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WO2025193386A1 - Schémas thérapeutiques pour maladie auto-immune à l'aide de cellules immunitaires modifiées ciblant cd19 - Google Patents

Schémas thérapeutiques pour maladie auto-immune à l'aide de cellules immunitaires modifiées ciblant cd19

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Publication number
WO2025193386A1
WO2025193386A1 PCT/US2025/016015 US2025016015W WO2025193386A1 WO 2025193386 A1 WO2025193386 A1 WO 2025193386A1 US 2025016015 W US2025016015 W US 2025016015W WO 2025193386 A1 WO2025193386 A1 WO 2025193386A1
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WO
WIPO (PCT)
Prior art keywords
car
cells
targeting
patient
immune cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/016015
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English (en)
Inventor
Socorro OLIVEIRA-PORTELLA
Enrique Zudaire
Sarbani J. BHADURI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Caribou Biosciences Inc
Original Assignee
Caribou Biosciences Inc
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Filing date
Publication date
Application filed by Caribou Biosciences Inc filed Critical Caribou Biosciences Inc
Publication of WO2025193386A1 publication Critical patent/WO2025193386A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • 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
    • A61K2239/39—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by a specific adjuvant, e.g. cytokines or CpG

Definitions

  • the invention relates to therapies utilizing engineered T-cells expressing a chimeric antigen receptor (CAR-T cells).
  • CAR-T cells chimeric antigen receptor
  • the present invention describes a lymphodepletion regimen for treatment of autoimmune diseases in human patients with anti-CD19 allogeneic CAR-T cells.
  • Adoptive cells CAR-T cells
  • CAR-T cells Adoptive cells
  • Initial treatment attempts were performed with autologous (patient- derived) T cells.
  • the next frontier in cell therpy involves allogeneic (donor-derived) T cells.
  • allogeneic cells face a greater risk of destruction by the patient’s immune sytem.
  • the invention is a method of treating an autoimmune disease in a patient, the method comprising: administering to the patient a lymphodepletion regimen including 20-60 mg/kg cyclophosphamide; and administering to the patient a composition comprising CD19-targeting engineered immune cells.
  • the lymphodepletion regimen includes 20 mg/kg cyclophosphamide for 2 days.
  • the lymphodepletion regimen includes 30 mg/kg cyclophosphamide for 2 days.
  • the lymphodepletion regimen includes 60 mg/kg cyclophosphamide for 2 days.
  • the lymphodepletion regimen further comprises administering fludarabine at 25mg/m 2 per day for up to 5 days.
  • the composition comprising CD 19- targeting immune cells contains 60-100 million (6 x 10 7 -l 0 8 ) cells.
  • the composition comprising CD19-targeting immune cells contains 60-100 million (6 x 10 7 -10 8 ) viable CAR-expressing cells.
  • the composition comprising CD19-targeting immune cells contains 80 million (8 x 10 7 ) cells.
  • the composition comprising CD19-targeting immune cells contains 80 million (8 x 10 7 ) viable CAR-expressing cells.
  • the autoimmune disease is systemic lupus erythematosus (SLE) (including lupus nephritis), rheumatoid arthritis (RA), type 1 diabetes (T1D), Sjogren's syndrome, multiple sclerosis (MS), systemic sclerosis (SSc) (including resulting interstitial lung disease (ILD)), dermatomyositis, psoriatic arthritis, neuromyelitis optica spectrum disorders (NMOSD), or related autoimmune central nervous system (CNS) diseases.
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • T1D type 1 diabetes
  • Sjogren's syndrome multiple sclerosis
  • MS systemic sclerosis
  • SSc systemic sclerosis
  • ILD interstitial lung disease
  • dermatomyositis including psoriatic arthritis, neuromyelitis optica spectrum disorders (NMOSD), or related autoimmune central nervous system (CNS)
  • the CD19-targeting engineered immune cells are allogeneic CAR-T cells expressing an anti-CD19 chimeric antigen receptor (CAR).
  • the anti-CD19 CAR comprises an anti-CD19 scFv, a transmembrane domain and an intracellular stimulatory domain.
  • the anti-CD19 CAR further comprises a signal peptide and a hinge.
  • the anti-CD19 CAR comprises FMC63, a CD8 hinge, a CD8 transmembrane domain, a 4- IBB co-stimulatory domain and a CD3 zeta signaling domain.
  • the engineered immune cells are engineered using a CRISPR nuclease and a nucleic acid-targeting nucleic acid (NATNA).
  • NATNA nucleic acid-targeting nucleic acid
  • the method further comprises assessing the patient for successful lymphodepletion by assessing reduction in lymphocyte count. In some embodiments, the method further comprises assessing the patient for adverse effects of lymphodepletion selected from prolonged cytopenia and suppression of osteoblasts. In some embodiments, the method further comprises assessing the patient for occurrence of toxic events selected from cytokine release syndrome (CRS), neurotoxicity (immune effector cell-associated neurotoxicity syndrome, ICANS), and graft versus host disease (GvHD).
  • CRS cytokine release syndrome
  • ICANS immune effector cell-associated neurotoxicity syndrome
  • GvHD graft versus host disease
  • Figure 1 depicts a nucleic acid expression construct encoding the CD 19-targeting chimeric antigen receptor (CAR).
  • therapeutic benefit refers to an effect that improves the condition of the patient with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • treatment of autoimmune disease may be measured by allevialtion of symtoms or achievement and duration of remission according to a disease-specific scale (i.e., DORIS criteria for SLE, American College of Rheumatology-European League against Rheumatism (ACR-EULAR), European Scleroderma Trials and Research Group (EUSTAR) activity index, and the like).
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic, or other deleterious reaction in a patient.
  • pharmaceutically and pharmacologically acceptable preparations should meet the standards set forth by the FDA Office of Biological Standards.
  • aqueous solvents e.g., water, aqueous solutions of alcohols, saline solutions, sodium chloride, Ringer's solution, etc.
  • non-aqueous solvents e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters
  • dispersion media coatings, surfactants, gels, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, stabilizers, binders, disintegration agents, lubricants, sweetening agents, flavoring agents, and dyes.
  • concentration and pH of the various components in a pharmaceutical composition are adjusted according to well-known parameters for each component.
  • domain refers to one region in a polypeptide which is folded into a particular structure independently of other regions.
  • adoptive cell refers to a cell that can be genetically modified for use in a cell therapy treatment.
  • adoptive cells include macrophages, and lymphocytes including T cells and natural killer (NK) cells.
  • cell therapy refers to the treatment of a disease or disorder that utilizes genetically modified cells.
  • ACT adaptive cell therapy
  • examples of ACT include T-cell therapies, CAR-T cell therapies, natural killer (NK) cell therapies and CAR-NK cell therapies.
  • Lymphocyte refers to a leukocyte that is part of the vertebrate immune system. Lymphocytes include T-cells such as CD4 + and/or CD8 + cytotoxic T cells, alpha/beta T cells, gamma/delta T cells, and regulatory T cells. Lymphocytes also include natural killer (NK) cells, natural killer T (NKT) cells, cytokine induced killer (CIK) cells, and antigen presenting cells (APCs), such as dendritic cells. Lymphocytes also include tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • an effective amount and “therapeutically effective amount” of a composition refer to a sufficient amount of the composition to provide the desired response in the patient to whom the composition is administered.
  • the effective amount of each therapeutic compound in the combination may be different from the effective amount of each therapeutic compound administered alone.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • CRISPR-Cas CRISPR-associated protein
  • CRISPR system refers to the genome editing tool derived from prokaryotic organisms and comprising a nucleic acid guide molecule and a sequence-specific nucleic acid-guided endonuclease capable of cleaving a target nucleic acid strand at a site complementary to a sequence in the nucleic acid guide.
  • NATNA nucleic acid targeting nucleic acid
  • dual guide including a CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA).
  • NATNA may be comprised a single nucleic acid targeting polynucleotide (“single guide”) comprising crRNA and tracrRNA connected by a fusion region (linker).
  • the crRNA may comprise a targeting region and an activating region.
  • the tracrRNA may comprise a region capable of hybridizing to the activating region of the crRNA.
  • targeting region refers to a region that is capable of hybridizing to a sequence in a target nucleic acid.
  • activating region refers to a region that interacts with a polypeptide, e.g., a CRISPR nuclease.
  • the present invention describes a lymphodepletion regimen for treatment of autoimmune disease in human patients with anti-CD19 allogeneic CAR-T cells.
  • the autoimmune disease is selected from systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type 1 diabetes (T1D), Sjogren's syndrome, multiple sclerosis (MS), systemic sclerosis (SSc), dermatomyositis, psoriatic arthritis, neuromyelitis optica spectrum disorders (NMOSD), and related central nervous system (CNS) diseases.
  • the autoimmune disease is refractory, i.e., has not responded to a prior treatment.
  • the autoimmune disease is or has become refractory to two or more treatments. In some embodiments, the autoimmune disease has relapsed, i.e., has recurred after a prior treatment. In some embodiments, the autoimmune disease has relapsed after each of two or more treatments. In some embodiment, at least one prior treatment is CD19-targeting therapy.
  • the invention comprises a lymphodepletion regimen to be administered to a patient prior to the patient receiving autologous or allogeneic CAR-T cells.
  • the lymphodepletion regimen comprises administering 20-60 mg/kg cyclophosphamide.
  • the lymphodepletion regimen comprises administering 20-60 mg/kg cyclophosphamide for several consecutive days.
  • the lymphodepletion regimen comprises administering 20 mg/kg cyclophosphamide for 2 days.
  • the lymphodepletion regimen comprises administering 30 mg/kg cyclophosphamide for 2 days.
  • the lymphodepletion regimen comprises administering 60 mg/kg cyclophosphamide for 2 days.
  • the lymphodepletion regimen further comprises administration of fludarabine, e.g., at 25mg/m 2 per day for up to 5 days.
  • the lymphodepletion is assessed or monitored by determining reduction in lymphocyte count. Lymphocyte count can be determined e.g., by flow cytometry or a rapid point-of-care method. In some embodiments, the lymphodepletion regimen is adjusted in response to the assessment or monitoring of the lymphocyte count. In some embodiments, the patient is monitored for adverse effects of the lymphodepletion on the bone marrow such as prolonged cytopenia and suppression of osteoblasts.
  • the regimen in response to results of the monitoring, is adjusted by decreasing the amount of lymphodepleting agent being administered or the number of days that the lymphodepleting agent is administered. In some embodiments, the regimen is adjusted by increasing the amount of lymphodepleting agent being administered or the number of days that the lymphodepleting agent is administered.
  • the invention utilizes T cells isolated from a healthy donor.
  • the T cells are obtained from a blood sample of a healthy donor via leukapheresis. Techniques for isolating lymphocytes are well known in the art, see, e.g., Smith, I.W. ( 1997) Apheresis techniques and cellular immunomodulation, Ther. Apher. 1:203-206.
  • isolated lymphocytes are characterized in terms of specificity, frequency of each subtype, and function.
  • the isolated lymphocyte population is enriched for specific subsets of T cells, such as CD8 + , CD25 , or CD62L + . See, e.g., Wang etal., (2016) Mol. Therapy - Oncolytics 3: 16015.
  • a quality control measure or a characterization step is applied to the cell-containig composition.
  • the quality control measure or characterization step is determining the percentage of CD8 + cells in the composition by flow cytometry.
  • lymphocytes are activated in order to promote proliferation and differentiation into specialized lymphocytes.
  • T cells can be activated using soluble CD3/28 activators, or magnetic beads coated with anti-CD3/anti-CD28 monoclonal antibodies.
  • the invention includes the use of T cells expressing a CD19-targeting (anti-CD19) receptor.
  • the CD 19-targeting immune cell is selected from a T cell, a natural killer (NK) cell, an iNK cell.
  • the immune cell is selected from a CAR-T cell, a CAR-NK cell.
  • the CD 19-targeting protein is an anti-CD19 chimeric antigen receptor (CAR).
  • the CAR comprises an extracellular domain including a CD19-binding region, a transmembrane domain and one or more intracellular co-activation (costimulatory) and activation (stimulatory) domains.
  • the CD19-binding region of the CAR is derived from a monoclonal antibody.
  • the CD19-binding region comprises a fragment of the variable portion of the heavy chain (VH) or a fragment of the variable portion of the light chain (Vi.) of a single-chain variable fragment (scFv) or a camelid single domain antibody (VHH). These fragments may be derived from a monoclonal antibody.
  • the single-chain variable fragment has the ability to bind CD 19.
  • the scFv is comprised of the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) linked via a spacer sequence.
  • the CD19-binding scFv is FMC63, see Nicholson et al., (1997) Construction and characterization of a functional CD 19 specific single chain Fv fragment for immunotherapy ofB lineage leukaemia and lymphoma, Mol. Immunol. 34:1157.
  • the transmembrane domain of the CAR is derived from a membrane-bound or transmembrane protein.
  • the transmembrane domain of the CAR may be the transmembrane domain of a T cell receptor alpha-chain or beta-chain, a CD3-zeta chain, CD28, CD3-epsilon chain, CD2, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, DNAM1, NKp44, NKp46, NKG2D, 2B4, or GITR.
  • the transmembrane domain of the CAR is the CD8 transmembrane domain.
  • the transmembrane domain of the CAR is the CD8A transmembrane domain
  • the intracellular signaling domain of a CAR is responsible for activation of one or more effector functions of the immune cell expressing the CAR.
  • the intracellular signaling domain of the CAR comprises a part of or the entire sequence of the CD3- zeta chain, CD3-epsilon chain, CD2, CD28, CD27, OX40/CD134, 4-1BB/CD137, ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, DNAM1, TLR1, TLR2, TLR4, TLR5, TLR6, MyD88, CD40 or a combination thereof.
  • the intracellular domain of the CAR consists of 4- IBB and CD3 zeta chain.
  • the CAR comprises a hinge domain.
  • the hinge domain of the CAR is the CD8 hinge domain.
  • the hinge domain of the CAR is the CD8A hinge domain.
  • the CAR comprises a signal sequence, an antiCD19 scFv, a CD8 hinge domain, a transmembrane domain, a 4-1BB and CD3- zeta intracellular domains.
  • the CAR is a fully human protein or is humanized to reduce immunogenicity in human patients.
  • the nucleic acid sequence encoding the CAR is optimized for codon usage in human cells.
  • a nucleic acid encoding the CAR may be introduced into a cell as a genomic DNA sequence or a cDNA sequence.
  • the cDNA sequence comprises the open reading frame for translation of the CAR and in some embodiments, further comprises untranslated elements that improve for example, the stability or the rate of translation of the CAR mRNA.
  • the CAR-expressing sequence is stably integrated into the genome of the immune cell.
  • the anti-CD19 CAR is encoded by a nucleic acid construct comprising a coding sequence for the CD 19-targeting CAR, and a promoter.
  • the CD 19-targeting CAR expression construct is introduced via an expression vector.
  • the vector is a viral vector (e.g., a retroviral vector, adenoviral vector, adeno-associated viral vector, or lentiviral vector). Suitable vectors are nonreplicating in the target cells.
  • the vector is selected from or designed based on SV40, EBV, HSV, or BPV.
  • the vector incorporates the protein expression sequences.
  • the expression sequences are codon-optimized for expression in mammalian cells.
  • the vector also incorporates regulatory sequences including transcriptional activator binding sequences, transcriptional repressor binding sequences, enhancers, introns, and the like.
  • the viral vector supplies a constitutive or an inducible promoter.
  • the promoter is selected from EFla, PGK1, MND, Ubc, CAG, CaMKIIa, and P-Actin promoter.
  • the promoter is selected from the SV-40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, the P-interferon promoter, the hsp70 promoter and EF-la promoter.
  • the promoter is an EF-l promoter.
  • the promoter is an MND promoter.
  • the viral vector supplies a transcription terminator or a polyadenylation signal.
  • the transcription terminator or polyadenylation signal is the BGH transcription terminator and polyadenylation signal.
  • the vector is a plasmid selected from a prokaryotic plasmid, a eukaryotic plasmid, and a shuttle plasmid.
  • the expression vector comprises one or more selection marker.
  • the selection markers are antibiotic resistance genes or other negative selection markers.
  • the selection markers comprise proteins whose mRNA is transcribed together with the CD19-targeting CAR mRNA and the polycistronic transcript is cleaved prior to translation.
  • the expression vector comprises polyadenylation sites.
  • the polyadenylation sites are SV-40 polyadenylation sites.
  • the coding sequence of the CD19-targeting CAR is introduced into the cells via a viral vector, such as e.g., AAV vector (AAV6) or any other suitable viral vector capable of delivering an adequate payload.
  • AAV vector AAV6
  • the coding sequence is joined to homology arms located 5’ (upstream or left) and 3 ’ (downstream or right) of the insertion site in the desired insertion site in the genome.
  • the homology arms are about 500 bp long.
  • the sequence coding for the CD19-targeting CAR together with the homology arms are cloned into a viral vector plasmid. The plasmid is used to package the sequences into a virus.
  • nucleic acid construct is shown in Figure 1.
  • the construct comprises an EFla promoter, left homology arm (LHA) and a right homology arm (RHA).
  • the homology arms comprise sequences capable of hybridizing to a desired locus in the cellular genome and thereby facilitating homologous recombination.
  • the immune cell is contacted with a viral vector so that the genetic material delivered by the vector is integrated into the genome of the target cell and then expressed in the cell or on the cell surface.
  • Transduced and transfected cells can be tested for transgene expression using methods well known in the art such as fluorescence-activated cell sorting (FACS), microfluidics-based screening, ELISA, or Western blot.
  • FACS fluorescence-activated cell sorting
  • ELISA ELISA
  • Western blot Western blot.
  • a composition comprising the engineered CD 19-targeting immune cells is assessed for the number or viable cells or viable CAR-expressing (CAR-positive) cells.
  • the coding sequence for the CD 19-targeting CAR is introduced into the immune cell as “naked” nucleic acid by electroporation as described e.g., in U.S. Patent No. 6,410,319.
  • an engineered CRISPR system is introduced into the immune cell for the purpose of introducing genome modifications in the cell.
  • the CRISPR system comprises a nucleic acid-guided endonuclease and nucleic acid-targeting nucleic acid (NATNA) guides (e.g., a CRISPR guide RNAs selected from tracrRNA, crRNA or a single guide RNA incorporating the elements of the tracrRNA and crRNA in a single molecule).
  • NATNA nucleic acid-guided endonuclease and nucleic acid-targeting nucleic acid
  • NATNA is selected from the embodiments described in U.S. Patent No. 9,260,752.
  • a NATNA can comprise, in the order of 5' to 3', a spacer extension, a spacer, a minimum CRISPR repeat, a single guide connector, a minimum tracrRNA, a 3' tracrRNA sequence, and a tracrRNA extension.
  • a nucleic acid-targeting nucleic acid can comprise, a tracrRNA extension, a 3' tracrRNA sequence, a minimum tracrRNA, a single guide connector, a minimum CRISPR repeat, a spacer, and a spacer extension in any order.
  • the NATNA is a single guide NATNA.
  • the NATNA comprises a spacer sequence which can be engineered to hybridize to the target nucleic acid sequence.
  • the NATNA further comprises a CRISPR repeat comprising a sequence that can hybridize to a tracrRNA sequence.
  • the NATNA can have a spacer extension and a tracrRNA extension. These elements can include elements that can contribute to stability of NATNA.
  • the CRISPR repeat and the tracrRNA sequence can interact, to form a base-paired, double-stranded structure. The structure can facilitate binding of the endonuclease to the NATNA.
  • the single guide NATNA comprises a spacer sequence located 5' of a first duplex which comprises a region of hybridization between a minimum CRISPR repeat and minimum tracrRNA sequence.
  • the first duplex can be interrupted by a bulge.
  • the bulge facilitates recruitment of the endonuclease to the NATNA.
  • the bulge can be followed by a first stem comprising a linker connecting the minimum CRISPR repeat and the minimum tracrRNA sequence.
  • the last paired nucleotide at the 3' end of the first duplex can be connected to a second linker connecting the first duplex to a mid-tracrRNA.
  • the mid-tracrRNA can comprise one or more additional hairpins.
  • the NATNA can comprise a double-guide nucleic acid structure.
  • the double-guide NATNA comprises a spacer extension, a spacer, a minimum CRISPR repeat, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and a tracrRNA extension.
  • the double guide NATNA does not include the single guide connector. Instead, the minimum CRISPR repeat sequence comprises a 3' CRISPR repeat sequence and the minimum tracrRNA sequence comprises a 5' tracrRNA sequence and the double guide NATNAs can hybridize via the minimum CRISPR repeat and the minimum tracrRNA sequence.
  • the NATNA is an engineered guide RNA comprising one or more DNA residues (CRISPR hybrid RDNA or chRDNA).
  • NATNA is selected from the embodiments described in U.S. Patent Nos. 9,650,617, 9,580,701, 9,688,972, 9,771,601, 9,868,962, 10,519,468, 10,988,781, 11,236,364, and 11,459,588.
  • some chRDNA for use with a Type II CRISPR system may be composed of two strands forming a secondary structure that includes an activating region composed of an upper duplex region, a lower duplex region, a bulge, a targeting region, a nexus, and one or more hairpins.
  • a nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA.
  • Other chRDNA may be a single guide D(R)NA for use with a Type II CRISPR system comprising a targeting region, and an activating region composed of and a lower duplex region, an upper duplex region, a fusion region, a bulge, a nexus, and one or more hairpins.
  • a nucleotide sequence immediately downstream of a targeting region may comprise various proportions of DNA and RNA.
  • the targeting region may comprise DNA or a mixture of DNA and RNA
  • an activating region may comprise RNA or a mixture of DNA and RNA.
  • the single molecule and dual molecule chRDNA embodiments described above may be Cas9 chRDNAs or Casl2a chRDNAs.
  • the components of the CRISPR system are introduced into the cell in the form of nucleic acids.
  • the components of the CRISPR system are introduced into the cell in the form of DNA coding for the nucleic acid-guided endonuclease and the NATNA.
  • the gene coding for the nucleic acid-guided endonuclease e.g., a CRISPR nuclease selected from Cas9 and Casl2a
  • the gene coding for the NATNA guides is inserted into a plasmid capable of propagating in the cell.
  • the components of the CRISPR system z.e., the nucleic acid- guided endonuclease and NATNA guides are introduced into the cell in the form of RNA, e.g., the mRNA coding for the nucleic acid-guided endonuclease along with the NATNA guides.
  • the components of the CRISPR system i.e., the nucleic acid- guided endonuclease and the NATNA guides are introduced into the cell as a preassembled ribonucleoprotein complex (RNP).
  • the components of the CRISPR system, z.e., the nucleic acid-guided endonuclease and the NATNA guides are introduced into the cell via any combination of different means, e.g., the endonuclease is introduced as the DNA via a plasmid containing the gene encoding the endonuclease while the guides are introduced in its final format as RNA (or RNA containing DNA nucleotides).
  • the components of the CRISPR system z.e., the nucleic acids encoding the nucleic acid-guided endonuclease and NATNA guides or a preassembled RNP are introduced into the cell via electroporation.
  • the components of the CRISPR system i.e., the nucleic acids coding for the nucleic acid-guided endonuclease are introduced into the cell in the form of mRNA as described e.g., in the U.S. patent No. 10,584,352 via electroporation of viral pseudotransduction as described therein.
  • the coding sequence for the CD19-targeting CAR is inserted into a double-strand break in the genome of the immune cell.
  • the doublestrand break is introduced in a gene targeted for inactivation in the cell.
  • genome engineering comprises a gene knock-out and a simultaneous gene knock-in.
  • the gene targeted for inactivation by CAR insertion is selected from TRAC, CBLB, PDCD1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLECI0, and 2B4.
  • the CD19-targeting CAR sequence is inserted into the T cell receptor alpha constant (TRAC) gene.
  • the invention includes the use of T cells engineered to express an anti-CD19 CAR and further engineered to possess a second genome modification increasing survival and cytotoxic activity of the T cells.
  • the second genome modification comprises transcriptionally silencing or disrupting the coding sequence of one or more immune checkpoint genes.
  • the one or more immune checkpoint gene is selected from PDCD1 (encoding PD1), CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, and 2B4 (see e.g., U.S. Patent No. 11,304,975 Methods for engineering allogeneic and highly active T cell for immunotherapy or U .S. PatentNo. 9,889,160 Genetically-modified cells comprising a modified human T cell receptor alpha constant region gene)
  • the second genomic modification is disruption of the PDCD1 gene.
  • the invention comprises compositions including immune cells expressing a CD19-targeting protein, e.g., an anti-CD19 CAR, where the cells are stored in a cryopreservation solution comprising one or more of adenosine, dextrose, dextran-40, lactobionic acid, sucrose, mannitol or a combination thereof.
  • the compositions also include one or more pharmaceutically acceptable excipients.
  • excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Excipients suitable for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • carbohydrate excipients include, for example, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphat
  • the composition further comprises an antimicrobial agent for preventing or deterring microbial growth.
  • the antimicrobial agent is selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.
  • the composition further comprises an antioxidant added to prevent the deterioration of the lymphocytes.
  • the antioxidant is selected from ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
  • the composition further comprises a surfactant.
  • the surfactant is selected from polysorbates, sorbitan esters, lipids, such as phospholipids (lecithin and other phosphatidylcholines), phosphatidylethanolamines, fatty acids and fatty esters; steroids, such as cholesterol.
  • the composition further comprises a freezing agent such as 3% to 12% dimethylsulfoxide (DMSO) or 1% to 5% human albumin.
  • a freezing agent such as 3% to 12% dimethylsulfoxide (DMSO) or 1% to 5% human albumin.
  • DMSO dimethylsulfoxide
  • the number of adoptive cells, such as CAR-T cells or CAR-NK cells, in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose per vial or container.
  • the invention includes a step of administering to the patient a composition comprising CD 19-targeting immune cells.
  • the CD-19 targeting immune cells further comprise inactivated immune checkpoint gene, i.e., PDCD1.
  • the CD- 19 targeting immune cells are CAR-T cells.
  • the CD- 19 targeting immune cells are allogeneic. Information about administering CD 19-targeting CAR-T cells with disrupted PDCD1 gene (“CB-010”) can be found in the public record of the clinical trial identified as NCT04637763.
  • the dose of allogeneic anti-CD19 CAR-T cells is between 60 and 100 million (6 x 10 7 -10 8 ) cells. In some embodiments, the number “60 and 100 million (6 x 10 7 -10 8 ) cells” refers to viable CAR-expressing cells (CAR+ cells). In some embodiments, the dose of allogeneic anti-CD19 CAR-T cells is 80 million (8 x 10 7 ) cells. In some embodiments, the does is 80 million (8 x 10 7 ) viable CAR+ cells.
  • the therapeutic composition is administered to a patient by a route selected from intravenous, parenteral, intrathecal, local, and intramuscular.
  • the administration is by infusion and the infusion is selected from a single sustained dose, a prolonged continuous infusion, and multiple infusions.
  • the administration is a single infusion.
  • the method further comprises after the step of administering the CD 19-targeting immune cells, the step or steps of monitoring the patient for acute toxi cities such as cytokine release syndrome (CRS), neurotoxicity (immune effector cell-associated neurotoxicity syndrome, ICANS), and graft versus host disease (GvHD), and a further step of treating the patient for the symptoms of the toxicities.
  • acute toxi cities such as cytokine release syndrome (CRS), neurotoxicity (immune effector cell-associated neurotoxicity syndrome, ICANS), and graft versus host disease (GvHD)

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Abstract

L'invention comprend des méthodes et des compositions pour le traitement d'une maladie auto-immune, le schéma comprenant une lymphodéplétion avec une dose optimale prédéterminée d'agents de lymphodéplétion suivie de l'administration de cellules immunitaires ciblant CD19.
PCT/US2025/016015 2024-03-13 2025-02-14 Schémas thérapeutiques pour maladie auto-immune à l'aide de cellules immunitaires modifiées ciblant cd19 Pending WO2025193386A1 (fr)

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