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WO2025235605A1 - Activateur multispécifique des cellules immunitaires et ses utilisations - Google Patents

Activateur multispécifique des cellules immunitaires et ses utilisations

Info

Publication number
WO2025235605A1
WO2025235605A1 PCT/US2025/028138 US2025028138W WO2025235605A1 WO 2025235605 A1 WO2025235605 A1 WO 2025235605A1 US 2025028138 W US2025028138 W US 2025028138W WO 2025235605 A1 WO2025235605 A1 WO 2025235605A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
engager
protein
binding domain
immune cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/028138
Other languages
English (en)
Inventor
Jim QIN
Christopher Nicolai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Umoja Biopharma Inc
Original Assignee
Umoja Biopharma Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Umoja Biopharma Inc filed Critical Umoja Biopharma Inc
Publication of WO2025235605A1 publication Critical patent/WO2025235605A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • This disclosure relates generally to cell biology, immunology, and medicine - more particularly to multispecific immune cell engagers for us in medical treatments.
  • Multispecific antibodies recognize distinct antigens with each of their antigen-binding domains, in contrast to conventional antibodies (Abs) that recognize the same antigen with both Fab arms.
  • Bispecific T cell engagers targeting various antigens have been either approved or are in clinical development. These include blinatumomab (CD19), glofitamab (CD20), mosunetuzumab (CD20), solitomab (EpCAM), talquetamab (GPRC5D), tebentafusp (gplOO), and tarlatamab (DLL3). While these existing therapeutic options show promise for patients, improved methods and compositions for enhancing T cell activation combined with the targeting specificity of multispecific antibodies are needed.
  • a multispecific immune cell engager that includes an adhesion molecule, such as a binding domain that binds CD2 and/or activates CD2 signaling in a target cell (e.g., a T cell or other immune cell), and a target cell binding domain may be used to activate cells (e.g., T cells or other immune cells) and bind target cells.
  • a target cell e.g., a T cell or other immune cell
  • the concurrent binding of the adhesion molecule and the target cell binding domain may allow the activate tumor cell to kill the target cell.
  • the engager may be soluble.
  • the multispecific immune cell engager may be enhanced by fusing an adhesion molecule to a costimulatory molecule, an immune cell binding domain, or both in combination with a target cell binding domain.
  • the multispecific immune cell engager described herein allows for the concurrent activation of immune cells and targeting of cells, e.g., cancer cells.
  • the present disclosure provides a multispecific immune cell engager, comprising a binding domain that specifically binds to a cell-surface antigen present on immune cells (immune cell-binding domain) and a binding domain that specifically binds a cell-surface antigen present on target cell (target cell-binding domain), wherein the immune cell-cell engager further comprises: a binding domain specific to CD2 (CD2-binding domain); or both a CD2-binding domain and a binding domain specific to CD28 (CD28-binding domain).
  • CD2 CD2-binding domain
  • CD28-binding domain CD28-binding domain
  • Some embodiments relate to a multispecific immune cell engager, comprising: a CD2-binding domain; an immune cell-binding domain that binds to a cell-surface antigen on an immune cell; and a target cell-binding domain that binds a cell-surface antigen of a target cell.
  • the CD2-binding domain comprises a CD58 extracellular domain, optionally comprising a polypeptide sequence according to SEQ ID NO: 136, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.
  • the CD2-binding domain comprises a CD48 extracellular domain, optionally, optionally comprising a polypeptide sequence according to SEQ ID NO: 283, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.
  • the CD28-binding domain comprises a CD86 extracellular domain optionally comprising a polypeptide sequence according to SEQ ID NO: 135, or a sequence at least 80 at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.
  • the engager comprises a polypeptide chain comprising the immune cell-binding domain, the CD2-binding domain, the CD28-binding domain (if present), IgGl CH2 and CH3 domains; and the target cell-binding domain.
  • the polypeptide chain comprises, in N- to C- terminal order, the CD58 extracellular domain, the anti-CD3 antibody, the CD80 extracellular domain or the CD86 extracellular domain, IgGl CH2 and CH3 domains, and the target cell-specific binding domain.
  • the polypeptide chain comprises, in N- to C- terminal order, the CD80 extracellular domain or the CD86 extracellular domain, the anti-CD3 antibody, the CD58 extracellular domain, IgGl CH2 and CH3 domains, and the target cell binding domain.
  • the polypeptide chain comprises a polypeptide sequence disclosed in Table 6, or a sequence at least 80%, at least 90%, at least 95% identical thereto.
  • the present disclosure provides an engager, comprising a binding domain that specifically binds to a cell-surface antigen present on immune cells (immune cell-binding domain) and a binding domain that specifically binds a cell-surface antigen present on target cell (target cell-binding domain), wherein the engager further comprises: an adhesion molecule, optionally selected from CD58, CD48, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112, an extracellular domain thereof, or a functional fragment thereof; or both an adhesion molecule and a costimulatory molecule, optionally wherein the costimulatory molecule is CD80, CD86, CD40L (also known as CD154), GITRL, OX40L, 41BBL, ICOSL, CD70, CD30L, LIGHT, LTalpha, MICA, and MICB, or a functional fragment thereof.
  • an adhesion molecule optionally selected from CD58, CD48, ICAM-1,
  • the polypeptide chain comprises a polypeptide sequence disclosed in Table 6, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.
  • the immune cell-binding domain and/or the target cell-binding domain is an antibody, optionally fragment antigen binding (Fab), a fragment antigen-binding 2 (F(ab’)2), a single chain Fab (scFab), a single chain variable fragment (scFv), or a single domain antibody (sdAb) (e.g., a nanobody or VHH antibody)
  • Fab fragment antigen binding
  • F(ab’)2 fragment antigen-binding 2
  • scFab single chain Fab
  • scFv single chain variable fragment
  • sdAb single domain antibody
  • the target cell is a cancer cell or an immune cell (e.g., a natural killer cell or a B cell).
  • the target cell-binding domain is an antibody specific to CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, BCMA, ACTN4, BAGE-1, BCR-ABL, beta-catenin, CA 125, CA 15-3, CA 195, CA 242, CA 50, CAM43, Casp-8, CDC27, CDK4, CDKN2A, CEA, COA-1, DEK-CAN fusion protein, EBNA1, EF2, an Epstein Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, HSP70-2, KIAA0205, MART2, MUM-1, MUM- 2, MUM-3, neo-PAP, myosin class I proteins, OS-9, PML-RARa fusion protein, PTPRK, K
  • the immune cell-binding domain comprises a CD3 binding domain specific to CD3s.
  • the CD3-binding domain comprises an anti-CD3 antibody, optionally a CD3 single chain variable fragment.
  • a polynucleotide comprises a polynucleotide sequence encoding an engager, optionally wherein the polynucleotide sequence is operatively linked to a promoter or to recombination sites for gene editing, optionally comprising a polynucleotide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence disclosed in Table 7.
  • a vector comprises a vector genome comprising a polynucleotide comprising a polynucleotide sequence encoding an engager, optionally wherein the polynucleotide sequence is operatively linked to a promoter or to recombination sites for gene editing, and optionally comprising a polynucleotide sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence disclosed in Table 7.
  • a host cell is configured to express an engager and/or comprising a polynucleotide encoding the engager.
  • a composition such as a pharmaceutical composition.
  • a pharmaceutical composition comprises an engager and a pharmaceutically acceptable diluent or excipient.
  • kits comprises an engager or a pharmaceutical composition thereof and instructions for use in a method of the disclosure.
  • the engager binds concurrently to a T-cell and a target cell thereby allowing the T cell to kill the target cell.
  • the target cell is a cancer cell.
  • the target cell is an immune cell.
  • the target cell is a B cell.
  • the target cell is a CD20+ B cell.
  • the present disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multispecific immune cell engager.
  • the disease or disorder is a cancer.
  • the cancer is a B-cell malignancy, diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma.
  • B-cell malignancy diffuse large B-cell lympho
  • the disease or disorder is an autoimmune disease.
  • the autoimmune disease is caused or exacerbated by B cells.
  • the present disclosure provides a method of inducing cytokine product by a T cell, comprising contacting the T cell with an effective amount of a multispecific immune cell engager.
  • FIG. 1A is a diagram showing examples of bispecific antibodies that may include an anti- CD3 antibody and a CD20 antibody fused together via an FC region.
  • the CD3 antibody can be removed and an immune cell engager comprising a CD58 extracellular domain, anti-CD3 antibody, and a CD80 extracellular domain can be added.
  • the multispecific T cell engager can concurrently bind T cells via the immune cell engager and CD20+ tumor cells via the CD20 antibody.
  • FIG. 2 A is a schematic that shows an illustrative fusion protein comprising a CD58 extracellular region and a-CD3 scFv fused to the N-terminus of a CD80 protein via a linker.
  • the construct is termed “498.”
  • FIG. 2B is a schematic that shows an illustrative fusion protein comprising a CD58 extracellular region fused to an N-terminus of a CD80 protein via a linker.
  • the construct is termed “455.”
  • a-CD3 scFv is expressed as a separate polypeptide in the producer cells.
  • FIG. 3A includes diagrams illustrating example fusion molecules that may be included as part of an immune cell engager.
  • FIG. 3B includes diagrams illustrating example fusion molecules that may be included as part of an immune cell engager.
  • FIG. 4 shows a FACs plot quantifying CD20 expression in Naim 6 and Raji cells lines. A geometric mean of Mean Fluorescent Intensify (gMFI) of CD20 expression is also shown.
  • GMFI Mean Fluorescent Intensify
  • FIG. 5 shows percentages of dual positive CD3+CD25+, CD4+CD25+, or CD8+CD25+ immune cells in co-cultures with Naim 6 tumor cells 3 days after treatment with increasing concentrations of a multispecific T cell engager, Rituximab or Mosunetuzumab
  • FIG. 6 shows geometric means of Mean Fluorescent Intensify (gMFI) of CD25 in dual positive CD3+CD25+, CD4+CD25+, or CD8+CD25+ immune cells in co-cultures with Naim 6 tumor cells 3 days after treatment with increasing concentrations of a multispecific T cell engager, Rituximab or Mosunetuzumab in co-cultures with Naim 6 tumor cells.
  • MFI Mean Fluorescent Intensify
  • FIG. 7 shows a FACs plot a quantification of immune cell proliferation, after treatment with increasing concentrations of a multispecific T cell engager, Rituximab or Mosunetuzumab. Cell trace violet was used to track numbers of cell divisions.
  • FIG. 8A shows percents of Naim 6 tumor cells in co-culture with immune cells at day 4 after treatment with increasing concentrations of a multispecific T cell engager, Rituximab or Mosunetuzumab.
  • FIG. 8B shows percentages of Naim 6 tumor cell lysis at 4 days after co-culture with immune cells and treatment with 1000 ng/ml or 100 ng/ml of a multispecific T cell engager, Rituximab or Mosunetuzumab.
  • FIG. 9 shows numbers of Naim 6 tumor cells at indicated time points after co-culture with immune cells and treatment with 1000 ng/ml or 100 ng/ml of a multispecific T cell engager, Rituximab or Mosunetuzumab.
  • FIG. 11 shows concentrations (pg/mL) of cytokines (IFNy, 11-2 or TNFa) produced by immune cells after co-culture with Naim 6 tumor cells and treatment with 100 ng/ml a multispecific T cell engager, Rituximab or Mosunetuzumab.
  • cytokines IFNy, 11-2 or TNFa
  • FIG. 12 shows concentrations (pg/mL) of cytokines (IFNY, H-2 or TNFa) produced by immune cells after co-culture with Naim 6 tumor cells and treatment with increasing concentrations of a multispecific T cell engager, Rituximab or Mosunetuzumab.
  • cytokines IFNY, H-2 or TNFa
  • a multispecific immune cell engager is used to enhance activation of immune cells and kill CD20+ tumor cells.
  • An antibody or antibody fragment against a component of a T cell receptor, such as cluster of differentiation 3 (CD3), may be incorporated into the multispecific immune cell engager to target the engager to T cells.
  • CD3 cluster of differentiation 3
  • engagement of CD3 by the multispecific immune cell engager comprising a binding domain targeting CD3 may cause the T cells to activate via a primary activation signal.
  • Improved therapeutics may provide a more robust activation signal compared with existing bispecific therapeutic options due to the presence of costimulatory domains.
  • Incorporation of one or more ligands for a co-receptor into the multispecific such as CD28, a molecule expressed by T cells, may enhance T cell activation and effector functions by providing a secondary costimulatory signal.
  • the present disclosure relates generally to an immune cell engager for use in the binding and activation of target cells, such as immune cells, or specifically T cells.
  • target cells such as immune cells, or specifically T cells.
  • the disclosure provides, an immune cell engager for binding and activation of target cells, comprising an adhesion molecule linked to a costimulatory molecule, an immune cell binding domain, or both.
  • the immune cell engager of the present disclosure combines an adhesion molecule with a costimulatory molecule or an immune cell binding domain, or both.
  • the term “transduction” is used in its broadest sense to mean delivery of an agent to a cell, such as a therapeutic agent.
  • the agent may be a small molecule, polynucleotide, or polypeptide.
  • a combination of agents may be delivered, such as several polynucleotides or a protein-nucleic acid complex (e.g., a gene-editing nuclease in complex with guide nucleic acid).
  • the term “particle” includes but is not limited to viral particles (e.g., a virion), lipid nanoparticles (LNPs), lipoplexes, liposomes, and nanocarriers.
  • an immune cell engager may be engineered to incorporate any of the adhesion molecules or costimulatory molecules; extracellular fragments thereof; or functional fragments thereof discussed in this application.
  • Extracellular portions of these molecules may be identified in databases such as UniProt, which is available at www.uniprot.org, or may be predicted using methods, such as a method implemented by the TMHMM 2.0 program available at services.healthtech.dtu.dk.
  • functional fragments of each are identified in scientific literature or they may be identified using laboratory methods. For example, one may identify the fragments of a protein likely to form well-folded domains.
  • Fragments may be tested in binding assays against a cognate molecule or used in pull-down assays compared to the full molecule.
  • Functional assays such as expression of a fluorescence reporter under the control of a promoter activated by T-cell signaling (e.g., the NKkB promoter) when a T cell is contacted with a cell, an immune cell engager or particle expressing a putative functional fragment.
  • the sequence of the adhesion molecule, costimulatory molecule, or immune cell binding domain may be varied to identify and use variants that retain function. For example, conservative mutations may be made to a molecule, or a molecule may be randomly mutated with the function of the variant confirmed experimentally.
  • a molecule may be incorporated as a full-length form, without its native transmembrane domain.
  • the extracellular portion of the molecule may be replaced with a heterologous protein.
  • Each immune cell engager may be a monomer, bound to another engager to form a dimer or bound to an additional antigen binding domain.
  • an adhesion molecule, costimulatory molecule, and immune cell binding domain may be linked in any order with only the most N-terminal or C-terminal of the molecules.
  • the immune cell engager comprises or is associated with another membrane-associated molecule.
  • the term “display” is used, in a broad sense, to mean positioned on the surface of the immune cell engager such that the molecule may contact cognate molecules on the target cell. It is further contemplated that the immune cell engager may be loaded onto a solid support either by association with a component of the particle (e.g. , a capsid protein) or by the addition of an additional molecule with affinity for the solid support (e.g., as an immune cell engager comprising a capsid protein).
  • a component of the particle e.g. , a capsid protein
  • an additional molecule with affinity for the solid support e.g., as an immune cell engager comprising a capsid protein.
  • an “engager” refers to a binding molecule or a complex of binding molecules capable a binding to a target cell or target cells.
  • the target cell may be an immune cell, in which case the engager may be termed an “immune-cell engager.”
  • the target cell may be an tumor cell, in which case the engager may be termed a “tumor cell engager.”
  • the engager may be a T-cell engager.
  • the immune-cell engager may include a fusion molecule.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, or a cell-binding molecule.
  • the fusion molecule may include an adhesion molecule.
  • the fusion molecule may include a costimulatory molecule.
  • the fusion molecule may include an immune cell binding domain.
  • the fusion molecule may include a tumor cell binding domain.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, and an immune cell binding domain.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, and a tumor cell binding domain.
  • the fusion molecule may include an adhesion molecule and an immune cell binding domain.
  • the fusion molecule may include an adhesion molecule and a tumor immune cell binding domain.
  • the fusion molecule may include a costimulatory molecule and an immune cell binding domain.
  • the fusion molecule may include a costimulatory molecule and a tumor cell binding domain.
  • the fusion molecule may be or include a fusion protein.
  • the immune cell engager may be isolated.
  • the immune cell engager may be soluble.
  • the immune cell engager may be or include a soluble fusion protein.
  • the immune cell engager (or the multispecific immune cell engager) may be used in a method described herein.
  • the immune cell engager may include an adhesion molecule such as a CD2-binding domain.
  • the CD2-binding domain may specifically bind CD2.
  • the immune cell engager may include an immune cell-binding domain.
  • the immune cell-binding domain may bind to a cell-surface antigen on an immune cell. The binding may be specific.
  • the immune cell engager may include a target cellbinding domain.
  • the target cell-binding domain may bind a cell-surface antigen of a target cell.
  • the immune cell engager includes: a CD2-binding domain; an immune cell- binding domain that binds to a cell-surface antigen on an immune cell; and a target cell-binding domain that binds a cell-surface antigen of a target cell.
  • the immune cell engager includes: an adhesion molecule; an immune cell-binding domain that specifically binds to a cell-surface antigen on an immune cell; and a target cell-binding domain that specifically binds a cell-surface antigen on a target cell.
  • the immune cell engager may include a costimulatory molecule such as a CD28-binding domain.
  • the disclosure provides an immune cell engager comprising a combination of an adhesion molecule, a costimulatory molecule, and an immune cell binding domain (e.g., a TCR-binding molecule), thereof each component linked directly or indirectly to the other components.
  • the immune cell engager comprises adhesion molecule, a costimulatory molecule, and an immune cell binding domain (e.g., a TCR-binding molecule).
  • the immune cell engager comprises adhesion molecule and a costimulatory molecule, but not a TCR-binding molecule.
  • the immune cell engager comprises an adhesion molecule and an immune cell binding domain (e.g., a TCR-binding molecule), but not a costimulatory molecule.
  • the immune cell engager may further comprise one or more additional adhesion molecules, costimulatory molecules, or immune cell binding domains (e.g., TCR-binding molecules).
  • the disclosure provides a tumor cell engager comprising a combination of an adhesion molecule, a costimulatory molecule, and a tumor cell binding domain, thereof each component linked directly or indirectly to the other components (such as a target cell binding domain).
  • the tumor cell engager comprises adhesion molecule, a costimulatory molecule, and a tumor cell binding domain.
  • the immune cell engager comprises an adhesion molecule and a tumor cell binding domain, but not a costimulatory molecule.
  • the tumor cell engager may further comprise one or more additional adhesion molecules, costimulatory molecules, or immune cell binding domains (e.g., TCR-binding molecules).
  • An immune cell engager may be soluble.
  • An immune cell engager may exclude a transmembrane domain.
  • the term “soluble” refers to any molecule that does not contain a transmembrane domain.
  • the engager is a soluble macromolecule, substantially free of lipids.
  • the soluble engager is not part of a viral particle.
  • the term “macromolecular complex” refers to a stable assembly of two or more fusion proteins.
  • the immune cell engager may be a monomer.
  • the immune cell engager may comprise an Fc domain.
  • the immune cell engager may comprise an Fc domain on the C terminus.
  • the immune cell engager may comprise an Fc domain on the N terminus.
  • the Fc domain of the immune cell engager may facilitate dimer formation. In some embodiments, the dimer is a homodimer.
  • the term “transmembrane domain” refers to the portion of a protein that spans the phospholipid bilayer of a biological membrane, such as the plasma membrane of a cell.
  • the immune cell engager may be comprised of proteins without then- transmembrane domain.
  • the immune cell engager may be comprised of proteins without their transmembrane domain.
  • the transmembrane domain is replaced with a heterologous protein.
  • fusion molecule refers to any molecule having multiple components linked together, directly or indirectly, covalently or non-covalently.
  • the fusion molecule may be soluble.
  • the fusion molecule may be made up of more than one protein.
  • the immune cell engager may be made up of one or more fusion molecules. When those proteins are linked together into a single molecule by peptide bonds, the fusion molecule is termed a “fusion protein.”
  • the fusion molecule may be made using various linkers, including chemical (covalent) bonds (e.g., by click chemistry) or by peptide bounds.
  • the linker between each component of the fusion protein may be a single peptide bound (e.g. , a direct C- to N- peptide bound in a polypeptide chain) or via a polypeptide linker.
  • Illustrative polypeptide linkers may include, but are not limited to, the glycine-serine linkers, such as GGSGGS (SEQ ID NO: 304), GSSGSS (SEQ ID NO: 305), or others.
  • the fusion molecule is or includes a fusion protein.
  • the fusion protein may comprise an adhesion protein, a polypeptide linker, and a costimulatory portion.
  • the fusion protein comprises an adhesion molecule, a costimulatory molecule, and an immune cell binding domain.
  • the adhesion molecule is N-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is N-terminal to the immune cell binding domain. In some embodiments, the adhesion molecule is C-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is C-terminal to the immune cell binding domain.
  • the costimulatory molecule is N-terminal to the immune cell binding domain. In some embodiments, the costimulatory molecule is N-terminal to the adhesion molecule. In some embodiments, the costimulatory molecule is C-terminal to the immune cell binding domain. In some embodiments, the costimulatory molecule is C-terminal to the adhesion molecule.
  • Some embodiments of the fusion protein include a linker. Some embodiments include multiple linkers. In some embodiments, a linker directly connects the costimulatory molecule with the adhesion molecule. In some embodiments, a linker directly connects the costimulatory molecule with the immune cell binding domain. In some embodiments, a linker directly connects the adhesion molecule with the immune cell binding domain.
  • an N terminal end of the costimulatory molecule is juxtaposed (via a polypeptide bond or a polypeptide linker sequence) with an end of the adhesion molecule.
  • a C terminal end of the costimulatory molecule is juxtaposed with an end of the adhesion molecule.
  • an N terminal end of the costimulatory molecule is juxtaposed with an end of the immune cell binding domain.
  • a C terminal end of the costimulatory molecule is juxtaposed with an end of the immune cell binding domain.
  • an N terminal end of the immune cell binding domain is juxtaposed with an end of the adhesion molecule. In some embodiments of the fusion protein, a C terminal end of the immune cell binding domain is juxtaposed with an end of the adhesion molecule. In some embodiments of the fusion protein, an N terminal end of the immune cell binding domain is juxtaposed with an end of the costimulatory molecule. In some embodiments of the fusion protein, a C terminal end of the immune cell binding domain is juxtaposed with an end of the costimulatory molecule.
  • an N terminal end of the adhesion molecule is juxtaposed with an end of the costimulatory molecule.
  • a C terminal end of the adhesion molecule is juxtaposed with an end of the costimulatory molecule.
  • an N terminal end of the adhesion molecule is juxtaposed with an end of the immune cell binding domain.
  • a C terminal end of the adhesion molecule is juxtaposed with an end of the immune cell binding domain.
  • the fusion protein comprises a Fc fusion.
  • the transmembrane domain is replaced with a Fc domain from IgG. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgA. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgE. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgM. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgGl . In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG2.
  • the transmembrane domain is replaced with a Fc domain from IgG3. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG4. In some embodiments, the Fc domain allows the fusion protein to form a monomer. In some embodiments, the Fc domain allows the fusion protein to form a dimer. In some embodiments, the Fc domain allows the fusion protein to form a multimer. In some embodiments, the Fc domain allows the fusion protein to form an oligomer.
  • the transmembrane domain is replaced with an additional binding domain.
  • the additional binding domain can be an antibody or scFv. Incorporation of the antibody or scFv into the fusion protein may allow for targeting the fusion protein to specific cell types.
  • the transmembrane domain is replaced with a cytokine.
  • Cytokines are small proteins that are crucial in controlling the growth and activity of immune system cells and bloods cells.
  • the cytokine is an interleukin.
  • the cytokine is an interferon.
  • the cytokine is a tumor necrosis factor.
  • the cytokine is a growth factor.
  • Linking the fusion protein (or immune cell engager) to a cytokine may allow the cytokine to bind to its cognate receptor upon binding of the fusion protein to a cell. The addition of the cytokine to the fusion protein may allow for the fusion protein to further activate immune cells in vivo and in vitro.
  • the fusion protein may comprise, in any order, a CD80, a CD80 extracellular domain, or a functional fragment of cluster of differentiation 80 (CD80); a cluster of differentiation 58 (CD58), a CD58 extracellular domain; or a functional fragment of CD58; an immune cell binding domain (e.g., a TCR-binding molecule); and polypeptide linkers.
  • the fusion protein may comprise, in any order, a cluster of differentiation 58 (CD58), a CD58 extracellular domain; or a functional fragment of CD58; a tumor cell binding domain; and polypeptide linkers.
  • the fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular domain; or a functional fragment of CD58.
  • the fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular domain, or a functional fragment of CD80.
  • the fusion protein may comprise, in N- to C-terminal order, an immune cell binding domain (e.g., a TCR-binding protein); a polypeptide linker; CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular domain; or a functional fragment of CD58.
  • an immune cell binding domain e.g., a TCR-binding protein
  • CD58 e.g., CD58 extracellular domain
  • the fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; and an immune cell binding domain (e.g., a TCR-binding protein).
  • the fusion protein may comprise, in N- to C-terminal order, an immune cell binding domain (e.g., a TCR-binding protein); a polypeptide linker; CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular domain, or a functional fragment of CD80.
  • an immune cell binding domain e.g., a TCR-binding protein
  • the fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; and an immune cell binding domain (e.g., a TCR-binding protein).
  • An illustrative fusion protein comprises a CD58 extracellular domain and anti-CD3 scFv fused to the N-terminus of a CD80 via a linker; this construct is termed a tri-fusion polypeptide and/or termed “498.”
  • An illustrative fusion protein comprises a CD58 extracellular domain fused to the N- terminus of CD80 via a linker; this construct is termed a bi-fusion polypeptide and/or termed “455.” In this construct, an anti-CD3 scFv is expressed as a separate polypeptide in the producer cells.
  • the polypeptide linker may be optional. It may be omitted by directly linking a protein molecule to the next via a peptide bound. Although one may generate fusion proteins through chemical synthesis, fusion proteins are made by expressing the fusion protein from a single polynucleotide comprising a polynucleotide sequence encoding the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art.
  • the fusion molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).
  • a polynucleotide e.g., a DNA or RNA polynucleotide
  • the disclosure provides polynucleotides encoding such fusion proteins.
  • the polynucleotide may be an isolated polynucleotide, or it may be part of a vector (e.g., a plasmid) or it may be introduced into and propagated in a host cell.
  • Polypeptide sequences of illustrative dual CD58+CD80 fusion proteins are provided in Table 1.
  • an optional signal peptide is shown in parentheses.
  • the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 1.
  • the fusion protein may comprise a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 1.
  • an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues. Diagrams of example fusions are provided in FIG. 2A- 3B. Any aspect shown in any of these figures may be included in a immune cell engager. Table 1
  • polypeptide sequences of illustrative triple CD58+CD80+anti-CD3scFv fusion proteins are provided in Table 2.
  • the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 2.
  • the fusion protein may comprise a polypeptide less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 2.
  • an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues.
  • the present disclosure provides an engager, comprising: a cell binding domain that specifically binds a receptor on a target cell comprising a target immune cell or a target tumor cell; and an immune cell adhesion molecule that specifically binds a reciprocal adhesion molecule on the target cell or on an immune cell.
  • Concurrent binding of the cell binding domain and the adhesion molecule may form a supramolecular adhesion complex.
  • the supramolecular adhesion complex may be formed on the target cell.
  • the supramolecular adhesion complex may be formed on an immune cell.
  • the target cell may be an immune cell.
  • An example immune cell is a T cell.
  • An example immune cell is a natural killer (NK) cell.
  • the target cell may be a B cell.
  • the target cell may be a cancer cell.
  • the target cell may be a tumor cell.
  • some embodiments relate to or include an engager, comprising: a cell binding domain that specifically binds a receptor on a target tumor cell; and an immune cell adhesion molecule that specifically binds a reciprocal adhesion molecule on an immune cell.
  • the target cell may be a malignant immune cell.
  • the target cell may be a malignant B cell.
  • the engager may comprise an additional target cell binding domain.
  • the engager may include a cell binding domain that specifically binds an antigen on a target cell.
  • the engager may include an immune cell adhesion molecule that specifically binds a reciprocal adhesion molecule of an immune cell. Binding of the cell binding domain to the immune cell may activate the immune cell. Binding of the cell binding domain to the antigen on the target cell and binding of the immune cell adhesion molecule to the immune cell may activate the immune cell.
  • the engager may be soluble.
  • cell engagers comprising: a cell binding domain that specifically binds an antigen on a target cell; and an immune cell adhesion molecule that specifically binds a reciprocal adhesion molecule of an immune cell, wherein binding of the immune cell adhesion molecule to the immune cell activates the immune cell, and wherein the cell engager is soluble.
  • the target cell is a tumor cell.
  • the antigen comprises CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, B-cell maturation antigen, alpha-actinin- 4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29 ⁇ BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, an Epstein Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA- All, hsp70-2, KIAAO205, Mart2, Mum-1, Mum-2, Mum-3, neo-PAP, myosin class I, OS-9, pml- RARa fusion protein, PTPRK, K-ras, N-ras, triosephosphate is
  • the target cell is the immune cell or another immune cell. In some embodiments, the target cell is the immune cell. In some embodiments, the target cell is another immune cell. In some embodiments, the antigen comprises CD3. In some embodiments, the reciprocal adhesion molecule comprises CD2. In some embodiments, the immune cell adhesion molecule comprises a CD58 protein. Some embodiments include an immune cell costimulatory molecule. In some embodiments, the immune cell costimulatory molecule binds CD28. In some embodiments, the immune cell costimulatory molecule comprises a CD80 or CD86 protein. The engager may further comprise an additional target cell binding domain.
  • adhesion molecules may be included as part of a fusion protein.
  • the adhesion molecule may be included as part of an immune cell engager.
  • An engager may include an adhesion molecule such as a CD2-binding domain.
  • a CD2-binding domain may be specific for CD2.
  • An adhesion molecule may be or include an adhesion domain.
  • adhesion molecule refers, in a broad sense, to a molecular component of a SMAC or other immune synapse, other than an immune cell binding domain (e.g., TCR-binding agent) or a costimulatory molecule, which contributes to adhesion of a particle to target cells.
  • Adhesion molecules from natural sources may be molecules expressed, natively, on antigen- presenting cells and adapted for use here on particles. Both naturally occurring adhesion molecules, and their variants, and artificial adhesion molecules, such as antibodies, or fragments thereof, are contemplated.
  • adhesion molecule specifically binds a conjugate molecule with affinity sufficient to cause increased adhesion between the immune cell engager and the target cell compared to the adhesion of a reference immune cell engager lacking the adhesion molecule to the same or similar target cell.
  • adhesion molecule includes but is not limited to CD58, a CD58 extracellular domain, and functional fragments of CD58.
  • the term “functional fragment” is used herein to a fragment of a polypeptide, or other molecule, that retains the desired function of the polypeptide.
  • a functional fragment of CD58 is a fragment of CD58 that specifically binds cluster of differentiation 2 (CD2).
  • the adhesion molecule may be a protein, termed herein an “adhesion protein.”
  • the adhesion molecule comprises a cluster of differentiation 58 (CD58) protein, an intercellular adhesion molecule 1 (ICAM-1) protein, an intercellular adhesion molecule 2 (ICAM-2) protein, an intercellular adhesion molecule 3 (ICAM-3) protein, an intercellular adhesion molecule 4 (ICAM-4) protein, an intercellular adhesion molecule 5 (ICAM- 5) protein, a junctional adhesion molecule A (JAM-A) protein, a cluster of differentiation 155 (CD155) protein, or a cluster of differentiation 112 (CD112) protein.
  • CD58 cluster of differentiation 58
  • ICM-1 intercellular adhesion molecule 1
  • ICM-2 intercellular adhesion molecule 2
  • ICAM-3 intercellular adhesion molecule 3
  • ICM-4 intercellular adhesion molecule 4
  • ICM-5 intercellular adhesion molecule 5
  • JAM-A junctional adhesion molecule A
  • JAM-A junctional adhesion molecule A
  • the CD58 protein, ICAM-1 protein, ICAM-2 protein, ICAM-3 protein, ICAM-4 protein, ICAM-5 protein, JAM-A protein, CD 155 protein, or CD112 protein is or includes an extracellular domain or functional fragment of CD58, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112.
  • the adhesion molecule is CD58, cluster of differentiation 48 (CD48), ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112, or a functional fragment thereof.
  • a functional fragment may be or include a binding fragment.
  • Some functional fragments include or relate to a CD2 binding fragment.
  • the adhesion molecule is a binding domain that specifically binds CD2.
  • the costimulatory and/or adhesion molecule comprises an amino acid sequence 100% identical to a sequence in Table 3A or Table 3B. In some embodiments, the costimulatory and/or adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 3 A or Table 3B.
  • the costimulatory and/or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 3A or Table 3B.
  • adhesion molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 3 A, or functional fragments thereof.
  • Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 (or any range thereof) amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays that may be known in the art.
  • Some embodiments include an immune cell adhesion molecule.
  • Some examples of immune cell adhesion molecule that include a CD58 protein fragment are provided in Table 3C.
  • a CD58 protein may bind CD2.
  • a CD58 protein that binds CD2 may be or include a CD58 protein fragment.
  • an adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence in Table 3C.
  • the immune cell adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 312. In some embodiments, the immune cell adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 313.
  • the adhesion molecule is CD58.
  • CD58 is also known as lymphocyte function-associated antigen 3 (LFA-3).
  • LFA-3 lymphocyte function-associated antigen 3
  • CD58 binds to CD2 (LFA-2) on T cells.
  • the extracellular portion of CD58 is residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10): FSQQIYGWYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVS GSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNS HRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSR HR (SEQ ID NO: 10)
  • the polypeptide sequence of CD58 shares at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to SEQ ID NO: 248: FSQQIYGWYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYN LTSSDEDEYEMESPNITDTMKFFLYVLESL (SEQ ID NO: 248)
  • CD58 A crystal structure of CD58 is described in Ikemizu et al. PNAS USA 96(8):4289-94 (1999).
  • the extracellular portion of CD58 has a ligand-binding domain and a second extracellular domain.
  • the ligand-binding domain may be used as the functional fragment of CD58 — e.g., without the second extracellular domain.
  • the adhesion molecule (or immune cell engager) comprises the polypeptide sequence of SEQ ID NO: 1 or 10, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to SEQ ID NO: 1 or 10.
  • the adhesion molecule (or immune cell engager) comprises a sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 1 or 10.
  • the adhesion molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).
  • the adhesion molecule may be encoded by the polynucleotide sequence of CD58, SEQ ID NO: 11, or by a subsequence encoding the extracellular portion or a functional fragment.
  • SEQ ID NO: 11 (5’ to 3’): ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTGCTGCAC TGCTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGGGAATGTAACTTT CCATGTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAACAAAAGGATAAAGTTGC AGAACTGGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTAAAAATAGGGTTTATTTAGACACT GTGTCAGGTAGCCTCACTATCTACAACTTAACATCATCAGATGAAGATGAGTATGAAATGGAA TCGCCAAATATTACTGATACCATGAAGTTCTTTATGTGCTTGAGTCTCTCTTCCATCTCCAT
  • the polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to SEQ ID NO: 11, or a suitable subsequence, which may be used to express the adhesion molecule.
  • CD58 may be used.
  • homologs of CD58 from other species may be identified and tested for use in transducing human, or non-human, target cells. It is expected that at least some non-human homologs will retain adhesion molecule function when used with human target cells.
  • the adhesion molecule binds to CD2.
  • CD2 is also known as Ti l, LFA-2, and the erythrocyte rosette receptor. In its native state, CD2 is a surface protein expressed on T lymphocytes and NK cells. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, engagement of CD2 by CD58 provides a costimulatory signal that may enhance activation and effector functions.
  • the immune cell engager comprises an adhesion molecule that binds to CD2, which may be CD58 or a fragment thereof. In some embodiments, the immune cell engager comprises an antibody, single domain antibody, antibody fragment, and/or nanobody specific for CD2.
  • the adhesion molecule may comprise any polypeptide sequence in Table 1, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to a sequence in Table 1, to an extracellular portion thereof, or to a functional fragment thereof.
  • costimulatory molecules may be included as part of a fusion molecule.
  • the costimulatory molecule may be included as part of an immune cell engager.
  • An engager may include a costimulatory molecule such as a CD28-binding domain.
  • the fusion molecule comprising the immune cell engager may include a costimulatory molecule. However, in some embodiments, the fusion molecule does not include a costimulatory molecule.
  • the fusion molecule may incorporate a costimulatory molecule as a separate molecule, or the fusion molecule may lack any costimulatory molecule.
  • the costimulatory molecule may be a protein, termed herein a “costimulatory protein.”
  • costimulatory molecule refers to a molecule capable of providing a costimulatory signal to target cells.
  • the binding of the T cell receptor by an antigen can provide the primary stimulatory signal to the cell.
  • So-called costimulatory signals are provided by accessory molecules.
  • An example costimulatory signal is the signal provided by binding of CD28 on T cells by a ligand.
  • Some examples of ligands of cluster of differentiation 28 (CD28) include CD80 and cluster of differentiation 86 (CD86).
  • engager includes an immune cell costimulatory molecule that provide a costimulatory signal to the immune cell.
  • Illustrative costimulatory molecules include, but are not limited to, cluster of differentiation 80 (CD80), cluster of differentiation 86 (CD86), CD40 ligand (CD40L also known as CD 154), glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 4 (TNFSF4 also known as OX40L), tumor necrosis factor superfamily member 9 (TNFSF9 also known as 41BBL), inducible T cell costimulatory ligand (ICOSL), cluster of differentiation 70 (CD70), CD30 ligand (CD30L), tumor necrosis factor superfamily member 14 (TNFSF14 also known as LIGHT), lymphotoxin-alpha (LTalpha), MHC class I polypeptide-related sequence A (MICA), or MHC class I polypeptide-related sequence B (MICB) and anti-CD28.
  • CD80 cluster of differentiation
  • costimulatory molecule may be employed as a full-length protein, an extracellular domain, or functional fragment.
  • the costimulatory molecule is or includes a binding domain that specifically binds CD28.
  • the costimulatory molecule is or includes CD80, CD86, CD40L (also known as CD154), GITRL, OX40L, 41BBL, ICOSL, CD70, CD30L, LIGHT, LTalpha, MICA, or MICB, or a functional fragment thereof.
  • the costimulatory molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any sequence in Table 4, or functional fragments thereof.
  • the costimulatory molecule comprises a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or 100% sequence identity to any sequence in Table 4, or a functional fragment thereof.
  • Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays known in the art.
  • the costimulatory molecule is or includes CD80. In some embodiments, the costimulatory molecule is or includes a molecule that binds CD28. CD80 binds to CD28. The extracellular portion of CD80 includes residues 35-230 of SEQ ID NO: 12, which includes an Ig-like V-type domain (SEQ ID NO: 25) and an Ig-like C2-type domain (SEQ ID NO: 26), either or both of which may be included to form the costimulatory molecule.
  • CD80 also known as B7-1
  • the extracellular portion of CD80 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD80.
  • the costimulatory molecule is or includes CD86.
  • CD86 binds to CD28.
  • the extracellular portion of CD86 includes residues 33-225 of SEQ ID NO: 13, which includes an Ig-like V-type domain (SEQ ID NO: 27) and an Ig-like C2-type domain (SEQ ID NO: 28), either or both of which may be included to form the costimulatory molecule.
  • SEQ ID NO: 27 an Ig-like V-type domain
  • SEQ ID NO: 28 Ig-like C2-type domain
  • CD86 also known as B7-1
  • the extracellular portion of CD86 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD86.
  • CD80 or CD86 may be used.
  • homologs of CD80 or CD86 from other species may be identified and tested for use in transducing human, or non-human, target cells. It is expected that at least some non-human homologs will retain costimulatory molecule function when used with human target cells.
  • the costimulatory molecule comprises the polypeptide sequence of one or more of SEQ ID NO: 12-13 and 25-28, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to one or more of SEQ ID NO: 12-13 and 25-28.
  • the costimulatory molecule comprises a CD80 protein.
  • the CD80 protein may be or include the polypeptide sequence of SEQ ID NO: 250, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 250.
  • the costimulatory molecule (or immune cell engager) comprises a polypeptide sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to one or more of SEQ ID NO: 12-13 and 25- 28.
  • the costimulatory molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).
  • the costimulatory molecule may be encoded by the polynucleotide sequence of CD80 (SEQ ID NO: 29) or CD86 (SEQ ID NO: 30), or by a subsequence encoding the extracellular domain or a functional fragment.
  • the polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to SEQ ID NO: 29 or 30, or a suitable subsequence, which may be used to express the costimulatory molecule.
  • costimulatory molecules useful in the practice of the present disclosure may include any molecule that specifically binds CD28.
  • the costimulatory molecule may be a molecule that comprises an antibody, or antigen-binding fragment thereof, specific to CD28.
  • CD28 is a receptor expressed on T cells that provide costimulatory signal. T cell costimulation through CD28, resulting in, for example, the production of various interleukins (in particular IL-6).
  • the costimulatory molecule is an antibody, or fragment thereof, that specifically binds to CD28. Examples of such antibodies include 15E8, TGN1412, CD28.2, and 10F3, as well as humanized variants thereof.
  • 15E8 is a mouse monoclonal antibody to human CD28. Its complementarity determining regions (CDRs) are as follows:
  • CDRH1 GFSLTSY (SEQ ID NO: 36)
  • CDRH2 WAGGS (SEQ ID NO. 37)
  • CDRL1 RASESVEYYVTSLMQ (SEQ ID NO. 39)
  • CDRL2 AASNYES (SEQ ID NO. 40)
  • CDRL3 QQTRKVPST (SEQ ID NO. 41)
  • TGN1412 also known as CD28-SuperMAB
  • CD28-SuperMAB is a humanized monoclonal antibody that not only binds to, but also is a strong agonist for, the CD28 receptor. Its CDRs are as follows.
  • CDRH1 GYTFSY (SEQ ID NO. 42)
  • CDRH3 SHYGLDWNFDV (SEQ ID NO. 44)
  • CDRL1 HASQNIYVLN (SEQ ID NO. 45)
  • CDRL2 KASNLHT (SEQ ID NO. 46)
  • CDRL3 QQGQTYPYT (SEQ ID NO. 47)
  • the costimulatory molecule (or the immune cell engager) may comprise any polypeptide sequence of in Table 4, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, or 100% identity to a sequence in Table 4, to an extracellular portion thereof, or to a functional fragment thereof.
  • the costimulatory molecule is a VHH, an scFv, DARPin or any other antigen binding protein.
  • immune cell binding domains may be included as part of an immune cell engager.
  • An example of an immune cell binding domain may include a TCR-binding molecule.
  • An engager may include an immune cell binding domain such as a CD3 binding domain (e.g. an anti-CD3 antibody or antibody fragment).
  • the immune cell-binding domain comprises a CD3-binding domain.
  • the CD3-binding domain may be or include a binding domain specific to CD3s.
  • the immune cell engager may include an immune cell binding domain (e.g., a TCR-binding molecule) or other subunit that provides an activation signal to a target cell. However, in some embodiments, the immune cell engager does not include a TCR-binding molecule or other activation domain.
  • the immune cell engager may incorporate a TCR-binding molecule as a separate molecule, or the immune cell engager may lack any TCR-binding molecule.
  • the TCR-binding molecule may be a protein, termed herein a “TCR-binding protein.”
  • the immune cell binding domain may be or include an activation protein.
  • TCR-binding molecule refers to a molecule capable of directly binding the extracellular portion of the T cell receptor (TCR) by contacting one or more components of the TCR or otherwise providing a primary or “signal 1” activation signal to a target cell (e.g., a T cell or NK cell).
  • TCR T cell receptor
  • a target cell e.g., a T cell or NK cell.
  • TCR-binding molecules may include an antibody, or antigen binding domain, that specifically binds CD3 (an anti-CD3 monoclonal antibody, or antigen binding fragment thereof).
  • the immune cell binding domain comprises an antibody, single domain antibody, antibody fragment, nanobody, or other binding protein specific for CD3.
  • Illustrative antibodies include OKT3 (also known as Muromonab-CD3), otelixizumab, teplizumab and visilizumab.
  • the complementarity determining regions of OKT3 are as follows in Table 5. Table 5
  • the immune cell binding domain e.g., TCR-binding molecule
  • the immune cell binding domain may be a single chain variable fragment (scFv) incorporated into the fusion protein (or immune cell engager).
  • scFv single chain variable fragment
  • OKT3 in scFv format may be used.
  • the immune cell binding domain may include an anti-CD3 antibody or antibody fragment (such as a scFv) that includes a heavy chain variable domain (VH) domain.
  • the VH domain may be or include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 314.
  • the immune cell binding domain may include an anti-CD3 antibody or antibody fragment (such as a scFv) that includes a light chain variable domain (VL) domain.
  • the VL domain may be or include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 315.
  • the immune cell binding domain (e.g. TCR-binding molecule) is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to the anti-CD3 scFv of SEQ ID NO: 31 , which includes a variable light (VL) and variable heavy (VH) domain with a 3 x GGGS (SEQ ID NO: 307) linker:
  • the immune cell binding domain (e.g. TCR-binding molecule) is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to the anti-CD3 scFv of SEQ ID NO: 249, which includes a variable light (VL) and variable heavy (VH) domain with a 3 x GGGS (SEQ ID NO: 307) linker:
  • CDRH1 RYTMH (SEQ ID NO: 54)
  • CDRH2 YINPSRGYTNYNQKVKD (SEQ ID NO: 55)
  • CDRH3 YYDDHYCLDY (SEQ ID NO: 56)
  • CDRL1 SASSSVSYMN (SEQ ID NO: 57)
  • CDRL2 DTSKLASG (SEQ ID NO: 58)
  • CDRL3 QQWSSNPFT (SEQ ID NO: 59)
  • immune cell binding domains may comprise the binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen presenting cells.
  • SMAC supramolecular activation complex
  • CD3, 0X40, CD27, ICOS, 41BB, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC.
  • the immune cell engager disclosed herein may comprise portions of these proteins or domains that bind to these proteins.
  • T cells may express one or both of CD4 and/or CD8 and the fusion molecules disclosed herein may comprise domains that engage with either or both of CD4 and/or CD8.
  • an immune cell engager targeting NK cells may comprise domains that engage with proteins found on NK cells.
  • these proteins include CD2, CD 16, NKp46, NKp30, and NKG2D.
  • immune cell engager intended to target and/or activate NK cells may comprise domains that bind to CD2, CD 16, NKp46, NKG2D, etc.
  • the immune cell binding domain may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).
  • a polynucleotide e.g., a DNA or RNA polynucleotide
  • an engager such as an immune cell engager or a multispecific immune cell engager may include a tumor cell binding domain.
  • the tumor cell binding domain may contain any polypeptide that binds a desired antigen (e.g. antigen expressed on a tumor of interest).
  • the domain may comprise a scFv, a portion of an antibody or an alternative scaffold.
  • scFvs, llama VHH antibodies, other VH only antibody fragments, peptides, or small protein binding domains e.g. D domains
  • the tumor cell binding domain may comprise an scFv or VHH that binds to a target tumor antigen or the tumor cell binding domain may comprise a natural ligand, for example, folate.
  • the antigen to which the tumor cell binding domain binds can be any antigen of interest, e.g., can be an antigen on a tumor cell.
  • the tumor cell may be, e.g., a cell in a solid tumor, or a cell of a blood cancer.
  • the antigen can be any antigen that is expressed on a cell of any tumor or cancer type, e.g., cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glio
  • said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymph
  • the B cells of the CLL have a normal karyotype. In some embodiments, in which the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL carry a 17p deletion, an 1 Iq deletion, a 12q trisomy, a 13q deletion or a p53 deletion.
  • the antigen is expressed on a B-cell malignancy cell, relapsed/refractory CD19-expressing malignancy cell, diffuse large B-cell lymphoma (DLBCL) cell, Burkitt’s type large B-cell lymphoma (B-LBL) cell, follicular lymphoma (FL) cell, chronic lymphocytic leukemia (CLL) cell, acute lymphocytic leukemia (ALL) cell, mantle cell lymphoma (MCL) cell, hematological malignancy cell, colon cancer cell, lung cancer cell, liver cancer cell, breast cancer cell, renal cancer cell, prostate cancer cell, ovarian cancer cell, skin cancer cell, melanoma cell, bone cancer cell, brain cancer cell, squamous cell carcinoma cell, leukemia cell, myeloma cell, B cell lymphoma cell, kidney cancer cell, uterine cancer cell, adenocarcinoma cell, pancreatic cancer cell, chronic myelogen
  • the tumor cell binding domain comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD3, BCMA and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD19 and CD20, CD 19 and CD22, BCMA and GPRC5D, or CD20 and CD22.
  • the tumor cell binding domain comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA.
  • Some example antibodies useful for target cellbinding domains may include anti-mucin monoclonal antibody CAM 17.1, and M0vl8 which is an IgE antibody that targets folate receptor alpha.
  • the tumor cell binding domain binds to an antigen selected from the group consisting of: CD19, EpCAM, Her2/neu, EGFR, CD66e, CD33, EphA2, or MCSP. In some embodiments, the tumor cell binding domain binds to an antigen selected from the group consisting of: CD 19, EpCAM, CD20, CD 123, BCMA, B7-H3, CDE, or PSMA. In some embodiments, the tumor cell binding domain binds to a myeloid cell or dendritic cell antigen. In some embodiments, the tumor cell binding domain binds to CD33, DC-SIGN, CDllb, CDllc, or CD18.
  • the tumor cell binding domain comprises CCK2R ligand, a ligand bound by CCK2R-positive cancer cells (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).
  • CCK2R-positive cancer cells e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics. 2013. ePublication).
  • the tumor cell binding domain comprises folate, folic acid, or an analogue thereof, a ligand bound by the folate receptor on cells of cancers that include cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, E.I. et al., Cancer Metastasis Rev. 2008. 27(4):655-64).
  • the tumor cell binding domain comprises an NK-1R ligand.
  • Receptors for NK-1R the ligand are found, for example, on cancers of the colon and pancreas.
  • the NK-1R ligand may be synthesized according the method disclosed in Int’l Patent Appl. No. PCT/US2015/044229, incorporated herein by reference.
  • the tumor cell binding domain comprises a peptide ligand
  • the ligand may be a peptide ligand that is the endogenous ligand for the NK1 receptor.
  • the ligand may be a regulatory peptide that belongs to the family of tachykinins which target tachykinin receptors.
  • regulatory peptides include Substance P (SP), neurokinin A (substance K), and neurokinin B (neuromedin K), (see Hennig et al., International Journal of Cancer: 61, 786-792).
  • the tumor cell binding domain comprises a CAIX ligand.
  • Receptors for the CAIX ligand found, for example, on renal, ovarian, vulvar, and breast cancers.
  • the CAIX ligand may also be referred to herein as CA9.
  • Polypeptides described herein may form multimers such as dimers, trimers and higher oligomers, e.g., consisting of more than one polypeptide molecule.
  • Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical.
  • the corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc.
  • a multimerization polypeptide when fused to a second molecule, such as a heterologous polypeptide sequence, facilitates the formation of dimers, trimers, tetramers, pentamers or higher order oligomers or mixtures thereof among the polypeptides.
  • Fusing a multimerization polypeptide to an antigen binding protein such as an antigen binding polypeptide (e.g., antibody) can be used to increase the number of antigen binding sites via oligomer formation. Increasing the number of antigen binding sites can in turn increase antibody avidity for the antigen, which is useful in any therapeutic or diagnostic antibody application, particularly where it is desirable or advantageous to increase antibody avidity.
  • an antigen binding polypeptide e.g., antibody
  • the multimerization domain is the self-assembling tetramerization domain (TD) from the tumor suppressor gene p53.
  • the multimerization domain is the SpyCather-SpyTag system.
  • the multimerization domain is a self-associating peptide.
  • the self-associating peptide is RHCC derived from a right-handed coiled-coil peptide of an archaebacterium.
  • the self-associating peptide is COMPcc from human cartilage oligomeric matrix protein.
  • the self-associating peptide is C4bpa derived from human plasma C4-binding protein a-chain.
  • the disclosure provides a method of treating a disease or condition in a subject comprising administering to the subject a multispecific immune cell engager comprising a binding domain that specifically binds to a cell-surface antigen present on immune cells (immune cell engager) and a binding domain that specifically binds a cell surface antigen present on a target cell (target cell binding domain).
  • the immune cell engager further comprises a binding domain specific to CD2 (CD2 binding domain).
  • the immune cell binding domain further comprises a CD2 binding domain and a binding domain specific to CD28 (CD28- binding domain).
  • a multispecific immune cell engager may include an immune cell engager described herein and a target cell binding domain.
  • the multispecific immune cell engager can incorporate any fusion protein described herein and a target cell binding domain.
  • FIG. 1A-1B Some example immune cell engagers are shown in FIG. 1A-1B. Aspects shown in these figures may be included in a immune cell engager. Some embodiments include any aspect in in FIG. 2A-3B.
  • an engager such as a multispecific engager includes a tumor cellbinding domain and a target cell-binding domain. In some embodiments, an engager includes a tumor cell-binding domain or a target cell-binding domain. In some embodiments, an engager does not include a tumor cell-binding domain.
  • the multispecific immune cell engager can be used to treat a disease or condition in a subject.
  • the malignancy is a B-cell malignancy, a myeloma, or a solid tumor malignancy.
  • the disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leuk
  • the condition is an autoimmune disease or disorder.
  • the disclosure also provides a method of treating an autoimmune disease in a subject, comprising administering to the subject the engager the macromolecular complex or pharmaceutical composition of the disclosure.
  • an autoimmune disease may include systemic lupus erythematosus, Sjogren’s Syndrome, ANCA-associated vasculitis and autoimmune hemolytic anemia, rheumatoid arthritis, systemic sclerosis, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, pemphigus vulgaris, dermatomyositis, adult-onset Still’s disease, inflammatory bowel disease, type 1 diabetes mellitus, graft vs.
  • the immune cell-binding domain and/or the target cell-binding domain is an antibody, optionally fragment antigen binding (Fab), a fragment antigen-binding 2 (F(ab’)2), a single chain Fab (scFab), a single chain variable fragment (scFv), or a single domain antibody (sdAb) (e.g., a nanobody or VHH antibody).
  • Fab fragment antigen binding
  • F(ab’)2 fragment antigen-binding 2
  • scFab single chain Fab
  • scFv single chain variable fragment
  • sdAb single domain antibody
  • an engager includes an immunoglobulin domain.
  • the immunoglobulin domain may include an IgGl domain.
  • the immunoglobulin domain may include a constant domain.
  • the immunoglobulin domain may include an IgGl constant domain.
  • the immunoglobulin domain may include a CH2 domain.
  • the immunoglobulin domain may include an IgGl CH2 domain.
  • the immunoglobulin domain may include a CH3 domain.
  • the immunoglobulin domain may include an IgGl CH3 domain.
  • the immunoglobulin domain may include multiple constant domains.
  • the immunoglobulin domain may include an CH2 domain and a CH3 domain.
  • the immunoglobulin domain may include an CH2-CH3 domain.
  • the immunoglobulin domain may include an CH3-CH2 domain.
  • the immunoglobulin domain may include an IgGl CH2 domain and an IgGl CH3 domain.
  • an engager includes an IgGl CH2 domain.
  • an engager includes an IgGl CH3 domain.
  • an engager includes IgGl CH2 and CH3 domains.
  • the engager comprises a polypeptide chain comprising the CD2- binding domain, the immune cell-binding domain, a CD28-binding domain, IgGl CH2 and CH3 domains, and the target cell-binding domain.
  • the engager comprises a polypeptide chain comprising the adhesion molecule, the immune cell-binding domain, a costimulatory molecule, IgGl CH2 and CH3 domains, and the target cell-binding domain.
  • the polypeptide chain comprises, in N- to C- terminal order: the CD2- binding domain comprising a CD58 extracellular domain, the immune cell-binding domain comprising an anti-CD3 antibody, the CD28-binding domain comprising a CD80 or CD86 extracellular domain, IgGl CH2 and CH3 domains, and the target cell-binding domain.
  • polypeptide chain comprises, in N- to C- terminal order: the adhesion molecule comprising a CD58 extracellular domain, the immune cell-binding domain comprising an anti-CD3 antibody, the costimulatory molecule comprising a CD80 or CD86 extracellular domain, IgGl CH2 and CH3 domains, and the target cell-binding domain.
  • the polypeptide chain comprises, in N- to C- terminal order: the CD28-binding domain comprising a CD80 or CD86 extracellular domain, the immune cell-binding domain comprising an anti-CD3 antibody, the CD2-binding domain comprising a CD58 extracellular domain, IgGl CH2 and CH3 domains, and the target cell binding domain.
  • polypeptide chain comprises, in N- to C- terminal order: the costimulatory molecule comprising a CD80 or CD86 extracellular domain, the immune cell-binding domain comprising an anti-CD3 antibody, the adhesion molecule comprising a CD58 extracellular domain, IgGl CH2 and CH3 domains, and the target cell binding domain.
  • the polypeptide chain comprises, in N- to C- terminal order: the immune cell-binding domain comprising an anti-CD3 antibody, the CD2-binding domain comprising a CD58 extracellular domain, the CD28-binding domain comprising a CD80 or CD86 extracellular domain, IgGl CH2 and CH3 domains, and the target cell-binding domain.
  • polypeptide chain comprises, in N- to C- terminal order: the immune cell-binding domain comprising an anti-CD3 antibody, the costimulatory molecule comprising a CD80 or CD86 extracellular domain, the adhesion molecule comprising a CD58 extracellular domain, IgGl CH2 and CH3 domains, and the target cell binding domain.
  • polypeptide chain comprises the sequence of SEQ ID NO: 278 or 279, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.
  • the polypeptide chain comprises a polypeptide sequence disclosed in Table 6, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical thereto.
  • a polynucleotide comprises a polynucleotide sequence encoding an engager, optionally operatively linked to a promoter or to recombination sites for gene editing, optionally comprising a polynucleotide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence disclosed in Table 7.
  • a vector comprises a vector genome comprising a polynucleotide comprising a polynucleotide sequence encoding an engager, optionally operatively linked to a promoter or to recombination sites for gene editing, and optionally comprising a polynucleotide sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence disclosed in Table 7.
  • Table 7 Table 7
  • an engager may include an immune cell-binding domain.
  • a target-cell binding domain may specifically bind a cellsurface antigen on a target cell.
  • a target cell-binding domain is or includes a tumor cell-binding domain.
  • a target cell-binding domain does not include a tumor cell-binding domain.
  • the target cell binding domain comprises a receptor, or a portion of a receptor, which binds to said antigen.
  • the target cell binding domain comprises, or is, an antibody or an antigen-binding portion thereof.
  • the single-chain Fv domain can comprise, for example, a VL linked to VH by a flexible linker, wherein said VL and VH are from an antibody that binds said antigen.
  • the target cell is a cancer cell or an immune cell (e.g., a natural killer cell or a B cell). In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a CD 19+ B cell. In some embodiments, the target cell is a CD20+ B cell. In some embodiments, the target cell is a CD22+ B cell.
  • the antigen to which the target cell binding domain of the polypeptide binds can be any antigen of interest, e.g., can be an antigen on a tumor cell.
  • the tumor cell may be, e.g., a cell in a solid tumor, or a cell of a blood cancer.
  • the antigen can be any antigen that is expressed on a cell of any tumor or cancer type.
  • compositions and methods described herein can be used to treat a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a fibrom
  • said lymphoma can be chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extra nodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extra nodal NK/T lymphocyte lymph
  • the B cells of the CLL have a normal karyotype. In some embodiments, in which the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL carry a 17p deletion, an l lq deletion, a 12q trisomy, a 13q deletion or a p53 deletion.
  • the antigen is expressed on a B-cell malignancy cell, relapsed/refractory CD19-expressing malignancy cell, diffuse large B-cell lymphoma (DLBCL) cell, Burkitt’s type large B-cell lymphoma (B-LBL) cell, follicular lymphoma (FL) cell, chronic lymphocytic leukemia (CLL) cell, acute lymphocytic leukemia (ALL) cell, mantle cell lymphoma (MCL) cell, hematological malignancy cell, colon cancer cell, lung cancer cell, liver cancer cell, breast cancer cell, renal cancer cell, prostate cancer cell, ovarian cancer cell, skin cancer cell, melanoma cell, bone cancer cell, brain cancer cell, squamous cell carcinoma cell, leukemia cell, myeloma cell, B cell lymphoma cell, kidney cancer cell, uterine cancer cell, adenocarcinoma cell, pancreatic cancer cell, chronic myelogen
  • the antigen is CD 19.
  • a target cell binding domain comprises an extracellular domain comprising a FMC63 scFv binding domain for CD 19 binding.
  • a polypeptide encoding a target cell binding domain comprises an anti-CD19 scFv (CD19 VL linked to a CD19 VH) that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 93.
  • CDR complementary determining regions
  • RASQDISKYLN RASQDISKYLN
  • CDR- Ll HTSRLHS
  • CDR-L2 HTSRLHS
  • QQGNTLPYT CDR-L3
  • SEQ ID NO: 96 HTSRLHS
  • QQGNTLPYT CDR-L3
  • SEQ ID NO: 96 DYGV
  • VIWGSETTYYNSALKS CDR-H2; SEQ ID NO: 98
  • HYYYGGSYAMDY CDR-H3; SEQ ID NO: 99.
  • a polypeptide encoding a target cell binding domain comprises a polynucleotide encoding a CAR whose extracellular domain comprises an anti-CD19 scFv having these CDRs, wherein optionally the anti-CD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 93.
  • the polypeptide encoding a target cell binding domain comprises an anti-CD19 scFv having these CDRs, wherein optionally the anti-CD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 93 or 100.
  • the polynucleotide encoding a target cell binding domain comprises an anti-CD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 102.
  • the polypeptide encoding a target cell binding domain comprises an anti-CD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 103.
  • the polynucleotide encoding a target cell binding domain comprises an anti-CD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 104.
  • the polypeptide encoding a target cell binding domain comprises an anti-CD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 100.
  • CDR complementary determining regions
  • RASQDISKYLN RASQDISKYLN
  • CDR- Ll HTSRLHS
  • CDR-L2 HTSRLHS
  • QQGNTLPYT CDR-L3
  • SEQ ID NO: 96 HTSRLHS
  • QQGNTLPYT CDR-L3
  • SEQ ID NO: 96 DYGV
  • VIWGSETTYYNSALKS CDR-H2; SEQ ID NO: 98
  • HYYYGGSYAMDY CDR-H3; SEQ ID NO: 99.
  • the polypeptide encoding a target cell binding domain comprises an anti-CD19 scFv having these CDRs, wherein optionally the anti-CD19 scFv shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 100.
  • the polynucleotide encoding a target cell binding domain comprises an anti-CD19 scFv that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 105.
  • the polypeptide encoding a target cell binding domain comprises an anti-CD20 scFv and comprises an amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 309.
  • the anti-CD20 scFv VL comprises an amino acid sequence at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 176.
  • the anti-CD20 scFv VL comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 175.
  • the anti-CD20 scFv linker comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 177.
  • the anti-CD20 scFv VH comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 178.
  • the anti-CD20 scFv spacer comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 207.
  • the polynucleotide encoding a target cell binding domain comprises an anti-CD19 scFv comprising a nucleic acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% constrain at least 99%, or 100% identity to SEQ ID NO: 194.
  • the polypeptide encoding a target cell binding domain comprises an anti-CD19 scFv comprising an amino acid sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 93.
  • the anti-CD19 scFv VL comprises an amino acid sequence a at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 206.
  • the anti-CD19 scFv VH comprises an amino acid sequence at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 208.
  • the anti-CD19 scFv spacer comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 332.
  • the target cell antigen is BCMA.
  • Antibody based therapies targeting BCMA have been approved by the FDA and include teclistamab-cqyv, elranatamab- bcmm, idecabtagene vicleucel, and ciltacabtagene autoleucel. Binding domains targeting BCMA are described, for example, in US Publication No. 2020/0246381; US Patent No. 10,918,665; US Publication No. 2019/0161553; and US Publication No. 2022/0033509 each of which is herein incorporated by reference.
  • the target cell antigen is G protein-coupled receptor class C group 5 member D (GPRC5D). Binding domains targeting GPRC5D are described, for example, in US Publication No. 2018/0118803; US Publication No. 2020/0231686, and US Publication No. 2021/10393689, each of which is herein incorporated by reference.
  • GPRC5D G protein-coupled receptor class C group 5 member D
  • the target cell antigen is Fc Receptor-like 5 (FcRL5). Binding domains targeting FcRL5 are described, for example, in US Publication No. US 2017/0275362, which is herein incorporated by reference.
  • the target cell antigen is receptor tyrosine kinase-like orphan receptor 1 (ROR1). Binding domains targeting ROR1 are described, for example, in US Publication No. 2022/0096651, which is herein incorporated by reference.
  • the target cell antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • the tumor-associated antigen or tumor-specific antigen is B cell maturation antigen (BCMA), B cell Activating Factor (BAFF), GPRC5D, FCRL5/FCRH5, ROR1, LI -CAM, CD22, folate receptor, carboxy anhydrase IX (CAIX), claudin 18.2, FAP, mesothelin, IL-13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4 L six family member 1 (TM4SF1), epithelial cell adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, glypican-3 (GPC3), CD133, CD147, EGFR, MUC1,
  • the target cell binding domain comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD3, BCMA and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD 19 and CD20, CD 19 and CD22, BCMA and GPRC5D, or CD20 and CD22.
  • the target cell binding domain comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA.
  • the target cell-binding domain may bind cluster of differentiation 19 (CD 19), cluster of differentiation 20 (CD20), cluster of differentiation 22 (CD22), cluster of differentiation 27(CD27), cluster of differentiation 30 (CD30), cluster of differentiation 70 (CD70), cluster of differentiation 123 (CD123), cluster of differentiation (CD133), B-cell maturation antigen (BCMA), alpha-actinin- 4 (ACTN4), B melanoma antigen 1 (BAGE-1), breakpoint cluster region (BCR) - Abelson murine leukemia (ABL) fusion protein (BCR-ABL), beta-catenin, cancer antigen 125 (CA 125), cancer antigen 15-3 (CA 15-3, also known as CA 27.29), cancer antigen 195 (CA195), cancer antigen 242 (CA 242), cancer antigen 50 (CA 50), tumor associated antigen defined by CT43 monoclonal antibody (CAM43), Caspase-8 (Casp-8), cell division cycle protein 27 (CDC27), cyclin-dependent kina
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an immune cell engager according to the disclosure and a pharmaceutically acceptable carrier.
  • compositions of the present disclosure may comprise a combination of any number of immune cell engagers, and optionally one or more additional pharmaceutical agents (viral particles, polypeptides, polynucleotides, compounds etc.) formulated in pharmaceutically acceptable or physiologically-acceptable compositions for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
  • additional pharmaceutical agents viral particles, polypeptides, polynucleotides, compounds etc.
  • the one or more additional pharmaceutical agents further increases transduction efficiency of viral particles.
  • the formulations and compositions of the present disclosure may comprise a combination of any number of immune cell engagers, and optionally one or more nanocarriers.
  • Illustrative nanocarriers include, but are not limited to, micelles, polymers, liposomes, and lipid nanoparticles (LNPs).
  • compositions comprising a therapeutically-effective amount of an immune cell engager, as described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the composition further comprises other agents, such as, e.g., cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents.
  • compositions and formulations of the immune cell engager used in accordance with the present disclosure may be prepared for storage by mixing an immune cell engager having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • one or more pharmaceutically acceptable surface-active agents surfactant
  • buffers isotonicity agents
  • salts amino acids
  • sugars stabilizers and/or antioxidant
  • Suitable pharmaceutically acceptable surfactants comprise but are not limited to polyethylen-sorbitan-fatty acid esters, polyethylene-polypropylene glycols, polyoxyethylenestearates, and sodium dodecyl sulphates.
  • Suitable buffers comprise but are not limited to histidine- buffers, citrate-buffers, succinate-buffers, acetate-buffers, and phosphate-buffers.
  • Isotonicity agents are used to provide an isotonic formulation.
  • An isotonic formulation is liquid, or liquid reconstituted from a solid form, e.g., a lyophilized form and denotes a solution having the same tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum.
  • Suitable isotonicity agents comprise but are not limited to salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars including but not limited to glucose, sucrose, trehalose or and any component from the group of amino acids, sugars, salts, and combinations thereof.
  • isotonicity agents are generally used in a total amount of about 5 mM to about 350 mM.
  • Non-limiting examples of salts include salts of any combinations of the cations sodium potassium, calcium or magnesium with anions chloride, phosphate, citrate, succinate, sulphate, or mixtures thereof.
  • Non-limiting examples of amino acids comprise arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, or proline.
  • Non-limiting examples of sugars according to the disclosure include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine (also referred to as “meglumine”), galactosamine and neuraminic acid and combinations thereof.
  • Non-limiting examples of stabilizer includes amino acids and sugars as described above as well as commercially available cyclodextrins and dextrans of any useful kind and molecular weight.
  • Non-limiting examples of antioxidants include excipients such as methionine, benzylalcohol or any other excipient used to minimize oxidation.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Except insofar as any media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions comprising a carrier are suitable for parenteral administration, e.g., intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration.
  • pharmaceutically acceptable carriers may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Except insofar as any media or agent is incompatible with the transduced cells, use thereof in the pharmaceutical compositions of the present disclosure is contemplated.
  • compositions may further comprise one or more polypeptides, polynucleotides, vector genomes comprising same, compounds that increase the transduction efficiency of vector genomes, formulated in pharmaceutically acceptable or physiologically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
  • compositions of the present disclosure may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents.
  • agents such as, e.g., cytokines, growth factors, hormones, small molecules, or various pharmaceutically active agents.
  • compositions comprising an expression cassette or vector (e.g., therapeutic vector) and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • the pharmaceutical composition comprises an immune cell engager and a lentiviral vector comprising an expression cassette disclosed herein, e.g., wherein the expression cassette comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptor (CARs) and variants thereof.
  • CARs chimeric antigen receptor
  • the pharmaceutical compositions that contain immune cell engagers may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intra-arterial, intra-renal, intraurethral, epidural, intrathecal, intraperitoneal, or intramuscular administration.
  • the immune cell engager can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with pharmaceutical supports, to animals and human beings.
  • the pharmaceutical composition comprises cells transduced ex vivo with immune cell engagers and any of the vector genomes according to the present disclosure.
  • the immune cell engagers is effective when administered systemically.
  • the immune cell engagers of the disclosure in some cases, may be administered intravenously to subject (e.g., a primate, such as a non-human primate or a human).
  • the immune cell engagers administered in combination with the viral vectors of the disclosure are capable of inducing expression of CARs in various immune cells when administered systemically (e.g., in T-cells, dendritic cells, NK cells).
  • the pharmaceutical compositions contain vehicles (e.g., carriers, diluents, and excipients) that are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles e.g., carriers, diluents, and excipients
  • excipients include a poloxamer.
  • Formulation buffers for immune cell engagers may contain salts to prevent aggregation and other excipients (e.g., poloxamer) to reduce stickiness of the immune cell engagers.
  • the formulation is stable for storage and use when frozen (e.g., at less than 0 °C, about -60 °C, or about -72 °C). In some embodiments, the formulation is a cryopreserved solution.
  • compositions of the present disclosure formulation of pharmaceutically acceptable excipients and carrier solutions may be useful to those of skill in the art, such as for development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.
  • compositions disclosed herein parenterally, intravenously, intramuscularly, or intraperitoneally for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, incorporated herein by reference in its entirety).
  • the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Fluidity may be maintained, for example, by use of a coating, such as lecithin, by maintenance of a useful particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered if useful or necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • these particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the solution intended for subcutaneous administration includes hyaluronidase.
  • a sterile aqueous medium that can be employed may be useful.
  • One dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g.
  • Some embodiments include a composition that includes a gene therapy vector.
  • the composition may further include an immune cell engager herein.
  • the gene therapy vector may be included in a method herein, such as a method of treatment or use.
  • the method may include use of a gene therapy vector with an immune cell engager.
  • the method may include use of a particle with an immune cell engager, or administration of a gene therapy vector and an immune cell engager.
  • a gene therapy vector may be included in or with a particle.
  • the viral vector may for example be derived from an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or an adenovirus.
  • AAV derived vectors may comprise an AAV genome or a derivative thereof.
  • Some embodiments relate to a method of using an engager such as an immune cell engager herein.
  • the method may be an in vivo method.
  • a method may be or include a method of treatment.
  • the method may be an ex vivo method.
  • the method may be an in vitro method.
  • the lymph nodes for administration are the inguinal lymph nodes.
  • the immune cell engagers are administered by injection of the immune cell engagers into tumor sites e.g., intratumoral). In some embodiments, the immune cell engagers are administered subcutaneously. In some embodiments, the immune cell engagers are administered systemically. In some embodiments, the immune cell engagers are administered intravenously. In some embodiments, the immune cell engagers are administered intra-arterially. In some embodiments, the immune cell engagers are co-administered with a viral particle.
  • the immune cell engagers are administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the immune cell engagers are administered by intraperitoneal injection. In some embodiments, the immune cell engagers are administered by subcutaneous injection. In some embodiments, the immune cell engagers are administered by intranodal injection.
  • the transduced immune cells comprising a polynucleotide of the present disclosure are administered to the subject.
  • the disclosure provides a method of treating a condition in a subject comprising administering to the subject the immune cell engager or the pharmaceutical composition of the disclosure.
  • the disclosure provides a method of treating a malignancy in a subject, comprising administering to the subject the immune cell engagers and a viral particle comprising a nucleic acid encoding a CAR or pharmaceutical composition of the disclosure.
  • the malignancy is a B-cell malignancy, a myeloma, or a solid tumor malignancy.
  • the disclosure provides a method of treating diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, medulloblastoma, or sarcoma in a subject, comprising administering to the subject the immune cell engager and viral particles or pharmaceutical composition of the disclosure.
  • the condition is an autoimmune disease or disorder.
  • the disclosure also provides a method of treating an autoimmune disease in a subject, comprising administering to the subject immune cell engager and viral particles or pharmaceutical composition of the disclosure.
  • an autoimmune disease may include systemic lupus erythematosus, Sjogren’s Syndrome, ANCA-associated vasculitis and autoimmune hemolytic anemia, rheumatoid arthritis, systemic sclerosis, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, pemphigus vulgaris, dermatomyositis, adult-onset Still’s disease, inflammatory bowel disease, type 1 diabetes mellitus, graft vs. host disease, a myasthenia gravis, multiple sclerosis, Immune dysregulation, Polyendocrinopathy Enteropathy X-linked (IPEX) or autoimmune arthritis.
  • IPEX Immune dysregulation
  • the immune cell engager disclosed herein may be used with viral particles in a method to generate engineered cells in vivo.
  • the lentiviral particles incorporating the immune cell engager e.g., a fusion protein as disclosed herein
  • TCM engineered central memory T cells
  • administering lentiviral particles via one or more lymph nodes may contribute to generation of a predominately TCM engineered cell phenotype.
  • TCM may be characterized by expression of certain surface markers, for example, TCM may be CD62L+. TCM may also be CCR7+. TCM may also be characterized as CD45RA-, CD45RO+, and/or CD27+.
  • TCM are characterized as CCR7+, CD45RA-, CD45RO+, CD62L+, and CD27+. In some embodiments, TCM are characterized as CD45RA-, CCR7+.
  • engineered TCM may persist for a longer time in vivo and may show improved effector function compared with engineered effector memory (TEM) or similar effector cell types.
  • TEM engineered effector memory
  • the present disclosure further provides a method of generating predominately engineered TCM in vivo. Similar observations may be found in the ex-vivo setting, so the present disclosure further provides a method of generating predominately engineered TCM ex vivo, using one or more of the methods disclosed here. For example, via extracorporeal delivery or ex-vivo manufacturing.
  • Some embodiments include a method of inducing cytokine production by an immune cell. Some embodiments include contacting an immune cell with an engager. Some embodiments include a method of inducing cytokine production by an immune cell, comprising contacting the immune cell with an effective amount of an engager herein.
  • the cytokine is a chemokine, an interleukin, an interferon, a tumor necrosis factor or a colony stimulating factor.
  • the cytokine is interferon gamma (IFNy).
  • the cytokine is interleukin-2 (IL-2).
  • the cytokine is tumor necrosis factor alpha (TNFa).
  • the cytokine is interleukin- 15 (IL- 15). In some embodiments, the cytokine is interleukin- 12 (IL-12).
  • the disclosure provides a method of making an immune cell engager, comprising introducing a polynucleotide encoding an immune cell engager into a host cell comprising a polynucleotide encoding an immune cell engager (or fusion protein) as described herein.
  • the immune cell engager (or fusion protein) is expressed by the host cell.
  • the disclosure provides an in vivo method of transducing target cells in a subject in need thereof, comprising administering to the subject an immune cell engager and a particle or pharmaceutical composition of the disclosure.
  • the immune cell engager and particle may be administered by intranodal, intravenous, or subcutaneous injection.
  • Various disease or disorders may be treated using an immune cell engager and particles comprising a nucleic acid encoding a CAR as disclosed herein, or pharmaceutical composition comprising them.
  • the immune cell engager and particles may be administered to a subject suffering from or at risk for a B-cell malignancy, relapsed/refractory malignancy, diffuse large B-cell lymphoma (DLBCL), Burkitt’s type large B-cell lymphoma (B-LBL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cell carcinoma, leukemia, myeloma, B cell lymphoma, kidney cancer, uterine cancer, adenocar
  • Immune cell engagers of the present disclosure may enhance particle to T cell binding. Immune cell engagers of the present disclosure may enhance T cell activation. Immune cell engagers of the present disclosure may enhance immune cell expansion. Immune cell engagers of the present disclosure may enhance immune cell transduction. [0244] Some embodiments include a method of making an adhesion molecule, a costimulatory molecule, an immune cell binding domain, or a fusion protein (e.g., immune cell engager).
  • the method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes a protein comprising the adhesion molecule, costimulatory molecule, immune cell binding domain, or fusion molecule (e.g., immune cell engager).
  • a nucleic acid such as a DNA or RNA
  • a protein comprising the adhesion molecule, costimulatory molecule, immune cell binding domain, or fusion molecule (e.g., immune cell engager).
  • kits Disclosed herein, in some embodiments, are kits.
  • the kit includes an adhesion molecule.
  • the kit includes a costimulatory molecule.
  • the kit includes an immune cell binding domain.
  • the kit includes a fusion molecule.
  • the kit includes an immune cell engager.
  • the kit includes a particle.
  • the kit includes a composition described herein. The kit may include instructions for use, such as instructions for use in a method herein.
  • the disclosure provides a kit comprising the immune cell engager and instructions for use in treatment of a subject.
  • the kit may include a pharmaceutically acceptable carrier and/or an injection device.
  • the kit may further include suitable tubing for administering the immune cell engager.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • determining may be used interchangeably herein to refer to forms of measurement.
  • the terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • a sequence herein may include a conservative amino acid substitution.
  • a “conservative amino acid substitution” may refer to a substitution of an amino acid residue with another amino acid residue without abolishing a protein's desired properties. Such a substitution may be with an amino acid of a same amino acid class.
  • amino acid class examples include amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, or tyrosine; amino acids having basic side chains, such as lysine, arginine, or histidine; amino acids having acidic side chains, such as asparatic acid or glutamic acid; or amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine.
  • a conservative amino acid substitution may include substitution with a chemically derivatized residue or non-natural amino acid that does not abolish a protein's desired properties.
  • a percent sequence identity may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the % sequence identity.
  • a sequence identity may include a sequence identity to a reverse complement.
  • thymine (T) and uracil (U) may be interchangeable.
  • T and U may be interchangeable when describing an oligonucleotide.
  • Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T.
  • a subject can be an animal.
  • a subject can be a mammal.
  • a subject can be a primate.
  • the subject can be a human.
  • a subject may be healthy.
  • a subject may have a disease.
  • a subject may be diagnosed or suspected of being at high risk for a disease.
  • the term “about” a number refers to that number plus or minus 15% of that number.
  • the term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.
  • administering may include introducing a compound or a composition into a subject.
  • the term may encompasses external or internal administration.
  • Internal administration can refer to, for example, where a molecule is generated inside the subject’s body.
  • administering an RNA may include administering a plasmid DNA encoding the RNA, or a vehicle comprising said plasmid DNA, upon which the RNA may be expressed from the plasmid DNA in a cell of the subject.
  • treatment may be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • beneficial or desired results may include a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit may be achieved by eradication or amelioration of one or more of the physiological symptoms associated with an underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • a prophylactic effect may includes delaying, preventing, or eliminating appearance of a disease or condition, delaying or eliminating onset of a symptom, slowing, halting, or reversing progression of a disease or condition, or a combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • example multispecific immune cell engagers can activate immune cells.
  • the multispecific immune cell engagers in the example included an adhesion molecule comprising a CD28-binding domain, an immune cell-binding domain, a costimulatory molecule comprising a CD28-binding domain, and a target cell-binding domain.
  • This example specifically shows that an example multispecific immune cell engager comprising sMDFl or sMDF2 and a CD20 antibody can potently activate T cells.
  • the CD20 antibody is useful for allowing the engager to concurrently interact with a T cell (via the CD80 extracellular domain, the anti-CD3 antibody fragment, the CD58 extracellular domain) and a CD20+ tumor cell.
  • sMDFl is a fusion protein comprising, in N- to C-terminal order, a CD58 extracellular domain, an anti-CD3 antibody, and a CD80 extracellular domain (FIG. 1A-1B).
  • sMDF2 is a fusion protein comprising, in N- to C-terminal order, a CD80 extracellular domain, an anti-CD3 antibody fragment, and a CD58 extracellular domain (FIG. 1A-1B).
  • FACs was performed to analyze CD20 expression levels in Nalm6 and Raji cell lines. Naim 6 cells expressed a low level of CD20 and Raji cells expressed a high level of CD20 relative to a CD20- cell line (FIG. 4).
  • PBMCs from 3 donors were thawed and allowed to rest for 1 hour in Xvivo + 5% Human Serum + Glutamax.
  • 200,000 PBMCs were co-cultured with 20,000 Nalm6 cells (CD20+) and increasing concentrations of multispecific immune engagers (10,000ng/ml - 1 ng/ml) was added to the co-cultures.
  • Rituximab CD20 antibody
  • Mosunetuzumab Bi-specific CD3 and CD20 antibody
  • the percent of dual positive CD3+ CD25+, CD4+CD25+ or CD8+CD25 immune cells was quantified via FACs.
  • MDF1 CD20, MDF2 CD20 and Mosunetuzumab were able to potently activate CD3+, CD4+ and CD8+ T cells.
  • Rituximab did not generate appreciable CD25+ populations in any of the immune cell populations (FIG. 5).
  • example multispecific immune cell engagers may induce immune cell proliferation.
  • example multispecific immune cell engagers comprising sMDFl or sMDF2 and a CD20 antibody strongly induced T cell proliferation.
  • PBMCs were thawed, allowed to recover, and co-cultured with Raji cells and increasing concentrations of multispecific immune cell engager, as described above.
  • Rituximab and Mosunetuzumab were used as controls as described above.
  • PBMCs Prior to co-culture the PBMCs were labeled with a cell tracer dye to evaluate proliferation.
  • Cell trace violet is used to monitor distinct generations of proliferating cells by dye dilution. Live cells are covalently labeled with a very bright, stable dye. Every generation of cells appears as a different peak on a flow cytometry histogram.
  • T cell proliferation was measured via FACs.
  • MDF1 CD20, MDF2 CD20 and Mosunetuzumab were able to robustly induce T cell proliferation (FIG. 7).
  • Rituximab did not induce T cell proliferation in this example.
  • This example shows that a multispecific immune engager comprising sMDFl or sMDF2 and a CD20 antibody can induce T cell proliferation.
  • the ability to stimulate T cell proliferation was dependent on the concurrent binding of CD3 and CD20 by MDF1 CD20, MDF2 CD20 and Mosunetuzumab.
  • Rituximab did not induce robust T cell proliferation and it does not contain a CD3 binding domain.
  • This example shows that a multispecific immune engager can induce immune cells to kill target cells.
  • a multispecific immune engager comprising sMDFl or sMDF2 and a CD20 antibody can induce T cells to kill CD20+ tumor cells.
  • PBMCs were thawed, allowed to recover, and co-cultured with Nalm6 cells and increasing concentrations of multispecific immune engagers as described above.
  • Rituximab and Mosunetuzumab were used as controls as described above. Tumor cell growth was measured over 5 days using an IncuCyte imaging system.
  • the PBMCs and Nalm6 cells were grown at a 10: 1 effector to target cell ratio.
  • MDF1 CD20 and MDF2 CD20 were able to robustly induce T cell mediated lysis of CD20+ tumor cells in a dose dependent manner (FIG. 8A).
  • This example shows that a multispecific immune cell engager comprising sMDF 1 or sMDF2 and a CD20 antibody induce T cells to kill CD20+ tumor cells. T cell engagers were not able to induce tumor cell death in the absence of T cells (FIG. 10).
  • This example shows that a multispecific immune engager can induce immune cells to generate cytokine production.
  • a multispecific immune engager comprising sMDFl or sMDF2 and a CD20 potently induce T cell cytokine production.
  • PBMCs were thawed, allowed to recover, and co-cultured with Nalm6 cells and increasing concentrations of multispecific immune cell engagers, as described above.
  • Rituximab and Mosunetuzumab were used as controls as described above.
  • Cytokine production was measured after 3 days of culture using a cytokine and chemokine analysis kit from Meso Scale Design. At 100 ng/mL MDF1 CD20 and MDF2 CD20 robustly induced IFNy, IL-2 and TNFa production (FIG. 11).
  • MDF1 CD20 and MDF2 CD20 are able to robustly induce IFNy, IL-2 and TNFa production in a dose dependent manner (FIG. 12).
  • Rituximab did not induce IFNy, IL-2 and TNFa production in T cells.
  • This example shows that a multispecific immune engager comprising sMDFl or sMDF2 and a CD20 antibody is able to potently induce T cell cytokine production.

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Abstract

L'invention concerne des activateurs des cellules immunitaires et des activateurs multispécifiques des cellules immunitaires. Certains de ces modes de réalisation comprennent une molécule d'adhérence liée à une molécule costimulatrice ou un domaine de liaison à une cellule immunitaire et un domaine de liaison à une cellule cible. La divulgation concerne également des vecteurs tels que des vecteurs lentiviraux, des cellules et des procédés qui peuvent concerner ou comprendre de tels activateurs. Les activateurs multispécifiques des cellules immunitaires peuvent incorporer une molécule de fusion comprenant un domaine extracellulaire CD58, un anticorps anti-CD3, un domaine extracellulaire CD80 ou CD86 et un domaine de liaison à une cellule cible.
PCT/US2025/028138 2024-05-08 2025-05-07 Activateur multispécifique des cellules immunitaires et ses utilisations Pending WO2025235605A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020236792A1 (fr) * 2019-05-21 2020-11-26 Novartis Ag Molécules de liaison à cd19 et utilisations de celles-ci
WO2023215848A1 (fr) * 2022-05-06 2023-11-09 Umoja Biopharma, Inc. Particule virale à molecules de stimulation de surface

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020236792A1 (fr) * 2019-05-21 2020-11-26 Novartis Ag Molécules de liaison à cd19 et utilisations de celles-ci
WO2023215848A1 (fr) * 2022-05-06 2023-11-09 Umoja Biopharma, Inc. Particule virale à molecules de stimulation de surface

Non-Patent Citations (3)

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Title
LU HAIHUI ET AL: "PIT565, a First-in-Class Anti-CD19, Anti-CD3, Anti-CD2 Trispecific Antibody for the Treatment of B Cell Malignancies", vol. 140, no. Supplement 1, 15 November 2022 (2022-11-15), AMSTERDAM, NL, pages 3148 - 3148, XP093233060, Retrieved from the Internet <URL:https://ashpublications.org/blood/article/140/Supplement%201/3148/487267/PIT565-a-First-in-Class-Anti-CD19-Anti-CD3-Anti> DOI: 10.1182/blood-2022-168904 *
MAZZA IRIT AVIVI ET AL: "A Phase 1 Study of PIT565, a First-in-Class, Anti-CD3, Anti-CD19, Anti-CD2 Trispeci c Antibody in Patients with Relapsed and/or Refractory B-Cell Malignancies", vol. 142, no. Suppl. 1, 2 November 2023 (2023-11-02), pages 3099 - 3101, XP093306521, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S000649712309701X> *
WONG W M ET AL: "Rheumatoid arthritis T cells produce Th1 cytokines in response to stimulation with a novel trispecific antibody directed against CD2, CD3, and CD28", SCANDINAVIAN JOURNAL OF RHEUMATOLOGY, ALMQVIST & WIKSELL PERIODICAL CO., STOCKHOLM, SE, vol. 29, no. 5, 1 January 2000 (2000-01-01), pages 282 - 287, XP009179418, ISSN: 0300-9742, DOI: 10.1080/030097400447651 *

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