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WO2025235862A1 - Récepteur de cellule immunitaire modifiée comprenant un domaine de liaison à la cible et le domaine extracellulaire de cd16a - Google Patents

Récepteur de cellule immunitaire modifiée comprenant un domaine de liaison à la cible et le domaine extracellulaire de cd16a

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Publication number
WO2025235862A1
WO2025235862A1 PCT/US2025/028598 US2025028598W WO2025235862A1 WO 2025235862 A1 WO2025235862 A1 WO 2025235862A1 US 2025028598 W US2025028598 W US 2025028598W WO 2025235862 A1 WO2025235862 A1 WO 2025235862A1
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cells
cell
protein
seq
domain
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Inventor
Nikola IVICA
Istvan Kovacs
John W. BEADLE
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Inndura Therapeutics Inc
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Inndura Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/17—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4212—CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4221—CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4231—Cytokines
    • A61K40/4232—Tumor necrosis factors [TNF] or CD70
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • 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
    • 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
    • C07K16/2875—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 NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • 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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present disclosure relates to novel constructs, particularly modified immune cell receptor proteins and polynucleotides encoding said modified immune cell receptor proteins, and uses of said constructs.
  • cytotoxic and phagocytic lymphocytes to eliminate diseased cells is essential for the proper physiological functioning and overall health of an organism 1,2 .
  • cytotoxic lymphocytes including various subsets of T cells, Natural Killer (NK) cells, NKT cells, and others, play a pivotal role in identifying and killing diseased cells that have been infected by a virus.
  • phagocytes such as monocytes and macrophages
  • engulfing infected and apoptotic cells effectively facilitating their removal from the body.
  • These essential functions extend beyond viral infections to encompass other infectious diseases, cancer, and autoimmune disorders, where the complete elimination of diseased cells is essential for resolving the pathology 3,4,5 .
  • cytotoxic and phagocytic cells discern the diseased target cell and trigger their functionality vary across cell types, they share certain fundamental features. All cytotoxic and phagocytic cells express surface receptors that serve as sensors for markers indicative of a diseased state 6,7 .
  • T cells Upon binding to their ligands, these receptors initiate an intracellular signaling cascade that activates the immune cell and initiates its effector functions.
  • T cells express a T-cell receptor (TCR) that recognizes foreign antigens in the context of the major histocompatibility complex (MHC), thereby sensing foreign peptides expressed by virally infected cells or cancer cells harboring oncogenic mutations.
  • TCR T-cell receptor
  • MHC major histocompatibility complex
  • NK cells express germline-encoded receptors such as CD16A, NKG2D, NKp30, NKp44, and NKp46, which recognize bound antibodies or stress ligands on the surface of senescent, virus-infected, or cancer cells.
  • Monocytes and macrophages express CD16A and CD64, which recognize antibody-opsonized targets, along with other surface receptors recognizing damage-associated molecular patterns such as CD91, TIM4, and others 8 .
  • the goal of precision medicine has long aimed to achieve the safe and precise removal of targeted cell types from the human body.
  • a significant step towards this objective was realized through the development of chimeric antigen receptor (CAR)-T, CAR-NK, and CAR-macrophage cell therapies, leveraging the evolved functionalities of these cells to selectively kill or phagocytose specific target cells 9,10,11,12 .
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor was engineered as a surface receptor with the capacity to target a particular cell type via single-chain fragment variable (scFv) binding to a cognate surface antigen, concurrently delivering activating signal through fused domains derived from naturally occurring stimulatory or co-stimulatory receptors.
  • a typical CAR construct thus comprises a scFv antigen-binding domain, a hinge domain, a transmembrane domain and intracellular stimulatory or costimulatory domains derived from 4-1BB, CD28 and CD3 ⁇ signaling proteins.
  • CAR Crohn's disease
  • autoimmune diseases 13,14,15,16 autoimmune diseases 13,14,15,16 .
  • CAR construct presents significant limitations.
  • CAR exhibits propensity for trogocytosis, wherein it extracts the bound cognate antigen from the target cell membrane, leading to prolonged activation, exhaustion and the potential risk of fratricide 17,18 or activation-induced cell death (AICD).
  • AICD activation-induced cell death
  • Strategies addressing CAR trogocytosis involve the development of fast-off scFv's and enhanced CAR recycling rates 19,20 .
  • tonic signaling poses a challenge, whereby CAR initiates signal activation even in the absence of antigen binding.
  • CAR modalities such as 4-1BB versus CD28 co-stimulation, and diverse scFv's, like anti-CD19 versus anti-GD2, exhibit varying propensities for tonic signaling 21,22 .
  • Tonic signaling has been associated with early T cell exhaustion and premature differentiation, though some reports suggest that 4-1BB-containing CAR tonic signaling may improve CAR-T cell survival and proliferation 23 .
  • the CAR construct is limited to targeting only one or two antigens simultaneously, which poses a challenge for targeting solid tumors that are characterized by the absence of homogeneously expressed tumor-associated markers.
  • CD16A is unique in its ability to transmit strong activating signals through high-affinity interactions with the F C domain of target-bound IgG1, IgG3, and to a lesser extent IgG4 antibodies 24 .
  • CD16A exhibits binding to the antibody F C domain with the dissociation constant in the nanomolar range 25 .
  • the naturally occurring F176V CD16A allele demonstrates even stronger binding affinity for F C domains of antibodies, with dissociation constant in the low nanomolar range 26 .
  • CD16A The high-affinity interactions exhibited by CD16A suggest the existence of evolutionary adaptations to mitigate the issue of trogocytosis, particularly in the case of viral infections that can lead to uptake of viral particles.
  • CD16A lacking its own signaling domain, forms a multimeric complex with endogenously expressed signaling adaptor proteins Fc ⁇ RI ⁇ (FCER1G) and CD3 ⁇ (CD247) 29,30 .
  • CD16A is dependent upon CD3 ⁇ and/or FCER1G for the surface expression 32 .
  • CD3 ⁇ can be downregulated in several immune effector cells, including tumor-infiltrating lymphocytes, T cells, NK cells and macrophages, in diseases including cancer and autoimmunity 33-36 .
  • T cells tumor-infiltrating lymphocytes
  • NK cells NK cells
  • macrophages diseases including cancer and autoimmunity 33-36 .
  • the chimeric antigen receptor construct has demonstrated considerable efficacy in redirecting effector functionality of T cells, NK cells, and macrophages.
  • inherent limitations, including trogocytosis, tonic signaling, exhaustion, AICD and restricted targeting versatility persist.
  • EVE16 Engineered Valency-Enhanced CD16A
  • EVE16 exhibits the capacity to redirect cytotoxic and phagocytic immune cells towards a desired target cell, mitigating trogocytosis and tonic signaling, while simultaneously facilitating versatile targeting via binding to antibody FC domain.
  • EVE16 has a dual mechanism of action, via scFv-mediated antigen-specific targeting, and via F C -mediated targeting (antibody-dependent cellular cytotoxicity or ADCC).
  • the signaling capacity of the EVE16 construct is contingent upon precise target engagement facilitated by its target-binding domain and FC-binding domain (see Figure 1A).
  • Optimal EVE16 functionality requires its correct folding, high expression levels, and efficient trafficking of the full construct to the cell surface, while avoiding steric hindrances between different EVE16 domains.
  • robust signaling capacity that promotes cytotoxicity, cytokine secretion, and effector cell proliferation is essential for therapeutic intervention.
  • the presence of high concentration of serum antibodies, including afucosylated antibodies poses potential challenges, as they may bind to EVE16, resulting in steric occlusions that impede proper target engagement through the target-binding domain.
  • EVE16 can further contribute to steric occlusions.
  • EVE16 relies on endogenous CD3 ⁇ for trafficking to the cell surface, necessitating competition with other CD3 ⁇ binders.
  • the inventors discovered that varying the length and amino acid sequence of the linker domain that connects the target-binding domain and FC-binding domain significantly influenced EVE16 surface expression and target engagement. While the precise mechanism remains unclear, it is postulated that the linker domain folds beneath the V-shaped ectodomain of CD16A, extending into the extracellular space from one of the sides of CD16A (see Figure 2). For optimal target binding, this requires a minimum length linker with sufficient flexibility.
  • linker domain length and sequence are imperative to ensure proper target engagement and immune effector functions.
  • Suitable flexibility of the linker can be achieved through the optimal incorporation of certain amino acids including glycine, serine and by reducing the content of cysteine and proline.
  • the linker domain should exhibit minimal immunogenicity.
  • linker sequences derived from human proteins are preferred.
  • the inventors have identified human-derived linker domains of sufficient length and flexibility that support high-level EVE16 surface expression and functional activity. Furthermore, the inventors discovered that both the choice of leader peptide and the orientation of the single-chain variable fragment (scFv) affect the expression and surface localization of the EVE16 construct.
  • EVE16 constructs were consistently lower than that of CAR constructs containing the same scFv, potentially because of competition for CD3 ⁇ binding between various surface receptors, including endogenous CD16A, NKp30, NKp46 and TCR.
  • Co-expression of EVE16 with the full- length CD3 ⁇ protein significantly improved the surface expression of EVE16 confirming that CD3 ⁇ is limiting for EVE16 plasma membrane trafficking.
  • the present invention encompasses: A modified immune cell receptor protein comprising: - a target-binding domain, - the extracellular domain of CD16A, wherein the extracellular domain comprises the FC-binding domain and the hinge domain of CD16A, and - a transmembrane domain, wherein the modified immune cell receptor protein also comprises a flexible linker positioned between the target-binding domain and the extracellular domain of CD16A, wherein the linker is at least 10 amino acids in length.
  • the invention also encompasses: - A nucleic acid encoding the protein of the invention. - An immune cell or population of immune cells comprising the protein of the invention or the nucleic acid of the invention.
  • a pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid of the invention or the immune cell(s) of the invention.
  • the immune cell of the invention, or the pharmaceutical composition of the invention for use in therapy.
  • a method of treating a subject with cancer comprising administering to the subject the immune cell of the invention or the pharmaceutical composition of the invention.
  • a method of treating a subject with autoimmune disease comprising administering to the subject the immune cell of the invention or the pharmaceutical composition of the invention.
  • a method of treating a subject with a transplant comprising administering to the subject the immune cell of the invention or the pharmaceutical composition of the invention.
  • Figure 1 is a series of images showing schematic representations highlighting the similarities and differences between the endogenous human CD16A protein, the Engineered Valency-Enhanced CD16A (EVE16) receptor, and the standard chimeric antigen receptor (CAR) receptor.
  • EVE16 receptor is composed of the target-binding domain, the linker domain, the F C -binding domain, the hinge domain, the transmembrane domain, and the cytoplasmic domain ( Figure 1A).
  • the cytoplasmic domain of EVE16 comprises 4-1BB and DAP10 co- stimulatory sequences, although other co-stimulatory sequences can also be used.
  • Figure 1B exemplifies the open-reading frame (ORF) composition and the primary protein structure of an EVE16 receptor that contains FMC63 scFv target-binding domain (targeting CD19) and 4-1BB/DAP10 co-stimulatory domain.
  • Figure 2. comprising Figures 2A-G, is a series of images showing primary and tertiary structures of proteins important for the assembly of the Engineered Valency-Enhanced CD16A (EVE16) receptor.
  • Figures 2A and 2B depict primary and tertiary structures of human CD3zeta (A) and FCER1G (B) proteins, essential for the EVE16 plasma membrane localization.
  • Figures 2C and 2D depict primary and tertiary structures of human CD16A protein (C) as well as its predicted leader peptide cleavage site (D).
  • Figure 2E depicts primary and tertiary structures of human CD27 protein, with an outlined ectodomain sequence that functions as a CD70 ligand.
  • Figure 2F depicts primary and tertiary structures of human CD8A protein, with outlined leader peptide sequence that can be used for the expression of various plasma membrane proteins, and particularly the EVE16 receptor.
  • Figure 2G depicts primary and tertiary structures of an example EVE16 protein containing CD8A leader peptide and target-binding domain composed of CD27 ectodomain.
  • Figure 3. comprising Figures 3A-B, is a series of images showing that the linker domain of EVE16 is necessary for the surface expression of the full receptor, but its presence alone is not entirely sufficient for proper surface expression.
  • Figure 3A depicts the open-reading frame composition of EVE16 receptors containing various linker domain constructs. Each EVE16 receptor included the CD8 ⁇ leader peptide and CD27 ectodomain as the target- binding domain, while the cytoplasmic domain consisted of the native CD16A cytoplasmic domain.
  • FIG. 4 is a series of images showing that the expression of the CD27-EVE16 receptor containing a flexible linker domain is functional in cytokine- induced memory-like Natural Killer (CIML NK) cells.
  • CIML NK cytokine- induced memory-like Natural Killer
  • Figure 4A shows flow cytometry plots of untransduced CIML NK cells, or CIML NK cells transduced with lentiviral particles harboring indicated EVE16 receptors and stained for the surface expression of CD27 and CD16A.
  • Figure 4B shows the result of a 6-hour cytotoxicity assay of the indicated CIML NK cells against OCI-AML3 cell line (CD70-expressing) at the indicated effector-to-target ratios.
  • Figure 5. comprising Figures 5A-B, is a series of images showing a library of linker domains used for the optimization of EVE16 surface expression.
  • Figure 5A depicts open- reading frame composition of each linker domain used for the assembly of CD27-EVE16 receptors.
  • Figure 5B shows amino acid sequence and some of the biochemical characteristics of linker domains used for the optimization of EVE16 surface expression.
  • Figure 6, comprising Figures 6A-C, is a series of images showing the optimization of EVE16 surface using various linker domains.
  • Figure 6A shows flow cytometry plots
  • Figure 6B shows mean fluorescence intensities of Jurkat cell lines stably expressing indicated CD27-EVE16 receptors. Cells were stained for surface expression of CD27 and CD16A.
  • Figure 6C shows the binding of recombinant human CD70 to the Jurkat cells stably expressing indicated CD27-EVE16 receptors.
  • Each CD27-EVE16 receptor is bis- cistronic, containing an eGFP marker and utilizing a P2A self-cleavable peptide.
  • Figure 7. is a series of images showing that the CD27-EVE16 receptor containing the optimized linker domain is functional in CIML NK cells.
  • CIML NK cells were transduced with a bis-cistronic expression vector containing the indicated CD27-EVE16 receptor and the eGFP marker.
  • CIML NK cells were stained for surface CD27 and CD16A, and flow cytometry plot was generated from eGFP-positive gated cells.
  • FIG. 8 is a series of images showing that the FC-binding domain is necessary for the proper surface expression of the EVE16 receptor.
  • Jurkat cells were stably transduced with indicated FMC63-EVE16 receptor mutants and stained for surface FMC63 scFv and CD16A.
  • Each FMC63-EVE16 receptor is bis-cistronic, containing an eGFP marker, and utilizing a P2A self-cleavable peptide.
  • Flow cytometry plots of stained Jurkat cells were gated on live, eGFP-positive cells.
  • Figure 9. is a series of images showing that certain cytoplasmic domains enhance the signaling capacity of the EVE16 receptor.
  • the signaling capacity of CD27-EVE16 receptors with indicated cytoplasmic domains was evaluated in a cytotoxicity assay (A and C 6-hour assay, B 16-hour assay) using transduced CIML NK cells against OCI-AML3 cell line at the indicated effector-to-target ratio.
  • Each CD27-EVE16 receptor is bis-cistronic, containing an eGFP marker and utilizing a P2A self-cleavable peptide.
  • FIG. 9D shows flow cytometry data of isolated human CD3+ T cells transduced with either an anti-CD19 CAR (scFv derived from FMC63) or EVE16 variants containing the indicated intracellular signaling domains. The same T cell populations were evaluated in an overnight cytotoxicity assay against Raji cells to assess functional activation (bottom panel).
  • Figure 9E displays the in vivo antitumor activity of T cells transduced with FMC63-based EVE16 variants harboring different cytoplasmic domains and the optimized NKp44 long linker domain, as described above.
  • Figure 9F shows the in vivo efficacy of T cells transduced with anti-CD20 EVE16 variants (Rituximab-derived scFv) containing the indicated cytoplasmic signaling domains.
  • EVE16 variants Rostab-derived scFv
  • Figure 10 is a series of images showing that the co-expression of CD3zeta enhances the surface expression of the EVE16 receptor.
  • Jurkat cells were stably transduced with FMC63-EVE16 receptor containing 4-1BB costimulatory domain as a bis-cistronic construct co-expressing either the eGFP marker or the full-length human CD3zeta protein (note the open-reading frame composition for each construct).
  • FMC63-EVE16 surface expression was evaluated using surface CD16A staining and flow cytometry analysis.
  • Figure 11 is an image demonstrating that the optimized EVE16 receptor can be directed against multiple target surface antigens.
  • Human CD3+ T cells were isolated and transduced with EVE16 variants incorporating an optimized NKp44 long hinge-derived linker domain, an OX40-derived intracellular signaling domain, and single-chain variable fragments (scFvs) specific for CD19 (FMC63), CD20 (Rituximab), CD70 (Cusatuzumab), or CD22 (Pinatuzumab).
  • the immunoactivation potential of each EVE16 variant was evaluated in an overnight cytotoxicity assay using the Raji cell line, which endogenously expresses CD19, CD20, CD22, and CD70.
  • Figure 12 represents data demonstrating that both the leader peptide sequence and scFv orientation are critical determinants of EVE16 surface expression.
  • Figure 12A shows the open reading frames (ORFs) of a library of anti-CD20 EVE16 variants generated for the screen, each incorporating one of twelve distinct leader peptides. Single-chain variable fragments (scFvs) were derived from four anti-CD20 monoclonal antibodies: Rituximab, Ofatumumab, Ocrelizumab, and Ublituximab.
  • Figure 12B displays flow cytometry analysis of 293T cells transfected with the indicated EVE16 constructs, assessing surface expression using antibodies specific for CD16 and the G4S linker.
  • Figure 12C presents flow cytometry data from Jurkat cells transduced with the same EVE16 variants, showing surface staining with anti-CD16 and anti-G4S linker antibodies, as well as binding to CD20-GFP fusion virus-like particles.
  • Figure 12D compares the expression and functional activity of a Rituximab-based CAR construct and two Rituximab-derived EVE16 variants in isolated human CD3+ T cells. Functional activation was assessed in an overnight cytotoxicity co-culture assay using Raji target cells.
  • DETAILED DESCRIPTION All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
  • a value refers to that value but within a reasonable degree of scientific error.
  • a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x.
  • the terms “comprising” or “comprises” are intended to mean including but not limited to.
  • the phrase “...a modified immune cell receptor protein comprising a binding domain” should be interpreted to mean that the modified immune cell receptor protein comprises a binding domain, but the modified immune cell receptor protein may also comprise further features, such as the extracellular domain of CD16A and a transmembrane domain.
  • the words “comprising” or “comprises” are replaced with the phrases “consisting of” or “consists of”.
  • the phrases “consisting of” or “consists of” are intended to be limiting.
  • nucleic acid refers to a polymer of nucleotides, each of which are organic molecules consisting of a nucleoside (a nucleobase and a five-carbon sugar) and a phosphate.
  • nucleotide includes nucleosides that have a ribose sugar (i.e., a ribonucleotide that forms ribonucleic acid, RNA) or a 2’-deoxyribose sugar (i.e., a deoxyribonucleotide that forms deoxyribonucleic acid, DNA).
  • Nucleotides serve as the monomeric units of nucleic acid polymers or polynucleotides.
  • the four nucleobases in DNA are guanine (G), adenine (A), cytosine (C) and thymine (T).
  • RNA The four nucleobases in RNA are guanine (G), adenine (A), cytosine (C) and uracil (U).
  • Nucleic acids are linear chains of nucleotides (e.g., at least 3 nucleotides) chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar (i.e., ribose or 2’-deoxyribose) in the adjacent nucleotide.
  • the sequences for the modified immune cell receptor proteins may be encoded on a single nucleic acid or may be encoded by more than one nucleic acid (a set of nucleic acids).
  • antigen refers to an entity at least a portion of which is present on the surface of a cell, such as a cancer or immune cell.
  • Antigens may be proteins, peptides, peptide-protein complexes (e.g., a peptide bound to an MHC molecule), protein- carbohydrate complexes (e.g., a glycoprotein), protein-lipid complexes (e.g., a lipoprotein), protein-nucleic acid complexes (e.g., a nucleoprotein), etc.
  • EVE16 Engineered Valency-Enhanced CD16A receptor
  • a modified immune cell receptor protein comprising an active fragment of an immune cell activating receptor CD16A and a target-binding domain.
  • the latter redirects the specificity of the activating receptor to render it specific for a desired or intended target, for example such that activation and signaling occurs after binding a disease target, such as CD19, CD33, CD7, CD123, CD20, CD22, BCMA, GPRC5D, Mesothelin, claudin18.2, PSMA, B7-H6, CD3, CD4, BAFF-R, EGFR, HER2, gp120, gp41, or CD70.
  • a disease target such as CD19, CD33, CD7, CD123, CD20, CD22, BCMA, GPRC5D, Mesothelin, claudin18.2, PSMA, B7-H6, CD3, CD4, BAFF-R, EGFR, HER2, gp120, gp41, or CD70
  • Activating receptor components refers to elements of the modified immune cell receptor protein that are derived from (such as fragments of) an activating receptor.
  • An “activating receptor” as employed herein refers to a cell surface receptor that sends an activating signal to an immune cell, in particular to generate or enhance cytotoxic activity.
  • Activating receptors as employed herein include Killer Activation Receptors (KARs) are receptors expressed on the plasma membrane of Natural Killer cells (NK cells). KARs work together with inhibitory receptors (abbreviated as KIRs in the text), which inactivate them in order to regulate the NK cell function on host or transformed cells. These two kinds of specific receptors have some morphological features in common, such as being transmembrane proteins.
  • KARs and KIRs can have tyrosine containing activating or inhibitory motifs in the intracellular part of the receptor molecule (they are called ITAMs and ITIMs, respectively).
  • the activating receptors employed herein may comprise several domains, for example an immunoglobulin (Ig)-like domain, a hinge region, a transmembrane domain and a cytoplasmic domain. It is thought that the hinge, transmembrane, and cytoplasmic domains are important for signaling.
  • An active fragment of an immune cell activating receptor as employed herein refers to a fragment of the receptor that is able to induce signaling after a binding event.
  • Naturally occurring variants of CD16A present in the human population may be independently selected from F176V, and 48-L/R/H, and others.
  • An alternative name used for the F176V mutation, or polymorphism, in scientific literature is F158V.
  • Adaptor proteins as employed herein refers to signal transducing adaptor protein (STAPs). These are proteins that are accessory to main proteins in a signal transduction pathway. Adaptor proteins contain a variety of protein-binding modules that link protein-binding partners together and facilitate the creation of larger signaling complexes. These proteins tend to lack any intrinsic enzymatic activity themselves, instead mediating specific protein–protein interactions that drive the formation of protein complexes.
  • adaptor proteins examples include MYD88, Grb2, SHC1, DAP10, DAP12, as well as Fc ⁇ RI ⁇ and CD3 ⁇ .
  • Adaptor signaling proteins usually contain several domains within their structure (e.g., Src homology 2 (SH2) and SH3 domains) that allow specific interactions with several other specific proteins.
  • SH2 domains recognize specific amino acid sequences within proteins containing phosphotyrosine residues and SH3 domains recognize proline-rich sequences within specific peptide sequence contexts of proteins.
  • SH2 domains recognize specific amino acid sequences within proteins containing phosphotyrosine residues
  • SH3 domains recognize proline-rich sequences within specific peptide sequence contexts of proteins.
  • There are many other types of interaction domains found within adaptor and other signaling proteins that allow a rich diversity of specific and coordinated protein–protein interactions to occur within the cell during signal transduction.
  • Adaptor proteins contribute to the selection, differentiation, and activation of Natural Killer (NK) cells, T cells, Natural Killer T (NKT) cells and monocytes. Adaptor proteins are also expressed in a subset of T cells. DAP10, for example, is expressed in NK cells, a subset of T cells and myeloid cells. DAP12, for example, is expressed in NK cells, in peripheral blood monocytes, macrophages, and dendritic cells. Fc ⁇ RI ⁇ and CD3 ⁇ , for example, are expressed on at least NK cells, T cells, and monocytes.
  • the immune cell employed in the present disclosure is independently selected from one or more of a NK cell, a NKT cell, a T cell, a monocyte, macrophage, and a dendritic cell.
  • a NK cell a NKT cell
  • T cell a monocyte, macrophage, and a dendritic cell.
  • Endogenously present in the cell as employed herein refers to an entity that is native to the cell, in particular it has NOT been introduced by recombinant techniques and/or artificially introduced.
  • Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events.
  • Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside.
  • Signal transduction occurs as a result of a ligand binding to the outside region of the receptor (the ligand does not pass through the membrane).
  • Ligand-receptor binding induces a change in the conformation of the inside part of the receptor, a process sometimes called “receptor activation”. This results in either the activation of an enzyme domain of the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.
  • Gene activations and metabolism alterations are examples of cellular responses to extracellular stimulation that require signal transduction. Gene activation leads to further cellular effects, since the products of responding genes include instigators of activation; transcription factors produced as a result of a signal transduction cascade can activate even more genes.
  • an initial stimulus can trigger the expression of a large number of genes, leading to physiological events like the increased uptake of glucose from the blood stream and the migration of immune cells to sites of infection.
  • the set of genes and their activation order to certain stimuli is referred to as a genetic program.
  • the signaling from the modified immune cell receptor protein will generate activation of an immune cell, for example to generate cytotoxic activity, inflammatory responses, proliferation, phagocytic responses or similar.
  • the term “cytokine” is widely recognized in the field and encompasses a diverse group of proteins pivotal in cellular signaling. Within the cytokine family, notable members include various interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors.
  • cytokine receptors which are multi-chain receptors. Upon binding with their respective cytokines, these receptors initiate an intracellular signaling cascade, resulting in a change of the cell’s functional state.
  • cytokine receptors can also be activated through the use of antibodies and various fusion proteins, thereby facilitating functional binding analogous to that achieved through direct cytokine binding.
  • memory-like signifies a cellular state, specifically in an immune cell, marked by heightened cytotoxicity, proliferative capacity, and other effector functions, such as cytokine secretion or surface marker expression, in comparison to the non-activated cell state.
  • cytokine-induced memory-like pertains to a cellular state, particularly within an immune cell, that has undergone cytokine treatment. This state is characterized by increased cytotoxicity, proliferative capacity, and other effector functions, including cytokine secretion or surface marker expression, relative to the non-activated cell state.
  • a cytokine-induced memory-like T cell refers to a T cell treated with cytokines, demonstrating heightened effector functions upon activation.
  • the phrase “cytokine-induced memory-like state” or “CIML” is intended to mean a trained immunity, or activated state achieved by immune cells in response to stimulation by cytokines.
  • CIML immune cells have undergone transcriptional, epigenetic and metabolic reprogramming and have enhanced effector functions and increased persistence. Examples of CIML immune cells include T cells, CD4+ T cells, CD8+ T cells, ⁇ T cells, NK cells, NKT cells, dendritic cells, monocytes and macrophages.
  • cytokine-induced memory-like or, equivalently, “CIML” in reference to the immune cells described herein, means having a “memory” or “memory-like” phenotype and produced using a priming agent.
  • the phrase “immune cell” or “immune effector cell” refers to a cell that may be part of the innate or adaptive immune system and executes a particular effector function such as T cells, ⁇ T cells, NK cells (including memory-like NKs, ML NKs, and CIML NKs), NKT cells (including iNKT cells), B cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, ⁇ T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes and macrophages.
  • ILC innate lymphoid cells
  • CIK cytokine-induced killer
  • LAK lymph
  • Preferred immune cells are cells with cytotoxic effector function such as ⁇ T cells, NK cells (including memory-like NKs, ML NKs, and CIML NKs), NKT cells (including iNKT cells), ILC, CIK cells, LAK cells or ⁇ T cells.
  • cytotoxic effector function means a specialized function of a cell, e.g., in an NK cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.
  • immune cells include T cells, CD4+ T cells, CD8+ T cells, ⁇ T cells, NK cells, NKT cells, dendritic cells, monocytes and macrophages.
  • Natural killer (NK) cells constitute a group of innate immune cells, which are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cellular cytotoxicity via target-directed release of granzyme, granulysin and perforin. Most NK cells have a specific cell surface marker profile (e.g., CD3-, CD56+, CD16+) in addition to a collection of various activating and inhibitory receptors. While more recently NK cells have become a significant component of certain cancer treatments, generation of significant quantities of NK cells (and especially autologous NK cells) has been a significant obstacle as the fraction of NK cells in whole blood is relatively low.
  • NK Natural killer
  • cytokine-induced memory-like NK cell or “ML NK cell” refers to a NK cell derived from an NK cell which has been activated ex vivo with at least one cytokine and maintains an enhanced memory-like function after challenge in the absence of the same cytokines.
  • CIML NK cell refers to a NK cell derived from an NK cell which has been activated with at least one cytokine and exhibits enhanced activation and interferon-gamma responses.
  • Human cytotoxic and phagocytic lymphocytes constitute vital components of the immune system, equipped with the capacity to eliminate diseased cells within the human body either through the directed delivery of cytotoxic granules or by leveraging phagocytic function.
  • the cytotoxic subset of lymphocytes encompasses various immune cell types, including Natural Killer (NK) cells, Natural Killer T (NKT) cells, and cytotoxic T cells such as CD8+ T cells, and gamma-delta T cells, among others.
  • Monocytes and macrophages exemplify human phagocytic cells, adept at selectively engulfing and degrading other cells.
  • T cells While both cytotoxic and phagocytic immune cell types share the common goal of removing target cells from the body, they exhibit significant differences in their targeting mechanisms and interactions with respective target cells.
  • T cells distinguished by a unique somatically rearranged T-cell receptor (TCR), selectively recognize foreign antigen peptides within the context of major histocompatibility complex (MHC) proteins.
  • MHC major histocompatibility complex
  • NK cells, monocytes, and macrophages utilize a set of germline-encoded receptors to engage ligands on the surface of target cells or to recognize antibody-opsonized targets.
  • T cells envisaged to be used in the invention may be any type of T cell.
  • the T-cell may be a CD4+ T cell, or helper T cell (Th cell), such as a Th1, Th2, Th3, Th17, Th9, or Tfh cell.
  • the T cell may be a CD8+ T cell, or cytotoxic T cell.
  • the T cell may be a CD4+ or CD8+ memory T cell, such as a central memory T cell or an effector memory T cell.
  • the T cell may be a regulatory T cell (Treg).
  • the T cell may be, for example, a na ⁇ ve T cell or a T memory stem cell.
  • Na ⁇ ve T cells are precursors for effector and memory T cell subsets.
  • T stem cell-like memory (T SCM ) cells are a subset of memory lymphocytes endowed with the stem cell–like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.
  • the term “monocyte” refers to a type of immune cell that is made in the bone marrow and travels through the blood to tissues in the body where it becomes a macrophage or a dendritic cell.
  • Macrophages surround and kill microorganisms, ingest foreign material, remove dead cells, and boost immune responses.
  • amino acid 1 is considered to be the first Methionine of the leader peptide, i.e. the N-terminal amino acid of the full- length protein, with amino acids being counted from that residue.
  • “Signal peptide” and “leader peptide” may be used interchangeably herein.
  • the modified immune cell receptor protein of the present invention comprises: - a target-binding domain, - the extracellular domain of CD16A, wherein the extracellular domain comprises the F C -binding domain and the hinge domain of CD16A, and - a transmembrane domain, wherein the modified immune cell receptor protein also comprises a flexible linker positioned between the target-binding domain and the FC-binding domain of the extracellular domain of CD16A, wherein the linker domain is at least 10 amino acids in length.
  • the modified immune cell receptor protein of the present invention comprises: - a target-binding domain, - the extracellular domain of CD16A, wherein the extracellular domain comprises the FC-binding domain and the hinge domain of CD16A, and - a transmembrane domain, and - a cytoplasmic domain comprising the co-stimulatory domain of CD3 ⁇ , wherein the modified immune cell receptor protein also comprises a flexible linker positioned between the target-binding domain and the FC-binding domain of the extracellular domain of CD16A, wherein the linker domain is at least 10 amino acids in length.
  • the modified immune cell receptor protein of the present invention comprises: - a target-binding domain, - the extracellular domain of CD16A, wherein the extracellular domain comprises the F C -binding domain and the hinge domain of CD16A, and - a transmembrane domain, and - a cytoplasmic domain comprising the co-stimulatory domain of Fc ⁇ RI, wherein the modified immune cell receptor protein also comprises a flexible linker positioned between the target-binding domain and the F C -binding domain of the extracellular domain of CD16A, wherein the linker domain is at least 10 amino acids in length.
  • the modified immune cell receptor protein of the present invention comprises: - a target-binding domain, - the extracellular domain of CD16A, wherein the extracellular domain comprises the FC-binding domain and the hinge domain of CD16A, and - a transmembrane domain, and - a cytoplasmic domain comprising at least one of the following co-stimulatory domains: 4-1BB, CD28, OX40 and DAP10; wherein the modified immune cell receptor protein also comprises a flexible linker positioned between the target-binding domain and the F C -binding domain of the extracellular domain of CD16A, wherein the linker domain is at least 10 amino acids in length.
  • the modified immune cell receptor protein of the present invention comprises: - a target-binding domain, - the extracellular domain of CD16A, wherein the extracellular domain comprises the FC-binding domain and the hinge domain of CD16A, and - a transmembrane domain, and - a cytoplasmic domain comprising at least one of the following co-stimulatory domain combinations: 4-1BB and CD3 ⁇ , 4-1BB and CD28, OX40 and CD28, 4-1BB and DAP10, or OX40 and DAP10; wherein the modified immune cell receptor protein also comprises a flexible linker positioned between the target-binding domain and the FC-binding domain of the extracellular domain of CD16A, wherein the linker domain is at least 10 amino acids in length.
  • the target-binding domain targets a receptor or ligand on a cell, the killing of which cell is desirable. In one embodiment of the invention, the target-binding domain targets an antigen or other ligand on a cell. In one embodiment of the invention, the target-binding domain targets an antigen or other ligand on a cancer cell. In one embodiment of the invention, the target-binding domain targets a cognate receptor or cognate ligand of a cancer antigen on an immune cell. In one embodiment of the invention, the target-binding domain targets a receptor or other ligand on an immune cell. In one embodiment of the invention, the immune cell is a B cell or T cell.
  • the killing of the cell by target-binding domain directed targeting and/or antibody-dependent cellular cytotoxicity is desirable.
  • ADCC may be mediated by naturally occurring antibodies (including antibodies directed at cancer neoantigens), therapeutic antibodies (including IgG1, IgG3 and IgG4 monoclonal antibodies), and cell engagers that bind to the extracellular domain of CD16A.
  • the cancer antigen is any one of the cancer antigens provided herein.
  • the target-binding domain binds a receptor or other ligand on a B cell.
  • the target-binding domain binds any one of the receptors or other ligands on a B cell provided herein. In one embodiment of the invention, the target-binding domain binds a receptor or other ligand on a T cell. In one embodiment of the invention, the target-binding domain binds any one of the receptors or other ligands on a T cell provided herein. In one embodiment of the invention, the target-binding domain comprises an antibody fragment. In one embodiment of the invention, the target-binding domain comprises a single-chain variable antibody fragment (scFv). The target-binding domain may bind to an antigen. In some embodiments, the target- binding domain is an antibody fragment.
  • scFv single-chain variable antibody fragment
  • the target-binding domain is a single-chain variable antibody fragment (scFv) that includes a variable light (VL) and a variable heavy (VH) domain that may be derived from an immunoglobulin that binds the antigen.
  • scFv single-chain variable antibody fragment
  • VL variable light
  • VH variable heavy domain
  • the term “derived from” as used herein when referring to protein or nucleic acid sequences refers to a sequence that originates from another, parent sequence.
  • a sequence derived from a parent sequence may be identical, may be a portion of the parent sequence, or may have at least one variant from the parent sequence.
  • Variants may include substitutions, insertions, or deletions.
  • an amino acid sequence derived from a parent sequence may be identical for a specific range of amino acids of the parent but does not include amino acids outside that specific region.
  • the scFv may be in an VH-VL orientation in a N to C terminal direction.
  • the scFv may be in an VL-VH orientation in a N to C terminal direction.
  • the antigen is on a cancer (e.g., tumor) cell or a cognate receptor or cognate ligand of the antigen on an immune cell.
  • the cancer antigen may be “tumor-associated” or “tumor-specific” antigen.
  • Tumor-associated antigen refers to antigens that are expressed at a higher level on a cancer, tumor or neoplastic cell as compared to a normal cell derived from the same tissue or lineage as the cancer, tumor or neoplastic cell, or at a level where, while not exclusive to the cancer, tumor or neoplastic cell, allows for targeting of the cancer, tumor or neoplastic cell at a level to treat the cancer.
  • TAA Tumor-associated antigen
  • Cancer and tumor antigens include, without limitation, EGFR, CD19, CD20, CD22, NKG2D ligands, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, FAB, WT-1, PSMA, NY-ESO1, AFP, CSPG-4, IGF1-R, Flt-3, CD276, CD123, CD133, PD-L1, BCMA, GPRC5D, 41BB, CTAG1B, and CD33.
  • the antigen on the cancer cell that binds the target-binding domain is the cognate ligand for a receptor naturally present on an immune cell. Therefore, occupying the antigen by the target-binding domain can prevent the antigen’s binding the receptor, keeping the immune cell in an active state.
  • Examples of these “checkpoint” antigens present on cancer cells include PD-L1, epidermal growth factor receptor (EGFR), and HLA-E.
  • the target-binding domain binds the cognate ligand or cognate receptor naturally present on an immune cell. Therefore, occupying the cognate ligand or cognate receptor by the target binding domain can also prevent the antigen’s binding, keeping the immune cell in an active state.
  • Examples of these “checkpoint” cognate receptors or cognate ligands of antigens present on immune cells include transforming growth factor ⁇ (TGF ⁇ ), EGF, NKG2A (CD159), and NKG2D.
  • the target binding domain binds AFP, ALPP, AXL, B7-H3, B-cell maturation antigen (BCMA), GPRC5D, By0H3, CD7, CD19, CD20, CD22, CD33, CD44v6, CD70, CD117, CD147, CD123, CD126, CD171, CAIX, Chlorotoxin, CLDN, CEA, CLDN6, c-Met, c-Met, CPC3, DLL3, EPCAM, EphA2, FAP, FRA, FR ⁇ , GD2 ganglioside, GFR ⁇ 4, GLV, GP100, GPC3, GUCY2C, ERB-B2 receptor tyrosine kinase 2 (HER2), ICAM-1, IL13R ⁇ 2, KLK2, KNG2DL, LeY, LMP1, mesothelin, MG7, major histocompatibility complex, class I, E (HLA-E), MHC Class I polypeptide-related sequence A (MICA), ERB-
  • the target-binding domain binds CD19.
  • CD19 is an attractive target for cancer therapy because it is normally limited to cells of the B-cell lineage. Furthermore, it is expressed on the vast majority of B-cell malignancies, including 80% of acute lymphoblastic leukemias (ALLs), 88% of B-cell lymphomas, and 100% of B-cell leukemias. Therefore, CD19 is a suitable TAA against which to target anti-cancer agents. In contrast to CD20, CD19 is expressed throughout B-cell development, from B-cell precursors through to mature B cells before expression is lost when mature B cells become plasma cells.
  • the target-binding domain is a scFv that binds CD19.
  • the target-binding domain is derived from the sequence of a commercially available anti-CD19 antibody, antibody fragment, or derivative thereof.
  • Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of the sequences in Table 1.
  • Table 1 Amino acid sequences of representative anti-CD19 antibody fragments Polypeptide Sequence loncastuximab 1 qvqlvqpgae vvkpgasvkl scktsgytft snwmhwvkqa pgqglewige idpsdsytny heavy chain 61 nqnfqgkakl tvdkststay mevsslrsdd tavyycargs npyyyamdyw gqgtsvtvss light chain 181 sstltlskad yekhkvyace vthqglsspv tksfnrgec (SEQ ID NO: 10) target-binding domain is a scFv that binds to CD20.
  • the target- binding domain is derived from the sequence of a commercially available anti-CD20 antibody, antibody fragment, or derivative thereof.
  • Representative amino acid sequences of heavy and light chains of anti-CD20 antibodies are set forth in Table 2. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of the sequences in Table 2.
  • the target-binding domain is a scFv that binds CD20 which comprises the 3 CDR sequences found within SEQ ID NO: 17 and the 3 CDR sequences found within SEQ ID NO: 18. In some embodiments, the target-binding domain is a scFv that binds CD20 which comprises SEQ ID NO: 17 and SEQ ID NO: 18. In some embodiments, the target-binding domain is rituximab present in a VH to VL orientation, in a N terminal to C terminal direction. In some embodiments, the target- binding domain is rituximab present in a VL to VH orientation, in a N terminal to C terminal direction.
  • the target-binding domain is rituximab present in a VH to VL orientation, in a N terminal to C terminal direction, in combination with a NKp44 leader peptide as described herein. In some embodiments, the target-binding domain is rituximab present in a VL to VH orientation, in a N terminal to C terminal direction, in combination with a CD8A leader peptide as described herein. In some embodiments, the target-binding domain is ofatumumab present in a VH to VL orientation, in a N terminal to C terminal direction. In some embodiments, the target-binding domain binds CD22 (also known as Siglec-2).
  • the target-binding domain is a scFv that binds to CD22.
  • the target-binding domain is derived from the sequence of a commercially available anti-CD22 antibody, antibody fragment, or derivative thereof. Representative amino acid sequences of heavy and light chains of anti-CD22 antibodies are set forth in Table 2a. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of the sequences in Table 2a.
  • the target-binding domain is a scFv that binds CD22. In some embodiments, the target-binding domain is a scFv that binds CD22 such as Pinatuzumab.
  • the target-binding domain is a scFv that binds CD22 which comprises the 3 CDR sequences found within SEQ ID NO: 112 and the 3 CDR sequences found within SEQ ID NO: 113. In some embodiments, the target-binding domain is a scFv that binds CD22 which comprises SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, the target-binding domain binds B-cell maturation antigen (BCMA, also known as CD269 and TNFRSF17). In some embodiments, the target-binding domain is a scFv that binds to BCMA.
  • BCMA B-cell maturation antigen
  • the target-binding domain is derived from the sequence of a commercially available anti-BCMA antibody, antibody fragment, or derivative thereof.
  • Representative amino acid sequences of heavy and light chains of anti-BCMA antibodies are set forth in Table 2b. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of the sequences in Table 2b.
  • the target-binding domain binds CD117.
  • the target-binding domain is a scFv that binds to CD117.
  • Anti-CD117 antibodies and binding domains thereof are known in the art. See, e.g., U.S. Patents 10,111,966, 10,882,915, and 10,899,843, and such sequences are incorporated herein by reference.
  • the target-binding domain binds mesothelin.
  • the target-binding domain is a scFv that binds mesothelin.
  • Anti-mesothelin antibodies and binding domains thereof are known in the art. See, e.g., U.S. Patents 8,481,7039,023,3519,416,1909,719,996, and 10,851,175 and U.S. Patent Application Publications 2019/0218294 and 2022/0056147, and such sequences are incorporated herein by reference.
  • the target-binding domain binds PD-L1.
  • the target-binding domain may be but does not need to be derived from an antibody fragment, in some cases the target binding domain can be derived from a cognate ligand of a checkpoint molecule.
  • the target-binding domain is derived from at least a portion of the PD1 extracellular domain.
  • the target-binding domain is derived from a commercially available anti-PDL1 antibody, antibody fragment, or derivative thereof, e.g., atezolizumab (Tecentriq®), avelumab (Bavencio®), or durvalumab (Imfinzi®), the amino acid sequences of the heavy and light chains of which are set forth in Table 5.
  • Tecentriq® atezolizumab
  • avelumab Bavencio®
  • durvalumab Imfinzi®
  • Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
  • Table 5 Amino acid sequences of representative anti-PD-L1 antibody fragments Polypeptide Sequence atezolizumab 1 evqlvesggg lvqpggslrl scaasgftfs dswihwvrqa pgkglewvaw ispyggstyy avelumab light 1 qsaltqpasv sgspgqsiti sctgtssdvg gynyvswyqq hpgkapklmi ydvsnrpsgv chain (SEQ ID 61 snrfsgsksg ntasltisgl qaedeadyyc ssytssstrv fgtgtkvtvl gqpkanptvt NO: 22) 121 lfppsseelq ankatlvcli sdfypgavtv aw
  • the target-binding domain comprises an anti-CD19 antibody or antibody fragment thereof, PD1 or an anti-PDL1 antibody of antibody fragment thereof, or CD27.
  • the target is a receptor or other ligand on an immune cell, such as a B cell or T cell.
  • Immune cells expressing the modified immune cell receptor protein of the invention can be used in, for example, B cell or T cell depletion therapy.
  • the target-binding domain binds a receptor or other ligand on a B cell where the receptor or other ligand is, for example, Siglec-10, LILRB/PIR-B, CD31, FcyRIIIB, CD19, CD20, CD22, CD25, CD32, CD40, CD47, CD52, CD80, CD86, CD267, CD268, CD268, IgM, IgD, IgG, IgA or IgE.
  • the receptor or other ligand is, for example, Siglec-10, LILRB/PIR-B, CD31, FcyRIIIB, CD19, CD20, CD22, CD25, CD32, CD40, CD47, CD52, CD80, CD86, CD267, CD268, CD268, IgM, IgD, IgG, IgA or IgE.
  • the target-binding domain binds a receptor or other ligand on a T cell
  • the receptor or other ligand is, for example, CD43, CD44, CD45, LFAI, CD4, CD8, CD3, LAT, CD27, CD96, CD28, TIGIT, ICOS, BTLA, HVEM, 4-1BB, OX40, DR3, GITR, CD30, 10 SLAM, CD2, 2B4, TIM I, TIM2, TIM3, CD226, CD160, LAG3, LAIRI, CD112R, CTLA-4, PD-I, PD-LI or PD-L2.
  • the target is a B cell maturation antigen, wherein the B cell maturation antigen is, for example, (BCMA), GPRC5D, CD19, CD20, CD27, CD70, or CD117, or mesothelin.
  • the target-binding domain binds CD70, CD19, CD33, CLL1, or IL-3 receptor.
  • the target- binding domain comprises CD27.
  • the target- binding domain comprises the sequence of SEQ ID NO: 25.
  • the target-binding domain comprises the sequence of SEQ ID NO: 25, or a variant thereof that retains the functionality of the CD27 ectodomain.
  • the target-binding domain comprises the sequence of SEQ ID NO: 25, or a variant thereof that retains the functionality of the CD27 ectodomain that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 25.
  • Assays to determine whether variant of the CD27 ectodomain are known to the skilled person, such as binding assays to determine whether the variant binds to CD70.
  • the target-binding domain binds CD70. In some embodiments, the target-binding domain is a scFv that binds CD70. In some embodiments, the target- binding domain binds CD19. In some embodiments, the target-binding domain is a scFv that binds CD19.
  • the target-binding domain is a scFv that binds CD19 such as FMC63. In some embodiments, the target-binding domain is a scFv that binds CD19 which comprises the 3 CDR sequences found within SEQ ID NO: 26 and the 3 CDR sequences found within SEQ ID NO: 27. In some embodiments, the target-binding
  • the target-binding domain binds CD33.
  • the target-binding domain is a scFv that binds CD33.
  • the target- binding domain is a scFv that binds CD33, such as gemtuzumab.
  • the target-binding domain is a scFv that binds CD33, which comprises the 3 CDR sequences found within SEQ ID NO: 28 and the 3 CDR sequences found within SEQ ID NO: 29. In some embodiments, the target-binding domain is a scFv that binds CD33, which comprises SEQ ID NO: 28 and SEQ ID NO: 29.
  • the target-binding domain binds CLL1.
  • the target-binding domain is a scFv that binds CLL1. In some embodiments, the target- binding domain binds the IL-3 receptor. In some embodiments, the target-binding domain is a scFv that binds the IL-3 receptor. In some embodiments, the target-binding domain is, or comprises, IL-3. In a preferred embodiment of the invention, the target-binding domain binds CD20, CD22 or CD70. In the modified immune cell receptor protein of the invention, a flexible linker is present between the target-binding domain and the extracellular domain of CD16A, wherein the linker is at least 10 amino acids in length.
  • linkers derived hinge domains from CD28, CD8, or IgG molecules is widespread in the chimeric antigen receptor field.
  • the rigidity of these hinges can vary greatly, from highly rigid (e.g. CD28 stalk) to highly flexible (CH3-CH2 domain from IgG) structures.
  • linker domain folds beneath the inverted V-shaped ectodomain of CD16A, extending into the extracellular space from one of the sides of CD16A (see Figure 2). For optimal target binding, this requires a minimum length linker with sufficient flexibility. Consequently, specific requirements regarding linker domain length and sequence are imperative to ensure proper target engagement and immune effector functions.
  • the linker domain of the modified immune cell receptor of the invention has sufficient flexibility to allow folding beneath the V-shaped ectodomain of CD16A and is at least 10 amino acids in length. Suitable flexibility of the linker can be achieved through the optimal incorporation of certain amino acids including glycine and serine.
  • Suitable peptide linkers for use in connecting, or bridging, or being positioned between, the target binding domain to the extracellular domain of CD16A are amino acid sequences include those that allow the domains to fold independently from one another and providing sufficient flexibility to allow the domains to retain their functionality.
  • the linker may be at least 10 to about 70 amino acids in length, such as from about 10 to about 30 amino acids, e.g. about 20 amino acids in length.
  • the linker is at least 10 to about 70 amino acids in length, at least 20 to about 70 amino acids in length, or at least 30 to about 70 amino acids in length.
  • the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids in length.
  • Suitable flexible linkers may comprise Serine and/or Glycine residues which may be contiguous or separated by one or more amino acid. Appropriate linking groups may be designed using conventional modelling techniques.
  • Flexible linkers may include a poly- Glycine-Serine sequence such as: GGGGSGGGGS (SEQ ID NO:30), GGGGSGGGGSGGGGS (SEQ ID NO:31), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:32), GSTSGSGKPGSGEGSTKG (SEQ ID NO:33), KESGSVSSEQLAQFRSLD (SEQ ID NO:34), EGKSSGSGSESKST (SEQ ID NO:35), or GSAGSAAGSGEF (SEQ ID NO:36).
  • the linker contains no more than one cysteine residue. Two Cysteine residues may form di-sulphide bridges which may reduce the flexibility of the linker containing said residues.
  • Proline is the most rigid of the 20 naturally occurring amino acids and is frequently introduced to rigidify flexible regions of protein to enhance thermostability.
  • the linker contains no more than 30% Proline residues.
  • the linker comprises an amino acid sequence derived from the hinge domain of NKp44, NKp46, CD8 ⁇ , CD8 ⁇ , KIR2DS1, KIR3DS1 or CD27.
  • Examples of preferred flexible linkers include, but are not limited to, the following: delta CD8 ⁇ : TTTPAPRPPTPAPTIASQPLSLRPEAGGGGS (SEQ ID NO: 37) NKp44 long: ASASTQTSWTPRDLVSSQTQTQSSVPPTAGARQAPESPSTIPVPSQPQNSTLRPGPA APGGGGS (SEQ ID NO: 38) NKp44 short: ASASTQTSWTPRDLVSSQTQTQSSVPPTAGAGGGGS (SEQ ID NO: NKp46 long: GDIENTSLAPEDPTFPADTWGTYLLTTETGLQGGGGS (SEQ ID NO: 40) KIR2DS1: SNSWPSPTEPSSETGNPRHLHGGGGS (SEQ ID NO: 41) KIR3DS1: SSSWPSPTEPSSKSGNLRHLHGGGGS (SEQ ID NO: 42) CD27 short: NPSLTARSSQALSPHPQPTHLPYVSEMLEARGGGGS (SEQ ID NO: 43
  • the invention encompasses variants of the above sequences, wherein the variant linker sequences are at least 10 amino acids in length, contain no more than one cysteine residue and no more than 30% proline residues and wherein the variant linker sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the one of the SEQ ID NOs set out above.
  • Extracellular domain of CD16A The modified immune cell receptor protein of the present invention comprises the extracellular domain of CD16A, or variants thereof that maintain the functionality of the extracellular domain of CD16A. The functionality of the extracellular domain of CD16A can be tested by experiments known to the skilled person, such as binding assays to determine whether antibody Fc domain binding to CD16A is maintained.
  • the extracellular domain of CD16A consists essentially of the FC-binding domain of CD16A and the hinge domain of CD16A.
  • the hinge domain of CD16A contains an ADAM17 cleavage site.
  • the hinge domain of CD16A is a variant that is not cleavable by ADAM17.
  • the hinge domain of CD16A is a variant that is not cleavable.
  • the extracellular domain of CD16A is considered to correspond to amino acids P25 to Y207 of CD16A.
  • ADAM17 originally referred to as tumor necrosis factor (TNF)- ⁇ -converting enzyme (TACE), is expressed on NK cells, and to a lesser extent on T cells, and is known to cleave multiple targets, including CD16A, CD62L, TNF- ⁇ , TNF receptor I, and TNF receptor II.
  • ADAM17 is expressed on NK cells generally, as well as the CD3-CD56 bright and CD3- CD56 dim NK cell subsets.
  • ADAM17 is also expressed on CD3 + CD56 + NKT cells, but ADAM17 is not highly expressed on CD3 + CD56- T cells (Romee et al., Blood 121(18):3599- 608 (2013) and Kato et al., Front. Cell. Dev. Biol.
  • Immune cell activation for example, via stimulation with phorbol myristate acetate, or IL-12 and IL-18, results in increased ADAM17 activity and therefore target shedding.
  • Activation of immune cells expressing the modified immune cell receptor protein as disclosed herein results in ADAM17-mediated protein cleavage within the hinge domain of the extracellular domain of CD16A, releasing the target-binding domain and the FC-binding domain into the extracellular space as a soluble protein.
  • the hinge domain contains the amino acid sequence AVSTI (SEQ ID NO: 45), or a variant thereof.
  • a “variant” is any molecule with the same desired activity (such as cleavable by ADAM17 wherein the variant is a cleavable variant) but which may be a truncated version or a version with a sequence % identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
  • any one of the domains or other molecules or entities may be a variant of any one of the relevant sequences provided herein.
  • the variant contains a serine at position 3, when numbered according to SEQ ID NO: 45 and one or more variants of amino acids at positions 1, 2, 4, and/or 5. In some embodiments, the variant contains a proline at position 3, when numbered according to SEQ ID NO: 45 and one or more variants of amino acids at positions 1, 2, 4, and/or 5. In some embodiments, the extracellular domain of CD16A has the amino acid sequence set forth below (SEQ ID NO:46), or a variant thereof.
  • CD16A extracellular domain SEQ ID NO: 46 PKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAA TVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNT ALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITI TQGLAVSTISSFFPPGYQ
  • the extracellular domain comprises a variant of SEQ ID NO: 46 such that it has a higher affinity for IgG as compared to the wild-type sequence.
  • the extracellular domain has a F176V substitution (i.e., a valine at position 176 in place of the phenylalanine).
  • the extracellular domain of CD16A comprises the amino acid sequence of SEQ ID NO:46, or a variant thereof that retains the functionality of the CD16A extracellular domain that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 46.
  • Assays to determine whether variant of the CD16A extracellular domain are known to the skilled person, such as binding assays to determine whether the variant maintains its F C binding capability.
  • the extracellular domain of CD16A protein variants described herein or encoded by any of the nucleic acid or set of nucleic acids provided herein retains all functionality of the extracellular domain of CD16A, including ADCC activity.
  • ADCC activity can be determined by tests known to the skilled person, such as employing cytotoxicity assays with monoclonal antibodies and target cancer cell lines.
  • Transmembrane domain The transmembrane domain can enable retention and controlled release of at least the target- binding domain if not both the target-binding domain and extracellular domain of the modified immune cell receptor protein from the cell surface after ADAM17-mediated cleavage.
  • the transmembrane domain generally localizes the modified immune cell receptor protein to the endoplasmic reticulum during translation and delivery to the cell surface.
  • the transmembrane domain interacts with signaling adaptor proteins CD3 ⁇ and Fc Fragment of IgE Receptor Ig (FCER1G).
  • the transmembrane domain comprises a transmembrane domain of a protein cleavable by ADAM17.
  • the transmembrane domain is of any one of the relevant proteins provided herein.
  • the transmembrane domain comprises any one of the relevant specific sequences provided herein.
  • the transmembrane domain may be derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16A, CD22, CD28, CD33, CD37, CD45, CD62L, CD64, CD80, CD86, CD134, CD154, 4-1BB (also known CD137 or TNF Receptor Superfamily Member 9 (TNFRSF9)), Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , ICOS, KIR2DS2, MHC class I, MHC class II, or NKG2D, which includes variants thereof.
  • the transmembrane domain is derived from CD16A or CD62L.
  • the transmembrane domain is derived from CD3 ⁇ , CD4, CD8 ⁇ , CD28, or CD137 (4-1BB).
  • Amino acid sequences of representative transmembrane domains are listed in Table 4. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
  • Table 4 Amino acid sequences of transmembrane domains Transmembrane domain Sequence
  • the transmembrane domain is derived from CD16A, or a variant thereof that maintains the activity of the transmembrane domain of CD16A.
  • CD16A transmembrane domain can be considered to correspond to V209 to V229 of CD16A, i.e. VSFCLVMVLLFAVDTGLYFSV (SEQ ID NO: 52).
  • the transmembrane domain comprises SEQ ID NO: 52, or a variant thereof that maintains the activity of the transmembrane domain of CD16A that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 52.
  • Cytoplasmic domain In some embodiments of any one of the compositions or methods provided herein, the modified immune cell receptor protein further comprises a cytoplasmic domain (CD) of any one of the proteins cleavable by ADAM17 provided herein, which includes variants thereof, which can be connected to the transmembrane domain.
  • CD domain can provide signaling capacities to the modified immune cell receptor protein.
  • the CD domain is derived from CD16A, which includes variants thereof.
  • the CD16A CD domain can interact with the adaptor proteins CD3 ⁇ and FCER1G, each which contain ITAMs for downstream signaling pathways that include the kinases Syk and ZAP70. See, Lanier, Curr. Opin. Immunol.15(3):308-14 (2003).
  • the cytoplasmic domain is derived from CD16A, but lacks the CD16A signal peptide/leader peptide.
  • the CD16A cytoplasmic domain is considered to correspond to K230 to K254 of CD16A, i.e. KTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO: 53).
  • the cytoplasmic domain is derived from CD16A, or a variant thereof that maintains the activity of the cytoplasmic domain of CD16A that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence of SEQ ID NO: 53.
  • Assays to determine the functionality of the cytoplasmic domain of CD16A are known to the skilled person, such as assays to determine whether the variant can interact with the adaptor proteins CD3 ⁇ and FCER1G.
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention may comprise the cytoplasmic domain of CD16A and one or a plurality, e.g., 2 or 3, co- stimulatory signaling domains described herein, e.g., selected from 4-1BB, CD3 ⁇ , Fc ⁇ RI, CD28, CD27, ICOS, DAP10, and OX40.
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention comprises one or a plurality, e.g., 2 or 3, co-stimulatory signaling domains described herein, e.g., selected from 4-1BB, CD3 ⁇ , Fc ⁇ RI, CD28, CD27, ICOS, DAP10, and OX40, but does not comprise the cytoplasmic domain of CD16A.
  • the cytoplasmic domain may include a CD3 ⁇ co-stimulatory signaling domain as a primary signaling domain.
  • the cytoplasmic domain may include a CD3 ⁇ co-stimulatory signaling domain as a primary signaling domain, and/or any of the following pairs of co-stimulatory signaling domains from the extracellular to the intracellular direction: 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40-CD28; CD28-OX40; 4-1BB-CD3 ⁇ ; CD3 ⁇ -4-1BB; CD28-CD3 ⁇ ; CD3 ⁇ -CD28; CD28-4-1BB; 4-1BB-CD28; OX40-DAP10; DAP10-OX40; 4-1BB-DAP10
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention comprises the CD3 ⁇ co-stimulatory signaling domain as set out in SEQ ID NO: 54, or variants thereof that retain the functionality of the CD3 ⁇ co-stimulatory signaling domain.
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention comprises the CD3 ⁇ co-stimulatory signaling domain as set out in SEQ ID NO: 54, or variants thereof the have 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 54 that retain the functionality of the CD3 ⁇ co-stimulatory signaling domain.
  • Assays to determine the functionality of the CD3 ⁇ co-stimulatory signaling domain variants are known to the skilled person, for example, increased cellular effector functionality upon immune cell receptor signaling due to target antigen engagement.
  • Some immune effector functions of relevance to this embodiment include cytotoxicity against target cells, increased immune cell proliferation, and increased cytokine secretion.
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention comprises one or more of the following co- stimulatory signaling domain combinations: 4-1BB and CD3 ⁇ , 4-1BB and CD28, OX40 and CD28, 4-1BB and DAP10, or OX40 and DAP10.
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention comprises the following co-stimulatory signaling domains: Fc ⁇ RI as set out in SEQ ID NO: 66, 4-1BB as set out in SEQ ID NO 63 and CD28 as set out in SEQ ID NO: 59, OX40 as set out in SEQ ID NO: 70 and CD28 as set out in SEQ ID NO: 59, 4-1BB as set out in SEQ ID NO 63 and DAP10 as set out in SEQ ID NO: 64, or OX40 as set out in SEQ ID NO: 70 and DAP10 as set out in SEQ ID NO: 64.
  • the cytoplasmic domain of the modified immune cell receptor protein of the invention comprises the following co-stimulatory signaling domains: 4-1BB as set out in SEQ ID NO 63 and CD28 as set out in SEQ ID NO: 59, OX40 as set out in SEQ ID NO: 70 and CD28 as set out in SEQ ID NO: 59, 4-1BB as set out in SEQ ID NO 63 and DAP10 as set out in SEQ ID NO: 64, or OX40 as set out in SEQ ID NO: 70 and DAP10 as set out in SEQ ID NO: 64, or variants thereof that have 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the SEQ ID NOs set out above, that retain the functionality of the co-stimulatory domains set out above.
  • the primary signaling domain is derived from CD3 ⁇ , Fc ⁇ RI, CD27, CD28, CD40, KIR2DS2, MyD88, 2B4, DAP10 or OX40.
  • the co- stimulatory signaling domain is derived from one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD40, CD45, CD68, CD72, CD80, CD86, CD137 (4-1BB), CD154, CLEC-1, 4-1BB, DAP10 (hematopoietic cell signal transducer ((HCST)), DAP12 (TYROBP), Dectin-1, Fc ⁇ RI, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70.
  • Amino acid sequences of representative signaling domains are listed in Table 5. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
  • Table 5 Amino acid sequences of representative co-stimulatory signaling domains Signaling domain Sequence CD3 ⁇ (SEQ ID NO:54) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG S G S S S DAP10 (SEQ ID CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL NO:64) P T G GI D P S L P G G P C L L D P Q Q P Q Q P CD30 (SEQ ID NO:75) CHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEP VAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRV D L C N E P V L T V P A I Leader peptid
  • leader peptide refers to a short (e.g., 5- 30 or 10-100 amino acids long) stretch of amino acids that directs the transport of the protein. Modified immune cell receptor proteins containing a leader peptide and transmembrane domain can be trafficked to the plasma membrane.
  • the leader peptide is derived from albumin, CD8 ⁇ , CD33, erythropoietin (EPO), IL-2, human or mouse Ig-kappa chain V-III (IgK VIII), tissue plasminogen activator (tPA), or secreted alkaline phosphatase (SEAP). Leader peptides may also be synthetic (i.e., non-naturally occurring).
  • leader peptides are listed in Error! Reference source not found.. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 6: Amino acid sequences of leader peptides Leader peptide Sequence Albumin (SEQ ID NO:87) MKWVTFISLLFLFSSAYS Further leader peptides envisaged by the present invention include those derived from Syncytin-1, CD8B, CD28, CD3zeta, CD16A, NKp30, NKp44 and NKp46. Leader peptides may also be synthetic (i.e., non-naturally occurring). Amino acid sequences of representative leader peptides are listed in Error!
  • nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
  • Table 7a Further amino acid sequences of leader peptides Leader peptide Sequence Syncytin-1 (SEQ ID NO:122)
  • MALPYHIFLFTVLLPSFTLT ion encompasses ⁇ -CD19 scFv (FMC63) fused to CD16A canonical isoform.
  • the modified immune cell receptor protein of the invention comprises, in a N-terminal to C -terminal direction: the CD8 ⁇ leader peptide as set out in SEQ ID NO: 89, the FMC63 scFv comprising SEQ ID NOs: 26 and 27, the linker as set out in SEQ ID NO: 38, the extracellular domain of CD16A as set out in SEQ ID NO: 46, the transmembrane domain of CD16A as set out in SEQ ID NO: 52, the co-stimulatory signaling domain of CD137 (4-1BB) as set out in SEQ ID NO: 63, and the co-stimulatory signaling domain of DAP10 set out in SEQ ID NO: 64.
  • the modified immune cell receptor protein-encoding nucleic acid(s) may be introduced to an immune cell by a suitable vector or set of vectors.
  • a vector or set of vectors can be configured to contain the elements necessary to effect transport into the immune cell and effect expression of the nucleic acid(s) after transformation.
  • Such elements include an origin of replication, a poly-A tail sequence, a selectable marker, and one or more suitable sites for the insertion of the nucleic acid sequences, such as a multiple cloning site (MCS), one or more suitable promoters, each promoter operatively linked to the insertion sites of the nucleic acid sequences and the selectable marker, and additional optional regulatory elements.
  • MCS multiple cloning site
  • promoter refers to a nucleic acid sequence that regulates, directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked, which in the context of the present disclosure, is a modified immune cell receptor protein-encoding sequence.
  • a promoter may function alone to regulate transcription, or it may act in concert with one or more other regulatory sequences (e.g., enhancers or silencers, or regulatory elements that may be present in the nucleic acid sequences or the vectors). Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5' region of the sense strand). Promoters typically range from about 100-1000 base pairs in length.
  • a nucleic acid sequence is spatially situated or disposed in the vector relative to another nucleic acid sequence, e.g., a promoter is operatively linked to drive the expression of a nucleic acid coding sequence (e.g., the modified immune cell receptor protein-encoding nucleic acid sequence).
  • a vector contains a single promoter operatively linked to a modified immune cell receptor protein encoding nucleic acid.
  • the vector has a strong mammalian promoter, for example a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, synthetic promoters (e.g., RPBSA (synthetic, from Sleeping Beauty), or CAG (synthetic, CMV early enhancer element, chicken ⁇ -Actin, and splice acceptor of rabbit ⁇ -Globin)) or promoters derived from the ⁇ -actin, phosphoglycerate kinase (PGK), or factor EF1 ⁇ genes.
  • the promoter may have a core region located close to the nucleic acid coding sequence.
  • the promoter is modified to remove methylation sensitive motifs (e.g., a cytosine nucleotide is followed by a guanine nucleotide, or “CpG”) or by the addition of a regulatory sequence that binds transcriptional factors that repress DNA methylation.
  • the vector includes A/T-rich, nuclear matrix interacting sequences, known as scaffold matrix attachment regions (S/MAR), which enhance transformation efficiency and improve the stability of transgene expression.
  • the vector is a viral vector, for example, a retroviral vector, a lentiviral vector, an adenoviral vector, a herpesvirus vector, an adenovirus, or an adeno- associated virus (AAV) vector.
  • lentiviral vector is intended to mean an infectious lentiviral particle.
  • Lentivirinae lentiviruses
  • retroviruses enveloped retrovirinae
  • An infectious lentiviral particle will be capable of invading a target host cell, including infecting, and transducing non-dividing cells and immune cells.
  • the vector containing RNA is a non-integrative and non-replicative recombinant lentivirus vector.
  • the construction of lentiviral vectors has been described, for example, in U.S. Patents 5,665,577, 5,981,276, 6,013,516, 7,090,837, 8,119,119 and 10,954,530.
  • Lentivirus vectors include a defective lentiviral genome, i.e., in which at least one of the lentivirus genes gag, pol, and env, has been inactivated or deleted.
  • a lentiviral vector can exhibit functions additional to, or different from, a naturally occurring lentivirus.
  • a lentiviral vector can be modified to change or reduce a lentivirus characteristic.
  • a lentiviral vector also can be modified to exhibit characteristics of one or more other retroviruses, retroviral vectors, host cells or heterologous cells. Modifications can include, for example, pseudotyping, modifying binding and/or fusion functions of the envelope polypeptide, incorporating heterologous, chimeric, or multifunctional polypeptides into the vector, incorporating non-lentivirus genomes, or incorporating heterologous genes into the lentiviral vector genome.
  • pseudotyped a vector bearing components (e.g., envelop or capsid) from more than one source.
  • the sources may be from a heterologous virus or non-viral proteins.
  • Non-viral proteins may include antibodies and antigen-binding fragments thereof.
  • a representative pseudotyped vector is a vector bearing non-glycoprotein components derived from a first virus and envelope glycoproteins derived from a second virus. The host range of a pseudotyped vector may thus be expanded or altered depending on the type of cell surface receptor bound by the glycoprotein derived from the second virus.
  • the lentiviral vector is pseudotyped with a baboon endogenous retroviral (BaEV) envelope glycoprotein (BaEV-gp).
  • BaEV baboon endogenous retroviral envelope glycoprotein
  • the nucleic acid sequence of a representative BaEV-gp is set forth below.
  • the nucleic acid sequence of a representative BaEV-gp is set forth below (SEQ ID NO:100).
  • BaEV is intimately related with the infectious feline endogenous retrovirus RD114.
  • the env gene from RD114 is thought to be originally derived from the BaEV envelope gp.
  • These two viruses are stable in human and macaque sera, giving them a great potential for in vivo gene therapy. They also recognize the sodium-dependent neutral amino acid transport (ASCT- 2) in human cells, but only BaEV also recognizes ASCT-1, giving BaEV a wider tropism.
  • ASCT-1 and -2 receptors have a 57% identical sequence, and they are expressed in a wide number of cells.
  • the lentiviral vector is pseudotyped with the feline endogenous retrovirus RD114 glycoprotein.
  • the vector is a pseudotyped lentiviral vector for the use of transduction in NK cells.
  • Lentivirus pseudotyped with glycoprotein G from vesicular stomatitis virus (VSV-G) binds to low density lipoprotein receptor (LDL-R), which is not normally expressed on NK cells.
  • BaEV-gp pseudotyped lentivirus (BaEV-LV) binds to ASCT2, which is expressed on NK cells, furthermore NK ASCT2 expression is upregulated after IL-12, IL-15, and IL-18 treatment (Dong et al., Proc. Natl. Acad. Sci. U.S.A.119(25):e2122379119 (2022)).
  • NK cells can be transduced with BaEV-LV, and IL-12, IL-15 and IL-18 pretreatment further improves transduction efficiency.
  • CD56 bright (CD56 bright ; CD56 br ) NK cells express higher levels of ASCT2 compared to CD56 low expressing cells (CD56 dim ) with and without IL-12, IL-15, and IL-18 treatment and showed significantly higher BaEV-LV transduction rate.
  • NK cells derived from human PBMCs as well as from mouse spleens express ASCT2 and can be transduced with BaEV-LV.
  • NK cells may be transduced with pseudotyped lentivirus vectors encoding a modified immune cell receipt protein that achieves 40-60% transduction efficiency.
  • the term “bright” as used herein in the context of marker expression refers to a cell having a signal that is higher or more intense than a comparative control cell, wherein a user or computer may differentiate two populations of cells based on the levels or intensity of the signal.
  • the vector is a non-viral vector, representative examples of which include plasmids, mRNA, circular RNA (circRNA), linear single-stranded DNA (ssDNA) or linear double-stranded DNA (dsDNA), minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac(PB)-based vectors.
  • the vector may include both viral and non-viral elements.
  • the vector is a plasmid.
  • the plasmid may also contain other elements e.g., that facilitate transport and expression of the nucleic acid in an immune cell.
  • the plasmid may be linearized with restriction enzymes, in vitro transcribed to produce mRNA, and then modified with a 5’ cap and 3’ poly-A tail.
  • a carrier encapsulates the vector.
  • the carrier may be lipid-based, e.g., lipid nanoparticles (LNPs), liposomes, lipid vesicles, or lipoplexes.
  • the carrier is an LNP.
  • an LNP includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and/or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels. Lipid carriers, e.g., LNPs may include one or more cationic/ionizable lipids, one or more polymer conjugated lipids, one or more structural lipids, and/or one or more phospholipids.
  • a “cationic lipid” refers to positively charged lipid or a lipid capable of holding a positive charge. Cationic lipids include one or more amine group(s) which bear the positive charge, depending on pH.
  • a “polymer conjugated lipid” refers to a lipid with a conjugated polymer portion.
  • Polymer conjugated lipids include PEGylated lipids, which are lipids conjugated to polyethylene glycol (PEG).
  • a “structure lipid” refers to a non-cationic lipid that does not have a net charge at physiological pH. Exemplary structural lipids include cholesterol, fecosterol, sitosterol, ergosterol, campesterol and the like.
  • a “phospholipid” refers to lipids that have a triester of glycerol with two fatty acids and one phosphate ion. Phospholipids in LNPs assemble the lipids into one or more lipid bilayers.
  • Lipoplexes, liposomes, and lipid nanoparticles may include a combination of lipid molecules, e.g., a cationic lipid, a neutral lipid, an anionic lipid, polypeptide-lipid conjugates, and other stabilization components.
  • Representative stabilization components include antioxidants, surfactants, and salts.
  • compositions and preparation methods of lipoplexes, liposomes, and lipid nanoparticles are known in the art. See, e.g., U.S. Patents 8,058,069, 8,969,353, 9,682,139, 10,238,754, U.S. Patent Application Publications 2005/0064026 and 2018/0291086, and Lasic, Trends Biotechnol.16(7):307-21 (1998), Lasic et al., FEBS Lett.312(2-3):255-8 (1992), and Drummond et al., Pharmacol. Rev. 51(4):691-743 (1999).
  • Host cells the nucleic acids and vectors described above can be expressed in a supporter or host cell line.
  • Mammalian cell lines such as Chinese hamster ovary (CHO) cells or 293T cells are particularly suitable for these purposes.
  • the nucleic acids and vectors described above can be expressed in an alternative host cell, such as a bacterial cell, for example E. coli.
  • the invention encompasses a cell comprising the nucleic acids and vectors described herein.
  • Immune cells One aspect of the present disclosure is a genetically modified (or transformed) immune cell containing any one of the nucleic acids or sets of nucleic acids or any one of the vectors or sets of vectors provided herein.
  • immune cell refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response.
  • a combination of different immune cells may be used.
  • T cells are used. Representative examples of T cells include cytotoxic lymphocytes, cytotoxic T cells (CD8 + T cells), T helper cells (CD4 + T cells), ⁇ T cells and/or ⁇ T cells, NK T (NKT) cells, and Th17 T cells.
  • the immune cells are CD8 + T cells.
  • the immune cells are CD4 + T cells.
  • the immune cells are a combination of CD8 + T cells and CD4 + T cells.
  • T cells may be primary T cells isolated from healthy patients and engineered to express a modified immune cell receptor protein. Certain types of T cells have preferential properties when it comes to in vivo persistence, expansion, and effector function against cancer cells.
  • Na ⁇ ve (TN) and stem cell-like memory T (TSCM) cells are characterized by surface expression of CD45RA and CD62L.
  • T N and T SCM cells have been shown to provide improved anti-cancer effect in clinical trials due to their low exhaustion state and the ability to differentiate into several types of effector T cells.
  • the invention encompasses an immune cell, such as a na ⁇ ve (TN) and stem cell-like memory T (TSCM) cell modified to express the modified immune cell receptor protein of the invention.
  • the invention encompasses an immune cell, such as a na ⁇ ve (T N ) and stem cell-like memory T (T SCM ) cells comprising a nucleic acid or vector encoding the modified immune cell receptor protein of the invention.
  • the immune cells are NK cells.
  • the immune cells are a NK cell line, primary NK cells, memory-like NK cells, or cytokine-induced memory-like (CIML) NK cells.
  • the immune cells are monocytes or macrophages.
  • the immune cells are part of a mixed population of immune cells comprising NK cells and T cells.
  • the NK cells within the mixed immune cell population comprise a NK cell line, primary NK cells, memory-like NK cells, or cytokine-induced memory-like (CIML) NK cells.
  • the T cells within the mixed immune cell population are na ⁇ ve (TN) and stem cell-like memory T (TSCM) cells, cytotoxic lymphocytes, cytotoxic T cells (CD8 + T cells), T helper cells (CD4 + T cells), ⁇ T cells and/or ⁇ T cells, NK T (NKT) cells, Th17 T-cells, and CIML T cells.
  • the T cells comprise CD8 + T cells.
  • the T cells comprise CD4 + T cells.
  • the T cells comprise a combination of CD8 + T cells and CD4 + T cells.
  • Immune cells include cells derived from stem cells.
  • the stem cells can be adult stem cells (e.g., induced pluripotent stem cells (iPSC)), embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.
  • the immune cells are derived from peripheral blood mononuclear cells (PBMC), cell lines, or cell bank cells. The collection, isolation, purification, and differentiation of cells from body fluids and tissues is known in the art.
  • the immune cells are iPSC derived cells.
  • the immune cells are Dendritic cells (DCs). NK cells are produced in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56 bright CD16- to CD56 dim CD16 + cells that represents the most abundant NK population in peripheral blood.
  • iPSCs may be induced to differentiate into NK cells as set forth in Ruiz et al., Stem Cell Res.41:101600- 26 (2019), Laskowski et al., Stem Cell Reports 7:139-48 (2016), Ni et al., Methods Mol. Biol.1029:33-41 (2013), and Euchner et al., Front. Immunol.12:640672-11 (2021).
  • the cells are NK cells derived from cord blood as set forth in Mehta et al., Front. Med. (Lausanne) 2:93-10 (2016)), Chabannon et al., Front.
  • the cells are NK cells obtained from PBMCs as set forth in Koehl et al., Front. Oncol.3:118-12 (2013)) and Becker et al., Cancer Immunol. Immunother.65:477-84 (2016)).
  • the cells are primary NK cells, also known as “conventional NK cells” (cNK).
  • cNK cells are CD56 + NK cells that may be isolated from human blood.
  • cNK cells may be isolated from a normal, healthy donor, with a known HLA type, and preferably with an HLA match (autologous) or partial HLA match (allogeneic or syngenic) to the subject in need thereof.
  • cNK cells are purified by depleting non-NK cells in the donor sample, e.g., PBMCs. Purification may be performed by any means known in the art, e.g., by using a Miltenyi NK cell isolation kit.
  • the cells are memory-like NK cells. Memory-like NK cells are produced, typically in vitro, from cNK cells, isolated from a subject, in some cases, from the same subject in need of ACT.
  • the cells are cytokine-induced memory-like (CIML) NK cells.
  • CIML NK cells are produced by stimulating NK cells with one or, more typically a combination, of IL-12, IL-15, and IL-18.
  • CIML NK cells produce IFN- ⁇ , a prototype NK cell functional readout, in response to leukemia target cells or after stimulation with IL-12, IL-15, and IL-18.
  • CIML NK cells Upon restimulation with cytokines or target tumor cells, a larger fraction of CIML NK cells produce higher levels of IFN- ⁇ as compared with cNK cells.
  • CIML NK cells adoptively transferred into leukemia-bearing mice inhibit tumor growth to a greater degree as compared to conventional NK cells.
  • the cells are allogeneic to the subject receiving the cells, that is, the cells have a complete or at least a partial HLA-match with the subject. In some embodiments, the cells are autologous.
  • autologous refers to any material (e.g., NK cells or T cells) derived from the same subject to whom it is later re- introduced.
  • allogeneic refers to any material derived from a different subject of the same species as the subject to whom the material is later introduced. Two or more individual subjects are allogeneic when the genes at one or more loci are not identical (typically the HLA loci).
  • the cells are engineered to down-regulate HLA-A/B/C molecules.
  • Various methods for down-regulation are recognized by skilled practitioners and encompass genetic manipulation of genes encoding HLA-A/B/C or Beta-2 microglobulin (B2M).
  • HLA-A/B/C or B2M protein expression can be down- regulated through the use of short-interfering RNA (siRNA) or similar systems (shRNAs, microRNAs, etc.) that target mRNA encoding HLA-A/B/C or B2M proteins.
  • siRNA short-interfering RNA
  • shRNAs shRNAs, microRNAs, etc.
  • MHC-I Major Histocompatibility Complex-I
  • the cells are from NK cell lines.
  • Suitable NK cell lines are known in the art and include NK-92, NKG, NKL, KHYG-1, YT, NK-YS, SNK-6, IMC-1, YTS, NKL cells, and high affinity NK (haNK, an NK/T cell lymphoma cell line).
  • NK cell lines enable cell-based immunotherapies within the context of allogeneic adoptive transfer and without or lessened risk of graph versus host disease (GvHD).
  • NK cells lines avoids the need for leukapheresis, facilitating cell procurement, and avoiding undesirable side-effects. See, e.g., Leung et al., Clin. Cancer Res.20:3390-400 (2014); Tonn et al., Cytotherapy 15:1563-70 (2013).
  • the immune cells express endogenous CD3 ⁇ .
  • the immune cells are modified to comprise an exogenous nucleic acid encoding CD3 ⁇ , as well as comprising a nucleic acid encoding the modified immune cell receptor of the invention.
  • the immune cells are transduced with a bis-cistronic nucleic acid construct comprising a nucleic acid encoding CD3 ⁇ and a nucleic acid encoding the modified immune cell receptor protein of the invention.
  • the immune cells express endogenous Fc ⁇ RI ⁇ .
  • the immune cells are modified to comprise an exogenous nucleic acid encoding Fc ⁇ RI ⁇ , as well as comprising a nucleic acid encoding the modified immune cell receptor of the invention.
  • an exogenous nucleic acid encoding Fc ⁇ RI ⁇ there is co-expression of Fc ⁇ RI ⁇ and the modified immune cell receptor of the invention, with Fc ⁇ RI ⁇ being overexpressed when compared with the expression level of Fc ⁇ RI ⁇ in an immune cell where the exogenous nucleic acid encoding Fc ⁇ RI ⁇ is not present.
  • the immune cells are transduced with a bis-cistronic nucleic acid construct comprising a nucleic acid encoding Fc ⁇ RI ⁇ and a nucleic acid encoding the modified immune cell receptor protein of the invention.
  • lentiviral vector is transduced into immune cells.
  • the method entails the use of gamma-retroviral vectors. See, e.g., U.S. Patents 9,669,049, 11,065,311, and 11,230,719.
  • the method entails the use of Adenovirus, Adeno-associated virus (AAV), dsRNA, ssDNA, or dsRNA to deliver the first, the second, and the third nucleic acids.
  • AAV Adeno-associated virus
  • the method entails the use of a transposase to integrate the vector-delivered nucleic acids into the immune cell’s genome. See, e.g., U.S. Patents 7,985,739, 10,174,309, 11,186,847, and 11,351,272. In some embodiments, the method entails the use of self-replicating episomal nano-vectors. See, e.g., U.S. Patents 5,624,820, 5,674,703, and 9,340,775. In some embodiments, a plasmid containing a modified immune cell receptor protein- encoding nucleic acid is transfected into immune cells.
  • the vector(s) containing the nucleic acid sequence(s) is delivered to an immune cell by lipofection.
  • Lipofection is described, for example, in U.S. Patent Nos.5,049,386, 4,946,787; and 4,897,355.
  • Pharmaceutical compositions of the disclosure include compositions comprising therapeutically effective numbers of genetically modified immune cells expressing the modified immune cell receptor protein of the invention and a pharmaceutically acceptable carrier.
  • therapeutically effective number of immune cells (which indirectly includes a corresponding amount of the modified immune cell receptor protein) as used herein refers to a sufficient number of the immune cells that contain the modified immune cell receptor protein-encoding nucleic acid(s) to provide a desired effect.
  • the number of immune cells administered to a subject will vary between wide limits, depending upon the location, type, and severity of the disease or disorder, the age, body weight, and condition of the individual to be treated, etc. A physician will ultimately determine appropriate number of cells and doses to be used. Typically, the immune cells will be given in a single dose. In some embodiments, the effective number of the genetically modified immune cells is between approximately 1 ⁇ 10 5 to approximately 1 ⁇ 10 10 cells per subject. In some embodiments, the effective number of genetically modified immune cells is between approximately 1 ⁇ 10 5 to approximately 6 ⁇ 10 8 cells per kilogram of subject body weight.
  • compositions may be provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH.
  • Liquid carriers include aqueous or non-aqueous carriers alike. Representative examples of liquid carriers include saline, phosphate buffered saline, a soluble protein, dimethyl sulfoxide (DMSO), polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
  • DMSO dimethyl sulfoxide
  • polyol e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like
  • the liquid carrier includes a protein dissolved or dispersed therein, representative examples include serum albumin (e.g., human serum albumin, recombinant human albumin), gelatin, and casein.
  • serum albumin e.g., human serum albumin, recombinant human albumin
  • gelatin e.g., gelatin
  • casein e.g., gelatin
  • the compositions are typically isotonic, i.e., they have the same osmotic pressure as blood.
  • Sodium chloride and isotonic electrolyte solutions e.g., Plasma-Lyte®
  • other excipients may be added, e.g., wetting, dispersing or emulsifying agents, gelling and viscosity enhancing agents, preservatives and the like as known in the art.
  • a pharmaceutical composition comprising a CIML immune cell expressing the modified immune cell receptor protein of the invention, or the population of CIML immune cells expressing the modified immune cell receptor protein of the invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • the solution should be RNAse free.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like.
  • compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Treatment applications The compositions and methods provided herein may be used for cell killing and, thus, can be useful for the treatment of any disease or disorder in which cell killing may confer a benefit.
  • diseases or disorders include cancer as well as diseases and disorders where B cell depletion, plasma cell depletion or T cell depletion may be beneficial.
  • the present disclosure is directed to treating a cancer in a subject.
  • the method entails administering to a subject in need thereof a therapeutically effective number of the immune cells containing nucleic acid(s) encoding a modified immune cell receptor protein as described herein.
  • cancer refers to a disease or disorder characterized by excess proliferation or reduced apoptosis in a subject.
  • Cancers that may be treated with the genetically modified immune cells disclosed herein include both hematopoietic cancers and cancers characterized by the presence of a solid tumor.
  • the cancer is a myelodysplastic syndrome (MDS).
  • MDS are a group of cancers in which immature blood cells in the bone marrow do not mature into healthy blood cells (e.g., red blood cells, white blood cells, or platelets).
  • Acute myeloid leukemia is an MDS and a cancer of the blood and bone marrow.
  • AML also known as myelogenous leukemia and acute nonlymphocytic leukemia
  • haplo-HCT haploidentical hematopoietic cell transplantation
  • the cancer is a hematopoietic cancer.
  • the hematopoietic cancer may be leukemia, lymphoma, or multiple myeloma.
  • the hematopoietic cancer may also be acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma or blastic plasmacytoid dendritic cell neoplasm.
  • the cancer is characterized by the presence of a solid tumor.
  • the cancer is a breast cancer, cervical carcinoma, kidney cancer (e.g., renal cell carcinoma (RCC), transitional cell cancer, or Wilms tumor), glioma, glioblastoma, neuroblastoma, skin cancer (e.g., melanoma, basal cell carcinoma, and squamous cell carcinoma of the skin), bladder cancer (e.g., transitional cell carcinoma, also called urothelial carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, including adenocarcinoma and squamous cell carcinoma of the lung), prostate cancer, colorectal cancer, colon cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck, laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral and oropharyngeal cancer, and salivary gland cancer), ovarian cancer (e.g., cysta
  • cysta
  • the present disclosure is directed to treating an autoimmune disease in a subject.
  • Autoimmune disease is a disease in which the immune system fails to recognize a subject’s own organs, tissues or cells as self, and produces an immune response to attack those organs, tissues or cells as if they were foreign antigens.
  • Autoimmune diseases are well known in the art; for example, as disclosed in The Encyclopedia of Autoimmune Diseases, Dana K. Cassell, Noel R. Rose, Infobase Publishing, 14 May 2014, the diseases of which are herein incorporated by reference.
  • autoimmune diseases examples include, without limitation, Achalasia, Addison’s disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Baló disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid, Castleman disease
  • compositions or methods provided herein may be used for a subject that has received a transplant.
  • transplant refers to an organ or tissue moved from a donor to a recipient for the purpose of replacing the recipient’s damaged or absent organ or tissue. Any one of the methods or compositions provided herein may be used for a subject that has undergone a transplant of an organ or tissue. In some embodiments, the subject may be one suspected of having or a likelihood of having transplant rejection. In some aspects, the compositions or methods provided herein may be used for a subject that has graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • GVHD is a complication that can occur after a pluripotent cell (e.g., stem cell) or bone marrow transplant in which the newly transplanted material results in an attack on the transplant recipient's body. In some instances, GVHD takes place after a blood transfusion.
  • Graft-versus-host-disease can be divided into acute and chronic forms. The acute or fulminant form of the disease (aGVHD) is normally observed within the first 100 days post-transplant and is a major challenge to transplants owing to associated morbidity and mortality. The chronic form of graft-versus-host-disease (cGVHD) normally occurs after 100 days. The appearance of moderate to severe cases of cGVHD adversely influences long-term survival.
  • subject includes all members of the animal kingdom prone (or disposed) to or suffering from the indicated disease or disorder.
  • the subject is a human. Therefore, a subject “having a” disease or disorder or “in need of” treatment according to the present disclosure broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to the disease or disorder (e.g., on account of the basis of prior medical history and/or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to the disease or disorder).
  • the terms “treat”, “treating”, and “treatment” as used herein refer to any type of intervention, process performed on, or the administration of an active agent to the subject in need thereof with the therapeutic objective (“therapeutic effect”) of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease or disorder.
  • the genetically modified immune cells are T cells, na ⁇ ve T(T N ) cells or stem cell-like memory T (TSCM) cells, NK cells, NKT cells, Dendritic cells (DC)s, or monocytes or macrophages.
  • the genetically modified immune cells are a combination of T cells and other types of genetically modified immune cells such as NK cells. In some embodiments, the genetically modified immune cells are a combination of different types of T cells, e.g., CD8 + T cells and CD4 + T cells. In some embodiments, the genetically modified immune cells are autologous with respect to the subject receiving the cells. In some embodiments, the genetically modified immune cells are allogeneic to the subject receiving the cells. Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing, or at risk of progressing to a later stage of, cancer.
  • the subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans, or other animals such as chickens.
  • the subject can be a human subject.
  • a safe and effective amount of a therapy is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • the mode of administration will likely be via injection or infusion.
  • Compositions containing a therapeutically effective number of the genetically modified immune cells may be administered to a subject for the treatment of a disease or disorder by any medically acceptable route.
  • the genetically modified immune cells are typically delivered intravenously, although they may also be introduced into other convenient sites (e.g., to an affected organ or tissue) or modes, as determined by an attending physician.
  • Expansion and differentiation agents can be provided prior to, during or after administration of the cells to increase differentiation, expansion, or persistence of the genetically modified immune cells (e.g., T cells and NK cells).
  • Administration can be autologous or allogeneic.
  • immune cells or progenitors thereof can be isolated from a tissue of body fluid from one subject prior to administration to the same subject (autologous) or a different, compatible subject (allogeneic).
  • the present invention encompasses the modified immune cell receptor proteins of the invention for use in therapy.
  • the present invention encompasses immune cells expressing the modified immune cell receptor proteins of the invention for use in therapy.
  • the present invention modified immune cell receptor proteins of the invention for use in the manufacture of a medicament for the treatment of disease.
  • the present invention encompasses immune cells expressing the modified immune cell receptor proteins of the invention for use in the manufacture of a medicament for the treatment of disease.
  • the therapeutic use or disease to be treated is envisage to be any of the diseases or conditions described herein.
  • Combination Therapy may include co-administration of another agent, such as an anti-cancer agent, antibody therapy, cell engager, immunotherapy, etc.
  • co-administered includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens.
  • the first of the two therapies is, in some cases, still detectable at effective concentrations at the site of treatment.
  • the sequence and time interval may be determined such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise).
  • the therapeutics may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion.
  • the terms are not limited to the administration of the active agents at exactly the same time.
  • Anti-cancer agents that may be used in combination with the inventive cells are known in the art. See, e.g., U.S. Patent No.9,101,622 (Section 5.2 thereof).
  • an "anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of cancerous cells. This process may involve contacting the cancer cells with recipient cells and the agent(s) or multiple factor(s) at the same time.
  • the immune cells of the present disclosure are used in conjunction with chemotherapeutic, radiotherapeutic, immunotherapeutic intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy.
  • the immune cells of the present disclosure are administered after the subject receives lymphodepletion chemotherapy.
  • the lymphodepletion chemotherapy includes melphalan.
  • the subject receives a stem cell transplant after the lymphodepletion chemotherapy.
  • Additional ACT potentiating treatments include melphalan.
  • Melphalan (Alkeran®, Evomela®) attaches alkyl groups to the N-7 position of guanine and N-3 position of adenine of DNA that leads to the formation of monoadducts, and DNA fragmenting when repair enzymes attempt to correct the apparent replication error.
  • Melphalan can also cause DNA cross-linking from the N-7 position of one guanine to the N-7 position of another, preventing DNA strands from separating for synthesis or transcription.
  • Melphalan an alkylating antineoplastic agent, is used for high- dose conditioning prior to hematopoietic stem cell transplant in patients with multiple myeloma, as well as for palliative treatment of multiple myeloma and for the palliation of non-resectable epithelial carcinoma of the ovary.
  • Melphalan is also used to treat AL amyloidosis, neuroblastoma, rhabdomyosarcoma, breast cancer, ocular retinoblastoma, some conditioning regiments before bone marrow transplant, and in some cases, malignant melanoma.
  • Melphalan may be administered in pill form by mouth. Typically, in 2 mg doses taken on an empty stomach.
  • Melphalan may be administered as an injection or intravenous infusion. Dosing depends on weight, height, disease and disease state, and the subject’s general health. Anti-cancer therapies also include a variety of combination therapies with both chemical and radiation-based treatments.
  • Combination chemotherapies include, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabien, Navelbine®, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the foregoing and also combinations
  • Anti-cancer therapies also include radiation-based, DNA-damaging treatments.
  • Combination radiotherapies include what are commonly known as gamma-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells which cause a broad range of damage on DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells and will be determined by the attending physician.
  • Radiotherapy may include external or internal radiation therapy. External radiation therapy involves a radiation source outside the subject’s body and sending the radiation toward the area of the cancer within the body.
  • Immunotherapy including immune checkpoint inhibitors may also be employed as another therapeutic in the methods provided herein.
  • Immune checkpoint molecules include, for example, PD1, PDL1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A.
  • Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®).
  • PD1 inhibitors include nivolumab (Opdivo®), pembrolizumab (Keytruda®), and cemiplimab (Libtayo®). Additional inhibitors that may be useful in the practice of the present disclosure are known in the art. See, e.g., U.S. Patent Application Publications 2012/0321637, 2014/0194442, and 2020/0155520. Antibody therapy, such as treatment with monoclonal antibodies may also be used in the methods provided herein.
  • Examples of monoclonal antibodies for treatment include, but are not limited to, Abagovomab, Abciximab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab, Anti-thymocyte globin, Apolizumab, Arcitumomab, Aselizumab, Atlizumab (tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Biciromab, Bivatuzumab mertansine, Blinatum
  • Antibodies optimized for enhanced ADCC activity may have a particularly synergistic activity when combined with EVE16 expressing cells.
  • NK cell engagers such as BiKE (bispecific killer cell engager) or TriKE (trispecific killer cell engager) are a novel class of antibody-based therapeutics that exhibit several advantages over other cancer immunotherapies harnessing NK cells. By bridging NK and tumor cells, NKCEs activate NK cells and lead to tumor cell lysis. A growing number of NKCEs are currently undergoing development, with some already in clinical trials 37 . NKCEs may have a particularly synergistic activity when combined with EVE16 expressing cells since many NKCEs will bind specifically to the extracellular domain of CD16A.
  • the immune cells are contemporaneously or sequentially administered in combination with an antibody preparation or a cell engager preparation that binds to the extracellular domain of CD16A, in order to induce a beneficial ADCC effect.
  • the separation between dosing of the immune cells and the antibody preparation or the cell engager preparation is at least 1 hour, 6 hours, 24 hours, 48 hours, 1 week or two weeks.
  • Gene encoding BaEV-Rless envelope glycoprotein was designed in-house, codon-optimized for expression in human cell lines, and synthesized as a gene block by Integrated DNA Technologies.
  • the BaEV-Rless gene block was cloned in-house via Gibson assembly (NEBuilder HiFi DNA Assembly, New England Biolabs, catalog number E2621S), replacing the VSV-G open-reading frame in the pMD2.G plasmid.
  • the self-inactivating (SIN) lentiviral transfer plasmid was generated in-house. In this plasmid, lentiviral HIV-1 RNA expression is driven by the CMV enhancer and promoter, while transgene expression is controlled by the EF1 ⁇ core promoter.
  • the transfer plasmid additionally contains the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), the bovine growth hormone polyadenylation signal (bGH polyA), and the SV40 origin of replication for proper vector function. All plasmids were transformed and propagated using NEB Stable competent E. coli (New England Biolabs, catalog number C3040H), followed by purification with the PureLink HiPure Plasmid Maxiprep Kit (Thermo Fisher, catalog number K210007). All geneblocks encoding EVE16 variants were codon-optimized, synthesized as gene blocks by IDT and subcloned into 3 rd generation lentiviral transfer plasmid described above.
  • WPRE woodchuck hepatitis virus post-transcriptional regulatory element
  • bGH polyA bovine growth hormone polyadenylation signal
  • SV40 origin of replication for proper vector function. All plasmids were transformed and propagated using NEB Stable competent E. coli (New England Bio
  • the 293T human embryonic kidney cell line (ATCC, catalog number CRL-3216) was expanded in high-glucose Dulbecco's Modified Eagle's Medium (DMEM, Gibco, catalog number 11-995-065), supplemented with 1 mM sodium pyruvate, 5 mM l- Glutamine, 10 mM HEPES pH 7.0, 100 U/mL penicillin, 100 ⁇ g /mL streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (complete DMEM).
  • DMEM Dulbecco's Modified Eagle's Medium
  • PEI (at a ratio of 1:1 w/w relative to the total plasmid DNA) was resuspended in 0.5 mL Opti-MEM and incubated at room temperature for 5 minutes. The PEI solution was then added to the DNA mixture, mixed gently, and incubated at room temperature for 15 minutes to allow complex formation. The 1 mL PEI/DNA complex was added dropwise to the cells in the 10 cm plate. After 6 hours, the medium was replaced with 10 mL of fresh, pre-warmed complete DMEM.48 hours post- transfection, the supernatant containing lentiviral particles was harvested and centrifuged at 600xg for 5 minutes to remove cellular debris.
  • the supernatant was then filtered through a 0.45 ⁇ m PVDF filter unit (Corning, catalog number 431220).
  • the filtered supernatant containing lentiviral particles was either used immediately for cell transduction or stored at 4°C for up to 24 hours before use.
  • For T cell transduction the supernatant was centrifuged at 4°C for 16 hours at 10,000xg to concentrate the lentiviral particles.
  • lentiviral vector resuspension buffer 100 mM NaCl, 1% w/v sucrose, 1% w/v mannitol, 20 mM Tris-HCl pH 7.3.
  • the resuspended lentiviral particles were aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C until use. Manufacturing and Purification of Lentiviral Particles Using Suspension 293T Cell Line.
  • the adherent 293T human embryonic kidney cell line (ATCC, catalog number CRL- 3216) was trypsinized, washed once with phosphate-buffered saline (PBS) to remove trypsin, and transferred to FreeStyle 293 Expression Medium (Gibco, catalog number 12- 338-018) supplemented with 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 2% (v/v) heat-inactivated fetal bovine serum, and 33 ⁇ M phenol red (complete FreeStyle medium).
  • PBS phosphate-buffered saline
  • the cells were adjusted to a density of 0.5 ⁇ 10 6 cells/mL and expanded in the presence of anti-clumping agent (ACA, used at a 2,000x dilution, Gibco, catalog number 0010057AE) at 37°C in a tissue-culture shaker with 8% CO2 (shaking at 120 revolutions per minute).
  • ACA anti-clumping agent
  • Cell cultures were expanded until they reached a maximum density of 3 ⁇ 10 6 cells/mL, at which point they were split into fresh complete FreeStyle medium at 0.5 ⁇ 10 6 cells/mL and continued to expand with ACA. Viability and media color were monitored every 2-3 days, with media exchanges performed as necessary. Cells were expanded using this protocol for at least one week prior to transfection with PEI.
  • DNA plasmids were mixed at the following molar ratios: 1 mol of psPAX2, 1.2 mol of the transfer plasmid, and 0.5 mol of the BaEV-Rless envelope plasmid, using 1.5 ⁇ g of total plasmid DNA per million cells.
  • Opti-MEM was added to the plasmid mixture up to 5% of the total cell suspension volume, and the DNA mixture was resuspended thoroughly.
  • PEI at a 1:1 w/w ratio relative to total plasmid DNA
  • Opti-MEM also at 5% of the total cell suspension volume, and incubated for 5 minutes at room temperature.
  • the PEI solution was then combined with the DNA mixture, gently mixed, and incubated at room temperature for 10 minutes to allow for complex formation.
  • the cell suspension was removed from the 37°C shaker, and the PEI/DNA complex mixture was added dropwise to the cells.
  • Lentiviral particles were concentrated by centrifugation at 10,000xg for 16 hours at 4°C. After centrifugation, the supernatant was discarded, and the pellet containing lentiviral particles was resuspended in 1 mL of lentiviral vector resuspension buffer (100 mM NaCl, 1% w/v sucrose, 1% w/v mannitol, 20 mM Tris-HCl pH 7.3). The resuspended lentiviral particles were aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C until use. Determination of Lentiviral Particle Infectious Titer using Jurkat Cell Line.
  • the Jurkat cell line (Clone E6-1, ATCC catalog number TIB-152) was cultured in RPMI 1640 Medium (ATCC modification, Gibco catalog number A1049101) supplemented with 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 10% (v/v) heat-inactivated fetal bovine serum (complete RPMI).
  • RPMI 1640 Medium ATCC modification, Gibco catalog number A1049101
  • 100 U/mL penicillin 100 ⁇ g/mL streptomycin
  • 10% (v/v) heat-inactivated fetal bovine serum complete RPMI.
  • cells were maintained in a 37°C CO2 tissue-culture incubator and split every 2-3 days, ensuring a maximum density of approximately 3 ⁇ 10 6 cells/mL. Cells were not used beyond 30 passages.
  • Jurkat cells were centrifuged at 600xg for 5 minutes and resuspended in fresh, pre-warmed complete RPMI at a density of 1 ⁇ 106 cells/mL.
  • a 2 mL aliquot of the cell suspension (2x10 6 cells) was transferred to each well of a 6-well tissue-culture plate (Corning catalog number 3516).
  • Various volumes of the lentiviral vector (LVV) particle suspension were added to the wells in a dilution series, typically 5 ⁇ L, 1 ⁇ L, 0.1 ⁇ L, 0.05 ⁇ L, and 0.025 ⁇ L, with one well left untreated to serve as a negative control.
  • Cells were transduced using spinoculation at 1,000xg for 1 hour at 37°C, after which the plate was returned to the 37°C CO2 incubator. The following day, 1.5 mL of medium was aspirated from each well and replaced with 1.5 mL of fresh, pre- warmed complete RPMI. Forty-eight hours post-transduction, cells from each well were transferred to a 96-well round-bottom plate, washed once with FACS buffer (Dullbeco’s phosphate-buffered saline supplemented with 5% v/v heat-inactivated fetal bovine serum), and resuspended in 200 ⁇ L of FACS buffer containing DAPI.
  • FACS buffer Dullbeco’s phosphate-buffered saline supplemented with 5% v/v heat-inactivated fetal bovine serum
  • Flow cytometry was used to determine the percentage of transduced Jurkat cells by gating on live singlets and comparing fluorescent cell populations to the negative control. The infectious titer was calculated only for groups where 5-20% of cells were fluorescently positive. The titer was determined by multiplying the percentage of transduced cells by the total starting number of cells (2x10 6 ) and dividing by the volume of lentivirus used. Generation and analysis of stably transduced Jurkat cell lines.
  • Jurkat cells (clone E6- 1) were obtained from the American Type Culture Collection (ATCC) and transduced with lentiviral particles harboring various indicated EVE16 variants followed by P2A-eGFP transgene.
  • EVE16 and CD3zeta co-expression constructs eGFP was replaced with codon-optimized full-length human CD3zeta cDNA.
  • Jurkat cells were stained with PE anti-human CD16 (clone 3G8, Cell Signaling Technology), APC anti-human CD27 (BD Biosciences) or APC anti-FMC63 (Miltenyi) antibodies. Cells were gated on live, eGFP-expressing singlets, and flow plots were generated using FlowJo analysis software. Binding of CD70 to EVE16-expressing Jurkat cell lines was performed using 1 ug biotin-labeled recombinant CD70 (BPS Bioscience).
  • NK cells were incubated with recombinant CD70 for 15 minutes on ice, washed and then stained with APC Streptavidin (BioLegend) and analyzed using flow cytometry. Isolation of NK cells. Fresh whole blood sample (150 mL total volume) was divided into 50 mL tubes and diluted 1:1 (v/v) with Buffer A (Phosphate-Buffered Saline (PBS) supplemented with 2% (v/v) Fetal Bovine Serum (FBS) and 1 unit/mL Heparin, pH 7.4). Peripheral Blood Mononuclear Cells (PBMCs) were separated by centrifugation using a density gradient medium, Lymphoprep (STEMCELL Technologies).
  • Buffer A Phosphate-Buffered Saline
  • FBS Fetal Bovine Serum
  • PBMCs were resuspended at a density 5x10 ⁇ 7 cells/mL in Buffer B (PBS, 2% FBS, and 1 mM EDTA, pH 7.4).
  • Buffer B PBS, 2% FBS, and 1 mM EDTA, pH 7.4
  • human NK and NKT cells were isolated from PBMCs utilizing the EasySep Human NK Cell Isolation Kit (STEMCELL) and the Easy 50 EasySep Magnet (STEMCELL) according to manufacturer instructions. The isolated cells were then resuspended at a density of 2-3x10 ⁇ 6 cells/mL in complete NK MACS media.
  • NK MACS media composition included NK MACS basal medium (Miltenyi Biotec), 10% Human AB serum (Sigma-Aldrich), 1% NK MACS supplement (part of the NK MACS basal medium kit), 100 ⁇ M nicotinamide (Sigma-Aldrich), and 1% Penicillin/Streptomycin (ThermoFisher Scientific).
  • NK cells were used to determine the purity of NK cells: Brilliant Violet 605 anti-human CD45 (clone 2D1, BioLegend), FITC anti-human CD3 (clone OKT3, BioLegend), APC anti-human CD19 (clone HIB19, BioLegend) and PE anti-human CD56 (clone QA17A16, BioLegend).
  • Live NK cells were defined as DAPI-, CD45+, CD56+, CD19-, CD3-.
  • Freshly isolated population of human NK cells was resuspended at 2x10 6 cells/mL in complete NK MACS media and placed in a sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well.
  • the media was then supplemented with 20 ng/mL IL-12 and 25 ng/mL IL-18 and the plate was placed in a 37°C tissue culture incubator for 6 hours. After 6 hours, the cells were washed three times with PBS to remove the cytokines and then resuspended at 2x10 6 cells/mL in complete NK MACS media and placed in a fresh, sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 10 ng/mL IL-15 and 50 ng/mL IL-18 and the plate was placed in a 37°C tissue culture incubator for 36 hours.
  • NK cells were transduced with lentiviral particles pseudotyped with BaEV-Rless glycoprotein 48 hours after isolation.
  • OCI-AML3 (acute myeloid leukemia) cell line was obtained from the AcceGen (ABC-TC179D) and transduced with lentiviral particles harboring Luciferase-P2A-mScarlet transgene.
  • Transduced cancer cells were purified using flow cytometry-assisted sorting of mScarlet hi cells and used in subsequent experiments.
  • CIML NK cells were added to each well at indicated effector: target ratio (for 1:1100,000 NK cells, etc.) in a 100 uL volume in complete NK MACS media supplemented with 2 ng/mL IL-15.
  • target ratio for 1:1100,000 NK cells, etc.
  • the plate was centrifuged at 300xg for 1 minute to allow cells to settle to the bottom of the well, and the plate was then placed in a 37°C tissue culture incubator for 16 hours.
  • the cells were washed twice using Annexin V Buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES pH 7.4) and resuspended in 200 ⁇ L of Annexin V Buffer supplemented with APC- Annexin V (BioLegend), DAPI, and Precision Count Beads (5,000 beads per well, BioLegend).
  • Annexin V Buffer 140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES pH 7.4
  • APC- Annexin V BioLegend
  • DAPI DAPI
  • Precision Count Beads 5,000 beads per well, BioLegend.
  • the percentage of live cancer cells was determined using flow cytometry, live cells defined as DAPI-AnnexinV-mScarlet + single cells, normalized to the number of counting beads per well, and divided by the number of cancer cells incubated without NK cells.
  • PBMCs Human Peripheral Blood Mononuclear Cells
  • Fresh whole blood samples from healthy donors were obtained from Research Blood Components (Watertown, MA).
  • the whole blood was diluted 1:1 with Dulbecco's Phosphate-Buffered Saline (DPBS) supplemented with 2% (v/v) heat- inactivated fetal bovine serum (FBS, Gibco).
  • DPBS Dulbecco's Phosphate-Buffered Saline
  • FBS heat- inactivated fetal bovine serum
  • the diluted blood was gently layered over a Lymphoprep medium in SepMate-50 tubes (STEMCELL Technologies) and centrifuged at 1,200xg for 20 minutes at room temperature to isolate peripheral blood mononuclear cells (PBMCs).
  • DPBS Dulbecco's Phosphate-Buffered Saline
  • FBS heat- inactivated fetal bovine serum
  • the plasma and PBMC layers were carefully transferred to a fresh 50 mL tube.
  • the cells were pelleted by centrifugation at 600xg for 5 minutes, the supernatant was discarded, and the cell pellet was washed once with DPBS/2% FBS buffer.
  • the cells were centrifuged again and resuspended in 10 mL of pre- warmed complete RPMI medium.
  • the total number of PBMCs was counted using Tuerk’s solution (Sigma-Aldrich, catalog number 1092770100), and a small volume of the PBMC suspension was used for flow cytometry to determine the percentage and viability of T cells, B cells, NK cells, and monocytes.
  • the cells were pelleted by centrifugation at 600xg for 5 minutes, and the supernatant was discarded. The cell pellet was then resuspended in CryoStor CS10 solution (BioLife Solutions, catalog number 210502) at a concentration of 50 million PBMCs per mL. The cell suspension was aliquoted into cryovials, frozen in a controlled-rate freezer, and stored in a liquid nitrogen container until further use.
  • Fc receptors Prior to antibody staining, Fc receptors were blocked using Human TruStain FcX (BioLegend, catalog number 422301). Isolation, Activation and Transduction of T cells from healthy donors Frozen peripheral blood mononuclear cells (PBMCs) stored in cryovials were thawed by placing the cryovial in a 37°C water bath for 2 minutes. The cell suspension was then transferred to a clean centrifuge tube, diluted 1:10 with pre-warmed complete RPMI, and centrifuged at 600xg for 5 minutes at room temperature. After centrifugation, the supernatant was completely aspirated, and the cell pellet was resuspended in 5 mL of complete RPMI. Viable cells were counted at this stage.
  • PBMCs peripheral blood mononuclear cells
  • CD3 MicroBeads To isolate T cells, CD3 MicroBeads and the MultiMACS Cell24 Separator (Miltenyi Biotec) were used. The PBMCs were centrifuged again at 600xg for 5 minutes and resuspended at a concentration of 100 ⁇ 10 6 cells/mL in cold Wash Buffer (Dulbecco’s phosphate-buffered saline without calcium and magnesium, pH 7.2, supplemented with 2 mM EDTA and 2% (v/v) heat- inactivated fetal bovine serum). CD3 MicroBeads (200 ⁇ L per 100 ⁇ 10 6 cells) were added to the suspension, mixed thoroughly, and incubated in the cold room for 15 minutes.
  • Cold Wash Buffer Dulbecco’s phosphate-buffered saline without calcium and magnesium, pH 7.2, supplemented with 2 mM EDTA and 2% (v/v) heat- inactivated fetal bovine serum.
  • the cells were washed twice with cold Wash Buffer and resuspended at 100 ⁇ 10 6 cells/mL.
  • the suspension was applied to a pre-equilibrated LS column (Miltenyi Biotec) using 1 LS column per 100 ⁇ 10 6 cells.
  • the column was washed with 15 mL of cold Wash Buffer, and the CD3+ T cells were eluted and washed off the column with additional Wash Buffer.
  • the eluted cells were centrifuged at 600xg for 5 minutes and resuspended in complete TexMACS medium (Miltenyi Biotec, catalog number 130-097-196) supplemented with 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 10% (v/v) heat- inactivated human male AB serum, 5 mM l-Arginine, 10 ng/mL IL-7, and 10 ng/mL IL-15 at a concentration of 1 ⁇ 10 6 cells/mL. The purity and viability of the isolated T cells were assessed using flow cytometry.
  • Isolated T cells were resuspended at a density of 1 ⁇ 106 cells/mL in complete TexMACS medium and seeded into sterile 6-well plates at 2 mL per well.
  • Cells were stimulated with CD3/CD28 agonist polymer (TransAct, Miltenyi Biotec, catalog #130-128-758) at a final concentration of 10 ⁇ L/mL and incubated at 37°C for 48 hours.
  • T cells were harvested by centrifugation, washed once with DPBS, and resuspended in fresh TexMACS medium at 1 ⁇ 106 cells/mL.
  • VSV-G pseudotyped lentiviral vector was added to each well at a multiplicity of infection (MOI) of 10, together with Vectofusin.
  • MOI multiplicity of infection
  • the cells were then spinoculated by centrifugation at 1,000 ⁇ g for 1 hour at 37°C. After spinfection, cells were returned to a tissue culture incubator, and the culture medium was replaced with fresh TexMACS every 24 hours. At 72 hours post-transduction, transduction efficiency was assessed by flow cytometry.
  • Transduced T cells were collected by centrifugation, resuspended in CryoStor10, and cryopreserved until further use.
  • T Cell Cytotoxicity Using Raji Cell Line T cells were purified, activated, and transduced as described above.
  • Raji (Burkitt’s lymphoma) cell line was obtained from the American Tissue Culture Collection (CCL-86) and maintained in complete RPMI. Raji cells were transduced with lentiviral particles harboring Luciferase-P2A-mScarlet transgene. Transduced cancer cells were purified using flow cytometry-assisted sorting of mScarlet hi cells and used in subsequent experiments. For cytotoxicity experiments using Raji cell line, 100,000 cancer cells per well were seeded in a 96-well round-bottom plate in complete RPMI media in 100 ⁇ L volume. T cells were added to each well at indicated effector-to- target ratio (for 1:1100,000 T cells, etc.) in a 100 uL volume in complete RPMI media.
  • the plate was centrifuged at 300xg for 1 minute to allow cells to settle to the bottom of the well, and the plate was then placed in a 37°C tissue culture incubator for 16 hours. After 16 hours, the cells were washed twice using Annexin V Buffer (140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES pH 7.4) and resuspended in 200 ⁇ L of Annexin V Buffer supplemented with APC-AnnexinV (BioLegend), FITC-anti-human CD19 (HIB19, BioLegend), DAPI, and Precision Count Beads (5,000 beads per well, BioLegend).
  • Annexin V Buffer 140 mM NaCl, 2.5 mM CaCl2, 10 mM HEPES pH 7.4
  • the percentage of live cancer cells was determined using flow cytometry, live cells defined as DAPI-AnnexinV-mScarlet + single cells, normalized to the number of counting beads per well, and divided by the number of cancer cells incubated without T cells. T Cell In Vivo Activity. All mouse experiments were conducted in compliance with institutional guidelines and approved IACUC protocols. Human T cells used in these studies were prepared as described above. Male NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice, aged 6–12 weeks, were obtained from The Jackson Laboratory and used in all experiments.
  • mice were intravenously (i.v.) injected with 1 ⁇ 106 Raji cells expressing a Luciferase-P2A-mScarlet transgene, suspended in USP-grade PBS. Where indicated, mice received intraperitoneal (i.p.) injections of human recombinant IL-2 (50,000 IU/injection) every 3 days. On Day 5, mice were administered 3 ⁇ 106 T cells via i.v. injection. Mice were monitored every 3 days for body weight and body condition (BC) scores. Where indicated, peripheral blood was collected via submandibular vein puncture for analysis of T cell persistence. A volume of 100 ⁇ L blood was collected, red blood cells were lysed, and the remaining leukocytes were stained for surface antigen expression.
  • Bioluminescent imaging was performed weekly using the IVIS system following intraperitoneal injection of luciferin.
  • mice were euthanized, and cardiac blood, spleen, and bone marrow were harvested and processed. Samples were stained with the following antibodies: Brilliant Violet 605 anti-human CD45 (BioLegend), APC anti-human CD3 (BioLegend), and PE anti-human CD56 (BioLegend), and analyzed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Flow cytometry data were processed using FlowJo Software (BD Biosciences). Immunophenotyping.
  • NK and T cells For cell surface antibody staining of NK and T cells, 50,000- 100,000 cells were washed and resuspended in 200 ⁇ L of FACS buffer (PBS supplemented with 2% FBS).5 ⁇ L of Human TruStain (BioLegend) was added to the sample and incubated for 5 min at room temperature to block non-specific binding of IgG antibodies. Cells were incubated with antibodies (2 ⁇ L each) on ice for 15-20 minutes, washed twice with 1 mL of FACS buffer, and after the final wash resuspended with 200 ⁇ L of FACS buffer supplemented with DAPI (1 ⁇ g/mL) and placed on ice before flow cytometry analysis.
  • FACS buffer PBS supplemented with 2% FBS
  • DAPI 1 ⁇ g/mL
  • the Flow Cytometry data was analyzed using Flow-Jo software (BD Biosciences).
  • the following antibodies were used to determine the purity of NK and T cells: Brilliant Violet 605 anti-human CD45 (clone 2D1, BioLegend), FITC anti-human CD3 (clone OKT3, BioLegend), APC anti-human CD19 (clone HIB19, BioLegend), PE anti-human CD56 (clone QA17A16, BioLegend), Brilliant Violet 605 anti-human CD4 (clone SK3, BioLegend), and APC anti-human CD8 (clone SK1, BioLegend).
  • Live NK cells were defined as DAPI-, CD45 + , CD56 + , CD19-, CD3- and live T cells were defined as DAPI-, CD45 + , CD56-, CD19-, CD3 + .
  • the following antibodies were used for the analysis of NK and T cell phenotype: FITC anti-human CD25 (clone BC96, Biolegend), PE anti-human CD71 (clone CY1G4, Biolegend), FITC anti-human NKp46 (clone 9E2, Biolegend), PE anti-human CD16 (clone 3G8, Cell Signaling Technology), FITC anti-human CD70 (clone 113-16, Biolegend), PE anti-human NKG2D (clone 1D11, Biolegend), APC anti-human CD94 (clone DX22, Biolegend), Brilliant Violet 605 anti-human 4-1BB (clone 4B4-1, Biolegend), APC anti- human CD69 (clone FN50,
  • NK and T cell viability were washed twice with Annexin V Buffer (10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ), and resuspended with 200 ⁇ L Annexin V Buffer supplemented with APC Annexin V (2 ⁇ L, BioLegend) and DAPI. Live cells were defined as Annexin V-, DAPI-.
  • Annexin V Buffer 10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2
  • APC Annexin V (2 ⁇ L, BioLegend
  • Live cells were defined as Annexin V-, DAPI-.
  • EXAMPLE 2 Schematic of CD16A, CAR and EVE16 receptors.
  • CD16A (also known as CD16-II, low affinity immunoglobulin gamma Fc region receptor III-A, IgG Fc receptor III-A, Fc ⁇ RIIIa, and FcR-10) is an activating receptor expressed on the surface of NK cells, NKT cells, monocytes, macrophages, neutrophils, and certain T cell subsets.
  • the extracellular domain consists of a membrane proximal hinge domain and membrane distal FC-binding domain, the latter composed of two Ig-like domains ( Figure 1B and Figure 2C).
  • the FC-binding domain is responsible for binding to the Fc chain of IgG1, IgG3 and IgG4 antibodies.
  • CD16A interacts with disulfide-linked homodimers or heterodimers of CD3zeta and/or the gamma subunit of the high-affinity IgE receptor (FCER1G), via specific transmembrane interactions.
  • FCER1G high-affinity IgE receptor
  • the activation of the receptor follows the classical ITAM-signaling cascade.
  • the antibody binding affinity of CD16A determines the strength of the cellular response. Human population possesses two major CD16A polymorphisms, with the V176 allotype exhibiting a higher affinity for antibodies compared to the F176 allotype.
  • EVE16 Engineered Valency-Enhanced CD16A
  • CD16A F176V
  • Figure 1A- B and Figure 2G the receptor consists of an extracellular target-binding domain (e.g. single-chain fragment variable, peptide ligand or other alternative moieties) fused to the CD16A molecule via a flexible linker domain.
  • target-binding domain e.g. single-chain fragment variable, peptide ligand or other alternative moieties
  • EVE16 receptors do not need to include the intracellular activation domain, as the recognition of target antigen results in signal transduction via the CD16A transmembrane region associated with CD3zeta or FCER1G chains (Figure 1A).
  • EXAMPLE 3 Optimizing the extracellular domain of EVE16 receptor improves its surface expression and signaling capacity. To dissect the role of the linker domain on EVE16 surface expression and function, we generated a series of EVE16 receptors with various linker domains ( Figure 3A). The effect of modifications was systematically analyzed by measuring EVE16 receptor expression on the surface of Jurkat cells as well as in cytokine-induced memory-like Natural Killer (CIML NK) cells.
  • CIML NK cytokine-induced memory-like Natural Killer
  • CD8A linker domain into the construct led to a reduction in surface expression of EVE16, as seen by the very dim expression of both CD16A and CD27 (Figure 3A-B, construct CD8A-G4SGMRTED).
  • the CD8A stalk domain contains two cysteine residues that are prone to intermolecular disulfide bond formation ( Figure 2F).
  • Modelling the structure of the CD8A-G4S linker containing EVE16 receptor demonstrated an unfavorable steric configuration of the CD8A molecule in the vicinity of CD16A extracellular stalk, resulting in steric hindrance and epitope masking of recognition domains.
  • FIG. 3A and B show that the removal of cysteine containing region from the construct rescued and significantly improved the surface expression of properly folded EVE16 ( ⁇ CD8 ⁇ hinge- G4S-GMRTEDL linker domain).
  • the CD16A preprotein contains a 16-amino acid long signal peptide/leader peptide at its N-terminus ( Figure 2C- D). To map out the exact position of the signal peptidase cleavage site SignalP prediction methodology was used ( Figure 2D).
  • CD70-targeting EVE16 receptors were transduced into CIML NK cells, and co-cultured with CD70-expressing OCI-AML3 acute myeloid leukaemia cells and the percentage of tumor cell lysis was determined using flow cytometry.
  • CIML NK cells transduced with the deltaCD8-G4S EVE16 construct demonstrated marginally improved cellular cytotoxicity compared to cells transduced with G4S EVE16 ( Figure 4B).
  • EXAMPLE 4 The Fc-binding domain is necessary for the surface expression of EVE16. To test whether the intact extracellular domain of CD16A is necessary for optimal surface expression of the retargeting construct, a series of molecules were generated ( Figure 8).
  • EVE16 a prototypic Engineered Valency-Enhanced CD16A receptor
  • FMC63 anti-CD19
  • scFv single-chain variable fragment
  • F176V enhanced binding affinity variant CD16A molecule
  • NKp44-derived linker N-terminally fusing the anti-CD19 (FMC63) single-chain variable fragment (scFv) with the enhanced binding affinity (F176V) variant CD16A molecule via an NKp44-derived linker.
  • FMC63 anti-CD19
  • scFv single-chain variable fragment
  • F176V enhanced binding affinity variant CD16A molecule via an NKp44-derived linker
  • a series of deletion mutants were generated, in which either one or both Ig-like domains were removed from the EVE16 molecule.
  • Jurkat cells were transduced with the relevant constructs. The surface expression was then measured using flow cytometry 7-days post-transfection.
  • EVE16 receptor can be redirected to an antigen of choice and can be utilized as a payload delivery molecule.
  • the CD27 ectodomain was interchanged with various single-chain variable fragments (e.g.
  • hinge domains derived from CD28, CD8, or IgG molecules is widespread in the chimeric antigen receptor field.
  • the rigidity of these hinges can vary greatly, from highly rigid (e.g. CD28 stalk) to highly flexible (CH3-CH2 domain from IgG) structures.
  • Proline is the most rigid of the 20 naturally occurring amino acids and is frequently introduced to rigidify flexible regions of protein to enhance thermostability.
  • Jurkat cells were transduced with the newly designed constructs and surface expression was compared to the stalkless (G4S linker containing only) and truncated CD8A containing EVE16 receptor variants. All EVE16 receptors with flexible, long linkers demonstrated a significantly higher level of surface expression, according to FACS analysis ( Figure 6A). In terms of CD27 and CD16A mean fluorescence, the NKp44 receptor variant with a longer stalk outperformed all the other evaluated constructs ( Figure 6A-B). The effect of the elevated surface expression of the EVE16 receptor on target antigen binding was examined by staining Jurkat cells with biotin-labeled recombinant human CD70 protein.
  • EXAMPLE 7 The incorporation of co-stimulatory domains can further improve EVE16 receptor-mediated effector activation and cytotoxicity in vitro. Restricted expansion and survival of T cells are key factors behind the limited efficacy of CAR-modified T cells. The inclusion of co-stimulatory molecules in the CAR designs has resulted in improved efficiency, proliferation, and durability of the cells, as exemplified by the presence of CD28 and 4-1BB costimulatory domain in second-generation CARs.
  • co-stimulatory molecules have been included in the receptor design, typically positioned between the transmembrane and CD3zeta activation domains, as illustrated in Figure 1.
  • Novel, alternative designs segregate the target recognition and intracellular signaling domains (such as those in split CAR designs) or include additional co- stimulatory domains in the form of chimeric co-stimulatory receptors (CCR).
  • CCR chimeric co-stimulatory receptors
  • the EVE16 receptor design enables the easy, plug and play integration of single or tandem co-stimulatory molecules, which are fused directly to the N-terminal cytoplasmic tail of the CD16A receptor.
  • a set of EVE16 receptors with co-stimulatory domains were developed.
  • CD3zeta enhances the surface expression of the EVE16 receptor.
  • CD16A surface expression is contingent upon its interaction with signaling adaptor molecules including CD3zeta and/or FCER1G.
  • the incorporation of the homo or heterodimeric signaling adaptors is a rate limiting step for the assembly and surface expression of functional CD16A receptor.
  • CD3zeta expression is stable within the cell, and it competes with multiple other receptor complexes such as TCR, NKp30, and NKp46, we hypothesized that the presence of EVE16 on the cell surface may rely on the availability of CD3zeta.
  • EVE16 was co-expressed with or without CD3zeta in Jurkat cells ( Figure 10). Analysis of the surface expression was performed by staining the cells with fluorochrome-conjugated anti-CD16 antibody and using flow cytometry. Consistent with our hypothesis that CD3zeta is rate-limiting for the surface expression of EVE16 receptors, Figure 10 shows that the over-expression of CD3zeta led to a significant increase in EVE16 surface expression, as indicated by higher mean fluorescent intensity of CD16A.
  • EXAMPLE 9 shows that the over-expression of CD3zeta led to a significant increase in EVE16 surface expression, as indicated by higher mean fluorescent intensity of CD16A.
  • leader peptides were paired with four anti-CD20 scFvs derived from Rituximab, Ofatumumab, Ocrelizumab, and Ublituximab. Each scFv was expressed in both VL–VH and VH–VL orientations, resulting in a total of 96 unique EVE16 constructs.
  • the EVE16 variants were cloned into a lentiviral expression vector co-expressing full- length human CD3 ⁇ under a bicistronic system using P2A self-cleaving peptide.
  • the EVE16 library was first evaluated in HEK 293T cells following transient transfection (Figure 12B).
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Abstract

La présente divulgation concerne de nouvelles constructions, en particulier des protéines de récepteur de cellule immunitaire modifiée et des polynucléotides codant pour lesdites protéines de récepteur de cellule immunitaire modifiée, et des utilisations desdites constructions. Ladite protéine de récepteur de cellule immunitaire modifiée est désignée par CD16A modifié par augmentation de valence (EVE16), qui est destiné à une expression sur la surface de diverses cellules immunitaires, comprenant, mais sans s'y limiter, des cellules tueuses naturelles (NK), des lymphocytes T tueuses naturelles (NKT), des lymphocytes T, des monocytes et autres. EVE16 est un récepteur de protéine chimérique conçu pour exploiter les voies de transduction de signal endogène inhérentes aux cellules immunitaires, notamment les cellules NK, les lymphocytes NKT, les lymphocytes T, les monocytes et autres, ce qui facilite la modulation de la signalisation cellulaire, en particulier pour initier une signalisation d'activation. La présente divulgation concerne des constructions comprenant des peptides leader optimisés, des domaines de liaison à une cible, des domaines lieurs ainsi que des domaines cytoplasmiques qui présentent une expression de surface et une signalisation d'activation améliorées d'EVE16, ce qui permet d'obtenir une cytotoxicité plus puissante et d'autres fonctions effectrices de cellules immunitaires. De plus, la présente divulgation concerne des vecteurs hébergeant les constructions susmentionnées (y compris des constructions d'expression), des cellules, telles que des cellules immunitaires humaines, en particulier des cellules NK, des lymphocytes NKT, des lymphocytes T, ou des monocytes exprimant lesdites constructions, et leur utilisation dans le traitement de maladies, en particulier le cancer et les maladies auto-immunes.
PCT/US2025/028598 2024-05-10 2025-05-09 Récepteur de cellule immunitaire modifiée comprenant un domaine de liaison à la cible et le domaine extracellulaire de cd16a Pending WO2025235862A1 (fr)

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