with a cognate barnase molecule.
  • barnase and barstar binding partners have been identified in many organisms including a number of bacterial species such as Bacillus amyloliquefaciens .
  • barnase (a portmanteau of "BActerial” “RiboNucleASE”) is a bacterial protein that consists of 110 amino acids and has ribonuclease activity.
  • Bamase is lethal to Bacillus amyloliquefaciens when expressed without its inhibitor barstar. The inhibitor binds to and occludes the ribonuclease active site, preventing bamase from damaging the cell's RNA after it has been synthesized but before it has been secreted.
  • the barnase/barstar complex is noted for its extraordinarily tight protein-protein binding, with an on-rate of 10 8 s -1 M -1 .
  • Structure and function of barnase have been well characterized in the art. See, e.g., Buckle etal., Biochemistry. 33 (30): 8878-8889, 1994; Serrano el al., J. Mol. Biol. 224 (3): 783 -804, 1992; Serrano el al., J. Mol. Biol. 224 (3): 805 818, 1992; Matouschek et al., J. Mol. Biol. 224 (3): 224 (3): 819-835, 1992; and Mossakowska el al., Biochem.
  • Barstar is a small protein synthesized by the bacterium Bacillus amyloliquefaciens. Its function is to inhibit the ribonuclease activity of its binding partner barnase, with which it forms an extraordinarily tightly bound complex within the cell until bamase is secreted. Expression of barstar is necessary to counter the lethal effect of expressed active bamase.
  • the structure of the bamase-barstar complex is known. See, e.g., Buckle et al., Biochemistry. 33 (30): 8878-89, 1994; Hartley, Trends Biochem. Sci. 14 (11): 450-454, 1989; and Hartley, J. Mol. Biol. 202 (4): 913-915, 1988.
  • bamase proteins known in the art may be employed in the construction of the switchable CAR-T system of the invention. These include the bamase enzyme of the first, identified barnase-barstar pair in Bacillus amyloliquefaciens . Hartley, J. Mol. Biol. 202 (4), 913-915, 1988. As used in the switch molecules exemplified herein, sequence of this bamase protein (without the N-terminal Met residue) is shown in SEQ ID NO: 1. As noted below, barnases from many other organisms are also known in the art. Similar to bamases, many barstar orthologs and variants known and well characterized in the art are suitable for the invention.
  • the barstar partner used for constructing the CAR of the CAR-T cell should preferably be from the same species or organism. This is just to ensure the bamase and barstar pair will provide the optimized binding activity that, is essential to targeted delivery of the CAR-T cell by the switch molecule.
  • the employed barstar moiety and/or barnase moiety are full length proteins, including wildtype proteins and mutants (e.g., Bacillus amyloliquefacien barstar C40A/C82A mutant exemplified herein).
  • the employed barstar moiety and/or barnase moiety can be a fragment of the full length protein. So long a barnase fragment (or a barstar fragment) maintains the ability to bind to the binding partner, it can be employed in the practice of the invention. Sequences of the various barnase/barstar proteins and functional characterizations have been reported in the literature.
  • barnase-binding region on Bacillus amyloliquefaciens barstar and the barstar-binding site on the cognate barnase protein are known. See, e.g., Jones et al., FEBS 331 : 165-172, 1993; and Sahu et al., PROTEINS: Structure, Function, and Genetics 41 :460-474, 2000. Suitable barnase fragments and/or barstar fragments can be readily designed and examined in accordance with such information that is well known in the art.
  • barnase and barstar molecules including natural or engineered variants thereof from any species may be employed in the practice of the invention.
  • some preferred embodiments of the invention utilize the cognate barnase and barstar pair from the same species to construct the switch molecule and the switchable CAR- T cells.
  • Bacillus amyloliquefacien barnase and barstar binding partners are exemplified herein (e.g., SEQ ID NOs: l and 2, respectively), barstar and barnase binding pairs from other organisms, including fragment sequences, can also be employed in the practice of the invention due to functional identity and structural similarities.
  • Xenorhabdus szentirmaii barnase (Accession PHM34870), Xenorhabdus budapestensis barnase (Accession PHM27417), Xenorhabdus beddingii barnase (Accession OTA 19270), Bacillus paralicheniformis barnase (Accession AJ020109), Bacillus stratosphericus barnase (Accession EMI12858), Pseudomonas aeruginosa barstar (Accession OPF35620), Bacillus subtilis barstar (Accession AOL98511), Bacillus licheniformis barstar (Accession
  • AOP 16948 Caudoviricetes sp. barstar (Accession DAF83522), Burkholderia cenocepacia barstar (Accession, CAG2486045), and Burkholderia pseudomallei barstar (Accession VCG90737).
  • the switchable CAR-T systems of the invention contain a switch molecule that can target the corresponding CAR-T cells to a target molecule of interest (e.g., a tumor antigen).
  • a target molecule of interest e.g., a tumor antigen
  • the invention also encompasses their components, including the switch molecules, the corresponding CAR sequences and inert CAR-T cells.
  • the switch molecule is typically a conjugation molecule (e.g., a fusion protein) having a targeting moiety that is fused to (e.g., covalently linked to) a barnase moiety or a barstar moiety.
  • the switch molecule contains a barnase moiety
  • the corresponding CAR-T has in the extracellular domain the cognate barstar moiety.
  • the switch molecule contains a barstar moiety
  • the corresponding CAR-T has in the extracellular domain the cognate barnase moiety.
  • the barnase moiety (or barstar moiety) is fused at its N-terminus to the targeting moiety.
  • the barnase moiety (or barstar moiety) may be fused at its C-terminus to the targeting moiety.
  • a linker or spacer is used to connect the targeting moiety to the barnase moiety (or the barstar moiety).
  • the switch molecule contains a targeting moiety that is linked to the barnase moiety (or the barstar moiety).
  • the targeting moiety specifically recognizes a target molecule (e.g., a tumor antigen) present on the surface of a target cell of interest (e.g., a tumor cell).
  • target molecule e.g., a tumor antigen
  • target cell of interest e.g., a tumor cell.
  • targeting agent or “targeting moiety” as used herein refers to a moiety that recognizes, binds or adheres to a target molecule located in a cell, tissue (e.g. extracellular matrix), fluid, organism, or subset thereof.
  • the target molecule can contain an antigen.
  • the target molecule can be a protein, a lipid moiety, a glycoprotein, a glycolipid, a carbohydrate, a polysaccharide, a nucleic acid, an MHC -bound peptide, or a combination thereof.
  • the target molecule is a molecule located on the surface of the target cell, esp. a tumor cell surface receptor.
  • the targeting moiety of the employed CAR-T switch binds to a target molecule that is present on the surface of a target tumor cell, e.g., Her2- or EpCAM-binding DARPins as exemplified herein.
  • a targeting moiety and its cognate target molecule represent a binding pair of molecules, which interact with each other through any of a variety of molecular forces including, for example, ionic, covalent, hydrophobic, van der Waals, and hydrogen bonding, so that the pair have the property of binding specifically to each other.
  • Specific binding means that the binding pair exhibit binding with each other under conditions where they do not bind to another molecule.
  • binding pairs are biotin-avidin, horm one- receptor, receptor-ligand, enzyme-substrate, IgG-protein A, antigen-antibody, and the like.
  • the targeting moiety and its cognate target molecule exhibit a significant association for each other.
  • the targeting moiety is a known or selected DARPin molecule that is capable of specifically binding to a target molecule, e.g., a tumor antigen such as Her2 or tumor-associated antigen EpCAMas as exemplified herein.
  • DARPins an acronym for designed ankyrin repeat proteins
  • DARPins are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat proteins.
  • DARPins consist of tightly packed ankyrin repeats, each forming a P-tum and two antiparallel a-helices. A single repeat typically consists of 33 amino acids, six of which form the binding surface.
  • these sites are used to introduce the codons of random amino acids, except for cysteine (to avoid the formation of disulfide bonds), as well as glycine and proline (since some amino acids are part of the a-helix).
  • DARPins are typically formed by two or three of the binding motifs contained between the N- and C-terminal motifs shielding the hydrophobic regions.
  • DARPins are suitable for the construction of CAR-T switch molecule of the invention. Their use in the switch molecules also provides other beneficial properties in addition to their small size.
  • the ease of production in bacteria allows one to create fusion proteins and add sequences for purification and labeling, while the absence of cysteine residues in the DARPin molecule allows one to introduce a unique additional cysteine for precise conjugation.
  • DARPins are small proteins that are extremely thermostable (their melting point (Tm) can reach 90°C) and resistant to proteases and denaturing agents. They can be produced in bacteria with a high yield of up to 200 mg of protein from 1 liter of liquid culture.
  • the DARPin in the switch molecule is specific to a target molecule (e.g., a cell surface receptor) that is expressed by a tumor cell , e.g., HER2, EGFR or EpCAM.
  • a target molecule e.g., a cell surface receptor
  • a tumor cell e.g., HER2, EGFR or EpCAM.
  • DARPins G3 SEQ ID NO:7
  • DARPin 9.29 SEQ ID NO:8
  • DARPin ECI SEQ ID NO: 9
  • the barnase and barstar based switchable CAR-T system of the invention can also utilize many other agents as the targeting moiety. These include, e.g., antibody or antigen-binding fragments such as Fab or scFv, other antibody mimetics, Anticalin® proteins and small molecular compounds.
  • the targeting moiety is a targeting polypeptide such as a targeting antibody or antigen-binding fragment thereof (e.g., an Fab).
  • the targeting antibody can be human, fully human, humanized, human engineered, non-human, and/or chimeric antibody.
  • a non-human antibody to be used in the CAR-T switch can be humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • the targeting antibody can specifically bind to different target molecules on tumor cells, e.g., CD19, Her2, CLL1, CD33, CD123, EGFR, EGFRvIII, CD20, CD22, CS1, BCMA, CEA or a fragment thereof.
  • the targeting moiety of the CAR-T switch is an antibody or antigen-binding fragment that contains humanized heavy and/or light chain sequences.
  • tumor-targeting antibody sequences that can be used in the switch molecules of the invention are described, e.g., in PCT/US2017/057460, PCT/US2014/060713, PCT/US2014/060684, PCT/US2016/024524, PCT/US2016/027997, and PCT/US2016/027990.
  • the barstar (or barnase) moiety and the targeting moiety are fused together in the switch molecule.
  • a structural component (e.g., a peptide terminus) of the barstar (or barnase) moiety is joined with or linked to a terminus of a polypeptide targeting moiety (e.g., a DARPin or an antibody).
  • a polypeptide targeting moiety e.g., a DARPin or an antibody.
  • the barstar (or barnase) moiety and the targeting moiety are fused together via a linker.
  • suitable linkers include peptide linker EFPKPSTPPGSSGGAP (SEQ ID NO:20) exemplified herein.
  • the barstar (or barnase) moiety is attached to the targeting moiety in a site-specific manner. Attachment in a site-specific manner may entail attaching the barstar (or barnase) moiety to a predetermined site on the targeting moiety. In some embodiments, site-specific attachment can entail attaching the barstar (or barnase) moiety to an unnatural amino acid in the targeting moiety.
  • the switchable CAR-T systems of the invention also contain a complementary CAR-T cell that is targeted by the switch molecule to a desired site (e.g., an antigen on a tumor cell) in therapeutic applications.
  • a desired site e.g., an antigen on a tumor cell
  • any CAR-T cell that can be bound by the employed CAR-T switch via barnase-barstar binding can be used in the methods of the invention.
  • the CAR-T cells contain a chimeric antigen receptor (CAR) that contains an extracellular domain, a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the extracellular domain also contains a barstar moiety (or alternatively, a barnase moiety).
  • a linker motif can be employed to connect the barstar moiety to the other motifs in the extracellular domain.
  • a GS liker containing tandem repeats of GGS, GGGS (SEQ ID NO: 10), or GGGGS (SEQ ID NO:21) can be inserted between the barstar sequence (or barnase sequence) and the hinge region in the extracellular domain of the CAR.
  • the linker motif contains at least about 12, 13, 14, 15, 16, 17, 18, 19, 20, or more residues, e.g., (GGGGS) 3 (SEQ ID NO: 13), (GGGGS) 4 (SEQ ID NO:22) or (GGGGS)s (SEQ ID NO:23).
  • GGGGS 3
  • GGGGS 4
  • GGGGS GGGGS
  • SEQ ID NO:23 a relatively long linker enables high surface expression, maximum interaction with barnase, and high cytotoxicity.
  • Other domains of the CAR sequence can also be separated by a suitable linker or peptide spacer, e.g., a peptide linker inserted between the extracellular domain and the transmembrane domain.
  • the CAR molecule is capable of specifically binding to the switch molecule that contains a barnase moiety (or a barstar moiety).
  • a barnase moiety or a barstar moiety
  • the barnase and barstar pair respectively present in the switch molecule and the CAR molecule are preferably cognate binding partners from the same organism.
  • the CAR molecule of the CAR-T cells also contains a transmembrane domain and an intracellular domain. These domains can be derived from any protein domains that have been routinely employed in the construction of CAR-T cells.
  • the transmembrane domain can be the transmembrane domain of CD8 or CD28.
  • the intracellular signaling domain can contain signaling domains such as CD3( ⁇ , FcR-y, and Syk-PT as well as co-signaling domains such as CD28, 4- IBB, and CD 134.
  • the intracellular signaling domain of the CAR can contain (a) a CD3-zeta domain, plus (b) a CD28 domain, a 4-1BB domain, or both a CD28 domain and a 4- IBB domain.
  • a hinge region can be present in the CAR to connect the extracellular domain with the transmembrane domain.
  • a barstar-containing CAR for producing CAR-T cells of the invention is shown in SEQ ID NO: 11. It contains at the N-terminus of the extracellular domain the B. amyloliquefaciens barstar C40A/C82A mutant sequence (SEQ ID NO:3), which is followed by a hinge region (SEQ ID NO: 14) and the IgG4 CH2-CH3 domain (SEQ ID NO: 15).
  • a GS linker e.g., (GGGGS) 3 ; SEQ ID NO: 13
  • the CAR sequence additionally contains a CD28 transmembrane and intracellular domain SEQ ID NO: 17), a 4-1BB intracellular domain (SEQ ID NO:18), and a CD3z intracellular domain (SEQ ID NO: 19).
  • a linker or spacer motif, GSTSGSGKPGSGEGSTKG (SEQ ID NO: 16) is positioned in the CAR sequence between the extracellular domain and thew transmembrane domain.
  • the CAR can additionally have a N-terminal signal peptide sequence, e.g., IL-2 signal peptide MYRMQLLSCIALSLALVTNS (SEQ ID NO: 12) as exemplified herein.
  • the complementary and inert CAR-T cells to be used in the invention can be prepared in accordance with methods well known in the art or specific protocols exemplified in, e.g., W02018/075807, WO2015057834, WO2015057852, and Marcu-Malina et al., Expert Opinion on Biological Therapy, Vol. 9, No. 5.
  • recombinant technology can be used to introduce CAR-encoding genetic material into any suitable T-cells, e.g., human T cells such as central memory T-cells.
  • the CAR-T cells to be used in the methods of the invention can be generated by transduction of human T cells with a lentiviral vector expressing the engineered CAR (e.g., a humanized CAR).
  • a lentiviral vector expressing the engineered CAR e.g., a humanized CAR
  • humanized CAR sequences and CAR-T cells harboring such humanized CAR sequences are known in the art. They can all be readily employed and/or adapted for use in the methods of the invention. See, e.g., W02018/075807; Li et al., Biomarker Research 8: 36, 2020; and Maude et al., Blood 128: 217, 2016.
  • the invention provides methods for using the switchable CAR-T compositions described herein for targeting CAR-T cells to a cellular site (e.g., a target molecule on the surface of a target tumor cell), and methods of using the compositions in treating various types of tumors or cancer.
  • a cellular site e.g., a target molecule on the surface of a target tumor cell
  • the CAR-T cell used in these therapeutic applications has a barstar moiety (or a bamase moiety) in the extracellular binding domain of its chimeric antigen receptor.
  • the CAR-T cell When the CAR-T cells is co-administered with a corresponding switch molecule containing a cognate barnase moiety (or a barstar moiety), the CAR-T cell is attached to the switch molecule due to the bamase-barstar binding activity, and is site-specifically delivered to a target molecule of interest (e.g., a tumor antigen) by the targeting moiety in the switch molecule.
  • a target molecule of interest e.g., a tumor antigen
  • the generic and inert CAR-T cells which can covalently bind to and be activated by the co-administered switch molecule can be prepared in accordance with methods well known in the art or specific protocols exemplified in, e.g., W02018/075807, WO2015057834, WO2015057852, and Marcu-Malina et al., Expert Opinion on Biological Therapy, Vol. 9, No. 5.
  • the barstar moiety (or barnase moiety) is linked to a synthetic molecule containing one or more of the following domains: an IgG Fc CH2-CH3 domain, a spacer or hinge region (e.g., a CD28 sequence or a IgG4 hinge-Fc sequence), a transmembrane region (e.g., a transmembrane canonical domain), and an intracellular T-cell receptor (TCR) signaling domain, thereby forming a chimeric antigen receptor (CAR) or T- body.
  • an IgG Fc CH2-CH3 domain e.g., a CD28 sequence or a IgG4 hinge-Fc sequence
  • a transmembrane region e.g., a transmembrane canonical domain
  • TCR intracellular T-cell receptor
  • Intracellular TCR signaling domains that can be included in a CAR (or T-body) include, but are not limited to, CD3( ⁇ , FcR-y, and Syk-PT signaling domains as well as the CD28, 4- IBB, and CD 134 co-signaling domains.
  • Recombinant technology can be used to introduce CAR-encoding genetic material into any suitable T-cells, e.g., human T cells such as central memory T-cells.
  • the CAR-T cells to be used in the methods of the invention can be generated by transduction of human T cells with a lentiviral vector expressing the engineered CAR.
  • the lentiviral vector can contain an expression cassette that fuses the sequences of a barstar moiety (or a barnase moiety) to a human IgG4 hinge, and optionally also a IgG Fc CH2-CH3 spacer to complete the extracellular domain.
  • the extracellular domain in the sequence encoded by the expression cassette is followed by a transmembrane segment of CD28 and the cytoplasmic signaling domains of 4-1BB and CD3( ⁇ .
  • the vector Downstream of the CAR and linked by a T2A ribosomal skip element, the vector can further encode a truncated epidermal growth factor receptor (EGFRt) sequence.
  • EGFRt epidermal growth factor receptor
  • the switch molecule and the otherwise inert CAR-T cells are coadministered to a subject in need of treatment, the CAR-T cells become bound by the switch molecule due to the barnase-barstar interaction, and are delivered to the cellular site recognized by the targeting moiety in the switch.
  • Administration of the switch and the CAR- T cells can be simultaneous or sequential. The administration can be performed in accordance with standard protocols of immunotherapy. In some preferred embodiments, switch and the CAR-T cells are administered to the subject by infusion.
  • the invention further provides pharmaceutical compositions that contain a switch molecule and/or a CAR-T cell described herein and a pharmaceutically acceptable carrier.
  • compositions can be prepared from any of the switch molecules and inert CAR-T cells described herein.
  • the pharmaceutically acceptable carrier can be any suitable pharmaceutically acceptable carrier. It can be one or more compatible solid or liquid fillers, diluents, other excipients, or encapsulating substances which are suitable for administration into a human or veterinary patient (e.g., a physiologically acceptable carrier or a pharmacologically acceptable carrier).
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the use of the active ingredient, e.g., the administration of the active ingredient to a subject.
  • the pharmaceutically acceptable carrier can be co-mingled with one or more of the active components, e.g., a switch molecule, and with each other, when more than one pharmaceutically acceptable carrier is present in the composition, in a manner so as not to substantially impair the desired pharmaceutical efficacy.
  • Pharmaceutically acceptable materials typically are capable of administration to a subject, e.g., a patient, without the production of significant undesirable physiological effects such as nausea, dizziness, rash, or gastric upset. It is, for example, desirable for a composition comprising a pharmaceutically acceptable carrier not to be immunogenic when administered to a human patient for therapeutic purposes.
  • compositions of the invention can additionally contain suitable buffering agents, including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • suitable buffering agents including, for example, acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt.
  • the compositions can also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.
  • Pharmaceutical compositions of the invention can be presented in unit dosage form and can be prepared by any suitable method, many of which are well known in the art of pharmacy. Such methods include the step of bringing the antibody of the invention into association with a carrier that constitutes one or more accessory ingredients.
  • composition suitable for parenteral administration conveniently comprises a sterile aqueous preparation of the inventive composition, which preferably is isotonic with the blood of the recipient.
  • This aqueous preparation can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3 -butane diol.
  • Suitable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed, such as synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid can be used in the preparation of injectables.
  • Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • compositions of the invention and their various routes of administration can be carried out in accordance with methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20 th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • the delivery systems useful in the context of the invention include time-released, delayed release, and sustained release delivery systems such that the delivery of the inventive composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated.
  • the inventive composition can be used in conjunction with other therapeutic agents or therapies.
  • release delivery systems can avoid repeated administrations of the inventive composition, thereby increasing convenience to the subject and the physician, and may be particularly suitable for certain compositions of the invention.
  • Suitable release delivery systems include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides
  • hydrogel release systems such as sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides
  • sylastic systems such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-di-and triglycerides
  • peptide based systems such as fatty acids or neutral fats
  • wax coatings such as those described in U.S.
  • pump-based hardware delivery systems can be used, some of which are adapted for implantation.
  • SEQ ID NO: 1 (Accession CAA31365; Bacillus amyloliquefaciens bamase) AQVINTFDGV ADYLQTYHKL PDNYITKSEA QALGWVASKG NLADVAPGKS IGGDIFSNRE GKLPGKSGRT WREADI NY I S GFRNSDR1LY SSDWLIYKTT DHYQTFTKIR
  • SEQ ID NO:2 wildtype Bacillus amyloliquefaciens barstar
  • SEQ ID NO: 3 (B. amyloliquefaciens barstar C40A/C82A mutant)
  • SEQ ID NO:4 B. amyloliquefaciens barstar I87E mutant
  • SEQ ID NO:5 (B. amyloliquefaciens barstar C40A/C82A/I87E mutant) KKAVINGEQ IRSISDLHQT LKKELALPEY YGENLDALWD ALTGWVEYPL VLEWRQFEQS KQLTENGAES VLQVFREAKA EGADITIELS
  • SEQ ID NO:6 (Accession OCB92323; B. amyloliquefaciens barstar variant)
  • DARPin G3 (SEQ ID NO:7) (Zahnd et al., J. Mol. Biol., 369: 1015-28, 2007) MDLGKKLLEAARAGQDDEVRILMANGADVNAKDEYGLTPLYLATAHGHLEIVEVL LKNGADVNAVDAIGFTPLHLAAFIGHLEIAEVLLKHGADVNAQDKFGKTAFDISIGN GNEDLAEILQKLN [0078] DARPin 9-29 (SEQ ID NO: 8) (Steiner et al., J. Mol.
  • DARPin ECI SEQ ID NO:9 (Stefan et al., J. Mol. Biol., 413: 826-43, 2011) MDLGKKLLEAARAGQDDEVRILMANGADVNAHDFYGITPLHLAANFGHLEIVEVL
  • IL-2 signal peptide SEQ ID NO: 12: MYRMQLLSCIALSLALVTNS
  • GS linker (SEQ ID NO: 13): GGGGSGGGGSGGGGS
  • IgG4 hinge (SEQ ID NO: 14): ESKYGPPCPSCP
  • IgG4 CH2-CH3 domain (SEQ ID NO: 15):
  • Linker 2 (SEQ ID NO: 16): GSTSGSGKPGSGEGSTKG
  • CD28 transmembrane and intracellular domain SEQ ID NO: 17:
  • Example 1 DARPins-bamase switching modules mediate HER2-specific targeting
  • Fusion proteins of anti-HER2 DARPins (G3 and 9.29) with barnase were used as molecular switches directing BsCAR T cells against HER2-positive tumor cells.
  • DARPins G3 and 9.29 were reported to interact with different domains of the HER2 receptor, which is frequently overexpressed in solid tumors.
  • G3- Bn and 9.29-Bn proteins specifically interact with HER2-positive BT-474 human aggressive ductal breast carcinoma cell line.
  • HER2 overexpression resulted in specific targeting of BT- 474 cells by G3-Bn and 9.29-Bn, which was not observed in human MDA-MB-231 epithelial breast cancer cells with normal HER2 expression and in HER2-negative hamster CHO cells.
  • phosphatidylserine exposure to the outer plasma membrane was analyzed by the Annexin V assay. The signs of apoptosis were detected after 48-72 h after incubation (Fig. 2).
  • HER2-specific DARPin-Bamase conjugates required both the HER2 targeting moiety and the functional RNAse component, as we did not observe similar cytotoxicity in a clonogenic assay with trastuzumab or unconjugated barnase at equimolar concentrations (Fig. 3).
  • Example 2 BsCAR T cells provide DARPin-Bn guided cytotoxicity
  • a panel of barstar mutants was created to optimize the extracellular bamase- binding module of CAR.
  • Barstar amino acid sequence contains unpaired cysteines (C40 and C82) and isoleucine 87 that may lead to homodimerization. If barstar dimerizes, it could block the bamase interaction site on CAR.
  • the interaction of DARPins-Bn with barstar in the context of CAR requires a certain flexibility, which could be achieved by inserting peptide linkers. To address these potential issues, a panel of BsCARs was designed.
  • BsCARs were based on wild type barstar and barstar point mutants having short (G4S) and long 3X(G4S) linkers (short linkers: BsCARvl, wild type; BsCARv2, C40A and C82A substitutions, and long linkers: BsCARv3, wild type; BsCARv4, C40A and C82A, BsCARv5, 187E, BsCARv6, C40A, C82A, and I87E) (Fig. 4). All CAR variants had a mutated IgG4 hinge with two point mutations (L235E; N297Q) to suppress interaction with FcyR and promote CAR T cells persistence.
  • CAR comprised a membrane and an intracellular part of CD28, intracellular cytoplasmic activation domain of 4-1BB (CD137), and CD3( ⁇ . All variants of BsCAR were detected on the T cell surface at similar levels, except for BsCARv5. BsCAR T cells were stained with DARPin 9.29-Bn-FITC to determine which BsCAR variant retained the ability to interact with bamase. Only four out of six BsCAR variants (v2, v3, v4, and v6) efficiently interacted with bamase, and BsCARv4 demonstrated the strongest binding.
  • the cytotoxicity of the BsCAR variants towards BT-474 cells was evaluated in the presence of 1 nM 9.29-Bn or G3-Bn.
  • the BsCAR variants v2, v3, v4 (p ⁇ 0.0001), and v6 (p ⁇ 0.05) exerted cytotoxic effects mediated by 9.29-Bn or G3-Bn switches.
  • the BsCARv4 T cells induced death of tumor cells more efficiently than other BsCAR variants (p ⁇ 0.0001).
  • BsCARv4 was chosen for the following experiments based on its surface expression, barnase binding, and high cytotoxicity.
  • the switchable BsCAR T cells enable targeting multiple tumor antigens.
  • the extracellular domain of the HER2 receptor has four subdomains.
  • the DARPins 9.29-Bn and G3-Bn fusions are specific to subdomains I and IV of HER2, respectively, that allows for targeting different sites of the tumor antigen by the same BsCAR and tuning the CAR T cell activity.
  • Incubating BT-474 target cells with BsCAR T cells with increasing concentrations of the 9.29-Bn or G3-Bn proteins resulted in dosedependent cytotoxicity.
  • a dose-response effect was observed by varying the 9.29-Bn and G3-Bn concentration and cell ratio (Effector to Target ratio, E:T) in the BsCAR T cell functional assays.
  • the modular approach of the switchable CAR T cells allows redirecting of the BsCAR T cells to other cancer antigens.
  • EpCAM-specific fusion based on DARPin ECI (Stefan et al., J. Mol. Biol. 413, 826-843, 2011) linked with barnase was designed to demonstrate antigen switching.
  • Example 4 BsCAR T cell therapy eradicates HER2-positive xenogeneic ductal carcinoma in vivo
  • DARPins-Bn and BsCAR T cells Tumor growth was inhibited by the combination of DARPins-Bn and BsCAR T cells .
  • the tumor was eliminated after the second cycle of 9.29-Bn administration (Fig. 9).
  • the DARPin G3- Bn that binds to the membrane-proximal domain of HER2 did not completely suppress tumor growth.
  • Monotherapy with the dose escalation cycles of 9.29-Bn or infusion of BsCAR T cells alone did not suppress tumor growth in vivo (Fig. 7).
  • Recruitment of CD8 T cells to the tumor was observed only in animals that received the combination of BsCAR T cells and 9.29-Bn or G3-Bn.
  • Tumor-infiltrating BsCAR T cells represented -18% of total cells in the tumors from the 9.29-Bn treated group (Fig. 8).
  • Example 5 Some exemplified materials and methods
  • Cell culture Cell lines were maintained in media (DMEM or RPMI 1640) supplemented with 10% FBS (Gibco), 10 mM HEPES, 100 U/mL penicillin, 100 ug/mL streptomycin, and 2 mM GlutaMAX (Gibco).
  • Cell lines of human ductal carcinoma BT-474 (HTB-2; ATCC), MDA-MB-231 (HTB-26; ATCC), HEK293T lentiviral packaging cell line (Clontech) and Chinese hamster ovary CHO (Russian Cell Culture Collection) were incubated in a humidified atmosphere with 5% CO2 at 37 °C.
  • the BT-474 cell line was transduced with lentiviruses to generate a stable line expressing Flue (pCDH-CMV-LUC- EF1 Hygro, #129437, addgene), and Flue-positive cells were enriched by Hygromycin B selection.
  • the cell lines were repeatedly tested for mycoplasma contamination (MycoReport Mycoplasma Detection Kit, Evrogen, Russia).
  • FITC conjugation FITC-labeled Bn, DARPins 9.29-Bn, G3-Bn, and Trastuzumab (Roche) were prepared as follows. 100 pg of the protein in 90 pL of PBS buffer was rapidly mixed with 10 pL FITC in DMSO at concentrations of 1 g/L, 1 g/L, 1 g/L, and 0.3 g/L, respectively. The proteins were incubated overnight at RT and purified from the unreacted FITC molecules using Zeba Spin Desalting Columns, 7k MWCO (Pierce, USA) according to the manufacturer’s recommendations.
  • Confocal laser scanning microscopy Protein binding was visualized by confocal laser scanning microscopy. Cells were incubated with 2 pg/mL of proteins and Hoechst 33342 (1 pg/mL) in PBS with 1% BSA on ice for 30 min, washed from unbound proteins, and imaged by confocal laser scanning microscopy using an LSM 980 (Zeiss) confocal microscope under the following conditions: excitation 488 nm, emission 492-550 nm for FITC detection and excitation 405 nm, emission 410-520 nm for Hoechst33342 detection.
  • Resazurin toxicity assay Protein cytotoxicity was tested using a resazurin-based assay. Cells were seeded on a 96-well plate at 5* 10 3 cells per well in 100 pL of DMEM medium supplemented with 10% FBS and cultured overnight. The proteins were added to wells in 100 pL of DMEM growth medium, and cells were incubated for 10 days. The medium was then removed, and 100 pL of resazurin solution (0.13 g/L in PBS) was added to the cells.
  • the IC50 value was determined by GraphPad Prism software.
  • Cytotoxicity assays The cytotoxicity of engineered T cells was evaluated using the standard lactate dehydrogenase release assay (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega) following the manufacturer’s recommendations. The 5* 10 3 BT-474 cells were cocultured with Mock T or BsCAR T cells for 12-16 and in the presence of 10 to 0.016 nM or 1 nM in case of E:T titration of DARPins-Bn in RPMI (Gibco) media supplemented with 40 U/ml of human IL-2 (R&D).
  • CAR T cell cytokine detection For the cytokine release assays, 5xl0 3 BT-474 cells were mixed with 5xl0 4 Mock T cells or BsCAR T cells in the 96-well plate for 24 h in the presence of different concentrations of DARPins-Bn in RPMI without IL-2. Basal levels of IFN-y and IL-2 were detected in non-stimulated CAR T samples. The supernatant was separated from cells by centrifugation (4 °C, 300g, 5 min), transferred to a new 96-well plate, and stored at -20°C. IL-2 and IFN-y secretion by human CAR T cells were analyzed by cytokine-specific ELISA kits (Vector-best, Russia) according to the manufacturer's instructions. The IC50 values were determined by GraphPad Prism software.
  • mice were housed under specific pathogen-free conditions in the Pushchino Animal Breeding Facility IBCh RAS (Bioresource collection "Collection of laboratory rodents SPF category for basic, biomedical and pharmacological research"). The experiments were conducted on six- to eight-week-old female and male NSG (NOD/SCID/IL2rynull) mice with an average weight of 16 to 20 g were used. All procedures were approved by the IBCh RAS Institutional Animal Care and Use Committee. The mice were inoculated subcutaneously with 2xl0 6 BT-474 FLuc HER2-overexpressing cancer cells in 30% Matrigel in 100 pL of complete culture medium.
  • IBCh RAS Bioresource collection "Collection of laboratory rodents SPF category for basic, biomedical and pharmacological research”
  • the experiments were conducted on six- to eight-week-old female and male NSG (NOD/SCID/IL2rynull) mice with an average weight of 16 to 20 g were used. All procedures were approved by the I
  • mice were randomly assigned to experimental and control groups. Tumor-bearing mice from all groups were injected intravenously with 10xl0 6 BsCAR T cells. Four hours after BsCAR T cells injection, different doses of the DARPins 9.29-Bn and G3-Bn switches were administered to the mice. The doses of DARPin-Bn were escalated according to the established protocol. Every other day animals were injected with increasing doses of switches (5, 50, and 500 nmol per kg, respectively). The mice were then rested for 7-8 days, and the treatment cycle was repeated two times. Tumors were monitored every 10 days using the IVIS Spectrum In vivo Imaging System (PerkinElmer) after intraperitoneal injection of D-luciferin (GoldBio).
  • IVIS Spectrum In vivo Imaging System PerkinElmer
  • Histology Formalin-fixed paraffin-embedded 10 pm sections were stained with anti-CD8 antibodies (1 :300, clone RPA-T8) conjugated with PE. Antigen was retrieved using a heated lOmM Tris-EDTA buffer (pH 9.0) before staining. For immunostaining, sections washed with PBS buffer (pH 7.4) were incubated in PBS containing 10% (vol/vol) fetal bovine serum, 0.3 mM glycine, and 0.5% (vol/vol) Triton X-100 for 1 h, and stained with an antibody at the appropriate dilution overnight at room temperature.
  • DARPin-Bn production The genes encoding DARPin 9.29-Bamase-His5, DARPin G3-Bamase-His5, and DARPin ECl-Bamase-His5 were cloned into the pET39b plasmid between restriction sites Ndel and Hindlll as described earlier for DARPin 9.29- Bamase-His5 (Shipunova et al., ACS Appl. Mater. Interfaces 10, 17437-47, 2018). The DARPin ECI nucleotide sequence was deduced from its amino acid sequence published by Stefan et al. (J. Mol. Biol. 413, 826-43, 2011).
  • the DARPin G3 gene nucleotide sequence was deduced from a DARPin G3 amino acid sequence deposited in PDB (accession number PDB: 2JAB), as described earlier (Vorobyeva et al., Sci Rep 9, 9405, 2019).
  • the plasmid also contained the barstar gene under its natural promoter.
  • Recombinant A coll strain BL21(DE3) transformed with the appropriate plasmid was grown in the ZYM-5052 autoinduction medium supplemented with kanamycin (50 mg/L) at 25°C on a shaker in 2.5 L flasks with no more than 300 ml of medium.
  • the biomass was resuspended in the solution containing 100 mM Tris-HCl, 250 mM sucrose, 0.5 mM EDTA, pH 8.0, 0.5 mM PMSF, and lysozyme was added to a final concentration of 30 pg/ml. After 30 min the cell suspension was sonicated for 5 times for 10 s at 60 W with 2-4 min intervals on an ice bath under stirring. The resulting cell lysate was centrifuged for 30 min at 18500 g. The supernatant containing the soluble fraction of the target protein was decanted and filtered through a 0.22 pm pore size membrane (MilliporeSigma).
  • the solution containing the target protein was applied on a 5 ml HisTrap HP column equilibrated with 10 volumes of solution I (20 mM NaPi, pH 7.5, 500 mM NaCl) with 30 mM imidazole at a speed of 5 ml/min
  • the column was washed with 10 volumes of the solution I containing 30 mM imidazole at a 5 ml/min rate.
  • the sorbent was washed with 25 volumes of solution I with 6 M guanidine hydrochloride at a 1 mL/min rate.
  • DARPin-Bn free from the barstar inhibitor was renatured directly on the column by a linear downward gradient (60 column volumes) of guanidine hydrochloride 6 M - 0 M at a 1 mL/min rate. After the renaturation step, the column was washed with 10 volumes of solution II (20 mM NaPi, pH 7.5, 100 mM NaCl) at a rate of 5 mL/min. DARPin-Bn was eluted from the column with the solution II containing 250 mM imidazole at the same rate.
  • the eluate obtained after affinity chromatography was diluted with solution III (20 mM NaPi, pH 7.5) by a factor of 5 and applied rate on a 5 ml HiTrap Q HP column, equilibrated with 10 volumes solution III at a 5 ml/min.
  • the column was washed with 10 volumes of Solution III, and then DARPin-Bn was eluted at a 5 mL/min rate with a linear gradient of NaCl (0-500 mM) in Solution III.
  • the volume of the gradient was 20 column volumes.
  • the fractions containing DARPin-Bn were pooled and sterilized by filtration through a 0.22 pm membrane.
  • RNAse activity The ribonuclease activity of proteins was determined by the acid-soluble residue method using yeast RNA, as described earlier (Rushizky et al., Biochemistry 2, 787-93, 1963). The protein sample under study was dissolved at a concentration of 2 pM in 0.125 M Tris-HCl, pH 8.5, and then a series of successive 3-fold dilutions of the sample was prepared in the same buffer. A 0.125 M Tris-HCl solution, pH 8.5, was used as a control sample. To 40 pL of protein in 0.125 M Tris-HCl, pH 8.5 was mixed with 160 pL of yeast RNA at a concentration of 2 g/L on ice.
  • the reaction mixture was incubated for 15 min at +37 °C.
  • the reaction was stopped by adding 200 pL of cold 6% perchloric acid and the mixture was incubated for 15 min at 0 °C.
  • perchloric acid large RNA fragments formed a precipitate, which was centrifuged at 16000 g for 10 min at 0 °C.
  • the supernatants were diluted 20-fold and the optical density was measured at 260 nm using an Infinite M1000 Pro (Tecan) plate reader in UV-Vis-transparent plates.
  • the ECso was calculated by GraphPad Prism software.
  • T-cell isolation, activation, expansion, and transduction Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by gradient density centrifugation on a Ficoll-Paque (GE Healthcare) according to a standard protocol. All healthy donors provided informed consent. Dynabeads Untouched Human T-cells Isolation Kit (Invitrogen) was used to isolate T cells from human PBMCs. Human T-cells were activated with CD3/CD28 Dynabeads (Thermo Scientific) in a medium containing 40 lU/ml recombinant IL-2 (Pan Biotech) at a 1 : 1 ratio for 24 h.
  • PBMCs Human peripheral blood mononuclear cells
  • Activated T cells were resuspended in the 1 ml of fresh media and 1 ml of lentiviral supernatants at a concentration of I x lO 6 ml/cells.
  • Cells were seeded on 6-well plates in the presence of Polybrene 10 mg/ml (MilliporeSigma). The plate was centrifuged at 1200x g for 90 min at 32°C and incubated for 8 hours at 37°C. The culture medium was changed every 2 days, cells were grown in flasks to a density of 1.0> ⁇ 10 6 /ml.
  • CAR T Cell Activation Luciferase Assay Jurkat-LuciaTM NF AT (Jurkat) cells, transduced to express BsCAR or Mock, were used for the luciferase test as effector cells. BT-474 were used as target cells in the amount of 10 4 cells per well in a 96 well plate. Jurkat CAR T cells were added in the amount of 10 5 cells per well. After 24 h of coincubation at 37°C, the supernatant was collected for measurement. 25 pl of supernatant was separated from cells by centrifugation (4°C, 300g, 5 min) and transferred to the opaque 96-well plate.
  • Activation of the reporter Jurkat-NFAT-Lucia CAR T cells was measured by the level of luciferase activity following reaction with QUANTI-LucTM substrate (Invivogen, France) according to the manufacturer's instructions.
  • the IC50 value was determined by GraphPad Prism software.
  • Ex vivo tumor analysis For analysis of tumor-infiltrating CAR T cells, preweighed fresh tumor fragments pellets underwent a red blood cell lysis step, washed with cold PBS and filtered through a 40 pm nylon cell strainer.
  • the resulting single-cell suspensions were stained by anti-IgG4 Goat anti-Human, DyLightTM 650 (SA510137, Invitrogen) and FITC labeled barnase (Bn-FITC) to detect BsCAR and analyzed by flow cytometry (NovoCyte 2060, ACEA Biosciences, USA) in BL1 channel (excitation laser 488 nm, emission filter 530/30 nm) and BL3 (excitation laser 635 nm, emission filter 660/20 nm). Data were analyzed with NovoExpress Software (ACEA Biosciences) and Flow Jo XI 0 (Flow Jo).
  • DARPins-Bn linker constructed de novo. We considered it as an intrinsically disordered region and felt free in choosing its representative conformation, so its shape was minimized by a sculpting tool in the pymol package (Schrodinger et al., The PyMOL Molecular Graphics System, Version 2, 2020).
  • Cell Apoptosis Detection 100 pL of BT-474 cells were seeded into 96-well plates at the concentration of 30- 103 cells/mL in phenol-red free DMEM medium with 10 % FBS. After overnight cultivation, 100 pL of 1200 nM DARP_9.29-Bamase was added to each well to achieve a final concentration of 600 nM. Cells were cultured for seven days and analyzed every day with the Molecular Probes Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & Propidium Iodide (PI) (Invitrogen) according to the manufacturer's recommendations.
  • PI Propidium Iodide
  • the medium was removed, cells from 3 wells for one sample were harvested from the plastic surface with 2 mM EDTA in PBS, stained with the Cell Apoptosis Kit, and analyzed using Accuri C6 flow cytometer (BD, USA) in the FL1 channel (excitation laser 488 nm, emission filter 530/30 nm) and in the FL3 channel (excitation laser 488 nm, emission filter 670LP nm).
  • Clonogenic assay 1500 BT-474 cells in 1 mL complete DMEM medium were incubated with proteins at different concentrations and grown for three weeks at 12-well plates. After the cultivation, the medium was removed, cells were washed with 1 mL of PBS, and incubated in 1 mL of 70% EtOH in PBS for 15 min. Next, cells were incubated in 1 mL 95% EtOH for 10 min. Next, 600 pL 1% crystal violet in water was added and incubated for 30 min at room temperature. Next, wells were washed 10 times with water, and the plates scanned using EPSON Perfection 2400 Photo scanner.
  • ELISA 0.5 pg of 9.29-Bamase was absorbed per well overnight at + 4 °C in 100 pL of carbonate buffer (4 mM Na2CO3, 50 mM NaHCOs, pH 9.2) in 96-well ELISA plates. Then the wells were washed twice with 200 pL of PBS with 0.05 % Tween-20. Diluted mouse blood serum in 100 pL PBS with 1 % BSA was then added to the wells and incubated for 1 h at room temperature. Then the wells were washed twice with 200 pL of PBS with 0.05 % Tween-20.
  • the wells were added with 100 pL of anti-mouse antibodies with alkaline phosphatase in 100 pL of PBS with 1 % BSA and incubated for 1 h at room temperature. Then the wells were washed three times with 200 pL of PBS with 0.05 % Tween-20. Then p-nitrophenyl phosphate was added to the wells at a concentration of 10 g/L in glycine buffer (0.1 M glycine, 1 mM MgC12, pH 2). The reaction was stopped with a 0.1 M NaCl solution, pH 10.4, and the absorption of the samples was recorded at a wavelength of 405 nm.

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Abstract

L'invention concerne des compositions de lymphocytes T porteurs d'un récepteur antigénique chimérique (CAR-T) pour le ciblage de cellules tumorales. Les compositions contiennent (a) une cellule CAR-T comprenant dans le domaine extracellulaire de son CAR une fraction barstar, et une molécule de commutation comprenant une fraction barnase correspondante fusionnée à une fraction de ciblage, ou (b) une cellule CAR-T comprenant dans le domaine extracellulaire de son CAR une fraction barnase, et une molécule de commutation comprenant une fraction barstar apparentée fusionnée à une fraction de ciblage. La fraction de ciblage reconnaît de manière spécifique une molécule de surface d'une cellule tumorale. Les compositions permettent une liaison solide entre le CAR et la molécule de commutation. Les fractions de ciblage utilisées dans les compositions peuvent être n'importe quel agent ou composé qui reconnaît de manière spécifique une molécule cible (par exemple, un récepteur de surface cellulaire ou un antigène) sur une cellule cible (par exemple, une cellule tumorale). L'invention concerne également des méthodes thérapeutiques d'utilisation des compositions de cellules CAR-T.
PCT/US2023/066644 2023-05-05 2023-05-05 Compositions de car-t commutables contenant des motifs barnase-barstar Pending WO2024232911A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "1BRS - PROTEIN-PROTEIN RECOGNITION: CRYSTAL <STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-ARESOLUTION", PROTEIN DATA BANK; VERSION 1.4, 29 November 2017 (2017-11-29), XP093239061, Retrieved from the Internet <URL:https://www.rcsb.org/structure/1BRS> DOI: 10.2210/pdb1BRS/pdb *
ANONYMOUS: "4HRN - Structural Basis for Eliciting a Cytotoxic Effect in HER2- Overexpressing Cancer Cells via Binding to the Extracellular Domain of HER2", PROTEIN DATA BANK; VERSION 1.1 (ACCESSED VIA THE WAYBACK MACHINE), 23 July 2019 (2019-07-23), XP093239068, Retrieved from the Internet <URL:https://web.archive.org/web/20190723103148/https://www.rcsb.org/structure/4HRN> DOI: 10.1016/j.str.2013.08.020 *
NICOLAS BERY, SANDRINE LEGG, JUDIT DEBRECZENI, JASON BREED, KEVIN EMBREY, CHRISTOPHER STUBBS, PAULINA KOLASINSKA-ZWIERZ, NATHALIE : "KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe", NATURE COMMUNICATIONS, vol. 10, no. 1, 1 December 2019 (2019-12-01), pages 1 - 10, XP055767692, DOI: 10.1038/s41467-019-10419-2 *
STEPANOV ALEXEY V., ROMAN S. KALININ, VICTORIA O. SHIPUNOVA, DING ZHANG, JIA XIE, YURI P. RUBTSOV, VALERIA M. UKRAINSKAYA, ALEXEY : "Switchable targeting of solid tumors by BsCAR T cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 119, no. 46, 7 November 2022 (2022-11-07), pages e2210562119, XP093239054, DOI: 10.1073/pnas.2210562119 *

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