WO2021232200A1 - Thérapie par cellules immunitaires armées à il-12 et leurs utilisations - Google Patents

Thérapie par cellules immunitaires armées à il-12 et leurs utilisations Download PDF

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WO2021232200A1
WO2021232200A1 PCT/CN2020/090885 CN2020090885W WO2021232200A1 WO 2021232200 A1 WO2021232200 A1 WO 2021232200A1 CN 2020090885 W CN2020090885 W CN 2020090885W WO 2021232200 A1 WO2021232200 A1 WO 2021232200A1
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cell
cells
vector
nucleic acid
human
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PCT/CN2020/090885
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English (en)
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Lu Zhang
Rui Chen
Josephine Anna TUELLER
Rhiannon SPENCER
Brooke WOLFF
Lixia ZHAO
Zhenbo SU
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Guangdong Tcrcure Biopharma Technology Co., Ltd.
Tcrcure Biopharma Corp.
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Priority to PCT/CN2020/090885 priority Critical patent/WO2021232200A1/fr
Priority to PCT/CN2021/094469 priority patent/WO2021233317A1/fr
Priority to TW110117939A priority patent/TW202208415A/zh
Publication of WO2021232200A1 publication Critical patent/WO2021232200A1/fr

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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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • This disclosure relates to IL-12 armored immune cell therapies and the use of IL-12 in combination with immune cell therapies.
  • Cancer is one of the most widespread cellular anomalies caused by biological and environmental factors, such as age, gender, genetic mutations, environmental exposure such as UV radiation, occupational risk factors, carcinogens, asbestos, radioactive materials, and viral infections (e.g., HPV, EBV, HBV, HCV, HTLV-1 and KSHV) (Margaret E et al., “Viruses Associated With Human Cancer, ” Biochimica et Biophysica Acta. 1782: 127–150 (2008) ) .
  • biological and environmental factors such as age, gender, genetic mutations, environmental exposure such as UV radiation, occupational risk factors, carcinogens, asbestos, radioactive materials, and viral infections (e.g., HPV, EBV, HBV, HCV, HTLV-1 and KSHV) (Margaret E et al., “Viruses Associated With Human Cancer, ” Biochimica et Biophysica Acta. 1782: 127–150 (2008) ) .
  • the present disclosure provides modified (e.g., membrane tethered) or unmodified IL-12, which can be used in combination with immune cell therapies (e.g., TCR-T, CAR-T, or TIL) to treat cancers (e.g., a solid tumor) .
  • immune cell therapies e.g., TCR-T, CAR-T, or TIL
  • the present disclosure also provides the use of IL-12 fused with a tumor-targeting antibody (e.g., NHS76) in combination with immune cell therapies (e.g., TCR-T, CAR-T, CAR-NK, or TIL) to treat cancers.
  • TCR-T tumor-targeting antibody
  • CAR-T CAR-T
  • CAR-NK CAR-NK
  • TIL tumor-targeting antibody
  • a cell expressing (a) an exogenous T cell receptor (TCR) , or a chimeric antigen receptor (CAR) ; and (b) IL-12.
  • TCR exogenous T cell receptor
  • CAR chimeric antigen receptor
  • the IL-12 is a membrane tethered IL-12.
  • the membrane tethered IL-12 comprises a CD4 transmembrane region.
  • the membrane tethered IL-12 comprises an immunoglobulin CH2 domain, an immunoglobulin CH3 domain, and a CD4 transmembrane region.
  • the membrane tethered IL-12 comprises two or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a CD4 transmembrane region.
  • the immunoglobulin CH2 domain is a wild-type immunoglobulin constant domain.
  • the amino acid at position 235 (EU numbering) of the immunoglobulin CH2 domain is Glu and the amino acid residue at position 297 (EU numbering) of the immunoglobulin CH2 domain is Gln.
  • the membrane tethered IL-12 further comprises an immunoglobulin hinge region.
  • the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain
  • the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain
  • the CD4 transmembrane region is a human CD4 transmembrane region.
  • the IL-12 is a soluble IL-12.
  • the IL-12 is linked to a tumor-targeting antibody or antigen-binding fragment thereof.
  • the tumor-targeting antibody or antigen binding fragment thereof is a single-chain variable fragment (scFv) .
  • the tumor-targeting antibody is NHS76.
  • the IL-12 is a human IL-12 or a mouse IL-12.
  • the TCR or CAR targets BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (IL-3R) , CEA, IL13Ra2, ALPP, EBV-related antigens (e.g., LMP2) , EGFR, EGFRvIII, GD2, GPC3, HER2, a HPV-related antigen (e.g., E6 or E7) , MAGE (e.g., MAGE-A3) , Mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.
  • the CAR comprises an extracellular domain
  • the extracellular domain is a single chain variable fragment (scFv) , a ligand (e.g., a receptor-binding ligand) , or an antibody mimetic.
  • scFv single chain variable fragment
  • ligand e.g., a receptor-binding ligand
  • antibody mimetic an antibody mimetic
  • a vector comprising: a) a first nucleic acid sequence encoding an IL-12 alpha subunit and an IL-12 beta subunit; b) a second nucleic acid sequence encoding one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a transmembrane region.
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a first linker sequence.
  • the first linker sequence encodes an immunoglobulin hinge polypeptide sequence.
  • the transmembrane region is a CD4 transmembrane region.
  • the transmembrane region is an immunoglobulin transmembrane region.
  • the IL-12 alpha subunit and the IL-12 beta subunit are linked by a linker peptide sequence.
  • the vector further comprises a sequence encoding a signal peptide.
  • the IL-12 alpha subunit is a human IL-12 alpha subunit
  • the IL-12 beta subunit is a human IL-12 beta subunit
  • the immunoglobulin CH2 domain is a human immunoglobulin CH2 domain
  • the immunoglobulin CH3 domain is a human immunoglobulin CH3 domain.
  • the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) , or a chimeric antigen receptor (CAR) .
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the third nucleic acid sequence is linked to the first nucleic acid by a second linker sequence.
  • the second linker sequence encodes a P2A sequence.
  • the first nucleic acid and the second nucleic acid are under control of a regulatory element (e.g., a promotor) .
  • a regulatory element e.g., a promotor
  • the first nucleic acid, the second nucleic acid, and the third nucleic acid are under control of a regulatory element (e.g., a promotor) .
  • a regulatory element e.g., a promotor
  • a vector comprising: a) a first nucleic acid sequence encoding a heavy chain variable region (VH) and a light chain variable region (VL) of a tumor-targeting antibody; b) a second nucleic acid sequence encoding an IL-12 alpha subunit and an IL-12 beta subunit.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a first linker sequence.
  • the tumor-targeting antibody targets a tumor-associated antigen.
  • the tumor-targeting antibody is NHS76.
  • the vector further comprises a nucleic acid encoding a signal peptide.
  • the signal peptide is a human signal peptide
  • the IL-12 alpha subunit is a human IL-12 alpha subunit
  • the IL-12 beta subunit is a human IL-12 beta subunit.
  • the vector further comprises a third nucleic acid sequence encoding a T cell receptor (TCR) , or a chimeric antigen receptor (CAR) .
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the third nucleic acid sequence is linked to 5’ of the first nucleic acid by a second linker sequence.
  • the second linker sequence encodes a P2A sequence.
  • the first nucleic acid and the second nucleic acid are under control of a regulatory element (e.g., a promotor) .
  • a regulatory element e.g., a promotor
  • the first nucleic acid, the second nucleic acid, and the third nucleic acid are under control of a regulatory element (e.g., a promotor) .
  • a regulatory element e.g., a promotor
  • a vector comprising, in a 5’ to 3’ direction a) a first nucleic acid sequence encoding an exogenous T cell receptor (TCR) , or a chimeric antigen receptor (CAR) ; b) a second nucleic acid sequence encoding a signal peptide, an IL-12 alpha subunit and an IL-12 beta subunit.
  • TCR exogenous T cell receptor
  • CAR chimeric antigen receptor
  • the first nucleic acid sequence and the second nucleic acid sequence are linked by a linker sequence.
  • the IL-12 alpha subunit and the IL-12 beta subunit are linked by a linker peptide sequence.
  • the linker sequence encodes a P2A sequence
  • a fusion polypeptide comprising a) a first region comprising an IL-12 alpha subunit, and an IL-12 beta subunit.
  • the IL-12 alpha subunit and the IL-12 beta subunit are linked by a linker peptide sequence.
  • a second region comprising one or more immunoglobulin CH2 domains, one or more immunoglobulin CH3 domains, and a transmembrane region.
  • the first region and the second region are linked by an immunoglobulin hinge peptide.
  • a fusion polypeptide comprising a) a first region comprising an IL-12 alpha subunit, and an IL-12 beta subunit.
  • the IL-12 alpha subunit and the IL-12 beta subunit are linked by a first linker peptide sequence.
  • a second region comprising a heavy chain variable region (VH) and a light chain variable region (VL) of a tumor-targeting antibody.
  • VH and the VL are linked by a second linker peptide sequence.
  • the first region and the second region are linked by a third linker peptide sequence.
  • the third linker polypeptide sequence has a sequence that is at least 80%identical to SEQ ID NO: 17.
  • nucleic acid encoding the polypeptide as described herein.
  • nucleic acid in one aspect, provided herein is a vector comprising the nucleic acid as described herein.
  • a cell comprising the vector as described herein.
  • the cell secrets a higher level of a cytokine as compared to a same cell except that the cell does not comprise the vector as described herein.
  • the cell stimulates one or more cells in the vicinity of the cell to secret a cytokine.
  • the cytokine is an IFN ⁇ .
  • the cell expresses a higher level of an early TCR activation marker as compared to a same cell except that the cell does not comprise the vector as described herein, and/or stimulates one or more cells in the vicinity of the cell to express the early TCR activation marker.
  • the activation maker is a CD69.
  • the cell is a cell line.
  • the cell is a primary cell obtained from a subject (e.g., a human subject) .
  • the cell is an immune cell (e.g., a lymphocyte) .
  • an immune cell e.g., a lymphocyte
  • the cell is a tumor-infiltrating lymphocyte (TIL) or a NK cell (e.g., a CAR-NK cell) .
  • TIL tumor-infiltrating lymphocyte
  • NK cell e.g., a CAR-NK cell
  • the cell is a T cell.
  • the T-cell is isolated from a human subject.
  • the T cell is CD8+.
  • the T cell is CD4+.
  • the vector is an expression vector, a viral vector, a retroviral vector, or a lentiviral vector.
  • the retroviral vector is pMP71.
  • a method for producing a cell comprising introducing the vector as described herein into the cell in vitro or ex vivo.
  • the vector is introduced into the cell by transduction.
  • provided herein is a method of treating a subject having a cancer, the method comprising administering to the subject in need thereof, an effective amount of the cell as described herein.
  • the cancer is a heterogeneous cancer. In some embodiments, the cancer is a homogeneous cancer.
  • the cell is isolated from peripheral blood mononuclear cells (PBMCs) of the subject.
  • PBMCs peripheral blood mononuclear cells
  • a method of treating a subject having a cancer comprising administering to the subject in need thereof, a) an effective amount of cells expressing a T cell receptor (TCR) , or a chimeric antigen receptor (CAR) ; and b) an effective amount of a protein comprising an IL-12 and a tumor-targeting antibody or antigen binding fragment thereof.
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the cell is isolated from peripheral blood mononuclear cells of the subject.
  • a method of treating a human subject having a cancer comprising providing cells collected from the human subject or a different human subject; introducing the vector as described herein in to the cells; culturing and expanding the cells; and administering an effective amount of composition comprising the cells to the subject.
  • the cancer is a heterogeneous cancer. In some embodiments, the cancer is a homogeneous cancer.
  • the cells are peripheral blood mononuclear cells (PBMC) .
  • PBMC peripheral blood mononuclear cells
  • the cells are tumor-infiltrating lymphocytes and the vector comprises a nucleic acid encoding IL-12.
  • the IL-12 is a membrane tethered IL-12.
  • the cells are T cells and the vector comprises a nucleic acid encoding TCR or CAR. In some embodiments, the vector further comprises a nucleic acid encoding IL-12. In some embodiments, the IL-12 is a membrane tethered IL-12.
  • the term “genetically engineered cell” or “genetically modified cell” refers to a cell with a modification of a nucleic acid sequence in the cell, including, but not limited to, a cell having a insertion, deletion, substitution, or modification of one or more nucleotides in its genome, and/or a cell with an exogenous nucleic acid sequence (e.g., a vector) , wherein the exogenous nucleic acid sequence is not necessarily integrated into the genome.
  • membrane tethered IL-12 refers to IL-12 in a modified form that is tethered to a cell membrane.
  • peripheral blood cells refers to cells normally found in the peripheral blood including, but is not limited to, eosinophils, neutrophils, T cells, monocytes, K cells, granulocytes, and B cells.
  • cancer refers to the cells dividing in an uncontrolled manner. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth.
  • the term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • the cancer cells can form the solid tumors or the excessive tumor cells in blood (e.g., hematologic cancer) . Alternatively or additionally it can include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon) , genitourinary tract (e.g., renal, urothelial cells) , prostate and pharynx.
  • Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • cancers that can be treated by the methods described herein include e.g., bone cancer, pancreatic cancer, skin cancer (e.g., melanoma) , cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic le
  • vector refers to a vehicle by which a polynucleotide sequence (e.g. a foreign gene) can be introduced into a host cell, in order to obtain the desired gene expression of the introduced nucleotide sequence.
  • Cloning vectors can include e.g., plasmids, phages, viruses, etc. Most popular type of vector is a "plasmid” , which refers to a closed circular double stranded DNA loop into which additional DNA segments comprising gene of interest can be ligated.
  • plasmid refers to a closed circular double stranded DNA loop into which additional DNA segments comprising gene of interest can be ligated.
  • viral vector in which a nucleic acid construct to be transported is ligated into the viral genome.
  • Viral vectors are capable of autonomous replication in a host cell into which they are introduced or may integrate themselves into the genome of a host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors" or simply "expression vectors” . In some embodiments, the vectors are viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) .
  • a "subject" is a mammal, such as a human or a non-human animal.
  • the subject e.g., patient, to whom the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human.
  • the primate is a monkey or an ape.
  • the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • the subject is a non-primate mammal, such as a dog, a cat, a horse, a rodent, a rat, or a mouse.
  • the term “about” refers to a measurable value such as an amount, a time duration, and the like, and encompasses variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%or ⁇ 0.1%from the specified value.
  • FIG. 1A is a schematic diagram showing mouse IL-12 expression vectors.
  • P2A encodes a 2A self-cleaving peptide
  • mSP encodes a mouse signal peptide
  • mP40 encodes mouse IL-12B (beta chain)
  • the linker encodes a peptide linker
  • mP35 encodes mouse IL-12A (alpha chain)
  • the hinge encodes an immunoglobulin hinge region
  • mCH2 encodes mouse immunoglobulin IgG4 heavy chain constant domain CH2
  • mCH3 encodes mouse immunoglobulin IgG4 heavy chain constant domain CH3
  • mCD4 TM encodes a mouse CD4 transmembrane region
  • NHS-76 vH encodes the heavy chain variable region (VH) of a tumor necrosis-targeting human IgG1 (NHS-76); and NHS-76 vL encodes the light chain variable region (VL) of NHS-76.
  • FIG. 1B is a schematic diagram showing human IL-12 expression vectors.
  • P2A encodes a 2A self-cleaving peptide
  • hSP encodes a human signal peptide
  • hP40 encodes human IL-12B
  • the linker encodes a peptide linker
  • hP35 encodes human IL-12A
  • the hinge encodes an immunoglobulin hinge region
  • hmCH2 human mutated CH2
  • hCH3 human CH3
  • hCD4 TM encodes a human CD4 transmembrane region
  • NHS-76 vH encodes the VH of NHS-76
  • NHS-76 vL encodes the VL of NHS-76.
  • FIG. 1C is a schematic diagram showing vectors expressing human IL-12 in combination with a TCR.
  • TCR encodes a T-cell receptor (e.g., L202) ;
  • P2A encodes a 2A self-cleaving peptide;
  • hSP encodes a human signal peptide;
  • hP40 encodes human IL-12B;
  • the linker encodes a peptide linker;
  • hP35 encodes human IL-12A;
  • the hinge encodes an immunoglobulin hinge region;
  • hmCH2 encodes mutated human immunoglobulin IgG4 heavy chain constant domain CH2;
  • hCH3 encodes human immunoglobulin IgG4 heavy chain constant domain CH3;
  • hCD4 TM encodes a human CD4 transmembrane region;
  • NHS-76 vH encodes the VH of NHS-76;
  • NHS-76 vL encodes the VL of NHS-76.
  • FIG. 1D is a schematic diagram showing vectors expressing human IL-12 in combination with a CAR.
  • CAR encodes a chimeric antigen receptor
  • P2A encodes a 2A self-cleaving peptide
  • hSP encodes a human signal peptide
  • hP40 encodes human IL-12B
  • the linker encodes a peptide linker
  • hP35 encodes human IL-12A
  • the hinge encodes an immunoglobulin hinge region
  • hmCH2 encodes mutated human immunoglobulin IgG4 heavy chain constant domain CH2
  • hCH3 encodes human immunoglobulin IgG4 heavy chain constant domain CH3
  • hCD4 TM encodes a human CD4 transmembrane region
  • NHS-76 vH encodes the VH of NHS-76
  • NHS-76 vL encodes the VL of NHS-76.
  • FIG. 2A is a histogram showing mouse IL-12 concentration in culture media as determined by ELISA (enzyme-linked immunosorbent assay) .
  • Purified mouse lymphocytes were untransduced (Sample Name: UT) , or transduced to express the L202 TCR alone (Sample Name: L202) , L202 plus mouse membrane-tethered IL-12 (Sample Name: mt12) , or L202 plus NHS76-IL-12 (Sample Name: NHS76) .
  • the lymphocytes were co-cultured with LLC-HLA-A2-Peplinker (LLW) (target cells) , or cultured alone (Control) .
  • LLC-HLA-A2-Peplinker LLC-HLA-A2-Peplinker
  • FIG. 2B is a histogram showing mouse IFN ⁇ concentration in the culture medium as determined by ELISA.
  • Purified mouse lymphocytes were untransduced (Sample Name: UT) , or transduced to express the L202 TCR alone (Sample Name: L202) , L202 plus mouse membrane-tethered IL-12 (Sample Name: mt12) , or L202 plus NHS76-IL-12 (Sample Name: NHS76) .
  • the lymphocytes were co-cultured with LLC-HLA-A2-Peplinker (LLW) (target cells) , or cultured alone (Control) .
  • LLC-HLA-A2-Peplinker LLC-HLA-A2-Peplinker
  • FIG. 3 is a histogram showing mouse IFN ⁇ concentration in the culture medium as determined by ELISA.
  • Purified mouse lymphocytes were untransduced (Sample Name: UT) , or transduced to express the L202 TCR alone (Sample Name: L202) , L202 plus membrane-tethered IL-12 (Sample Name: mt12) , or L202 plus NHS76-IL-12 (Sample Name: NHS76) .
  • the lymphocytes were co-cultured with Jurkat cells (target cells+mouse lymphocytes) , or cultured alone (Control) .
  • FIG. 4A is a graph showing cell surface IL-12 expression in untransduced primary mouse lymphocytes (Sample Name: untransduced) .
  • FIG. 4B is a graph showing cell surface IL-12 expression in primary mouse lymphocytes that were transduced to express L202 TCR (Sample Name: L202) .
  • FIG. 4C is a graph showing cell surface IL-12 expression in primary mouse lymphocytes that were transduced to express L202 TCR and human membrane-tethered IL-12 (Sample Name: L202+mtIL12) .
  • FIG. 4D is a graph showing cell surface IL-12 expression in primary mouse lymphocytes that were transduced to express L202 TCR and NHS76-IL12 (Sample Name: L202+NHS-IL12) .
  • FIG. 5A is a graph showing human Fab (hFab) expression in untransduced primary mouse lymphocytes (Sample Name: NHS76-12 UT) or primary mouse lymphocytes that were transduced to express NHS76-IL-12 (Sample Name: NHS76-12) .
  • NHS76-IL12 was detected by staining for human Fab.
  • FIG. 5B is a graph showing IL-12 expression in untransduced primary mouse lymphocytes (Sample Name: NHS76-12 UT) or primary mouse lymphocytes that were transduced to express NHS76-IL-12 (Sample Name: NHS76-12) .
  • NHS76-IL12 exhibited a peak shift when stained for IL-12.
  • FIG. 6A is a graph showing TCRb surface staining of untransduced human PBMCs (Sample Name: untransduced) .
  • FIG. 6B is a graph showing TCRb surface staining of human PBMCs that were transduced to express L202 TCR in combination with human long membrane-tethered IL-12 (Sample Name: L202+long mtIL12) .
  • FIG. 6C is a graph showing TCRb surface staining of human PBMCs that were transduced to express L202 TCR in combination with human membrane-tethered IL-12 (Sample Name: L202+mtIL12) .
  • FIG. 6D is a graph showing TCRb surface staining of human PBMCs that were transduced to express L202 TCR alone (Sample Name: L202) .
  • FIG. 6E is a graph showing human IL-12 surface staining of untransduced human PBMCs (Sample Name: untransduced) .
  • FIG. 6F is a graph showing human IL-12 surface staining of human PBMCs that were transduced to express L202 TCR in combination with long mt-IL-12 (Sample Name: L202+long mtIL12) .
  • FIG. 6G is a graph showing human IL-12 surface staining of human PBMCs that were transduced to express L202 TCR in combination with mt-IL-12 (Sample Name: L202+mtIL12) .
  • FIG. 6H is a graph showing human IL-12 surface staining of human PBMCs that were transduced to express L202 TCR alone (Sample Name: L202) .
  • FIG. 7A is a graph showing CD69 expression in human PBMCs co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • Samples included untransduced human PBMCs (Sample Name: UT A375LLW) ; human PBMCs transduced to express L202 TCR (Sample Name: L202 A375LLW) ; human PBMCs transduced to express L202 plus human membrane-tethered IL-12 (Sample Name: smt A375LLW) ; and human PBMCs transduced to express L202 plus human long membrane-tethered IL-12 (lmt) (Sample Name: lmt A375LLW) .
  • FIG. 7B is a graph showing CD69 expression in human PBMCs without co-culturing.
  • Samples included untransduced human PBMCs (Sample Name: UT) ; human PBMCs transduced to express L202 TCR (Sample Name: L202) ; human PBMCs transduced to express L202 plus human membrane-tethered IL-12 (Sample Name: smt) ; and human PBMCs transduced to express L202 plus human long membrane-tethered IL-12 (Sample Name: lmt) .
  • FIG. 8A shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in untransduced human PBMCs.
  • FIG. 8B shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in human PBMCs that were transduced to express L202 TCR.
  • FIG. 8C shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in human PBMCs that were transduced to express L202 TCR plus membrane-tethered IL-12.
  • FIG. 8D shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in human PBMCs that were transduced to express L202 TCR plus long membrane-tethered IL-12.
  • FIG. 8E shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in untransduced human PBMCs.
  • the PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • LLW A375-HLA-A2-peplinker
  • FIG. 8F shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in human PBMCs that were transduced to express L202 TCR.
  • the PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • LLW A375-HLA-A2-peplinker
  • FIG. 8G shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in human PBMCs that were transduced to express L202 TCR plus membrane-tethered IL-12.
  • the PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • LLW A375-HLA-A2-peplinker
  • FIG. 8H shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in human PBMCs that were transduced to express L202 TCR plus long membrane-tethered IL-12.
  • the PBMCs were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • LLW A375-HLA-A2-peplinker
  • FIG. 9A is a graph showing CD69 expression in the mTCRb+ population after creating a mixed population of 50%L202 TCR positive (mTCRb+) PBMCs (including untransduced PBMCs as a control) and 50%untransduced human PBMCs (mTCRb-) .
  • FIG. 9B is a graph showing CD69 expression in mTCRb-population after creating a mixed population of 50%L202 TCR positive (mTCRb+) PBMCs (including untransduced PBMCs as a control) and 50%untransduced PBMCs (mTCRb-) .
  • FIG. 10A shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from untransduced PBMCs
  • FIG. 10B shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from PBMCs that were transduced to express L202 TCR.
  • FIG. 10C shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from PBMCs that were transduced to express L202 TCR plus human membrane-tethered IL-12.
  • FIG. 10D shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from PBMCs that were transduced to express L202 TCR plus human long membrane-tethered IL-12.
  • FIG. 10E shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from untransduced PBMCs.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 10F shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from PBMCs that were transduced to express L202 TCR.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 10G shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from PBMCs that were transduced to express L202 TCR plus human membrane-tethered IL-12.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 10H shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb+ cells from PBMCs that were transduced to express L202 TCR plus human long membrane-tethered IL-12.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 11A shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from untransduced PBMCs
  • FIG. 11B shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from PBMCs that were transduced to express L202 TCR.
  • FIG. 11C shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from PBMCs that were transduced to express L202 TCR plus human membrane-tethered IL-12.
  • FIG. 11D shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from PBMCs that were transduced to express L202 TCR plus human long membrane-tethered IL-12.
  • FIG. 11E shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from untransduced PBMCs.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 11F shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from PBMCs that were transduced to express L202 TCR.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 11G shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from PBMCs that were transduced to express L202 TCR plus human membrane-tethered IL-12.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 11H shows a flow cytometry result showing human IFN ⁇ (hIFN ⁇ ) expression in mTCRb-cells from PBMCs that were transduced to express L202 TCR plus human long membrane-tethered IL-12.
  • the PBMCs were co-cultured with A375-LLW target cell.
  • FIG. 12A shows a flow cytometry result showing CD4 and CD8 expression in untransduced PBMCs (UT) .
  • FIG. 12B shows a flow cytometry result showing CD4 and CD8 expression in L202 TCR-T PBMCs (L202) .
  • FIG. 12C shows a flow cytometry result showing CD4 and CD8 expression in L202 TCR-T PBMCs expressing human membrane-tethered IL-12 (mt-IL12) .
  • FIG. 12D shows a flow cytometry result showing CD4 and CD8 expression in L202 TCR-T PBMCs expressing human long membrane-tethered IL-12 (lmt-IL12) .
  • FIG. 13 shows a flow cytometry result showing percentages of effector memory CD4+cells and central memory CD4+ cells in untransduced PBMCs.
  • FIG. 14A shows a flow cytometry result showing percentages of effector memory CD4+cells and central memory CD4+ cells in untransduced PBMCs (UT) .
  • FIG. 14B shows a flow cytometry result showing percentages of effector memory CD4+cells and central memory CD4+ cells in L202 TCR-T PBMCs (L202) .
  • FIG. 14C shows a flow cytometry result showing percentages of effector memory CD4+cells and central memory CD4+ cells in L202 TCR-T PBMCs expressing human membrane-tethered IL-12 (mt-IL12) .
  • FIG. 14D shows a flow cytometry result showing percentages of effector memory CD4+cells and central memory CD4+ cells in L202 TCR-T PBMCs expressing human long membrane-tethered IL-12 (lmt-IL12) .
  • FIG. 15A is an image of flow cytometry results showing percentages of effector memory CD8+ cells and central memory CD4+ cells in untransduced PBMCs (UT) .
  • FIG. 15B is an image of flow cytometry results showing percentages of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMCs (L202) .
  • FIG. 15C is an image of flow cytometry results showing percentages of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMCs expressing human membrane-tethered IL-12 (mt-IL12) .
  • FIG. 15D is an image of flow cytometry results showing percentages of effector memory CD8+ cells and central memory CD4+ cells in L202 TCR-T PBMCs expressing human long membrane-tethered IL-12 (lmt-IL12) .
  • FIG. 16 shows IL-12 concentrations in cell culture medium of untransduced human PBMCs (Sample Name: UT) , human PBMCs transduced to express human IL-12 (Sample Name: IL-12) , human PBMCs transduced to express human NHS76-IL-12 (Sample Name: NHS76-IL-12) , or human PBMCs transduced to express human membrane-tethered IL-12 (Sample Name: Mt-IL-12) .
  • FIG. 17 lists sequences that are described in the present disclosure.
  • the human immune system is capable of recognizing and eliminating cells that have become infected or damaged as well as those that have become cancerous.
  • Immune cell therapy takes advantage of the human immune system and is revolutionizing cancer therapy. It involves the transfer of immune cells into a patient. The cells are most commonly derived from the immune system and can originate from the patient or from another individual. In autologous cancer immunotherapy, immune cells are extracted from the patient, genetically modified and cultured in vitro, and returned to the same patient. Comparatively, allogeneic therapies involve cells isolated and expanded from a donor subject.
  • TIL tumor-infiltrating lymphocyte
  • TCR engineered T cell receptor
  • CAR chimeric antigen receptor
  • NK natural killer
  • TIL Tumor-infiltrating lymphocyte
  • TCR engineered T cell receptor
  • MHC major histocompatibility complex
  • CAR Chimeric antigen receptor
  • the cells are transfected by a vector encoding a chimeric antigen receptor.
  • the chimeric antigen receptor can bind to cancer antigens and does not require that the cancer antigens be presented by MHC.
  • Some other immune cells can also be used in these cell therapies.
  • natural killer cells can also be transfected with a vector encoding a chimeric antigen receptor.
  • IL-12 Interleukin-12
  • IL-12 administration is commonly associated with severe toxicity, including e.g., hematologic toxicities, anemia, lymphopenia, neutropenia, muscle and hepatic toxicities, and even in some cases, death.
  • the toxicities of IL-12 administration greatly limits its use in immune cell therapies.
  • the present disclosure provides an improved immune cell therapy, wherein the engineered cells (e.g., immune cells, T cells, NK cells, tumor infiltrating cells) produce IL-12 in a controlled fashion and deliver IL-12 directly to the target site, thereby significantly improving efficacy and safety.
  • the IL-12 is a membrane-tethered IL-12, which further limits the effects of IL-12 to the local target area.
  • the IL-12 is conjugated to scFv of NHS76, which is an antibody that targets a tumor’s necrosis center.
  • IL-12 further improves the efficacy of immune cell therapies.
  • Antigen escape and downregulation e.g., antigen loss
  • antigen loss have emerged as major issues impacting the durability of immune cell therapy.
  • antigens are expressed at lower levels and/or more heterogeneously. Because of the heterogeneity, certain tumor cells can escape immune cell therapy targeting a specific antigen by decreasing the expression of that antigen.
  • the immune pressure by the immune cell therapy can drive cancer cells to evolve by modulating expression of their target antigens, through either loss of detectable antigen or diminished expression of the antigen to a level below a threshold required for immune cell activity.
  • the expression of IL-12 in engineered immune cells can further increase immune response (e.g., stimulating surrounding immune cells) at the local tumor microenvironment, thereby killing surrounding cancer cells that do not express those antigens or express a low level of antigens, preventing any cancer cells from escaping the immune cell therapy.
  • immune response e.g., stimulating surrounding immune cells
  • the present disclosure relates to IL-12 armored immune cell therapies (e.g., TCR-T, CAR-T, CAR-NK or TIL) .
  • the IL-12 is modified by fusing to one or more (e.g., 1, 2, 3, 4, or 5) immunoglobulin constant domains and/or a membrane-tethering region (e.g., a transmembrane region) , such that the IL-12 is tethered to the cell membrane.
  • the IL-12 is modified by linking it to variable regions (e.g., VH and VL) of a tumor-targeting antibody.
  • IL-12 armored immune cell therapies can boost anti-tumor immunity or efficacy, induce host immune system activation, induce epitope spreading, and/or increase safety in humans.
  • the disclosure also provides vectors, cells comprising such vectors, and methods of making such vectors. Also provided are methods of using the IL-12 armored immune cell therapies of the disclosure, including but not limited to, treating various cancers and some other disorders in a human.
  • IL-12 is a heterodimeric molecule composed of an alpha chain (the p35 subunit) and a beta chain (the p40 subunit) covalently linked by a disulfide bridge to form the biologically active 70 kDa dimer.
  • IL-12 is an inflammatory cytokine that is produced in response to infection by a variety of cells of the immune system, including phagocytic cells, B cells and activated dendritic cells.
  • IL-12 plays an essential role in mediating the interaction of the innate and adaptive arms of the immune system, acting on T-cells and natural killer (NK) cells to enhance the proliferation and activity of cytotoxic lymphocytes and the production of other inflammatory cytokines, especially interferon-gamma (IFN-gamma) .
  • NK natural killer
  • IFN-gamma interferon-gamma
  • IL-12 plays an essential role in mediating the interaction of the innate and adaptive arms of the immune system, acting on T-cells and natural killer (NK) cells to enhance the proliferation and activity of cytotoxic lymphocytes and the production of other inflammatory cytokines, especially interferon-gamma (IFN-gamma) .
  • IL-12 can be found in, e.g., Colombo et al., “Interleukin-12 in anti-tumor immunity and immunotherapy. ” Cytokine &growth factor reviews 13.2 (2002) : 155-168; and Hamza et al
  • the IL-12 receptor is expressed in a constitutive or inducible manner in a variety of immune cells, including NK cells and T and B lymphocytes.
  • Ligand-bound IL-12R becomes phosphorylated on tyrosines, which provides harboring sites for two kinases, JAK2 and TYK2.
  • STAT4 is considered to be the most specific mediator of cellular responses elicited by IL-12.
  • a remarkable function of IL-12 is its ability to induce IFN ⁇ release from natural killer (NK) cells as well as CD4 + and CD8 + T cells.
  • IL-12 signaling via STAT-4 is critical for Th1 differentiation and the acquisition of cytolytic functions by CD8 + T cells.
  • IFN ⁇ in turn strongly modifies the tumor microenvironment.
  • the best studied beneficial mechanisms of IFN ⁇ are: 1) enhancing MHC I antigen presentation by tumor cells; 2) inducing the expression of CXCL9, 10, and 11 chemokines to attract NK, Th1, and CD8 + T cells; 3) transforming M2 macrophages into activated antitumor M1 macrophages; and 4) acting on endothelial cells to mediate anti-angiogenesis in a CXCR3-dependent fashion while also enhancing the expression of homing receptors for T-cell recruitment.
  • IL-12 IL-12
  • IL-12 administration also elicit severe side effects.
  • These side effects associated with systemic administration of IL-12 in clinical investigations and the very narrow therapeutic index of this cytokine have markedly limited the use of IL-12 as anti-cancer therapy.
  • a detailed description of IL-12 can be found, e.g., in Lasek et al., "Interleukin 12: still a promising candidate for tumor immunotherapy? . " Cancer Immunology, Immunotherapy 63.5 (2014) : 419-435; Berraondo et al., "Revisiting interleukin-12 as a cancer immunotherapy agent. " Clinical Cancer Research 24.12 (2018) : 2716-2718; which are incorporated herein by reference in their entirety.
  • IL-12 is expressed in immune cells that target cancer cells.
  • engineered cells e.g., immune cells, T cells, NK cells, tumor-infiltrating cells
  • the IL-12 is a membrane-tethered IL-12, which further limits the effects of IL-12 to the local target area.
  • the modified IL-12 proteins can be any of the fusion proteins described herein.
  • a fusion protein comprising an IL-12 alpha subunit and an IL-12 beta subunit.
  • the IL-12 proteins can be derived from any species.
  • the IL-12 alpha subunit is a human IL-12 alpha subunit.
  • the IL-12 alpha subunit is a mouse IL-12 alpha subunit.
  • the IL-12 beta subunit is a human IL-12 beta subunit.
  • the IL-12 beta subunit is a mouse IL-12 beta subunit.
  • the IL-12 alpha subunit and beta subunit are connected by a polypeptide linker sequence.
  • the amino acid sequence of the IL-12 subunits can be modified.
  • the IL-12 subunits includes one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of a wild-type molecule, e.g., any IL-12 subunits described herein.
  • Exemplary variants include those designed to improve the binding affinity to IL-12 receptors and/or other biological properties of the IL-12 subunits (e.g., protein stability) .
  • Amino acid sequence variants of an IL-12 subunit can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the IL-12 alpha and/or beta subunits, or by peptide synthesis.
  • modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues within the amino acid sequences of the IL-12 alpha and/or beta subunits. Any combination of such deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., specifically binding to the IL-12 receptor.
  • the IL-12 alpha subunit has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 15.
  • the IL-12 beta subunit has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 16.
  • the IL-12 alpha and beta subunits are connected by a linker peptide sequence GGGGSGGGGSGGGGS (SEQ ID NO: 17) .
  • the IL-12 fusion protein described herein has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 8.
  • the fusion protein described herein comprises a membrane-tethering region.
  • the membrane-tethering region is a transmembrane region of a transmembrane protein (e.g., CD4) .
  • the transmembrane region is a transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, B7 (e.g., B7-1) , CD3 epsilon, CD4, CD5, CD8, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.
  • the fusion protein comprise a transmembrane domain of a membrane immunoglobulin (mIg) .
  • the fusion protein comprises the transmembrane domain of CD28, CD3-zeta, CD3-alpha, or CD3-beta transmembrane.
  • the fusion protein described herein comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more constant domains of immunoglobulins.
  • the constant domains are heavy chain constant domains.
  • the constant domains are light chain constant domains.
  • the protein comprises 2 constant domains. In some embodiments, the 2 constant domains are CH2 and CH3 domains.
  • the 2 constant domains are CH1 and CH3 domains. In some embodiments, the 2 constant domains are CH1 and CH2 domains. In some embodiments, the 2 constant domains are both CH1 domains. In some embodiments, the 2 constant domains are both CH2 domains. In some embodiments, the 2 constant domains are both CH3 domains. In some embodiments, the protein comprises the entire Fc region of the immunoglobulin. In some embodiments, the protein comprises 3 constant domains. In some embodiments, the 3 constant domains are two CH2 and one CH3. In some embodiments, the 3 constant domains are linked sequentially as CH2-CH2-CH3. In some embodiments, the 3 constant domains are linked sequentially as CH1-CH2-CH3.
  • the protein comprises 1, 2, 3, 4, 5 or more CH2 domains. In some embodiments, the protein comprises 1, 2, 3, 4, 5 or more CH3 domains.
  • the immunoglobulin is a human immunoglobulin. In some embodiments, the immunoglobulin is a mouse immunoglobulin. In some embodiments, the immunoglobulin is an immunoglobulin G (IgG) , an IgM, an IgE, an IgA, or an IgD molecule. In some embodiments, the immunoglobulin is an IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin is a human IgG4. In some embodiments, the constant domains are from the same immunoglobulin class (e.g., IgG, IgM, IgA) . In some embodiments, the constant domains comprise CH2, CH3, and CH4 of IgM.
  • the constant domain is a wild-type constant domain from human. In some embodiments, the constant domain is a mutated human constant domain. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain. In some embodiments, the constant domain is a wild-type human immunoglobulin IgG4 heavy chain constant domain CH2. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain CH2. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids are mutated in the CH2 domain.
  • the constant domain is a wild-type human immunoglobulin IgG4 heavy chain constant domain CH3. In some embodiments, the constant domain is a mutated human immunoglobulin IgG4 heavy chain constant domain CH3. In some embodiments, the mutated constant domain described herein (e.g., hmCH2) has a reduced binding affinity to soluble Fc ⁇ R as compared to a corresponding wild-type constant domain.
  • the mutated constant domain described herein has an enhanced T cell persistence in vivo as compared to a corresponding wild-type constant domain.
  • the mutations in the CH2 domain are L235E and/or N297Q according to EU numbering.
  • the mutation includes one or more of the following: N297A, N297Q or N297G (EU numbering) .
  • the mutation is L234A/L235A (LALA) (EU numbering) .
  • the mutation is F234A and/or L235A.
  • the mutation includes one or more of the following: H268Q, V309L, A330S and/or P331S (EU numbering) . In some embodiments, the mutation includes one or more of the following: V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S.
  • V234A, G237A, P238S, H268A, V309L, A330S, and/or P331S Detailed descriptions can be found, e.g., in Jonnalagadda et al., "Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy. " Molecular Therapy 23.4 (2015) : 757-768; and U.S. Patent No. 9,914,909 B2 each of which is incorporated herein by reference in its entirety.
  • the CH2 domain described herein comprises the amino acid sequence set forth in any of SEQ ID NO: 41, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the CH2 domain described herein is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 42.
  • the CH3 domain described herein comprises the amino acid sequence set forth in any of SEQ ID NO: 43, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the CH3 domain described herein is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 44.
  • the constant domains are linked to the IL-12 subunits with a hinge region.
  • the amino acid sequence of the constant domain described herein can be modified.
  • the constant domain e.g., CH2 or CH3
  • the constant domain include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of a wild-type molecule, e.g., any constant domains described herein.
  • the fusion protein is a membrane-tethered protein.
  • the membrane-tethering region is fused to the constant domains as described herein.
  • the membrane-tethered IL-12 fusion protein described herein has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 10.
  • the membrane-tethered IL-12 fusion protein described herein has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 12.
  • the fusion protein is a soluble protein.
  • the fusion protein comprises a signal peptide (e.g., a human signal polypeptide) .
  • the signal peptide sequence is a secretion signal peptide.
  • Another strategy for improving the safety of IL-12 is to direct their delivery to tumors via fusion to a tumor-targeting antibody.
  • antibody-cytokine fusion proteins or “immunocytokines, ” have previously demonstrated the ability to enhance anti-tumor immunity in preclinical models.
  • the antibody selected as a vehicle usually binds specifically to a tumor-associated antigen.
  • antibodies directed against necrosis-associated antigens which are abundantly present in tumors but not in normal tissues, offer an attractive delivery approach.
  • these antibody-cytokine fusion proteins are expressed in tumor-targeting cells (e.g., immune cells, T cells, NK cells, tumor- infiltrating cells) ; thus, these antibody-cytokine fusion proteins are expressed specifically at the target site, which further improves their safety.
  • tumor-targeting cells e.g., immune cells, T cells, NK cells, tumor- infiltrating cells
  • the fusion protein comprises a tumor-targeting antibody or antigen-binding fragment thereof.
  • the tumor-targeting antibody or antigen binding fragment can deliver IL-12 to the target site, thereby further reducing the side effects of IL-12.
  • the fusion protein is not a membrane-tethered protein and/or does not have a transmembrane protein.
  • the tumor-targeting antibody or antigen-binding fragment can target a tumor-associated antigen.
  • tumor associated antigen refers to an antigen that is or can be presented on a tumor cell surface and that is located on or within tumor cells.
  • the tumor associated antigens can be exclusively expressed on tumor cells or may represent a tumor specific mutation compared to non-tumor cells. In some other embodiments, the tumor associated antigens can be found in both tumor cells and non-tumor cells, but is overexpressed on tumor cells when compared with non-tumor cells, or is more accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to non-tumor tissue. In some embodiments the tumor associated antigen is located on the vasculature of a tumor.
  • tumor associated surface antigens include CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135) , chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan) , Epidermal growth factor receptor (EGFR) , Her2neu, Her3, IGFR, IL3R, fibroblast activating protein (FAP) , CDCP1, Derlin1, Tenascin, frizzled 1-10, the vascular antigens VEGFR2 (KDR/FLK1) , VEGFR3 (FLT4, CD309) , PDGFR-alpha (CD140a) , PDGFR-beta (CD140b) , Endoglin, CLEC14, Tem1-8
  • a fusion protein comprising an IL-12 alpha subunit and an IL-12 beta subunit.
  • the protein further comprises a heavy chain variable region and a light chain variable region of a tumor-targeting antibody or antigen binding fragment thereof.
  • the tumor-targeting antibody or antigen binding fragment thereof is a scFv.
  • the tumor necrosis-targeting antibody is a human antibody.
  • the tumor necrosis-targeting antibody is a human IgG1.
  • the tumor necrosis-targeting antibody is NHS76.
  • NHS76 is a fully human, phage display-derived IgG1 antibody selected for its specific ability to bind to necrotic regions and thereby target to tumors in vivo. Detailed descriptions can be found, e.g., in Fallon et al., "The immunocytokine NHS-IL12 as a potential cancer therapeutic. " Oncotarget 5.7 (2014) : 1869; and Sharifi et al., "Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors. " Hybridoma and hybridomics 20.5-6 (2001) : 305-312; each of which is incorporated herein by reference in its entirety.
  • the antibody-cytokine fusion proteins has an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 14.
  • T cells are a type of lymphocyte which typically develops in the thymus gland and plays a central role in the adaptive immune response. T cells are distinguished from other lymphocytes by the presence of a T-cell receptor on the cell surface. Differentiated T cells have an important role in controlling the immune response.
  • CD8+ T cells also known as "killer cells” , are cytotoxic. Once they recognize a target cell, they are able to directly kill the target cell (e.g., virus-infected cells or cancer cells) .
  • CD8+ T cells also produce cytokines and recruit other cells (e.g., macrophages and natural killer (NK) cells) to mount an immune response.
  • NK natural killer
  • CD4+ T cells also known as "helper cells”
  • helper cells can indirectly kill target cells, e.g., by facilitating maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs) . Once activated, they divide rapidly and secrete cytokines that regulate or assist the immune response. Regulatory T cells are important for tolerance, thereby preventing or inhibiting autoimmune response.
  • the major role of regulatory T cells is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress autoreactive T cells that escaped the process of negative selection in the thymus.
  • T cells play an important role in cancer immunity where antigens from the cancer cells are taken up and presented on the cell surface of special immune cells called antigen-presenting cells (APCs) so that other immune cells can recognize the antigens of interest.
  • APCs antigen-presenting cells
  • the APCs activate the T-cells and activate them to recognize the tumor cells.
  • the activated T-cells can then travel via the blood vessels to reach the tumor, infiltrate it, recognize the cancer cells and kill them.
  • T cell receptor or “TCR” is a molecule that contains a variable a (or alpha) and b (or beta) chains (also known as TCR ⁇ and TCR ⁇ , respectively) or a variable g (or gamma) and d (or delta) chains (also known as TCR ⁇ and TCR ⁇ , respectively) , or antigen-binding portions thereof, and which is capable of specifically binding to an antigen, e.g., a peptide antigen or peptide epitope bound to an major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • binding molecules derived from TCR binding molecules derived from TCR.
  • the TCR is in the ab form.
  • TCRs that exist in ⁇ and ⁇ forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions.
  • a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens, such as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is an intact or full-length TCR, such as a TCR containing the a chain and b chain.
  • the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex.
  • an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds.
  • an antigen-binding portion contains the variable domains of a TCR, such as variable a (Va or V ⁇ ) chain and variable b (Vb or V ⁇ ) chain of a TCR, or antigen -binding fragments thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.
  • variable domains of the TCR contain complementarity determining regions (CDRs) , which generally are the primary contributors to antigen recognition and binding capabilities and specificity of the peptide, MHC and/or MHC-peptide complex.
  • CDRs complementarity determining regions
  • a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule.
  • the various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs) , which generally display less variability among TCR molecules as compared to the CDRs.
  • CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide-MHC complex.
  • the CDR1 of the alpha chain can interact with the N-terminal part of certain antigenic peptides.
  • CDR1 of the beta chain can interact with the C-terminal part of the peptide.
  • CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex.
  • the a-chain and/or b-chain of a TCR also can contain a constant domain, a transmembrane region and/or a short cytoplasmic tail.
  • each chain (e.g. alpha or beta) of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end.
  • a TCR for example via the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof.
  • a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
  • the intracellular tails of CD3 signaling subunits e.g. CD3 ⁇ , CD3 ⁇ , CD3e and CD3z chains
  • locus of a domain or region can vary depending on the particular structural or homology modeling or other features used to describe a particular domain. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization of a TCR are for illustrative purposes and are not meant to limit the scope of the embodiments provided. In some cases, the specific domain (e.g. variable or constant) can be several amino acids (such as one, two, three or four) longer or shorter. In some aspects, residues of a TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see e.g. www. imgt.
  • IMGT International Immunogenetics Information System
  • the a chain and b chain of a TCR each further contain a constant domain.
  • the a chain constant domain (Ca) and b chain constant domain (Cb) individually are mammalian, such as is a human or a non-human constant domain (e.g., a mouse constant domain) .
  • the constant domain is adjacent to the cell membrane.
  • the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, which variable domains each contain CDRs.
  • TCRs as descried herein can contain a human constant region, such as an alpha chain containing a human Ca region and a beta chain containing a human Cb regin. In some embodiments, the TCRs are fully human. In some embodiments, the expression and/or activity of TCRs, such as when expressed in human cells, e.g. human T cells, such as primary human T cells, are not impacted by or are not substantially impacted by the presence of an endogenous human TCR.
  • the engineered TCRs are expressed at similar or improved levels on the cell surface, exhibit the similar or greater functional activity (e.g. cytolytic activity) and/or exhibit similar or greater anti-tumor activity, when expressed by human cells that contain or express an endogenous human TCR, such as human T cells, as compared to the level of expression, function activity and/or anti-tumor activity of the same TCR in similar human cells but in which expression of the endogenous TCR has been reduced or eliminated.
  • cytolytic activity e.g. cytolytic activity
  • anti-tumor activity e.g. cytolytic activity
  • an engineered TCR as described herein when expressed in human T cells, is expressed on the cell surface at a level that is at least or at least about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%or 120%of the level of expression of the same TCR when expressed in similar human T cells but in which expression of the endogenous TCR has been reduced or eliminated.
  • each of the Ca and Cb domains is human.
  • the Ca is encoded by the TRAC gene (IMGT nomenclature) or is a variant thereof.
  • the variant of a Ca contains replacement of at least one non-native cysteine.
  • the TCR can be a heterodimer of two chains a and b that are linked, such as by a disulfide bond or disulfide bonds.
  • the constant domain of the TCR can contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR.
  • a TCR can have an additional cysteine residue in each of the a and b chains, such that the TCR contains two disulfide bonds in the constant domains.
  • each of the constant and variable domains contains disulfide bonds formed by cysteine residues.
  • the TCR comprises CDRs, Va and/or Vb and constant region sequences as described herein.
  • the TCR is a dimeric TCR (dTCR) .
  • a dTCR contains a first polypeptide wherein a sequence corresponding to a provided TCR a chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR a chain constant region extracellular sequence, and a second polypeptide wherein a sequence corresponding to a provided TCR b chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR b chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond.
  • a TCR can be cell-bound or in soluble form. In some embodiments, the TCR is in cell-bound form expressed on the surface of a cell.
  • the TCR is a single chain TCR (scTCR) .
  • the scTCR is a single amino acid strand containing an a chain and a b chain that is able to bind to MHC-peptide complexes.
  • a scTCR can be generated using methods known to those of skill in the art. These methods are described e.g., in WO 96/13593, WO 96/18105, W099/18129, WO 04/033685, W02006/037960, WO2011/044186; WO 2019 /195486; U.S. Patent No. 7,569,664; each of which is incorporated herein by reference in its entirety.
  • the TCR, antigen binding fragments thereof, and TCR-derived binding molecules can bind or recognize a peptide epitope associated with an antigen of interest (e.g., a tumor-associated antigen) .
  • the antigen can be a peptide epitope expressed on the surface of a cancer cell and/or a cell infected with a virus.
  • the antigen is presented in the context of an MHC molecule.
  • binding molecules include e.g., T cell receptors (TCRs) and antigen-binding fragments thereof, antibodies and antigen binding fragments thereof, and TCR-like CAR. They exhibit antigenic specificity for binding or recognizing such peptide epitopes.
  • engineered cells that express a provided binding molecule e.g. a TCR or antigen-binding fragment, exhibit cytotoxic activity against target cells expressing the peptide epitope, such as cancer cells or cells that are infected with a virus (e.g., HPV or EBV) .
  • a provided binding molecule e.g. a TCR or antigen-binding fragment
  • the TCR, antigen binding fragments thereof, and TCR-derived binding molecules recognize or bind to epitopes in the context of an MHC molecule, such as an MHC Class I molecule or an MHC class II molecule.
  • MHC Class I molecules or MHC class II molecules are human leukocyte antigens (HLA) . They play an important component of adaptive immune system.
  • HLA expression is controlled by genes located on chromosome 6. It encodes cell surface molecules specialized to present antigenic peptides to the T-cell receptor on T cells.
  • the TCR, antigen binding fragments thereof, and TCR-derived binding molecules recognize or bind to epitopes in the context of an MHC Class I molecule.
  • the MHC Class I molecule is a human leukocyte antigen (HLA) -A2 molecule, including any one or more subtypes thereof, e.g. HLA-A*020l, *0202, *0203, *0206, or *0207.
  • HLA-A2 human leukocyte antigen A2
  • HLA-A2 is among the most common human serotypes. In some cases, there can be differences in the frequency of subtypes between different populations.
  • HLA-A*020l For example, more than 95%of the HLA-A2 positive Caucasian population is HLA-A*020l, whereas in the Chinese population the frequency has been reported to be approximately 23%for HLA-A*020l, 45%for HLA-A*0207, 8%for HLA-A*0206 and 23%for HLA-A*0203.
  • the MHC molecule is HLA-A*020l.
  • the binding molecule e.g., TCR or antigen-binding fragment thereof or TCR-derived binding molecule
  • the binding molecule is isolated or purified, or is recombinant.
  • the binding molecule e.g., TCR or antigen-binding fragment thereof or TCR-derived binding molecule
  • the binding molecule is fully human.
  • the binding molecule is monoclonal.
  • the binding molecule is a single chain. In other embodiments, the binding molecule contains two chains.
  • the binding molecule e.g., TCR, antigen-binding fragment thereof or TCR-derived binding molecule, is expressed on the surface of a cell.
  • the TCR, antigen-binding fragment thereof, or TCR-derived binding molecules specifically binds to a tumor-associated antigen, e.g., BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R) , CEA, IL13Ra2, ALPP, EBV-related antigens (e.g., LMP2 ) , EGFR, EGFRvIII, GD2, GPC3, HER2, HPV-related antigens (e.g., E6 or E7) , MAGE antigens, Mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.
  • a tumor-associated antigen e.g., BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R) , CEA, IL13Ra2, ALPP, EBV-related antigens (e.g., LMP2
  • the TCR, antigen-binding fragment thereof, or TCR-derived binding molecules can have a Va and a Vb, or a region that is similar to Va and a region that is similar to Vb.
  • the TCR binds to latent membrane protein 2 (LMP2) of Epstein-Barr virus (EBV) .
  • LMP2 latent membrane protein 2
  • EBV Epstein-Barr virus
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 22, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO: 23, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 24, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO:25, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 26, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO: 27, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the TCR binds to tumor antigen E6 of human papilloma virus (HPV) .
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 28, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO: 29, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 32, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO: 33, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the TCR binds to tumor antigen E7 of human papilloma virus (HPV) .
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 30, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO: 31, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the TCR binds to NY-ESO-1 (Cancer/testis antigen 1, also known as LAGE2) .
  • the Va region comprises the amino acid sequence set forth in any of SEQ ID NO: 34, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Vb region comprises the amino acid sequence set forth in any of SEQ ID NO: 35, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the Va region comprises one or more Va CDR sequences as described herein.
  • the Vb region comprises one or more Vb CDR sequences as described herein.
  • nucleic acid encoding the alpha chain and the nucleic acid encoding the beta chain can be connected via a linker, such as any described elsewhere herein.
  • the TCR or antigen-binding fragment thereof, or TCR-derived binding molecules can activate T cells (e.g., by activating TCR signaling pathway) .
  • the activation can upregulate immune response, increase expression of cytokines (e.g., IFN ⁇ ) and/or CD107a, promote T-cell proliferation and T cell mediated killing.
  • the TCR or antigen-binding fragment thereof, or TCR-derived binding molecules as described herein can increase immune response, activity or number of T cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.
  • the TCR or antigen-binding fragment thereof, or TCR-derived binding molecules, when the antigen of interest is present can increase serum concentrations of IFN- ⁇ .
  • the activation can induce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds, 10 folds, 100 folds, or 1000 folds increase of the serum concentrations of IFN- ⁇ . In some embodiments, the activation can induce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 3 folds, 4 folds, or 5 folds increase of specific killing of target cells.
  • the provided recombinant TCRs include TCRs that are at least partially CD8-independent. In some aspects, the provided recombinant TCRs include TCRs that are at least partially CD8-dependent.
  • the TCR or antigen-binding fragment thereof, or TCR-derived binding molecules have a relatively high expression efficiency.
  • the expression efficiency for the TCR or antigen-binding fragment thereof, or TCR-derived binding molecules described herein can be at least 10%, 20%, 30%, 40%, 50%, or 100%higher than an reference molecule (e.g., an endogenous TCR) under the same conditions.
  • the binding molecule e.g. TCR
  • the binding molecule does not exhibit cross-reactive or off-target binding, such as undesirable off-target binding, e.g. off-target binding to antigens present in healthy or normal tissues or cells.
  • Chimeric antigen receptors combine many facets of normal T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell when an antigen is bound. CARs are typically composed of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T-cell signaling domain.
  • the antigen recognition domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. It interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule.
  • the antigen recognition domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scFv) .
  • An scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobulins, connected with a short linker peptide.
  • the linker between the two chains consists of hydrophilic residues with stretches of glycine and serine in it for flexibility as well as stretches of glutamate and lysine for added solubility.
  • the antigen binding domain specifically binds to a tumor associated antigen, e.g., BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R) , CEA, EBV-related antigens (e.g., LMP2) , EGFR, GD2, GPC3, HER2, HPV-related antigens (e.g., E6) , MAGE antigens, Mesothelin, MUC-1, NY-ESO-1, PSCA, PSMA, ROR1, WT1, or Claudin 18.2.
  • a tumor associated antigen e.g., BCMA, CD19, CD22, CD30, CD33, CD56, CD123 (also known as IL-3R) , CEA, EBV-related antigens (e.g., LMP2) , EGFR, GD2, GPC3, HER2, HPV-related antigens (e.g., E6) , MAGE antigens, Mesothelin, M
  • the hinge also called a spacer, is a small structural domain that sits between the antigen recognition region and the cell's outer membrane.
  • An ideal hinge enhances the flexibility of the scFv receptor head, reducing the spatial constraints between the CAR and its target antigen. This promotes antigen binding and synapse formation between the CAR-T cells and target cells. Hinge sequences are often based on membrane-proximal regions from other immune molecules including IgG, CD8, and CD28.
  • the transmembrane domain is a structural component, consisting of a hydrophobic alpha helix that spans the cell membrane. It anchors the CAR to the plasma membrane, bridging the extracellular hinge and antigen recognition domains with the intracellular signaling region. This domain is essential for the stability of the receptor as a whole. Generally, the transmembrane domain from the most membrane-proximal component of the endodomain is used, but different transmembrane domains result in different receptor stability.
  • the CD28 transmembrane domain is known to result in a highly expressed, stable receptor.
  • the transmembrane domain is a transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.
  • the intracellular T-cell signaling domain lies in the receptor's endodomain, inside the cell. After an antigen is bound to the external antigen recognition domain, CAR receptors cluster together and transmit an activation signal. Then the internal cytoplasmic end of the receptor perpetuates signaling inside the T cell.
  • Normal T cell activation relies on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic domain of CD3-zeta. To mimic this process, CD3-zeta's cytoplasmic domain is commonly used as the main CAR endodomain component. Other ITAM-containing domains have also been tried, but are not as effective.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • T cells also require co-stimulatory molecules in addition to CD3 signaling in order to persist after activation.
  • the endodomains of CAR receptors typically also include one or more chimeric domains from co-stimulatory proteins.
  • Signaling domains from a wide variety of co-stimulatory molecules have been successfully tested, including CD28, CD27, CD134 (OX40) , and CD137 (4 ⁇ 1BB) .
  • the CAR molecules specifically binds to a tumor-associated antigen, e.g., BCMA.
  • the CAR comprises the amino acid sequence set forth in any of SEQ ID NO: 37, 38, or 39, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the CAR molecules specifically binds to CD19.
  • the CAR comprises the amino acid sequence set forth in any of SEQ ID NO: 36, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the CAR molecules specifically targets to IL13Ra2.
  • the CAR comprises the amino acid sequence set forth in any of SEQ ID NO: 40, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • CAR molecules or target binding domains thereof are described e.g., WO2018200496A1, WO2019241686A1, WO2018085690A1, WO2018028647A1, and WO2018052828A1; each of which is incorporated herein by reference in its entirety.
  • Exemplary antigen receptors including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in Chandran et al., "T cell receptor ⁇ based cancer immunotherapy: Emerging efficacy and pathways of resistance. " Immunological reviews 290.1 (2019) : 127-147; Cartellieri, Marc, et al., “Chimeric antigen receptor-engineered T cells for immunotherapy of cancer. " BioMed Research International 2010 (2010) ; and PCT publication No. WO2017173256A1; US2002/131960, US2013/287748, US2013/0149337, U.S. 6,451,995, U.S. 7,446,190, U.S. 8,252,592; each of which is incorporated herein by reference in its entirety.
  • the CAR described herein comprises an antibody mimetic.
  • Antibody mimetics are compounds that behave in a similar fashion to antibodies, and bind to specific antigens, but not related to antibodies (e.g., antibody fragments) .
  • the antibody mimetic is a peptide, a nucleic acid, a small molecule, or combinations thereof.
  • the antibody mimetic is an affibody molecule (e.g., Z domain of Protein A) , an adnectin, a monobody (e.g., 10 th type III domain of fibronectin) , a peptide aptamer, a peptide affimer (e.g., cystatin) , an affilin (e.g., gamma-B crystallin or ubiquitin) , an affitin (e.g., Sac7d) , an alphabody (e.g., triple helix coiled coil) , an anticalin (e.g., a lipocalin) , an avimer (e.g., A domains of various membrane receptors) , a fynomer (e.g., SH3 domain of Fyn) , an armadillo repeat protein, a DARPin (e.g., an ankyrin repeat motif) ,
  • engineered cells e.g., immune cells, T cells, NK cells, tumor-infiltrating lymphocytes
  • IL-12 e.g., membrane-tethered IL-12
  • TCR TCR
  • CAR CAR
  • various proteins as described herein.
  • engineered cells can be used to treat various disorders or disease as described herein (e.g., virus infection, cancers, virus-induced disorders) .
  • the cell that is engineered can be obtained from e.g., humans and non-human animals.
  • the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species.
  • the cell is from humans, rats or mice.
  • the cells are mouse lymphocytes and engineered (e.g., transduced) to express the TCR, CAR, or antigen-binding fragment thereof.
  • the cell is obtained from humans.
  • the cell that is engineered is a blood cell.
  • the cell is a leukocyte (e.g., a T cell) , lymphocyte or any other suitable blood cell type.
  • the cell is a peripheral blood cell. In some embodiments, the cell is a tumor-infiltrating lymphocyte (TIL) . In some embodiments, the cell is a T cell, B cell or NK cell. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs) . In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells.
  • TIL tumor-infiltrating lymphocyte
  • the cells is a T cell, B cell or NK cell.
  • the cells are human peripheral blood mononuclear cells (PBMCs) . In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells.
  • the cell is a T cell.
  • the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell.
  • the cell surface receptor can be a wild type or recombinant T cell receptor (TCR) , a chimeric antigen receptor (CAR) , or any other surface receptor capable of recognizing an antigenic moiety that is associated with the target cell.
  • T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients) , with a viral vector.
  • the T cell is a TCR gene-modified or CAR-modified T cell.
  • the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells.
  • the T cells are T helper type 1 T cells and T helper type 2 T cells.
  • the T cell expressing this receptor is an ⁇ -T cell.
  • the T cell expressing this receptor is a ⁇ -T cell.
  • the T cells are central memory T cells.
  • the T cells are effector memory T cells.
  • the T cells are T cells.
  • the cell is an NK cell.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the binding molecule, e.g., TCR or CAR can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells are stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs) .
  • the cells can be primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the stem cells are cultured with additional differentiation factors to obtain desired cell types (e.g., T cells) .
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity-or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the genetic engineering generally involves introduction of a nucleic acid encoding the therapeutic molecule, e.g. TCR, CAR, e.g. TCR-like CAR, IL-12 (e.g., membrane-tethered IL-12) , polypeptides, fusion proteins, into the cell, such as by retroviral transduction, transfection, or transformation.
  • a nucleic acid encoding the therapeutic molecule, e.g. TCR, CAR, e.g. TCR-like CAR, IL-12 (e.g., membrane-tethered IL-12)
  • IL-12 e.g., membrane-tethered IL-12
  • gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical application.
  • a stimulus such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker
  • recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40) , adenoviruses, adeno-associated virus (AAV) .
  • recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors.
  • the retroviral vector has a long terminal repeat sequence (LTR) , e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV) , myeloproliferative sarcoma virus (MPSV) , murine embryonic stem cell virus (MESV) , murine stem cell virus (MSCV) , or spleen focus forming virus (SFFV) .
  • LTR long terminal repeat sequence
  • MoMLV Moloney murine leukemia virus
  • MPSV myeloproliferative sarcoma virus
  • MSV murine embryonic stem cell virus
  • MSCV murine stem cell virus
  • SFFV spleen focus forming virus
  • retroviral vectors are derived from murine retroviruses.
  • the retroviruses include those derived from any avian or mammalian cell source.
  • the retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including
  • the vector is a lentivirus vector.
  • recombinant nucleic acids are transferred into T cells via electroporation.
  • recombinant nucleic acids are transferred into T cells via transposition.
  • Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment and strontium phosphate DNA co-precipitation. Many of these methods are descried e.g., in WO2019195486, which is incorporated herein by reference in its entirety.
  • a humanized and/or a fully human recombinant TCR receptor when engineered into a human T cell, may compete with endogenous TCR complexes and/or can form mispairings with endogenous TCRa and/or TCRb chains, which may, in certain aspects, reduce recombinant TCR signaling, activity, and/or expression, and ultimately result in reduced activity of the engineered cells.
  • the engineered cell can be genetically modified.
  • the engineered cells can comprise a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
  • TTC T cell receptor alpha constant
  • TRBC T cell receptor beta constant
  • the TRBC gene is one or both of a T cell receptor beta constant 1 (TRBCJ) or T cell receptor beta constant 2 (TRBC2) gene.
  • TRBCJ T cell receptor beta constant 1
  • TRBC2 T cell receptor beta constant 2
  • the engineered cells do not express endogenous TCR a chain and/or TRC b chain.
  • non-human constant domains are used, e.g., rodent (e.g., mouse) constant domains. The use of non-human constant domains can effectively reduce the likelihood of mispairing.
  • populations of engineered cells, compositions containing such cells and/or enriched for such cells such as in which cells expressing the binding molecule make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as T cells, CD8+ or CD4+ cells.
  • the engineered cells are co-cultured with target cells (e.g., antigen presenting cells) for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, or longer, such that the engineered cells (e.g., TCR-T cells) can be activated.
  • the target cells are Jurkat cells.
  • the target cells are LLC-HLA-A2-Peplinker (LLW) melanoma cells.
  • the target cells are antigen-presenting cells.
  • the target cells express major histocompatibility complex (MHC) (e.g., class I, class II, and/or class III MHC) .
  • the target cells comprise human leukocyte antigen (HLA) system.
  • IL-12 and modified IL-12 can be expressed by the engineered cells.
  • the fusion protein comprising the modified IL-12 described herein can be expressed on cell surface of engineered cells, e.g., when the fusion protein is a membrane-tethered protein.
  • the fusion protein comprising modified IL-12 described herein can be expressed and secreted, e.g., when the fusion protein is a soluble protein.
  • IL-12 in the engineered cells provides some additional benefits. For example, it can increase production of IFN- ⁇ , which is the most potent mediator of IL-12 actions, from NK and T cells, stimulate of growth and cytotoxicity of activated NK cells, CD8+ and CD4+ T cells, shift differentiation of CD4+ Th0 cells toward the Th1 phenotype, increase antibody-dependent cellular cytotoxicity (ADCC) against tumor cells, and induce IgG and suppression of IgE production from B cells, e.g., by at least or about 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, or 20 folds.
  • IFN- ⁇ is the most potent mediator of IL-12 actions
  • NK and T cells stimulate of growth and cytotoxicity of activated NK cells, CD8+ and CD4+ T cells, shift differentiation of CD4+ Th0 cells toward the Th1 phenotype, increase antibody-dependent cellular cytotoxicity (ADCC) against tumor cells
  • ADCC antibody-dependent cellular cytotoxicity
  • co-culturing with the target cells can increase cytokine (e.g., IFN ⁇ ) secretion of the engineered cells by at least or about 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, 100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10000 folds, or more as compared to the cytokine secretion level of the engineered cell without co-culturing.
  • cytokine e.g., IFN ⁇
  • modified IL-12 expression can increase cytokine (e.g., IFN ⁇ ) expression or secretion of the engineered cells (e.g., TCR-T cells) by at least or about 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10000 folds, or more as compared to the cytokine expression or secretion level of an engineered cell without expressing the modified IL-12.
  • cytokine e.g., IFN ⁇
  • the engineered cells e.g., TCR-T cells
  • modified IL-12 expression in the engineered cells can stimulate cytokine (e.g., IFN ⁇ ) expression or secretion of immune cells in the vicinity of engineered cells by at least or about 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10000 folds, or more as compared to the cytokine expression or secretion level of cells in the vicinity of the engineered cells that do not express the modified IL-12.
  • cytokine e.g., IFN ⁇
  • modified IL-12 expression can increase expression of one or more early TCR activation markers (e.g., CD69) of the engineered cells (e.g., TCR-T cells) or immune cells in the vicinity of engineered cells by at least or about 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, or more as compared to the expression levels of the one or more early TCR activation markers of an engineered cell that does not express the modified IL-12 or immune cells in the vicinity of engineered cells that does not express the modified IL-12.
  • one or more early TCR activation markers e.g., CD69
  • the engineered cells e.g., TCR-T cells
  • immune cells in the vicinity of engineered cells by at least or about 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000 folds, or more as compared to the expression levels of the
  • the cells are human PBMCs and engineered (e.g., transduced) to express the IL-12, TCR, CAR, or antigen-binding fragment thereof.
  • the engineered cells can further express the modified IL-12 as described herein.
  • the modified IL-12 is tethered to the membrane of the engineered cells.
  • the membrane-tethered IL-12 can increase cytokine (e.g., IFN ⁇ ) expression or secretion of the engineered cells by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, 100 folds, or more as compared to the cytokine expression or secretion level of the engineered cells without expressing the membrane-tethered IL-12.
  • cytokine e.g., IFN ⁇
  • the membrane-tethered IL-12 when the engineered cells (e.g., PBMCs or TCRb-PBMCs) are co-cultured with target cells (e.g., antigen-presenting cells) , the membrane-tethered IL-12 can increase activated T cell population by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, or more as compared to the activated T cell population in the engineered cells without expressing the membrane-tethered IL-12.
  • the T cell activation status can be measured by CD69 expression levels.
  • the membrane-tethered IL-12 can increase CD4+ T cell population in the engineered cells (e.g., PBMCs) by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100 folds, or more as compared to the CD4+ T cell population in the engineered cells without expressing the membrane-tethered IL-12.
  • the engineered cells e.g., PBMCs
  • the membrane-tethered IL-12 can decrease CD8+ T cell population in the engineered cells (e.g., PBMCs) to less than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less as compared to the CD8+ T cell population in the engineered cells without expressing the membrane-tethered IL-12.
  • engineered cells e.g., PBMCs
  • the membrane-tethered IL-12 can increase effector memory cell population in the engineered cells (e.g., CD4+ PBMCs or CD8+ PBMCs) by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 6 folds, 7 folds, 8 folds, 9 folds, 10 folds, 20 folds, 50 folds, 100 folds, or more as compared to the effector memory cell population in the engineered cells without expressing the membrane-tethered IL-12.
  • the engineered cells e.g., CD4+ PBMCs or CD8+ PBMCs
  • the membrane-tethered IL-12 can increase effector memory cell population in the engineered cells (e.g., CD4+ PBMCs or CD8+ PBMCs) by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds,
  • the membrane-tethered IL-12 can decrease central memory cell population in the engineered cells (e.g., CD4+ PBMCs or CD8+ PBMCs) to less than or about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less as compared to the central memory cell population in the engineered cells without expressing the membrane-tethered IL-12.
  • the engineered cells e.g., CD4+ PBMCs or CD8+ PBMCs
  • the cells are engineered to express the membrane-tethered IL-12 fusion protein comprising a soluble portion.
  • the soluble portion comprises the IL-12A and/or the IL-12B.
  • the soluble portion comprises a sequence that is at least or about 50%, 60%, 70%, 80%, 90%, or 100%identical to SEQ ID NO: 8.
  • the membrane-tethered IL-12 fusion protein cannot be released from the cells.
  • the IL-12 level in the medium from the cells expressing the membrane-tethered IL-12 fusion protein is less than 10%, 5%, 4%, 3%, 2%, 1%, or less as compared to cells that are engineered to express a soluble IL-12.
  • the concentration of IL-12 in the medium as detected by ELISA is less than 300, 200, or 100 pg/ml.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleot
  • a vector is a construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-Atail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-Atail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • the present disclosure provides a recombinant vector comprising a nucleic acid construct suitable for genetically modifying a cell, which can be used for treatment of pathological disease or condition.
  • Any vector or vector type can be used to deliver genetic material to the cell.
  • vectors include but are not limited to plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs) , yeast artificial chromosomes (YACs) , and human artificial chromosomes (HACs) .
  • Viral vectors can include but are not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems.
  • AAV adeno-associated viral
  • Other vectors that are known in the art can also be used in connection with the methods described herein.
  • the vector is a viral vector.
  • the viral vector can be grown in a culture medium specific for viral vector manufacturing. Any suitable growth media and/or supplements for growing viral vectors can be used in accordance with the embodiments described herein.
  • a MP71 vector is used.
  • the vector used is a recombinant retroviral vector.
  • a retroviral vector is capable of directing the expression of a nucleic acid molecule of interest.
  • a retrovirus is present in the RNA form in its viral capsule and forms a double-stranded DNA intermediate when it replicates in the host cell.
  • retroviral vectors are present in both RNA and double-stranded DNA forms.
  • the retroviral vector also includes the DNA form which contains a recombinant DNA fragment and the RNA form containing a recombinant RNA fragment.
  • the vectors can include at least one transcriptional promoter/enhancer, or other elements which control gene expression.
  • Such vectors can also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used.
  • LTRs long terminal repeats
  • LTRs are identical sequences of DNA that repeat many times (e.g., hundreds or thousands of times) found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes.
  • the vectors can also include a signal which directs polyadenylation, selectable markers such as Ampicillin resistance, Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • retroviral vector used herein can also refers to the recombinant vectors created by removal of the retroviral gag, pol, and env genes and replaced with the gene of interest.
  • the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules.
  • promoters can be multicistronic (bicistronic or tricistronic) .
  • transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site) , which allows coexpression of gene products (e.g. encoding an alpha chain and/or beta chain of a TCR and a modified IL-12) by a message from a single promoter.
  • a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF) , two or three genes (e.g.
  • a self-cleavage peptide e.g., P2A or T2A
  • a protease recognition site e.g., furin
  • the ORF thus encodes a single polyprotein, which, either during (in the case of 2A e.g., T2A) or after translation, is cleaved into the individual proteins.
  • the peptide such as T2A
  • eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER. cells; and NSO cells.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the binding molecule.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • the disclosure relates to a cell comprising the vector or the pair of vectors as described herein.
  • the cell is a T cell.
  • TCRs may exhibit poor expression or activity in part due to mispairing and/or competition with endogenous TCR chains and/or other factors.
  • One method to address these challenges has been to design recombinant TCRs with mouse constant domains to prevent mispairings with endogenous human TCR a or b chains.
  • the use of recombinant TCRs with mouse sequences may present a risk for immune response.
  • a genetic disruption is introduced, e.g., by gene editing, at an endogenous gene encoding one or more TCR chains.
  • mouse and human IL-12 expression vectors comprising nucleic acid sequences encoding IL-12B (p40) and IL-12A (p35) .
  • the nucleic acid sequences encoding the IL-12B and IL-12A are linked by a linker sequence.
  • the vectors further comprise a sequence encoding an immunoglobulin hinge region, two or three constant domains (e.g., one or two CH2, and a CH3) , and a transmembrane region (e.g., a CD4 transmembrane region) , linked after IL-12A.
  • the vector encoding IL-12 further comprises a sequence encoding a heavy chain variable region (vH) and light chain variable region (vL) of a tumor-targeting antibody (e.g., NHS-76) before IL-12B.
  • vH heavy chain variable region
  • vL light chain variable region
  • the nucleic acid sequences encoding the vH and vL are linked by a linker sequence.
  • nucleic acid sequences encoding the vL and IL-12B are linked by a linker sequence.
  • the vector described herein comprises a sequence encoding a signal peptide sequence.
  • the sequence encoding the signal peptide sequence is linked to the sequence encoding IL-12B.
  • the sequence encoding the signal peptide sequence is linked to the sequence encoding vH of the tumor-targeting antibody.
  • the vector further comprises a sequence encoding a linker peptide sequence (e.g., P2A) before the signal peptide sequence.
  • the vector described herein further comprises a sequence encoding a T cell receptor (TCR) , or a chimeric antigen receptor (CAR) .
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the sequence encoding the TCR or the CAR is linked before the sequence encoding the signal peptide sequence (e.g., P2A) .
  • the present disclosure also provides nucleic acids that encodes human IL-12 fusion proteins.
  • the nucleic acid that encodes the IL-12 fusion protein described herein is set forth in SEQ ID NO: 7, or a nucleic acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the nucleic acid that encodes the membrane-tethered IL-12 fusion protein described herein is set forth in SEQ ID NO: 9, or a nucleic acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the nucleic acid that encodes the membrane-tethered IL-12 fusion protein described herein is set forth in SEQ ID NO: 11, or a nucleic acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • the nucleic acid that encodes the soluble IL-12 fusion protein described herein is set forth in SEQ ID NO: 13, or a nucleic acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • Linker refers to an oligo-or polypeptide region from about 1 to 100 amino acids in length, which links together any of the domains/regions.
  • Linkers can be composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.
  • Linkers can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (for example P2A, T2A) , 2A-like linkers or functional equivalents thereof and combinations thereof.
  • the linkers include the picornaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A) , Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof.
  • P2A porcine teschovirus
  • T2A Thosea asigna virus
  • Other linkers will be apparent to those of skill in the art and can be used in the methods described herein.
  • the linker peptide sequence comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker sequence comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues. In some embodiments, the linker sequence comprises or consists of both glycine and serine residues.
  • the linker sequence comprises or consists of a sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100%identical to SEQ ID NO: 17, GGGGSGGGGS (SEQ ID NO: 18) , or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19) .
  • the linker sequence comprises at least 1, 2, 3, 4, 5, or 6 repeats of GGGGS (SEQ ID NO: 20) .
  • nucleic acid sequence comprising a nucleotide sequence encoding any of the IL-12, modified IL-12, CAR, TCRs, antigen binding fragments thereof, and/or TCR-derived binding molecules (including e.g., functional portions and functional variants thereof, polypeptides, or proteins described herein) .
  • Nucleic acid as used herein can include “polynucleotide, ” “oligonucleotide, ” and “nucleic acid molecule, ” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources, which can contain natural, non-natural or altered nucleotides.
  • the nucleic acid comprises complementary DNA (cDNA) . It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it can be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • nucleic acids as described herein can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides.
  • the nucleotide sequence is codon-optimized.
  • the present disclosure also provides the nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.
  • the peptide that causes ribosome skipping is a P2A or T2A peptide.
  • the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.
  • the vector can additionally include a nucleic acid sequence that encodes a checkpoint inhibitor (CPI) (e.g., an inhibitory protein) .
  • CPI checkpoint inhibitor
  • the checkpoint inhibitor is e.g., any antibody or antigen binding fragment thereof as described herein.
  • the antibody or antigen binding fragments thereof can specifically bind to PD-1, PD-L1, PD-L2, 2B4 (CD244) , 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, butyrophilins, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47) , TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, or VISTA.
  • CD244 CD244
  • 4-1BB A2aR
  • the inhibitory protein is a scFv (e.g., an anti-PD-1 scFv) .
  • the vector can additionally include a nucleic acid sequence that encodes a bifunctional trap fusion protein.
  • the bifunctional trap protein targets both the PD-1 and TGF- ⁇ .
  • the bifunctional trap protein targets both the PD-L1 and TGF- ⁇ .
  • the bifunctional fusion protein designed to block PD-L1 and sequester TGF- ⁇ .
  • M7824 (MSB0011395C) comprises the extracellular domain of human TGF- ⁇ receptor II (TGF ⁇ RII) linked to the C-terminus of the human anti-PD-L1 scFv, based on the human IgG1 monoclonal antibody (mAb) avelumab.
  • the bifunctional fusion protein comprises the extracellular domain of human TGF- ⁇ receptor II (TGF ⁇ RII) linked to the C-terminus of the human anti-PD-1 scFv.
  • the IL-12 (e.g., membrane-tethered IL-12) , CAR, TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
  • the polypeptide comprises a signal peptide.
  • the polypeptide and/or the fusion protein is recombinant.
  • the disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.
  • the disclosure relates to nucleotide sequence that is
  • the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
  • the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the present disclosure provides a method or process for preparing, manufacturing and/or using the engineered cells for treatment of pathological diseases or conditions.
  • the cells for introduction of the protein described herein can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • a sample such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector) , washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs) , leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS) .
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated "flow-through” centrifuge.
  • a washing step is accomplished by tangential flow filtration (TFF) .
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca 2+ /Mg 2+ free PBS.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
  • the method comprises one or more steps of: e.g., isolating the T cells from a patient’s blood; transducing the population T cells with a viral vector including the nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and/or infusing the expanded cells into the patient, where the engineered T cells will seek and destroy antigen positive tumor cells.
  • the nucleic acid construct further includes a sequence encoding an inhibitory protein.
  • these engineered T cells can block PD-1/PD-L1 immunosuppression and strengthen the antitumor immune response.
  • the method further comprises: transfection of T cells with the viral vector containing the nucleic acid construct.
  • the methods involve introducing any vectors described herein into a cell in vitro or ex vivo.
  • the vector is a viral vector and the introducing is carried out by transduction.
  • the cell is transduced for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer.
  • the methods further involve introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
  • T cell receptor alpha constant TRAC
  • TRBC T cell receptor beta constant
  • the one or more agent is an inhibitory nucleic acid (e.g., siRNA) .
  • the one or more agent is a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease (e.g., a clustered regularly interspaced short palindromic nucleic acid (CRISPR) -associated nuclease) .
  • CRISPR clustered regularly interspaced short palindromic nucleic acid
  • the cell is a tumor infiltrating lymphocyte, and the cell is transfected with a vector encoding IL-12 or a modified IL-12.
  • the cell is a T cell, and the cell is transfected with a vector encoding IL-12 (e.g., a modified IL-12) and TCR or a vector encoding IL-12 (e.g., a modified IL-12) and CAR.
  • transfection of T cells can be achieved by using any standard method such as calcium phosphate, electroporation, liposomal mediated transfer, microinjection, biolistic particle delivery system, or any other known methods by skilled artisan.
  • transfection of T cells is performed using the calcium phosphate method.
  • the present disclosure provides a method to create a personalized anti-tumor immunotherapy.
  • Genetically engineered T cells can be produced from a patient’s blood cells. These engineered T cells are then reinfused into the patient as a cellular therapy product.
  • the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the disclosure features methods that include administering a therapeutically effective amount of engineered cells expressing IL-12 (e.g., modified IL-12) , TCR, CAR, antigen binding fragments thereof, and TCR-derived binding molecules to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) .
  • IL-12 e.g., modified IL-12
  • TCR e.g., modified IL-12
  • CAR e.g., antigen binding fragments thereof
  • TCR-derived binding molecules e.g., a subject having, or identified or diagnosed as having, a cancer
  • the subject has a solid tumor (e.g., a heterogeneous solid tumor or a homogeneous solid tumor) .
  • the subject has breast cancer (e.g., triple-negative breast cancer) , carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancy.
  • breast cancer e.g., triple-negative breast cancer
  • carcinoid cancer e.g., cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer
  • the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, or metastatic hormone-refractory prostate cancer.
  • NSCLC non-small cell lung carcinoma
  • SCLC small cell lung cancer
  • the cancer is cervical cancer, head and neck cancer, oropharyngeal cancers, anal cancer, penile cancer, vaginal cancer or vulvar cancer.
  • the subject has a heterogeneous cancer. In some embodiments, the subject has a homogeneous cancer. In some embodiments, the heterogeneous cancer has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more cancer cells expressing different antigens as compared to one or more antigens that are expressed by the majority (e.g., at least 50%, 60%, 70%, 80%, or 90%) of cancer cells in the heterogeneous cancer.
  • the majority e.g., at least 50%, 60%, 70%, 80%, or 90%
  • the TCR, CAR, antigen binding fragments thereof, and/or TCR-derived binding molecules can bind to one or more antigens that are expressed by a fraction (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or more) or all cells in the heterogeneous cancer.
  • the one or more antigens are not expressed by a fraction (e.g., less than 50%, 40%, 30%, 20%, 10%, or 5%) of cells in the heterogeneous cancer.
  • the one or more antigens are deactivated (e.g., cleaved, or through mechanisms such as immune escape) , such that the TCR, CAR, antigen binding fragments thereof, and/or TCR-derived binding molecules can no longer recognize.
  • a fraction of cells e.g., less than 50%, 40%, 30%, 20%, 10%, or 5%
  • immune checkpoint molecules e.g., PD-L1
  • the IL-12 (e.g., modified IL-12) expressed by the engineered cells described herein can provide improvement (e.g., killing cancer cells, or reducing tumor volume) of treating the heterogeneous cancer by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, 100 folds, or more as compared to similar engineered TCR or CAR cells that do not express the IL-12.
  • improvement e.g., killing cancer cells, or reducing tumor volume
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • the disclosure provides methods for treating infection or infection associated conditions in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of the disease. These methods generally involve administering a therapeutically effective amount of genetic engineered cells disclosed herein to a subject in need thereof.
  • the disease or condition treated is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Human Papilloma Virus (HPV) , Cytomegalovirus (CMV) , Epstein-Barr virus (EBV) , adenovirus, BK polyomavirus.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent and/or therapeutic compositions is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • the term "delaying development of a disease” refers to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer) .
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • a late stage cancer such as development of metastasis, can be delayed.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of compositions used.
  • Effective amounts and schedules for administrations may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the treatment, the route of administration, the particular type of therapeutic agents and other drugs being administered to the mammal. Guidance in selecting appropriate doses can be found in the literature. In addition, a treatment does not necessarily result in the 100%or complete treatment or prevention of a disease or a condition. There are multiple treatment/prevention methods available with a varying degree of therapeutic effect which one of ordinary skill in the art recognizes as a potentially advantageous therapeutic mean.
  • the present disclosure also provides methods of diagnosing a disease/condition in a mammal, wherein the TCRs, CARs, antigen binding fragments, TCR-derived binding molecules interact with the sample (s) obtained from a subject to form a complex, wherein the sample can comprise one more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen binding portions, blood, whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction thereof, wherein the detection of the complex is the indicative of presence of a condition in the mammal, wherein the condition is cancer or infection.
  • the sample can comprise one more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen binding portions, blood, whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction
  • the detection of the complex can be in any number of way known in the art but not limited to, ELISA, Flow cytometery, Fluorescence in situ hybridization (FISH) , Polymerase chain reaction (PCR) , microarray, southern blotting, electrophoresis, Phage analysis, chromatography and more.
  • the treatment methods can further include determining whether a subject can benefit from a treatment as disclosed herein, e.g., by determining whether the subject has infection or cancer.
  • the engineered cells and, and/or at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • at least two different engineered cells e.g., cells express different binding molecules
  • engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) .
  • engineered cells and the at least one additional therapeutic agent are administered in two different compositions.
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule.
  • the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • the one or more additional therapeutic agents can be administered to the subject prior to, concurrently with, or after administering the engineered cells to the subject.
  • one or more additional therapeutic agents can be administered to the subject.
  • the additional therapeutic agent can be a checkpoint inhibitor (CPI) .
  • CPI checkpoint inhibitor
  • the checkpoint inhibitor is an inhibitory protein, e.g., an antibody or antigen binding fragment thereof.
  • the checkpoint inhibitor can inhibit or block one or more immune checkpoints, including e.g., PD-1, PD-L1, PD-L2, 2B4 (CD244) , 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, butyrophilins, CD160, CD48, CTLA4, GITR, gp49B, HHLA2, HVEM, ICOS, ILT-2, ILT-4, KIR family receptors, LAG-3, OX-40, PIR-B, SIRPalpha (CD47) , TFM-4, TIGIT, TIM-1, TIM-3, TIM-4, VISTA and combinations thereof.
  • immune checkpoints including e.g., PD-1, PD-L1, PD-L2, 2B4 (CD244) , 4-1BB, A2aR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B
  • the inhibitory protein blocks PD-1 or PD-Ll.
  • the inhibitory protein comprises an anti-PD-1 scFv.
  • the inhibitory protein is capable of leading to reduced expression of PD-1 or PD-L1 and/or inhibiting upregulation of PD-1 or PD-L1 in T cells in the population and/or physically obstructing the formation of the PD-1/PD-L1 complex and subsequent signal transduction.
  • the inhibitory protein blocks PD-1.
  • the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.
  • the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-CD20 antibody (e.g., rituximab) , an anti-EGFR antibody (e.g., cetuximab) , an anti-CD319 antibody (e.g., elotuzumab) , or an anti-PD1 antibody (e.g., nivolumab) .
  • an anti-CTLA4 antibody e.g., ipilimumab
  • an anti-CD20 antibody e.g., rituximab
  • an anti-EGFR antibody e.g., cetuximab
  • an anti-CD319 antibody e.g., elotuzumab
  • an anti-PD1 antibody e.g., nivolumab
  • the additional therapeutic agent is a bifunctional trap fusion protein.
  • Bifunctional trap proteins can target both immune checkpoints and TGF- ⁇ negative regulatory pathways.
  • the tumor microenvironment contains other immunosuppressive molecules.
  • TGFB cytokine TGF- ⁇
  • TGF- ⁇ prevents proliferation and promotes differentiation and apoptosis of tumor cells early in tumor development.
  • tumor TGF- ⁇ insensitivity arises due to the loss of TGF- ⁇ receptor expression or mutation to downstream signaling elements.
  • TGF- ⁇ then promotes tumor progression through its effects on angiogenesis, induction of epithelial-to-mesenchymal transition (EMT) , and immune suppression.
  • EMT epithelial-to-mesenchymal transition
  • the bifunctional trap protein targets both the PD-1 and TGF- ⁇ . In some embodiments, the bifunctional trap protein targets both the PD-L1 and TGF- ⁇ . In some embodiments, the bifunctional fusion protein designed to block PD-L1 and sequester TGF- ⁇ .
  • M7824 (MSB0011395C) comprises the extracellular domain of human TGF- ⁇ receptor II (TGF ⁇ RII) linked to the C-terminus of the human anti-PD-L1 scFv, based on the human IgG1 monoclonal antibody (mAb) avelumab.
  • the bifunctional fusion protein comprises the extracellular domain of human TGF- ⁇ receptor II (TGF ⁇ RII) linked to the C-terminus of the human anti-PD-1 scFv.
  • bifunctional trap fusion proteins are described e.g., Knudson, et al., "M7824, a novel bifunctional anti-PD-L1/TGF ⁇ Trap fusion protein, promotes anti-tumor efficacy as monotherapy and in combination with vaccine. " Oncoimmunology 7.5 (2018) : e1426519, which is incorporated herein by reference in its entirety.
  • the subject is treated by cells that express TCR or antigen-binding molecules as described herein and one or more bifunctional trap fusion proteins.
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) .
  • an inhibitor of B-Raf an EGFR inhibitor
  • an inhibitor of a MEK an inhibitor of ERK
  • K-Ras an inhibitor of c-Met
  • ALK an inhibitor
  • the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) .
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, prala
  • therapeutic agents
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
  • TNF tumor necrosis factor
  • carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
  • the additional therapeutic agent is selected from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and/or combinations thereof.
  • compositions including pharmaceutical and therapeutic compositions
  • methods e.g., therapeutic methods for administrating the engineered cells and compositions thereof to subjects, e.g., patients.
  • compositions including the engineered cells for administration including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof are provided.
  • the pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carrier or excipient.
  • the composition includes at least one additional therapeutic agent.
  • a pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient.
  • the pharmaceutically acceptable carrier does not interfere with the active ingredient and is nontoxic to a subject.
  • a pharmaceutically acceptable carrier can include, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the pharmaceutical formulation refers to process in which different substances and/or agents are combined to produce a final medicinal product. The formulation studies involve developing a preparation of drug acceptable for patient. Additionally, a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the choice of carrier is determined in part by the particular cell (e.g., T cell or NK cell) and/or by the method of administration.
  • the pharmaceutical composition can contain preservatives. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001%to about 2%by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) .
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001%to about 4%by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005) .
  • the formulations can include aqueous solutions.
  • the formulation or composition can also contain more than one active ingredient useful for a particular indication, disease, or condition being treated with the engineered cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition can further include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • other pharmaceutically active agents or drugs such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutic agents e.g., asparaginase
  • the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • the desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
  • the cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous.
  • immunoresponsive T cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject after genetically modifying them in accordance with various embodiments described herein.
  • Peripheral blood derived immunoresponsive T cells or their progeny e.g., in vivo, ex vivo or in vitro derived
  • a therapeutic composition e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell
  • it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion) .
  • Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the cell populations are administered parenterally.
  • parenteral, ” as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can in some aspects be buffered to a selected pH.
  • sterile liquid preparations e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can in some aspects be buffered to a selected pH.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
  • Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • carriers can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as a suitable carrier, diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose) , pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts can in some aspects be consulted to prepare suitable preparations.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, and sorbic acid.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.
  • the methods described herein can reduce the risk of the developing diseases, conditions, and disorders as described herein.
  • the cells, populations, and compositions, described herein are administered to a subject or patient having a particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • adoptive cell therapy such as adoptive T cell therapy.
  • cells and compositions prepared by the provided methods such as engineered compositions and end-of-production compositions following incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition.
  • the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in cancer expressing an antigen recognized by the engineered T cells.
  • the cell therapy e.g., adoptive T cell therapy
  • the cell therapy is carried out by autologous transfer, in which the T cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the cell therapy e.g., adoptive T cell therapy
  • the cell therapy is carried out by allogeneic transfer, in which the T cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject.
  • the cells then are administered to a different subject, e.g., a second subject, of the same species.
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells.
  • the subject is refractory or non-responsive to the other therapeutic agent.
  • the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT.
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
  • the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time.
  • the subject has not relapsed.
  • the subject is determined to be at risk for relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
  • the subject has not received prior treatment with another therapeutic agent.
  • the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type (s) and/or a desired ratio of cell types.
  • the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio.
  • the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types.
  • the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
  • the populations or sub-types of cells are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells.
  • the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight.
  • the individual populations or sub-types are present at or near a desired output ratio (such as CD4+ to CD8+ ratio) , e.g., within a certain tolerated difference or error of such a ratio.
  • a desired output ratio such as CD4+ to CD8+ ratio
  • the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells.
  • the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.
  • the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations.
  • the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+ to CD8+ cells, and/or is based on a desired fixed or minimum dose of CD4+ and/or CD8+ cells.
  • the cells or individual populations of sub-types of cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values) , such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values) , and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells,
  • the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about 10 4 and at or about 10 9 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg, or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 6 T cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells are administered at or within a certain range of error of between at or about 10 4 and at or about 10 9 CD4+ and/or CD8+ cells/kilograms (kg) body weight, such as between 10 5 and 10 6 CD4+ and/or CD8+ cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 1.5 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 2 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, or 1 ⁇ 10 6 CD4+ and/or CD8+ cells/kg body weight.
  • body weight such as between 10 5 and 10 6 CD4+ and/or CD8+ cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 1.5 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 2 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, or 1 ⁇ 10 6 CD4+ and
  • the cells are administered at or within a certain range of error of, greater than, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 CD4+ cells, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 CD8+ cells, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 T cells.
  • the cells are administered at or within a certain range of error of between about 10 8 and 10 12 or between about 10 10 and 10 11 T cells, between about 10 8 and 10 12 or between about 10 10 and 10 11 CD4+ cells, and/or between about 10 8 and 10 12 or between about 10 10 and 10 11 CD8+ cells.
  • the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types.
  • the desired ratio can be a specific ratio or can be a range of ratios.
  • the desired ratio (e.g., ratio of CD4+ to CD8+ cells) is between at or about 1: 5 and at or about 5: 1 (or greater than about 1: 5 and less than about 5: 1) , or between at or about 1: 3 and at or about 3: 1 (or greater than about 1: 3 and less than about 3: 1) , such as between at or about 2: 1 and at or about 1: 5 (or greater than about 1: 5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9: 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5
  • the tolerated difference is within about 1%, about 2%, about 3%, about 4%about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%of the desired ratio, including any value in between these ranges.
  • the TCR described here provides improved expression and activity, thereby providing therapeutic effects even at a low effector to target (E: T) ratio.
  • Optimal response to therapy can depend on the ability of the engineered recombinant receptors such as TCRs, to be consistently and reliably expressed on the surface of the cells and/or bind the target antigen.
  • properties of certain recombinant receptors e.g., TCRs
  • TCRs can affect the expression and/or activity of the recombinant receptor, in some cases when expressed in a cell, such as a human T cell, used in cell therapy.
  • the level of expression of particular recombinant receptors, e.g., TCRs can be low, and activity of the engineered cells, such as human T cells, expressing such recombinant receptors, may be limited due to poor expression or poor signaling activity.
  • the desired ratio is between at or about 1: 10 and at or about 10: 1 (or greater than about 1: 10 and less than about 10: 1) , or between at or about 1: 1 and at or about 10: 1 (or greater than about 1: 1 and less than about 5: 1) , such as between at or about 2: 1 and at or about 10: 1.
  • the E: T ratio is greater than or about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.
  • the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician.
  • the compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
  • the cells described herein can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
  • the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the cells are administered prior to the one or more additional therapeutic agents.
  • the cells are administered after the one or more additional therapeutic agents.
  • the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence.
  • the methods comprise administration of a chemotherapeutic agent.
  • the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods.
  • Parameters to assess include specific binding of engineered T cells to the antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., "Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor.
  • the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD107a, IFN ⁇ , IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • Repeated dosing methods are provided in which a first dose of cells is given followed by one or more second consecutive doses.
  • the timing and size of the multiple doses of cells generally are designed to increase the efficacy and/or activity and/or function of engineered cells as described herein, when administered to a subject in adoptive therapy methods.
  • the methods involve administering a first dose, generally followed by one or more consecutive doses, with particular time frames between the different doses.
  • administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose, provided in multiple individual compositions or infusions, over a specified period of time (e.g., no more than 3 days) .
  • the first or consecutive dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time.
  • the first or consecutive dose is administered in multiple injections or infusions over a limited time period (e.g., no more than three days) , such as once a day for three days or for two days or by multiple infusions over a single day period.
  • a limited time period e.g., no more than three days
  • the cells of the first dose are administered in a single pharmaceutical composition.
  • the cells of the consecutive dose are administered in a single pharmaceutical composition.
  • the cells of the first dose are administered in a plurality of compositions, collectively containing the cells of the first dose.
  • the cells of the consecutive dose are administered in a plurality of compositions, collectively containing the cells of the consecutive dose.
  • additional consecutive doses can be administered in a plurality of compositions over a period of no more than 3 days.
  • the term “consecutive dose” refers to a dose that is administered to the same subject after the prior, e.g., first, dose without any intervening doses having been administered to the subject in the interim. Nonetheless, the term does not encompass the second, third, and/or so forth, injection or infusion in a series of infusions or injections comprised within a single split dose. Thus, unless otherwise specified, a second infusion within a one, two or three-day period is not considered to be a “consecutive” dose as used herein.
  • a second, third, and so-forth in the series of multiple doses within a split dose also is not considered to be an “intervening” dose in the context of the meaning of “consecutive” dose.
  • a dose administered a certain period of time, greater than three days, after the initiation of a first or prior dose is considered to be a “consecutive” dose even if the subject receives a second or subsequent injection or infusion of the cells following the initiation of the first dose, so long as the second or subsequent injection or infusion occurred within the three-day period following the initiation of the first or prior dose.
  • multiple administrations of the same cells over a period of up to 3 days is considered to be a single dose, and administration of cells within 3 days of an initial administration is not considered a consecutive dose and is not considered to be an intervening dose for purposes of determining whether a second dose is “consecutive” to the first.
  • multiple consecutive doses are given, in some aspects using the same timing guidelines as those with respect to the timing between the first dose and first consecutive dose, e.g., by administering a first and multiple consecutive doses.
  • the timing between the first dose and first consecutive dose, or a first and multiple consecutive doses is such that each consecutive dose is given within a period of time is greater than about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more.
  • the consecutive dose is given within a time period that is less than about 28 days after the administration of the first or immediately prior dose.
  • the additional multiple additional consecutive dose or doses also are referred to as subsequent dose or subsequent consecutive dose.
  • the size of the first and/or one or more consecutive doses of cells are generally designed to provide improved efficacy and/or reduced risk of toxicity.
  • a dosage amount or size of a first dose or any consecutive dose is any dosage or amount as described above.
  • the number of cells in the first dose or in any consecutive dose is between about 0.5 ⁇ 10 6 cells/kg body weight of the subject and 5 ⁇ 10 6 cells/kg, between about 0.75 ⁇ 10 6 cells/kg and 3 ⁇ 10 6 cells/kg or between about 1 ⁇ 10 6 cells/kg and 2 ⁇ 10 6 cells/kg.
  • first dose is used to describe the timing of a given dose being prior to the administration of a consecutive or subsequent dose. The term does not necessarily imply that the subject has never before received a dose of cell therapy or even that the subject has not before received a dose of the same cells or cells expressing the same recombinant receptor or targeting the same antigen.
  • multiple doses can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) .
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) .
  • Mouse and human IL-12 expression vectors were designed to contain IL-12B (p40) and IL-12A (p35) separated by a peptide linker.
  • IL-12B p40
  • IL-12A p35
  • membrane-tethered IL-12 an immunoglobulin hinge region with either two (membrane-tethered IL-12; mt-IL-12; or smt) or three (long membrane-tethered IL-12; long mt-IL-12; or lmt) constant domains (CH2 and CH3) together with a CD4 transmembrane region were added after IL-12A.
  • a vector expressing soluble IL-12 was also designed to contain both heavy chain variable region (VH) and light chain variable region (VL) of a tumor necrosis-targeting human IgG1, NHS-76 (NHS76-IL-12) .
  • an additional nucleic acid sequence encoding TCR (FIG. 1C) or CAR (FIG. 1D) was added to the expression vectors.
  • This additional sequence and the sequence encoding IL-12B, or NHS-76 vH, were separated by a P2A peptide sequence.
  • the mouse IL-12 construct is shown in FIG. 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 2 and SEQ ID NO: 1, respectively.
  • the mouse membrane-tethered IL-12 construct is shown in FIG. 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the mouse long membrane-tethered IL-12 construct is shown in FIG. 1A, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the human IL-12 construct is shown in FIG. 1B, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the human membrane-tethered IL-12 construct is shown in FIG. 1B, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
  • the human long membrane-tethered IL-12 construct is shown in FIG. 1B, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the human NHS76-IL-12 construct is shown in FIG. 1B, and its nucleic acid sequence and corresponding amino acid sequence are SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
  • L202 TCR L202 TCR
  • mt12 mouse membrane-tethered IL-12
  • NHS76 mouse NHS-76-IL-12
  • results in FIGS. 2A-2B indicate that both mouse IL-12 and IFN ⁇ were expressed at higher levels in mt12 and NHS76 cells, and cytokine concentrations were further increased upon co-culture with target cells.
  • Amino acid sequences of mouse membrane-tethered IL-12 and NHS76-IL-12 are shown in SEQ ID NO: 4 and SEQ ID NO: 14, respectively.
  • mouse lymphocytes isolated from lymph node were untransduced (Sample Name: UT) or transduced to express L202 TCR alone (Sample Name: L202) , L202 together with membrane-tethered IL-12 (Sample Name: mt12) , or L202 together with mouse NHS76-IL-12 (Sample Name: NHS76) .
  • the indicated lymphocyte populations were co-cultured overnight with Jurkat target cells.
  • Mouse IFN ⁇ was then measured in culture media using a mouse IFN ⁇ ELISA MAX kit (BioLegend) . As shown in FIG. 3, results indicate that the IL-12 expression stimulated lymphocyte IFN ⁇ production upon incubation with target cells and that IL-12 expression trans-activate lymphocytes.
  • EXAMPLE 4 IL-12 expression on the surface of armored TCR-T in mouse lymphocytes
  • EXAMPLE 5 Intracellular IL-12 expression of NHS-76-IL-12 mouse lymphocytes
  • Mouse lymphocytes were isolated from lymph nodes and then transduced for 2 days with NHS76-IL-12. As measured by staining with human Fab (FIG. 5A) or anti-IL12 antibody PE-Cy7 (FIG. 5B) , lymphocytes transduced with NHS76-IL-12 exhibited intracellular IL-12 expression.
  • EXAMPLE 6 TCRb and human IL-12 surface staining of human PBMCs
  • Human PBMCs were transduced with L202 TCR alone or in combination with human mt-IL-12 or human long mt-IL-12, as indicated. 12 days post-infection, TCR and IL-12 surface expression were measured by staining with anti-TCRb antibody (TCRb-PE) and anti-IL-12 antibody (IL-12-PE-Cy7) , respectively.
  • TCRb-PE anti-TCRb antibody
  • IL-12-PE-Cy7 anti-IL-12 antibody
  • mt-IL-12 human membrane-tethered IL-12
  • human long membrane-tethered IL-12 human long mt-IL-12
  • EXAMPLE 7 CD69 expression in PBMCs expressing mt-IL-12 co-cultured with target cells
  • FIG. 7A is a graph showing CD69 expression in human PBMCs co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • FIG. 7B is a graph showing CD69 expression in human PBMCs without co-culturing. As shown in FIGS. 7A-7B, PBMCs expressing IL-12 had stronger CD69 expression in comparison with L202 or untransduced T cells, when they were co-cultured with A375-HLA-A2-peplinker (LLW) melanoma target cells.
  • EXAMPLE 8 IFN ⁇ expression in IL-12 armored PBMCs
  • Untransduced human PBMCs, or human PBMCs transduced by L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (smt) , or L202 TCR-T plus human long membrane-tethered IL-12 (lmt) were co-cultured overnight with A375-HLA-A2-peplinker (LLW) target cells and then stained with hIFN ⁇ -FITC to measure T cell activation. As shown in FIGS.
  • EXAMPLE 9 CD69 expression in IL-12 armored mTCRb+ and mTCRb-cells
  • IL12 armored cells were co-cultured with A375-LLW melanoma target cells.
  • the L202 TCR transduced human PBMCs were either transduced by L202 alone, L202 plus human membrane-tethered IL-12 (smt) , or L202 plus human long membrane-tethered IL-12 (lmt) .
  • the L202 TCR has mouse TCRb.
  • the activation of CD3, CD8 double-positive T cells within the TCRb+ and TCRb-populations was measured by staining for CD69.
  • FIG. 9A the mTCRb+ population indicates cells with IL12 armor.
  • CD69 is a marker of activation.
  • the activation of TCRb+ cells was comparable for L202 TCR-T and armored L202 TCR-T cells (FIG. 9A) .
  • FIG. 9B the expression of CD69 on mTCRb-population indicates the activation of surrounding cells (unarmored cells) .
  • the unarmored cells were also activated.
  • the addition of membrane-tethered IL-12 further increased the activation of PBMCs that do not express mTCRb (FIG. 9B) .
  • Untransduced human PBMC cells human PBMC cells that were engineered to express L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (mtIL12) , or L202 TCR-T plus human long membrane-tethered IL-12 (long mtIL12) cells were mixed with untransduced human PBMC cells with a 1: 1 ratio.
  • EXAMPLE 11 IFN ⁇ activation in TCRb-cells is increased by the addition of membrane-tethered IL-12
  • Untransduced human PBMC cells human PBMC cells that were engineered to express L202 TCR-T, L202 TCR-T plus human membrane-tethered IL-12 (mtIL12) , or L202 TCR-T plus human long membrane-tethered IL-12 (long mtIL12) cells were mixed with untransduced human PBMC cells with a 1: 1 ratio.
  • EXAMPLE 12 Phenotypic analysis of IL-12 armored TCR-T cells
  • EXAMPLE 13 Flow cytometry staining to quantify central memory and effector memory T cells
  • EXAMPLE 14 Memory phenotype of IL-12 armored CD4+ TCR-T cells
  • CD4+ untransduced (UT) , L202 TCR-T (L202) , L202 TCR-T expressing human membrane-tethered IL-12 (mt-IL12) or L202 TCR-T expressing human long membrane-tethered IL-12 (lmt-IL12) PBMCs were stained for CD45RO and CCR7. As shown in FIGS. 14A-14D, results indicate that the expression of surface-bound IL-12 shifted CD4+ PBMCs toward an effector memory phenotype.
  • EXAMPLE 15 Memory phenotype of IL-12 armored CD8+ TCR-T cells
  • CD8+ untransduced (UT) , L202 TCR-T (L202) , L202 TCR-T expressing human membrane-tethered IL-12 (mt-IL12) or L202 TCR-T expressing human long membrane-tethered IL-12 (lmt-IL12) PBMCs were stained for CD45RO and CCR7. As shown in FIGS. 15A-15D, results indicate that the expression of surface-bound IL-12 shifted CD8+ PBMCs toward an effector memory phenotype.
  • EXAMPLE 16 IL-12 is not released from cells expressing membrane-tethered IL-12
  • Jurkat cells were either untransduced (UT) , or transduced to express IL-12, NHS76-IL-12, or membrane-tethered IL-12.
  • UT untransduced
  • 1 ⁇ 10 6 cells were plated in 1 ml of cell culture medium in a 12-well plate. After 24 hours, the cell cultures were centrifuged and supernatants were collected. Concentrations of IL-12 in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA) .
  • ELISA enzyme-linked immunosorbent assay

Abstract

La présente divulgation concerne des thérapies par cellules immunitaires armées à IL-12 et l'utilisation de l'IL-12 en association avec des thérapies par cellules immunitaires.
PCT/CN2020/090885 2020-05-18 2020-05-18 Thérapie par cellules immunitaires armées à il-12 et leurs utilisations WO2021232200A1 (fr)

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