WO2024102954A1 - Activation induced clipping system (aics) - Google Patents
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- WO2024102954A1 WO2024102954A1 PCT/US2023/079296 US2023079296W WO2024102954A1 WO 2024102954 A1 WO2024102954 A1 WO 2024102954A1 US 2023079296 W US2023079296 W US 2023079296W WO 2024102954 A1 WO2024102954 A1 WO 2024102954A1
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
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Definitions
- nucleic acids and related compositions and methods, encoding fusion proteins containing an a target binding domain, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain.
- ADAM Disintegrin and Metalloproteinase
- SUMMARY OF THE DISCLOSURE [0004] Chimeric antigen receptor (CAR)-expressing Natural Killer (NK) cells have shown promising clinical responses, such as during adoptive cell therapy (ACT) in some cancer types (Liu et al., N. Engl. J. Med. 382:545-553 (2020)). However, solid tumors have been difficult to treat with ACT.
- CAR Chimeric antigen receptor
- NK Natural Killer
- solid tumors Unlike dispersed hematologic malignancies (so called liquid tumors), solid tumors, especially late-stage solid tumors, exhibit an inhibitory tumor microenvironment (TME) due to, for example, hypoxia, low pH, suppressive cytokines, lactate, and prostaglandins. These inhibitory factors circumvent the ability of CAR-NK cells to infiltrate and kill cancerous cells (Martinez and Moon, Front. Immunol.10:128-21 (2019)).
- TEE tumor microenvironment
- immune checkpoint inhibitors are important therapies to prevent cancerous cells from using peripheral tolerance pathways to limit anti-tumor cytotoxic responses.
- immune checkpoint inhibitors results in toxicities and may cause inflammatory disorders of many different organs, diarrhea, polyneuropathy, inflammatory neuropathies, anemia, neutropenia, thromboyxtopenia, rashes, pulmonary, neurological, endocrinopathy, hepatic, and renal complications (Martins et al., Nat Rev. Clin. Oncol. 16:563- 580 (2019)).
- CAR T cells such as those directed against the CD19 antigen, have been introduced in therapy of autoimmune disease.
- autoreactive B cells play a key role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.
- B-cell-depleting monoclonal antibodies such as rituximab
- rituximab have poor therapeutic efficacy in autoimmune diseases, mainly due to the persistence of autoreactive B cells in lymphatic organs and inflamed tissues.
- CAR T cells have been found to induce rapid and sustained depletion of circulating B cells.
- many patients do not respond to currently used treatments.
- ADAM Disintegrin and Metalloproteinase
- a cleavable membrane-bound fusion protein with a target binding domain could be produced to provide controllable release of the target binding domain.
- Cell killing can also be enhanced with the fusion proteins as provided herein that target a first target and that also have a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17A and that allow for ADCC.
- ADAM Disintegrin and Metalloproteinase
- the fusion proteins provided herein allow for targeting of a CAR as well as ADCC.
- Cell killing can be useful in the treatment of a number of diseases and disorders, such as cancer, autoimmune disease, transplant rejection and graft versus host disease (GVHD).
- a first aspect of the present disclosure is directed to a nucleic acid or set of nucleic acids that together comprise a sequence that encodes a fusion protein, wherein the fusion protein comprises a target binding domain that binds a first target, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain, wherein the cleavage domain is located between the target binding domain and the transmembrane domain.
- the cleavage domain is of any one of the proteins provided herein cleavable by ADAM 17, such as CD16A.
- the cleavage domain comprises the extracellular domain of of any one of the proteins provided herein cleavable by ADAM 17, such as CD16A. In one embodiment of any one of the compositions or methods provided herein, the cleavage domain also comprises an Fc-binding domain. [0009] In one embodiment of any one of the compositions or methods provided herein, the cleavage domain comprises any one of the relevant specific sequences provided herein. In one embodiment of any one of the compositions or methods provided herein, the extracellular domain of the protein cleavable by ADAM 17 comprises any one of the relevant specific sequences provided herein. [0010] In one embodiment, a composition comprising any one of the nucleic acids or sets of nucleic acids provided herein is provided.
- 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 ADAM 17.
- 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 fusion protein further comprises an intracellular domain connected to the transmembrane domain, and the nucleic acid or set of nucleic acids encodes the intracellular domain.
- the intracellular domain is of a protein cleavable by ADAM 17.
- the intracellular domain is of any one of the relevant proteins provided herein.
- the intracellular domain comprises any one of the relevant specific sequences provided herein.
- the first target is a receptor or ligand on a cell, the killing of which cell is desirable.
- the first target is an antigen or other ligand on a cancer cell.
- the first target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell.
- the first target is a receptor or other ligand on an immune cell.
- the immune cell is a B cell or T cell.
- the killing of the cell by CAR targeting and/or antibody-dependent cellular cytotoxicity is desirable.
- 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. In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds any one of the receptors or other ligands on a B cell provided herein.
- the target binding domain binds a receptor or other ligand on a T cell. In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds any one of the receptors or other ligands on a T cell provided herein. [0018] In one embodiment of any one of the compositions or methods provided herein, the target binding domain comprises an antibody fragment. In one embodiment of any one of the compositions or methods provided herein, the target binding domain comprises a single-chain variable antibody fragment (scFv). [0019] In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds CD19, PDL1 or CD70.
- the target binding domain comprises an anti-CD19 antibody fragment, PD1 or an anti-PDL1 antibody fragment, or CD27.
- the nucleic acid or set of nucleic acids further comprise a sequence that encodes a CAR polypeptide, wherein the CAR polypeptide comprises a second target binding domain that binds a second target and a transmembrane domain.
- the first target and second target are different.
- the transmembrane domain is of a protein cleavable by ADAM 17.
- the transmembrane domain is of any one of the relevant proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain comprises any one of the relevant specific sequences provided herein. [0021] In one embodiment of any one of the compositions or methods provided herein, the CAR polypeptide further comprises an intracellular domain. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain is of a protein cleavable by ADAM 17. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain is of any one of the relevant proteins provided herein.
- the intracellular domain comprises any one of the relevant specific sequences provided herein.
- the second target is an antigen or other ligand on a cancer cell.
- the second target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell.
- the second target is a receptor or other ligand on an immune cell.
- the immune cell is a B cell or T cell.
- the cancer antigen is any one of the cancer antigens provided herein.
- the second target binding domain binds a receptor or other ligand on a B cell. In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds any one of the receptors or other ligands on a B cell provided herein. [0025] In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds a receptor or other ligand on a T cell.
- the second target binding domain binds any one of the receptors or other ligands on a T cell provided herein.
- the second target binding domain comprises an antibody fragment.
- the second target binding domain comprises a single-chain variable antibody fragment (scFv).
- scFv single-chain variable antibody fragment
- the second target binding domain binds CD19 or PDL1.
- the second target binding domain comprises an anti- CD19 antibody fragment, PD1 or an anti-PDL1 antibody fragment.
- the fusion protein is any one of the fusion proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the sequence of the fusion protein is any one of the specific relevant sequences provided herein. [0029] In one embodiment of any one of the compositions or methods provided herein, the fusion protein and CAR polypeptide is any one of the combinations of fusion proteins and CAR polypeptides provided herein. In one embodiment of any one of the compositions or methods provided herein, the sequences of the fusion protein and CAR polypeptide is any one of the combinations of specific relevant sequences provided herein.
- the first and second sequences are operatively linked to the same or different promoters.
- a vector or set of vectors comprising any one of the nucleic acids or sets of nucleic acids provided herein is provided.
- the vector is a viral vector.
- the vector is a non-viral vector.
- the non-viral vector is a plasmid.
- an immune cell or population of immune cells comprising any one of the nucleic acids or sets of nucleic acids or any one of the vectors or sets of vectors provided herein is provided.
- an immune cell that expresses any one or any one combination of the fusion proteins and CAR polypeptides provided herein is provided.
- the immune cell(s) express signaling adaptor proteins CD3 ⁇ and Fc Fragment of IgE Receptor Ig (FCER1G).
- the cell(s) is/are NK cell(s).
- the cell(s) is/are T cell(s). In one embodiment of any one of the compositions or methods provided herein, the cell(s) is a/are monocyte(s) or macrophage(s).
- a composition comprises any one of the immune cells or populations of immune cells provided herein is provided. In one embodiment of any one of such embodiments, the immune cell(s) are transformed with any one of the vector or sets of vectors encoding a fusion protein provided herein. In some embodiments of any one of such compositions, the immune cell(s) is transformed with a second vector containing a nucleic acid or set of nucleic acids that encode a CAR polypeptide.
- the composition further comprises a pharmaceutically effective carrier.
- the cell(s) is/are in a therapeutically effective amount.
- a method of killing cells comprising contacting cells with any one of the compositions provided herein is provided.
- the contacting may be in vitro or may be in vivo by administering any one of the compositions provided herein.
- a method of treating a subject with cancer comprising administering to the subject any one of the compositions provided herein is provided.
- a method of treating a subject with autoimmune disease comprising administering to the subject any one of the compositions provided herein is provided.
- a method of treating a subject with a transplant comprising administering to the subject any one of the compositions provided herein is provided.
- a method of treating a subject with GVHD comprising administering to the subject any one of the compositions provided herein is provided.
- the subject is one that has received or is to receive an antibody therapy.
- the method further comprises administering to the subject an antibody therapy.
- the immune cells are allogeneic but have a complete or partial HLA-match with the subject.
- the immune cells are autologous.
- the method further comprises isolating immune cells from a tissue or body fluid sample of the subject prior to the contacting or administering of any one of the compositions provided herein.
- the immune cells are isolated based on CD56 expression.
- FIG. 1 is a schematic illustration that shows a therapeutic avenue of an activation induced clipping system (AICS) in which NK cell activation via CAR engagement causes PL1- CD16 fusion protein shedding, release of PD1-containing target binding domain, binding of PDL1 on cancer cells, and subsequent blockade of PDL1-mediated inhibitory signals.
- AICS activation induced clipping system
- FIGs.2A – 2C are a set of flow cytometry and bar plots showing that NK92 MI cells express and shed the PD1-CD16 fusion protein.
- FIG.2A is set of flow cytometer plots that show PD1 and CD16 expression in cells with and without the PD1-CD16 fusion protein.
- FIG. 2B is a set of bar plots showing CD16 expression in NK92 MI cells expressing either CD19-CAR alone, PD1-CD16 fusion protein alone, PD1-CD16 fusion protein in combination with CD19-CAR, or PDL1scFv-CD16 fusion protein in combination with CD19-CAR exposed to different stimulations.
- FIG.2C is a set of bar plots showing PD1 expression in the same cells and conditions in FIG.2B.
- FIG.3 is a set of line plots showing that PD1-fusion protein expressing cells kill target cells.
- FIG. 3 is a set of line plots showing NK92 MI cells with and without expression of anti- CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16, or a combination thereof kill K562 target cells.
- FIG. 4 is a set of line plots of the same cells and conditions presented in FIG. 3 but graphed as a comparison between the different NK cell preparations.
- FIG.5 is a set of line plots showing that PD1-fusion protein expressing cells kill target cells.
- FIG.5 is a set of line plots showing primary NK (pNK) cells with and without expression of anti-CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16 fusion protein, or a combination thereof kill Raji target cells.
- FIGs. 6A – 6B are a set of bar plots showing the functional aspects of PD1-fusion protein expressing cells after co-incubation with target cells.
- FIG.6A is a set of bar plots showing expression of CD107a by pNK cells with and without expression of anti-CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16 fusion protein, or a combination thereof after co-incubation with K562 or Raji target cells.
- FIG. 6B is a set of bar plots showing IFN ⁇ expression under the same conditions as FIG.6A.
- FIG.7 is a set of bar plots showing the kinetics of CD16 shedding on NK92 MI cells.
- FIG.7 is a set of bar plots showing CD16 expression on NK92 MI cells expressing the PDL1scFv- CD16 fusion protein after co-incubation with K562 or Raji target cells with and without exogenous expression of PDL1.
- FIG. 8 is a set of bar plots showing the kinetics of PDL1 engagement on cancer cells after treatment with NK92 MI cells.
- FIG.8 is a set of bar plots showing PDL1 expression on K562 and Raji target cells with and without exogenous expression of PDL1 for different time points after co-culture with NK92 MI cells expressing the PDL1scFv-CD16 fusion protein and the CD19- CAR.
- FIG. 9 is a schematic illustration of embodiments comparing cell cytotoxicity in cells expressing a traditional CAR to those using a second therapeutic avenue in which cells express the CD19scFv-CD16 fusion protein without a CAR protein, where NK cell activation causes CD19scFv-CD16 fusion protein cleavage and direct killing of cancer cells.
- FIG. 10 is a set of line plots comparing the cytotoxicity ability of NK92 MI cells expressing either the CD19scFv-CD16 fusion protein or anti-CD19 CAR.
- FIG.10 is a set of line plots that show the cells tested against three cancer targets, cells from the Raji, Daudi, and Ramos cell lines.
- FIGs.11A – 11B are a set of bar plots showing the functional aspects of cells expressing the CD19scFv-CD16 fusion protein or anti-CD19 CAR after co-incubation with target cells.
- FIG. 11A is a set of bar plots showing CD107a expression on NK92 MI cells with and without expression of anti-CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16 fusion protein, or a combination thereof after co-incubation with K562 or Raji target cells with and without exogenous expression of PDL1.
- FIG.11B is bar plots showing IFN ⁇ expression under the same conditions as FIG.11A.
- FIG.12 is a set of flow cytometry plots showing T cells with and without transduction of CD19scFv-CD16 fusion protein or anti-CD19 CAR containing virus.
- FIG.12 is flow cytometry plots showing T cells transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and assayed for cell scatter, alive/dead, GFP (exogenous expression), CD16, and 4-1BBL.
- FIG. 13 is a set of line plots showing T cell killing of target cells.
- FIG. 13 is a set of line plots showing T cell killing of target cells.
- FIG. 13 is flow cytometry plots showing T cells transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and co-incubated with cells from the cancer cell lines Raji, Daudi, and Ramos.
- FIG.14 is a set of bar plots showing the functional aspects of T cells after co-incubation with target cells.
- FIG. 14 is a set of bar plots showing CD107a expressing T cells (all cells and gated on GFP+ cells) transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and co-incubated with cells from the cancer cell lines Raji, Daubi, and Ramos, or no target cell control.
- FIG.15 is a set of bar plots showing the functional aspects of T cells after co-incubation with target cells.
- FIG.15 is a set of bar plots showing IFN ⁇ expressing T cells (all cells and gated on GFP+ cells) transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and co-incubated with cells from the cancer cell lines Raji, Daubi, and Ramos, or no target cell control.
- FIG.16 is a schematic illustration of embodiments comparing cell cytotoxicity in cells expressing a traditional PDL1-CAR to those using a third therapeutic avenue in which cells express either a PDL1scFv-CD16 fusion protein or a PD1-CD16 fusion protein without a CAR protein, where NK cell activation causes fusion protein cleavage and direct killing of cancer cells.
- FIG.17 is a set of line plots showing cells expressing fusion proteins killing of target cells.
- FIG.18 is a set of line plots showing the cells and conditions in FIG.17 but comparing each cell condition separately.
- FIGs.19A – 19B are a set of bar plots showing the functional aspects of T cells after co-incubation with target cells.
- FIG. 20 is an illustration of how NK cells can kill cells, such as cancer cells, through various mechanisms.
- FIGs. 21A – 21C are a set of flow cytometry plots and line plots that show memory- like NK cells in the peripheral blood lymphocyte compartment after ACT.
- FIG. 21A – 21C are a set of flow cytometry plots and line plots that show memory- like NK cells in the peripheral blood lymphocyte compartment after ACT.
- FIG. 20A is a set of flow cytometry plots that show memory-like NK cells in the peripheral blood after ACT.
- FIG.20B is a plot that shows the percentage of NKG2A positive NK cell subsets.
- FIG.20C is line plot that shows the percentage of PD1 positive NK cell subsets.
- FIG.22 illustrates the hinge region of CD16 carries the ADAM17 cleavage site.
- FIG.23 illustrates the function of a regular CAR (for example, targeting CD19 antigen on cancer cells) versus a CD16-based fusion protein (for example, CD19scFv-CD16 fusion protein also targeting CD19 antigen on cancer cells).
- FIG.24 provides examples of construct designs. [0071] FIG.
- FIG. 25 shows that T cells expressing CAR or CD16-based receptor fusion proteins provided herein were comparable in their function.
- FIG.26 shows that T cells expressing CD16-based receptors fusion proteins provided herein were less activated than CAR T cells upon engagement of Raji cells.
- FIG.27 provides results that demonstrate that CAR T cells and T cells expressing fusion proteins provided herein were able to eliminate Raji cells more efficiently than untransduced T cells, however cells expressing fusion proteins provided herein were more potent than CAR T cells at later timepoints (day 12 and day 16) and lead to better clearance of Raji cells.
- FIG. 28 show the proportion and number of transduced T cells in the culture.
- FIG. 29 show the proportion and number of transduced CD8+ T cells in the culture. The results demonstrate that the expansion of the CD8+ subset of T cells expressing fusion proteins provided herein is more robust than the CD8+ subset of CAR T cells.
- FIG. 30 show the proportion and number of transduced CD4+ T cells in the culture. The results demonstrate that the expansion of the CD4+ subset of T cells expressing fusion proteins provided herein is more robust than that of CAR T cells.
- FIG.31 illustrates tumor evasion via antigen downregulation.
- FIG.32 illustrates regular CAR versus expressed CD16 fusion proteins provided herein.
- FIG.33 illustrates regular CAR versus expressed CD16 fusion proteins provided herein with CD19 antigen loss.
- FIG. 34 demonstrates the generation of CD19-deficient Raji cells as model of tumor escape.
- FIG. 35 shows results from co-culturing control antibody (IgG) or rituximab treated Raji and CD19-deficient Raji (CD19KO-Raji) with T cells expressing the indicated constructs for 24h. The results show that CD19-deficient Raji cells are resistant to CAR and SAR-mediated killing through CD19 targeting.
- IgG control antibody
- CD19KO-Raji CD19KO-Raji
- FIG.36 illustrates an experiment where T cells were co-cultured with CD19-deficient Raji cells (CD19KO-Raji cells) and re-challenged with CD19KO-Raji cells at day 8 and day 16 to study ADCC capability in a model of tumor escape where CD19 antigen was absent.
- the cells were cultured in the presence of IL-2 cytokine to promote T cell survival and proliferation.
- FIG.37 show the number of CD19KO-Raji cells remaining in the culture in the presence of control antibody (IgG) or rituximab (anti-CD20).
- IgG control antibody
- rituximab anti-CD20
- FIG. 38 show the viability of CD19KO-Raji cells (proportion of live cells and proportion of dead cells).
- FIG. 39 show the proportion and number of transduced T cells in the culture.
- FIG. 40 show the proportion and number of transduced CD4+ T cells in the culture.
- FIG. 41 show the proportion and number of transduced CD8+ T cells in the culture.
- FIG. 42 shows the mRNA expression levels of CD3 ⁇ (A) and Fc ⁇ RI ⁇ (B) in various human immune cells isolated from peripheral blood samples of healthy donors.
- CD3 ⁇ and Fc ⁇ RI ⁇ are essential plasma membrane signaling adaptor proteins that play a crucial role in the interaction with CD16A and the subsequent expression of CD16A on the cell surface.
- FIG.43 demonstrates the surface expression of the Activation Induced Clipping System (AICS) carrying the anti-CD19 single-chain fragment-variable (humanized FMC63 clone) in THP- 1, a monocyte cell line derived from an acute monocytic leukemia patient.
- FIG. 1 Activation Induced Clipping System
- FIG. 44 illustrates the importance of the FC-binding domain of CD16A in the proper surface expression of the Activation Induced Clipping System (AICS).
- AICS Activation Induced Clipping System
- Jurkat cells were transduced with CD27-AICS (A) or CD27-AICS constructs lacking the FC-binding domain (B and C), all linked to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide.
- the cells were subsequently stained with antibodies specific for the CD27 ectodomain and CD16, and the expression of CD27-AICS constructs was evaluated on eGFP-positive live cells using flow cytometry.
- FIG.45 illustrates wild-type CD16A as well as an examplary fusion protein.
- transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
- the transitional phrase “consisting of” excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of” is associated).
- the transitional phrase “consisting essentially of” limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.
- nucleic acid or set of nucleic acids that encode(s) a fusion protein comprising a target binding domain that binds a first target, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain.
- ADAM Disintegrin and Metalloproteinase
- 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 fusion proteins or combinations of fusion proteins and CAR polypeptides may be encoded on a single nucleic acid or may be encoded by more than one nucleic acid (a set of nucleic acids).
- the first target may be an antigen.
- the target binding domain is an 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
- 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. Thus, for example, 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 term “antigen” as used herein 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.
- 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, GD2, 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, PD-L1, BCMA, 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.
- occupying the antigen by the target binding domain can prevent the antigen’s binding the receptor, keeping the immune cell in an active state.
- 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.
- 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), 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), M
- 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 anticancer 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 301 nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre 361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr 421 wqqgnvfscs vmhealhnhy tqkslslspg k nts, the target binding domain is a scFv that
- 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.
- Table 2 Amino acid sequences of representative anti-CD20 antibody fragments Polypeptide Sequence ofatumumab 1 evqlvesggg lvqpgrslrl scaasgftfn dyamhwvrqa pgkglewvst iswnsgsigy heavy chain 181 slssvvtvps sslgtqtyic nvnhkpsntk vdkkaepksc dkthtcppcp apellggpsv (SEQ ID NO: 241 flfppkpkdt lmisrtpevt cvvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty 15) 301 rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt lpps
- 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. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- the target binding domain binds mesothelin. In some embodiments, 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.
- 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 3. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- Table 3 Amino acid sequences of representative anti-PD-L1 antibody fragments Polypeptide Sequence atezolizumab 1 evqlvesggg lvqpggslrl scaasgftfs dswihwvrqa pgkglewvaw ispyggstyy h h i 61 adsvkgrfti sadtskntay lqmnslraed tavyycarrh wpggfdywgq gtlvtvssas [ ] n some em o ments, t e us on prote n targets t e pat way y n ing EGFR.
- the target binding domain is a scFv that binds EGFR.
- the target binding domain is derived from the sequence of a commercially available anti-EGFR antibody, antibody fragment, or variant thereof, for example, cetuximab (Erbitux®), panitumumab (Vectibix®), necitumumab (Portrazza®), and amivantamab (Rybrevant®), the amino acid sequences of the heavy and light chains of which are set forth in Table 4. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- the fusion protein blocks the NKG2A/HLA-E pathway.
- target binding domain is a scFv that binds NKG2A or HLA-E.
- the target binding domain is derived from the sequence of a commercially available anti-NKG2A antibody, antibody fragment, or variant thereof, for example, monalizumab (formerly IPH2201) and humanized Z199; the amino acid sequences of the heavy and light chains of which are set forth in Table 5. Any one of the nucleic acids or sets of nucleiac acids provided herein may encode any one of such sequences.
- Table 5 Amino acid sequences of representative anti-NKG2A antibody fragments Polypeptide Sequence monalizumab 1 evqlvqsgae vkkpgeslki sckgsgysft sywmnwvrqm pgkglewmgr idpydsethy uch as a B cell or T cell.
- Immune cells expressing fusion proteins 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), CD19, CD20, CD27, CD70, or CD117, or mesothelin.
- B cell maturation antigen is, for example, (BCMA), CD19, CD20, CD27, CD70, or CD117, or mesothelin.
- ADAM 17 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.
- ADAM 17 is expressed on NK cells generally, as well as the CD3-CD56 bright and CD3-CD56 dim NK cell subsets.
- ADAM 17 is also expressed on CD3 + CD56 + NKT cells, but ADAM 17 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. 6:153 (2016)).
- Immune cell activation results in increased ADAM 17 activity and therefore target shedding, for example, stimulation with phorbol myristate acetate, IL-12, and IL- 18.
- Activation of immune cells expressing a fusion protein as disclosed herein results in ADAM 17-mediated fusion protein cleavage within the cleavage domain, releasing the target binding domain into the extracellular space as a soluble protein.
- the cleavage domain comprises any domain of a protein cleavable by ADAM 17, which includes variants thereof.
- proteins include, but are not limited to, CD16A, CD62L, TNF- ⁇ , TNF receptor I, and TNF receptor II.
- the cleavage domain contains the amino acid sequence AVSTI (SEQ ID NO: 37), or a variant thereof.
- a “variant” is any molecule with the same desired activity (such as cleavable by ADAM 17) 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: 37, and one or more variants of amino acids at positions 1, 2, 4, and/or 5.
- the cleavage domain contains the amino acid sequence KLDKSFSMIKEGDYN (SEQ ID NO: 38), or a variant thereof. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0117] In some embodiments of any one of the compositions or methods provided herein, the cleavage domain is derived from the extracellular (EC) domain of a protein cleavable by ADAM 17, which includes variants thereof. As used here, fusion proteins that in some embodiments comprise such an extracellular domain are also referred to herein as SAR, and cells, such as T cells, expressing such fusion proteins are referred to as SAR cells, such as SAR T cells.
- the cleavage domain is derived from the EC domain of CD16A and, thus, the fusion protein can comprise such an extracellular domain.
- the cleavage domain comprises the extracellular domain of CD16A.
- the cleavage domain is derived from the EC domain of CD62L.
- the ADAM 17 cleavage domain is embodied in a domain native to or derived from a CD16A isoform or a CD62L isoform. In some embodiments, this cleavage domain contains the extracellular domain of CD16A having the amino acid sequence set forth below (SEQ ID NO: 39), or a variant thereof.
- the cleavage domain comprises a variant of SEQ ID NO: 39 such that it has a higher affinity for IgG as compared to the wild-type sequence.
- the cleavage domain has a F176V substitution (i.e., a valine at position 176 in place of the phenylalanine, shown as a boxed amino acid in SEQ ID NO: 39).
- the cleavage domain has a Y158V substitution (i.e., a valine at position 158 of SEQ ID NO:39).
- the cleavage domain contains the extracellular domain of CD62L having the amino acid sequence set forth below (SEQ ID NO: 40), or a variant thereof.
- a signal peptide is included in the fusion protein, such as N-terminal to the target binding domain.
- the term “signal peptide” as used herein 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. Fusion proteins containing a signal peptide and transmembrane domain can be trafficked to the plasma membrane.
- the signal 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).
- EPO erythropoietin
- IL-2 human or mouse Ig-kappa chain V-III
- IgK VIII tissue plasminogen activator
- SEAP secreted alkaline phosphatase
- Amino acid sequences of representative signal peptides are listed in [0123] Table 6. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- the fusion protein comprises one or more linkers.
- a linker is present between sequences of the target binding domain (e.g., a linker disposed between the variable heavy (VH) and variable light (VL) domains of a scFv target binding domain).
- a linker is present between the target binding domain and the cleavage domain.
- a linker is present between a target binding domain and the extracellular domain of CD16A, CD62L, TNF- ⁇ , TNF receptor I, and TNF receptor II, such as when the fusion protein comprises a target binding domain and an extracellular domain of CD16A, CD62L, TNF- ⁇ , TNF receptor I, and TNF receptor II, which includes variants thereof.
- the linker comprises an amino acid having the sequence GGGX, GGGGX (SEQ ID NO: 54), or GSSGSX (SEQ ID NO: 55), where X is either cysteine (C) or serine (S), or a repeating sequence thereof.
- the linker has the amino acid sequence GGGGS (SEQ ID NO: 56), GGGGSGGGGS (SEQ ID NO: 57), GGGGSGGGGSGGGGS (SEQ ID NO: 58), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 59), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 60), KESGSVSSEQLAQFRSLD (SEQ ID NO: 61), EGKSSGSGSESKST (SEQ ID NO: 62), or GSAGSAAGSGEF (SEQ ID NO: 63).
- the transmembrane domain can be connected to the cleavage domain, which is generally between the transmembrane domain and the target binding 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 cleavage domain of the fusion protein from the cell surface after ADAM 17-mediated cleavage.
- the transmembrane domain generally localizes the fusion protein to the endoplasmic reticulum during translation and delivery to the cell surface.
- the transmembrane domain interacts with signaling adaptor protein CD3 ⁇ and Fc Fragment of IgE Receptor Ig (FCER1G).
- the transmembrane 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 7. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- Table 7 Amino acid sequences of transmembrane domains Transmembrane domain Sequence [0128]
- the fusion protein further comprises an intracellular domain (IC) of any one of the proteins cleavable by ADAM 17 provided herein, which includes variants thereof, which can be connected to the transmembrane domain.
- the IC domain can provide signaling capacities to the fusion protein.
- the IC domain is derived from CD16A, which includes variants thereof.
- the CD16A IC 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 CD62L IC domain is known to interact with ⁇ - actinin, calmodulin, ezrin, meosin, protein kinase C (PKC) isozymes and AP-1. See, Ivetic et al., Front. Immunol.10:1068 (2019).
- the intracellular domain is derived from CD16A, but lacks the CD16 signal peptide. In some embodiments, the intracellular domain is derived from CD62L.
- the nucleic acid or set of nucleic acids encode(s) a fusion protein containing the amino acid sequence set forth below (SEQ ID NO: 69), and which contains the features, from N-terminus to C-terminus, a PD1 single peptide (SP), a PD1 EC domain, a G4S linker, a CD16 EC domain, a CD16 transmembrane (TM) domain, and a CD16 intracellular (IC) domain (which lacks a CD16 signal peptide) set forth in Table 8.
- This latter portion of the larger fusion protein has the amino acid sequence SEQ ID NO: 74; the amino acid sequences of its components are set forth in Table 11.
- the CAR polypeptide is made up of a second target binding domain that binds to a second target and a transmembrane domain.
- the CAR polypeptide may also comprise an intracellular domain.
- the intracellular domain may comprise a signaling domain.
- this second target is different from the first target (of the fusion protein).
- the second target binding domain is an antibody fragment (e.g., a scFv).
- the second target may be any one of the targets provided herein, such as any one of the first targets provided herein.
- the second target is a cancer antigen as provided herein.
- the second target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell. In other embodiments, the second target is a receptor or other ligand on an immune cell. In some embodiments, the immune cell is a B cell or T cell. [0135] In other embodiments, the second target is BCMA, CD19, CD70, or PD-L1. In some embodiments, the CAR target binding domain is a scFv that binds BCMA. In some embodiments, the CAR target binding domain is derived from the sequence of a commercially available anti- BCMA antibody, antibody fragment, or derivative thereof. In some embodiments, the CAR target binding domain is derived from belantamab (Blenrep®) heavy and light chains.
- Blenrep® belantamab
- amino acid sequence of belantamab heavy chain is set forth below (SEQ ID NO: 88). 1 qvqlvqsgae vkkpgssvkv sckasggtfs nywmhwvrqa pgqglewmga tyrghsdtyy 61 nqkfkgrvti tadkststay melsslrsed tavyycarga iydgydvldn wgqgtlvtvs 121 sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vhtfpavlqs 181 sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkkve pkscdkth
- the CAR target binding domain is a scFv that binds CD70.
- Anti- CD70 antibodies and CD70-binding fragments thereof are known in the art. See, e.g., U.S.
- the CAR target binding domain is derived from a commercially available anti-CD38 antibody, CD38-binding fragments thereof, or derivative thereof, e.g., daratumumab (Darzalex®), isatuximab (Sarclisa®), and mezagitamab (TAK-079), the amino acid sequences of the heavy and light chains of which are set forth in Table 12.
- a commercially available anti-CD38 antibody CD38-binding fragments thereof, or derivative thereof, e.g., daratumumab (Darzalex®), isatuximab (Sarclisa®), and mezagitamab (TAK-079)
- daratumumab Darzalex®
- isatuximab Sarclisa®
- mezagitamab TAK-079
- Table 12 Amino Acid Sequences of anti-CD38 antibody fragments Polypeptide Sequence daratumumab 1 evqllesggg lvqpggslrl scavsgftfn sfamswvrqa pgkglewvsa isgsgggtyy 61 adsvkgrfti srdnskntly lqmnslraed tavyfcakdk ilwfgepvfd ywgqgtlvtv [ 0141]
- the CAR target binding domain e.g., a scFv, binds CD138.
- Anti- CD138 antibodies and CD138-binding fragments thereof are known in the art. See, e.g., U.S. Patents 9,221,914, 9,387,261, 9,446,146, and 10,975,158 and U.S. Patent Application Publications 2007/0183971, 2009/0232810, 2018/0312561, 2019/0100588, 2020/0384024, and 2020/0392241, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- the CAR target binding domain e.g., a scFv, binds FCRH5.
- Anti-FCRH5 antibodies and FCRH5-binding fragments thereof are known in the art, e.g., cevostamab, and U.S. Patents 8,466,260, 9,017,951, 10,323,094, 10,435,471, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0143] The amino acid sequence of a representative anti-FCRH5 heavy chain is set forth below (SEQ ID NO: 96).
- Anti-GPRC5D antibodies and GPRC5D-binding fragments thereof are known in the art, e.g., talquetamab, U.S. Patents 10,562,968 and 10,590,196, and U.S. Patent Application Publications 2019/0367612, 2020/0123250, 2020/0190205, 2020/0270326, and 2021/0054094, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0146] The amino acid sequence of a representative anti-FCRH5 antibody scFv fragment is set forth below (SEQ ID NO: 98).
- the antigen binding domain is derived from a commercially available anti- SLAMF7 antibody, SLAMF7-binding fragment, or derivative thereof, e.g., elotuzumab (Empliciti®).
- elotuzumab Empliciti®
- the amino acid sequence of an elotuzumab heavy chain is set forth below (SEQ ID NO: 99).
- the transmembrane domain of the CAR polypeptide can connect the CAR target binding domain to an intracellular signaling domain.
- the transmembrane domain is directly connected to the CAR target binding domain.
- the transmembrane domain of the CAR may be any one of the transmembrane domains provided herein, such as described for the fusion proteins.
- Amino acid sequences of representative transmembrane domains are listed in Table 7. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.Error! Reference source not found.
- the amino acid sequence of a naturally occurring transmembrane domain may be modified by an amino acid substitution to avoid binding of such regions to the transmembrane domain of the same or different surface membrane proteins to minimize interactions with other members of a receptor complex.
- the CAR polypeptide includes a hinge domain disposed between the CAR target binding domain and the transmembrane domain.
- a hinge domain may provide flexibility in terms of allowing the CAR target binding domain to obtain an optimal orientation for target binding, enhancing cell killing activities, etc.
- the hinge domain is derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the hinge domain is derived from CD3 ⁇ , CD4, CD8 ⁇ , CD28, IgG1, IgG2, or IgG4. Amino acid sequences of representative hinge domains of which are listed in Table 13. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 13: Amino acid sequences of representative hinge domains Hinge domain Sequence CD3 ⁇ (SEQ ID NO: 101) QSFGLLDPK Y ellular signaling and immune cell function.
- the signaling domain may include a primary signaling domain and/or a co-stimulatory signaling domain.
- the intracellular domain is capable of delivering a signal approximating that of natural ligation of an ITAM-containing molecule or receptor complex such as a TCR receptor complex.
- the intracellular signaling domain includes a plurality, e.g., 2 or 3, costimulatory signaling domains described herein, e.g., selected from 4-1BB, CD3 ⁇ , CD28, CD27, ICOS, and OX40.
- the intracellular signaling domain may include a CD3 ⁇ domain as a primary signaling domain, and 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 and 4-1BB-CD28.
- the primary signaling domain is derived from CD3 ⁇ , CD27, CD28, CD40, KIR2DS2, MyD88, 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.
- nucleic acid sequences of representative signaling domains are listed in Table . Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
- Table 14 Amino acid sequences of representative signaling domains Signaling domain Sequence CD3 ⁇ (SEQ ID NO: 108) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR R L T Y L V LLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPL GHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVW K N G N P R E E S
- the nucleic acid encodes a CAR containing the amino acid sequence set forth below (SEQ ID NO: 126), and which contains the features, from N-terminus to C-terminus, a CD8 SP, an anti-CD19 scFv, a
- This latter portion has the amino acid sequence SEQ ID NO: 130; the amino acid sequences of its components are set forth above.
- the sequences encoding the fusion protein and the CAR polypeptide are under the control of the same regulatory elements. In some embodiments, the sequences encoding the fusion protein and the CAR polypeptide are under the control of the different regulatory elements. In some embodiments of any one of the compositions or methods provided herein, the fusion protein-encoding nucleic acid(s) and the CAR-encoding nucleic acid(s) are separate nucleic acid molecules.
- Vectors [0160] The fusion protein-encoding nucleic acid(s) may be introduced to an immune cell by a suitable vector or set of vectors.
- the CAR-encoding nucleic acid(s) may be introduced into an immune cell by the same vector or set of vectors or by a separate 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 fusion protein-encoding sequence, a CAR polypeptide-encoding sequence, or a sequence encoding a fusion protein linked to a CAR polypeptide by a nucleic acid encoding a self-cleaving peptide.
- 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.
- operatively linked as used herein is to be understood that 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 fusion protein-encoding nucleic acid sequence).
- a vector contains a single promoter operatively linked to a fusion- protein encoding nucleic acid and/or a CAR-encoding nucleic acid.
- the fusion-protein encoding nucleic acid and the CAR-encoding nucleic acid are separated by a nucleic acid encoding a self-cleaving peptide.
- the fusion-protein encoding nucleic acid and the CAR-encoding nucleic acid are separated by nucleic acid encoding an internal ribosome entry site (IRES).
- IRS internal ribosome entry site
- a vector contains a first promoter operatively liked to a fusion- protein encoding nucleic acid and a second promoter operatively liked to a CAR-encoding nucleic acid.
- two vectors are provided, a first vector containing a promoter operatively linked to a fusion-protein encoding nucleic acid and a second vector containing a promoter operatively linked to a CAR-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.
- pseudotyping “pseudotyped”, “pseudotyped vector”, and “pseudotyped vector particle” are used herein to refer to 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 thusly 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 (SEQ ID NO: 132).
- 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.
- IL-18 pretreatment further enables transduction.
- 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 fusion protein that achieves 40-60% transduction efficiency.
- cytokine pretreatment of the immune cells followed by transduction with BaEV-LV results in selective expansion of NK cells without the expansion of T regulatory cells, and the expanded memory-like NK cells dominate the peripheral blood lymphocyte compartment in vivo (Shapiro et al., J. Clin. Invest. 132(11):e154334-17 (2022)).
- 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, linear single stranded (ss) DNA or linear double stranded (ds) DNA, 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.
- the vector multiple plasmids, a first plasmid encoding the fusion protein and a second plasmid encoding the CAR polypeptide.
- 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 a pegylated lipids, which are lipids conjugated to polyethylene glycol.
- 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. LNPs, their method of preparation, formulation, and delivery are disclosed in, e.g., U.S.
- 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.
- Cells [0180] 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.
- Representative examples of immune cells include those as shown in FIG. 45. Combination of different immune cells may be 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 fusion protein and, optionally, a CAR polypeptide.
- the immune cells are NK cells.
- the immune cells are a NK cell line, primary NK cells, memory-like NK cells, or induced memory like NK cells.
- the immune cells are monocytes or macrophages.
- 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.
- PBMC peripheral blood mononuclear cells
- the collection, isolation, purification, and differentiation of cells from body fluids and tissues is known in the art. See, for example, Brown et al., PloS One 5:e11373-9 (2010), Rivera et al., Curr. Protoc. Stem Cell Biol.
- 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 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. Immunol. 7:504-9 (2016), Shah et al., PLoS One 8:e76781-9 (2013); Zhao et al., Front Immunol 11:584099-8 (2020)).
- 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 cNsK cells.
- CIML NK cells adoptively transferred into leukemia-bearing mice inhibit tumor growth to a greater degree as compared to conventional NK cells. See, e.g., Cooper et al., Proc. Natl. Acad. Sci. USA 106:1915-9 (2009); Ni et al., J. Exp. Med. 209:2351-65 (2012); Keppel et al., J. Immunol. 190:4754-62 (2013); Romee et al., Sci. Transl. Med.8(357):357ra123-26 (2016).
- NK cells may be effectively used in ACT.
- NK cells grouped by visualization of t-Distributed Stochastic Neighbor Embedding (viSNE) represented the largest cell population after ACT, at the expense of CD3+ cells, CD8+ cells, and ungated cells before ACT (labeled ‘Day Pre’).
- viSNE t-Distributed Stochastic Neighbor Embedding
- Different NK cell subsets positive for NKG2A and PD1 were further tracked after NK ACT infusion (FIG. 20B – FIG. 20C). These data demonstrate this treatment regimen could be adapted to deliver AICS NK cells expressing a fusion protein.
- the cells are allogeneic to the subject receiving the cells, that is, the cells have a complete or at least partial HLA-match with the subject.
- 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 from NK cell lines.
- Suitable NK cell lines 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).
- GvHD graph versus host disease
- the use of NK cells lines avoids the need for leukapheresis, facilitating cell procurement, and avoiding undesirable side-effects. See, e.g., Leung et al., Clin.
- a lentiviral vector is transduced into immune cells.
- the method entails the use of gamma retroviral vectors.
- 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
- dsRNA dsRNA
- ssDNA dsRNA
- dsRNA dsRNA
- ssDNA dsRNA
- dsRNA dsRNA
- ssDNA ssRNA
- dsRNA to deliver the first, the second, and the third nucleic acids.
- the method entails ex vivo or in vivo delivery of linear, circular, or self-amplifying mRNAs. See, e.g., U.S.
- 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.
- compositions of the disclosure include compositions comprising therapeutically effective numbers of genetically modified immune cells and a pharmaceutically acceptable carrier.
- the term “therapeutically effective number of immune cells” refers to a sufficient number of the immune cells that contain the fusion protein-encoding nucleic acid(s) and, optionally, CAR polypeptide-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, and 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.
- 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 the genetically modified immune cells is between approximately 1 ⁇ 10 5 to approximately 6 ⁇ 10 8 cells per kg 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.
- 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.
- 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.
- the compositions are typically isotonic, i.e., they have the same osmotic pressure as blood.
- 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 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 fusion proteins and, optionally, a CAR polypeptide as described herein.
- the term “cancer” as used herein 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, 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), multiple myeloma, ovarian cancer
- RCC
- 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.
- 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, NK cells 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. Administration [0209] 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 methods may include co-administration of another agent, such as an anti-cancer agent, antibody therapy, 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.
- 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. [0219] 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
- EXAMPLE 1 Materials and Methods [0221] CAR vector design.
- the pHIV-CD19-CAR-GFP plasmid was obtained.
- the CD19- CAR consists of the CD8 signal peptide, an CD19 scFv, CD8 hinge, CD8 transmembrane domain, 4-1BB intracellular domain and CD3 ⁇ intracellular domain.
- the PD1-CD16 fusion protein consists of the PD1 signal peptide, the PD1 extracellular domain, a short linker of Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO: 56) and CD16 gene without the CD16 signal peptide.
- the PDL1scFv-CD16 fusion protein consists of the CD8 signal peptide, an anti-PDL1 scFv, a short linker (G4S) (SEQ ID NO: 56) and CD16 gene without the CD16 signal peptide.
- the anti-PDL1 scFv was generated using codon-optimized DNA sequences of the VL and VH regions of atezolizumab.
- the CD19scFv- CD16 fusion protein was generated by combining the CD19scFv from CD19-CAR with CD16 of PDL1scFv-CD16 fusion protein.
- Lentivirus production Lentivirus was produced by co-transfecting HEK293 with the transfer plasmids (pHIV-CD19-CAR-GFP, pHIV-PD1-CD16-CD19-CAR-GFP or pHIV- PDL1scFv-CD16-CD19-CAR-GFP) along with the packaging plasmids pCMV- ⁇ 8.9 and envelope (pCMV-VSVG or pCMV-BaEV) and pAdv plasmids.
- the pAdv plasmid is commercially available as pAdvAdvantageTM from Promega, and the nucleic acid sequence is provided at NCBI Accession No. U47294, version U47294.2, which sequence is incorporated herein by reference.
- Culture supernatants were collected at 24h and 36h, and lentivirus particles were pelleted by ultracentrifugation at 25,000 rpm for 2h at 4°C. Lentivirus particles were resuspended in 200 ⁇ l DMEM medium and stored at -80°C.
- Cell Lines K562, Raji, Daudi, Ramos and NK92 MI cell lines were obtained from ATCC.
- K562 and Raji cells were cultured in RP-10 medium (RPMI 1640 supplemented with 10% FBS, 1 ⁇ penicillin/streptomycin, 2mM L-glutamine, and 7.5 mmol HEPES).
- NK92 MI cells were cultured in X-VIVO TM 15 Hematopoietic serum-free culture media (Lonza) supplemented with 10% human AB serum (Millipore Sigma), 10% FBS, 1 ⁇ penicillin/streptomycin, and 2mM L-glutamine.
- K562 and Raji cells were transduced with VSVG- pseudotyped lentivirus encoding human PDL1 and/or mCherry-luciferase.
- NK92 MI cells were transduced using BaEV-pseudotyped lentivirus.
- Generation and transduction of human primary NK cells Human primary NK cells were isolated from leukoreduction system chamber from a healthy donor after incubation with RosetteSepTM Human NK Cell Enrichment Cocktail (StemCell) followed by Ficoll density gradient centrifugation. Cells were stimulated for 18 hours with 10ng/mL recombinant human IL- 12 (R&D Systems) and 50ng/mL recombinant human IL-18 (R&D Systems) in RP-10.
- NK cells were transduced with BaEV-pseudotyped lentivirus on RetroNectin-coated plates (Takara Bio) with the addition of Vectofusin-1 (Miltenyi). After spinfection, cells were rested for 2 days in RP- 10 with 1ng/mL recombinant human IL-15 (Miltenyi). NK cells were then expanded in NK MACS medium (Miltenyi) supplemented with 5% human serum and 100 U/mL recombinant human IL-2 (Miltenyi).
- NK cells were cultured in the presence of irradiated K562 feeder cells expressing mbIL-21, 4-1BBL and OX40L in RP-10 with 100U/mL recombinant human IL-2 (Miltenyi) for 5 days, then transduced as described above and cultured for another 10 days with the addition of feeders one day after transduction.
- Human primary NK cells were isolated from leukoreduction system chamber from a healthy donor after incubation with RosetteSepTM Human NK Cell Enrichment Cocktail (StemCell) followed by Ficoll density gradient centrifugation.
- NK cells were stimulated for 18 hours with 10ng/mL recombinant human IL- 12 (R&D Systems) and 50ng/mL recombinant human IL-18 (R&D Systems) in RP-10.
- NK cells were transduced with BaEV-pseudotyped lentivirus and rested for 2 days in RP-10 with 1ng/mL recombinant human IL-15 (Miltenyi).
- NK cells were then expanded in NK MACS medium (Miltenyi) supplemented with 5% human serum and 100 U/mL recombinant human IL-2 (Miltenyi).
- Target cells were cultured in RP-10 medium and labelled with 5uM of CellTrace Violet (Thermo Fisher Scientific) in PBS for 20 min at 37°C. Target cells and effector cells were washed twice with RP-10 and co- cultured at the indicated E:T ratios. To measure NK cell cytotoxicity, cells were co-cultured for 4h, then stained with 2 ⁇ L of PE-Annexin V (Biolegend) and 2 ⁇ L of 7-AAD (BD Biosciences) in 50uL Annexin V binding buffer (Biolegend) for 15 min at RT.
- PE-Annexin V Biolegend
- 7-AAD BD Biosciences
- NK cells were co-cultured for 1h, followed by the addition of 0.2uL BD GolgiPlugTM (BD Biosciences), 0.13uL BD GolgiStopTM (BD Biosciences) and 1uL of APC- CD107a (Biolegend). After an additional 5h of co-culture, cells were stained for intracellular IFN ⁇ using BD Cytofix/CytopermTM (BD Biosciences). Cells were acquired using BD LSRFortessaTM and analyzed using FlowJo (Tree Star). [0228] Cytotoxicity of NK cells in vivo. Six to eight week-old NSG mice were obtained from The Jackson Laboratories.
- NSG mice were injected intravenously with 1 million luciferase- expressing K562, Raji or Ramos cells. Three days after tumor inoculation, primary NK cells were injected intravenously. Mice were injected intraperitoneally with IL-2 (up to 75,000 IU/mouse) every 3 days. In vivo bioluminescent imaging (BLI) was performed once or twice a week to monitor tumor burden. Blood samples were collected from the tail vein. After 4-8 weeks, mice were euthanized for organ harvest for flow cytometric analysis. Mice were sacrificed when they became moribund and exhibited significant weight loss. [0229] Kinetics of AICS Shedding.
- NK cells were co-cultured with target cells and stained for surface expression of CD16 at different timepoints.
- CD16 shedding was analyzed by measuring soluble CD16 (or PDL1scFv-CD16 fusion protein) in the supernatant by ELISA or cytometric bead array (CBA).
- CBA cytometric bead array
- PDL1-expressing target cells were pre-treated with conditioned media containing released PDL1scFv-CD16 (from NK cell and target cell co-culture) before co-culture with PD1-expressing NK cells.
- Target cell death was then measured by Annexin V and 7AAD staining.
- NK cells were transduced to express CD19-CAR as well as both native PD1 protein and the PDL1scFv-CD16 fusion protein. NK cells were then co-cultured with PDL1-expressing target cells, and target cell death was measured by Annexin V and 7AAD staining.
- EXAMPLE 2 Activation Induced Clipping System (AICS) NK Cells Link Tumor Antigen Recognition to Regulatory Payload Delivery
- AICS Activation Induced Clipping System
- NK cells expressing an anti-PD1 scFV fusion protein and CD19 CAR enable the targetable delivery of the anti-PD1 payload after CD19 tumor antigen engagement (FIG.1).
- the inhibitory PD1/PDL1 pathway in the TME is the target in this example, which will allow these enhanced memory-like NK CAR cell’s effect on the TME to be examined in vitro.
- NK cell line NK92 MI does not normally express CD16 or PD1 as measured by Flow cytometry (FIG.2A, top panel), but transfection of the anti-PD1-CD16 fusion protein results in surface expression of both antigens (FIG.2A, bottom panel).
- NK cell activation can be measured by co-culture with cancer cell lines, with and without exogenous CD19 expression. Cancer cell lines include K562 leukemia cells, which do not express CD19, but can still activate NK cells by the NK activating receptor NKG2D and Raji B cell lymphoma cells, which do express CD19, and which will activate CD19 CAR expressing cells.
- NK92 MI cells expressing CD19 CAR, PD1-CD16 fusion protein, PD1-CD16 fusion protein and CD19 CAR, and CD19 CAR and PDL1scFv-CD16 fusion proteins were tested for CD16 expression with negative control (no stimulation), or positive control (PMA/Iono), and co- cultured with K562 ( ⁇ exogenous CD19) and Raji ( ⁇ exogenous CD19) cells (FIG. 2B).
- NK cells with Raji cells induced the shedding of PD1-CD16 fusion protein (based on downregulation of CD16 expression on the surface of NK cells), which was induced by CAR engagement (absence of CAR did not induce CD16 shedding upon co-culture with Raji cells).
- Cancer cells expressing PDL1 induced more downregulation of cell-associated CD16, which can be due to increased shedding or binding of the PD1-CD16 fusion protein with PDL1.
- the co- culture of NK cells with K562 cells also induced the shedding of PD1-CD16 fusion protein, and shedding is more pronounced when K562 express PDL1. Shedding of the entire fusion protein is confirmed when the same cells are stained for cell-surface PD1 (FIG.2C).
- NK cells kill cancer cells through antibody-dependent cell-mediated cytotoxicity (ADCC) when tumor specific antibodies are present.
- ADCC antibody-dependent cell-mediated cytotoxicity
- the inventive fusion protein-expressing NK cells were tested for their ability to kill target cells in the absence of antibodies (FIG.3).
- NK cells were co-cultured with K562 cells ( ⁇ exogenous CD19) and the killing of target cells by NK cells was measured.
- K562 cells ⁇ exogenous CD19
- PDL1+ K562 cells strongly inhibited the cytotoxic functions of control NK cells or CD19-CAR expressing NK cells (FIG.3, top left two panels).
- FIG. 4 shows a direct comparison of the different NK cell conditions as assayed in FIG. 3.
- Cells expressing the PDL1scFv-CD16 fusion protein induced the highest level of cytotoxicity. Similar results were obtained when the same NK cells were tested against Raji cells ( ⁇ exogenous CD19), as illustrated in FIG.5 and FIG.6.
- the activation of the NK92 MI cells were measured based on IFN ⁇ expression.
- PDL1scFv-CD16 fusion protein expressing the PDL1scFv-CD16 fusion protein along with CD19-CAR induced strong IFN ⁇ expression when stimulated with K562 ( ⁇ exogenous CD19) or Raji ( ⁇ exogenous CD19) cells (FIG. 7).
- NK92 MI cells expressing PDL1scFv-CD16 fusion protein and CD19-CAR were co-cultured with K562 ( ⁇ exogenous CD19) or Raji ( ⁇ exogenous CD19) cells.
- the PDL1+ cell lines were compared with the parental cell lines (PDL1-).
- NK cells were engineered to express both PDL1scFv-CD16 fusion protein and the CD19-CAR (PDL1scFv-CD16-CD19-CAR). These NK cells were co-cultured with K562 or Raji engineered to express exogenous PDL1.
- the PDL1+ cell lines (FIG. 8, right two panels) were compared with the parental cell lines (PDL1-, FIG.8, left two panels) for PDL1 expression over time.
- NK cells expressing PDL1scFv-CD16 fusion protein and CD19-CAR cocultured with either K562 or Raji cells expressing PDL1 induced the downregulation of PDL1 on cancer cells within 1 hour and continued to decrease over time, suggesting that PDL1 was quickly engaged by the PDL1scFv- CD16 fusion protein (FIG.8).
- EXAMPLE 3 CD19-CD16 Fusion Protein Killing [0238] A different approach is a CD16-based fusion protein with an ability to activate and result in CAR-independent cytotoxicity. Described herein are CD19-CD16 fusion protein-expressing cells, which are compared to CD19-CAR expressing cells (FIG.9).
- the CD19 portion of the fusion protein is an CD19 antibody fragment (e.g., scFv) targeting the CD19 antigen on cancer cells.
- CD19scFv-CD16 fusion protein can induce the killing of the target cells by NK cells.
- NK92 MI cells were engineered to express no CAR (negative control) (FIG. 10, circles), CD19-CAR (regular CAR, positive control) (FIG. 10, squares), and CD19scFv-CD16 fusion protein (FIG. 10, triangles).
- the NK92 cells were sorted to obtain a pure population (>95% positive for CD19-CAR or CD19scFv-CD16 fusion protein), based on GFP expression.
- NK cells were co-cultured with the B cell lymphoma cell lines Raji, Daudi, and Ramos, and the killing of target cells were measured by AnnexinV+ and 7AAD+ stained cells.
- Negative control, untransduced NK cells were effective at killing Raji cells (based on % cell death on y axis), but less so at killing Daudi and Ramos cells.
- NK cells expressing CD19scFv-CD16 fusion protein showed enhanced killing of the target cells as compared to negative control NK cells, and at a level similar to that of NK cells expressing the regular CD19- CAR as illustrated in FIG.10.
- NK cells expressing CD19scFv-CD16 fusion protein showed increased activation based on CD107a expression as compared to control NK cells and was similar to NK cells expressing the regular CD19-CAR.
- NK cells expressing CD19scFv-CD16 fusion protein also showed increased production of IFNg cytokine as compared to control NK cells, but less than NK cells expressing the regular CD19-CAR, suggesting that the regular CAR may be better than the CD16-based fusion protein at inducing cytokine production.
- CD19-CD16 fusion protein are also functional in T cells. T cells from healthy donor peripheral blood was engineered to express 1) No CAR (control), 2) CD19-CAR, 3) CD19scFv-CD16 fusion protein, 4) CD19scFv-CD16 fusion protein + 4-1BBL.
- the scFv of CD19-CAR was fused with the complete CD16 protein (excluding the signal peptide).
- the regular CD19-CAR contains the cytoplasmic domain of 4-1BB, a co- receptor that activates NK cells and T cells function and helps these cells persist in vivo.
- the CD19scFv-CD16 fusion protein does not contain the cytoplasmic domain of 4-1BB, CD19scFv-CD16 fusion protein and 4-1BBL, the ligand of 4-1BB, was co-expressed, these additional proteins may also help the cells persist in vivo.
- the results of transduction were analyzed by flow cytometry (FIG.12).
- the third rows of plots indicate the efficiency of transduction based on GFP expression after 9 days of culture.
- Control T cells (top row) were untransduced, hence they showed 0.03% of GFP+ cells.
- T cells transduced with regular CD19-CAR or CD19scFv-CD16 fusion protein were 18.8% and 17.7% GFP+, indicating that they are similar in the levels of transduction, and therefore a similar number of transduced T cells co-cultured with target cancer cells are in the assays, and thus a fair comparison in regard to the number of target cells killed can be made (the ratios of cancer cells versus T cells will be similar).
- T cells were co-cultured with B cell lymphoma cell lines (Raji, Daudi and Ramos) and the killing of target cells by T cells was measured (FIG. 13). Negative control, untransduced T cells were effective at killing Raji cells (based on % cell death on y axis), but less so at killing Daudi and Ramos cells.
- T cells expressing CD19scFv-CD16 fusion protein showed enhanced killing of the target cells as compared to control T cells, but at a lower level than that of T cells expressing the regular CD19-CAR.
- T cells expressing CD19scFv-CD16 fusion protein + 4-1BBL were less efficient at killing the target cells, however, only 2.68% of these cells were GFP+, and therefore transduced.
- CD107a a degranulation marker indicating the release of cytotoxic molecules by T cells
- the bottom graphs show the expression of CD107a on transduced T cells only (GFP+ only).
- T cells expressing the CD19scFv-CD16 fusion protein had increased activation based on CD107a expression as compared to control T cells, but less than T cells expressing the regular CD19-CAR (FIG.13).
- the activation of T cells based on the production of the IFN ⁇ cytokine (FIG.15) was similar to CD107a (FIG. 13).
- the top graphs show the production of IFN ⁇ on total T cells (untransduced and transduced, GFP- and GFP+), and the bottom graphs show the production of IFN ⁇ on transduced T cells only (GFP+ only).
- T cells expressing CD19scFv-CD16 fusion protein also showed increased activation based on their production of IFN ⁇ cytokine as compared to control T cells, but less than T cells expressing the regular CD19-CAR.
- the shedding of CD19scFv-CD16 fusion protein was measured. The right two columns of plots indicate the expression of CD16 on T cells (FIG. 17).
- CD19scFv-CD16 fusion protein Primary human T cells genetically engineered to express the CD19scFv-CD16 fusion protein were co-cultured with B cell lymphoma cell lines (Raji, Daudi and Ramos), and the expression of CD16 was measured.
- the co- culture of T cells with all cancer cells induced the downregulation of CD16 expression on the surface of T cells, which may indicate that the CD19scFv-CD16 fusion protein is being engaged by CD19 antigen on the cancer cells, or that the CD19scFv-CD16 fusion protein is being cleaved by the protease ADAM 17 and shed (released) from the cell surface.
- NK cells were engineered to express a PDL1-CD16 fusion protein, using the PDL1 repressive pathway ligand instead of the target antigen of CD19 (FIG. 16). The function of a regular CAR (targeting PDL1 antigen on cancer cells) versus the CD16-based fusion protein (PDL1scFv-CD16 or PD1-CD16 fusion proteins) was compared.
- NK cells were co-cultured with leukemia cell line cells with and without exogenous PDL1.
- NK92 MI cells were engineered to express 1) No CAR (negative control), 2) PDL1-CAR (standard CAR, positive control), 3) PDL1scFv-CD16 fusion protein, and 4) PD1-CD16 fusion protein (FIG.17).
- the scFv targeting PDL1 was replaced with the extracellular domain of PD1. It was expected that the PDL1scFv will bind with stronger affinity to PDL1 as compared with PD1.
- NK cells expressing PDL1scFv-CD16 fusion protein showed enhanced killing of the PDL1+ K562 cells as compared to control NK cells, and at a level similar to that of NK cells expressing the regular PDL1-CAR.
- NK cells expressing PD1-CD16 fusion protein also showed enhanced killing of the PDL1+ K562 cells as compared to control NK cells, but at a lower level than that of T cells expressing the regular CD19-CAR or PDL1scFv-CD16 fusion protein.
- FIG. 18 illustrates the data shown in FIG.17 but compares each NK cell line separately as a function of target cell killing (K562 with and without PDL1 expression).
- NK cells expressing PDL1scFv-CD16 fusion protein showed increased activation based on CD107a expression as compared to control NK cells and was similar to NK cells expressing the regular CD19-CAR.
- NK cells expressing PDL1scFv-CD16 fusion protein also showed decreased production of IFN ⁇ cytokine as compared to NK cells expressing the regular CD19-CAR.
- EXAMPLE 5 Comparing CD16-based Antigen Receptor (SAR) with Conventional CAR in Human Primary T Cells [0254] NK cells can kill cancer cells through various mechanisms. They can target cancer cells directly when engineered to express CAR, but they also have numerous intrinsic receptors that allow them to kill target cells. For example, NK cells express the activating receptor NKG2D that can recognize stress ligands on the surface of the cancer cells (e.g., MICA/B, ULBP1-6).
- NK cells can also recognize tumor antigens indirectly via a mechanism called ADCC where CD16A can bind the Fc portion of an antibody.
- Chimeric antigen receptors are synthetic receptors that allow cells, such as NK cells and T cells, to recognize a tumor antigen on the surface of the cancer cell and trigger the release of granules to kill the cancer cell.
- a subset of NK cells naturally express CD16A.
- NK cells can kill the cancer cell through CD16A by ADCC.
- Primary NK cells from the peripheral blood express high levels of CD16A.
- CD16A When NK cells are activated, with various stimuli, such as using PMA/ionomycin (activator of intracellular signaling pathways), IL-12 and IL-18 (cytokines), or through engagement with cancer cells (K562 leukemia cell line), CD16A can be cleaved and released from the cell surface. This phenomenon allows the NK cells to disengage the target cell and move on to 2 nd and 3 rd , etc. target cells allowing serial killing. CD16A shedding may also prevent NK cell exhaustion. The shedding of CD16A is mediated by the metalloprotease ADAM 17.
- IFN ⁇ cytokine production and degranulation (CD107a) of T cells upon co-culture with Raji lymphoma cells was also compared.
- CAR-T cells were co-cultured with Raji (B cell lymphoma cell line) for up to 24 days.
- the CAR-T cells were re-challenged with Raji cells at day 8 and day 16 to induce chronic stimulation of T cells and to study long-term persistence and expansion.
- T cells expressing the CD16-based receptors (SAR) were less activated than CAR-T cells upon engagement of Raji cells.
- the cells were cultured in the presence of IL-2 cytokine to promote T cell survival and proliferation.
- CAR-T cells and SAR-T cells were able to eliminate Raji cells more efficiently than untransduced T cells, however SAR-T cells were more potent than CAR-T cells at later timepoints (day 12 and day 16) and lead to better clearance of Raji cells. It was also found that SAR-T cells expanded more than CAR-T cells, as the numbers of SAR-T cells were about 5-fold higher than the number of CAR-T cells at day 12 and day 16, and about 10-fold higher at day 24. The results also demonstrate that the expansion of the CD4+ subset of SAR-T cells and the CD8+ subset of SAR-T cells is more robust than the CAR-T cells, respectively.
- CD16-based antigen receptor could also mediate antibody- dependent cell-mediated cytotoxicity (ADCC), which could limit tumor escape, by allowing the SAR to engage both CD19 (via scFv) and CD20 (via using a therapeutic antibody targeting CD20 called rituximab), was examined. In the presence of a tumor antigen specific antibody, it was found that cancer cells can be killed through CD16A through ADCC.
- a model of tumor escape was established using CRISPR-Cas9 technology to knockout (delete) the CD19 gene in Raji cells. These cells still express other markers such as CD20 and CD22 which can be targeted.
- gRNAs guide RNAs
- Cas9-RFP enzyme Cas9-RFP enzyme to form RNPs
- Raji cells were electroporated with RNPs.
- CD19 antigen was deleted in about 33% of Raji cells.
- the CD19 knockout Raji cells were sorted to obtain a pure population (>99%).
- Control antibody (IgG) or rituximab treated Raji and CD19-deficient Raji (CD19KO- Raji) were co-cultured with T cells expressing the indicated constructs for 24h.
- the results (FIG. 35) show that CD19-deficient Raji cells are resistant to CAR and SAR-mediated killing through CD19 targeting.
- T cells expressing CD19scFv-haCD16-GFP were able to kill CD19-deficient Raji cells treated with rituximab which targets CD20, thus inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
- CAR-T cells were co-cultured with CD19KO-Raji cells for up to 24 days.
- the CAR-T cells were re-challenged with CD19KO-Raji cells at day 8 and day 16 to study the ADCC capability of SAR in a model of tumor escape where CD19 antigen was absent.
- the cells were cultured in the presence of IL-2 cytokine to promote T cell survival and proliferation.
- the cells were also treated with either a non-targeting negative control antibody (IgG) or rituximab (anti- CD20) to induce ADCC.
- IgG non-targeting negative control antibody
- rituximab anti- CD20
- FIGS.37-39 The results show the number of CD19KO-Raji cells remaining in the culture in the presence of control antibody (IgG) or rituximab (anti-CD20).
- the results demonstrate that SAR-T cells were more potent at eliminating CD19KO- Raji cells in the presence of rituximab as compared to untransduced T cells or CAR-T cells, suggesting that the CD16 moiety of the SAR in capable of inducing ADCC.
- the high-affinity CD16-based SAR (CD19scFv-haCD16, variant V158) was slightly better than the low affinity version of SAR (CD19scFv-CD16, variant F158) at inducing killing of CD19KO-Raji cells in the presence of rituximab.
- the results demonstrate that SAR-T cells expand more than CAR-T cells when co-cultured with CD19KO-Raji cells in the presence of rituximab, as the numbers of SAR- T cells were about 3-fold higher than the number of CAR-T cells at day 20.
- FIG.40 show the proportion and number of transduced CD4+ T cells in the culture.
- FIG.41 show the proportion and number of transduced CD8+ T cells in the culture.
- CD16A interacts with adaptor signaling proteins CD3 ⁇ and FCER1G exclusively via transmembrane domains.
- both CD3 ⁇ and FCER1G adaptor signaling proteins are transmembrane entities characterized by extremely short extracellular domains
- CD16A is characterized by an extremely short intracellular domain, which has no known signaling function.
- the interaction of CD16A with these adaptor proteins is not only necessary but also sufficient for its surface expression and signaling, devoid of a predilection for selective interactions between the two entities.
- the functional capacity of CD16A to activate diverse immune cell types, such as NK cells, T cells, monocytes, and macrophages has been established.
- FIG.42 shows the full spectrum of immune cell types that express CD3 ⁇ and FCER1G, and expression of fusion proteins as provided herein in human NK cells, T cells, and monocytes has been demonstrated.
- FIG.42 (A) CD3 ⁇ mRNA and (B) FCER1G mRNA show expression in human peripheral blood-derived immune cells. Population-averaged gene expression data for purified human immune cells was acquired from the ImmGen Human Cell Atlas dataset. This dataset was generated through ultra-low-input RNA-sequencing using peripheral blood mononuclear cells from two healthy donors. The data is publicly accessible and can be found at immgen.org.
- FIG.43 demonstrates the surface expression of the Activation Induced Clipping System (AICS) carrying the anti-CD19 single-chain fragment-variable (humanized FMC63 clone) in THP-1, a monocyte cell line derived from an acute monocytic leukemia patient.
- AICS Activation Induced Clipping System
- Untransduced THP-1 cells (A), THP-1 cells expressing human CD16A (B), and FMC63-AICS (C), connected to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide, were subjected to staining with an isotype control, as well as antibodies specific for human CD16 and FMC63 scFv. Surface expression of the constructs was subsequently analyzed using flow cytometry.
- the THP-1 cell line (TIB-202) was procured from the American Type Culture Collection (ATCC) and maintained in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 5 mM L-glutamine, and 1% penicillin/streptomycin.
- THP-1 cells involved the use of VSV-G pseudotyped lentiviral particles to introduce two distinct genetic constructs: a biscistronic cDNA encoding human endogenous CD16A and enhanced GFP (eGFP) fused with a self-cleaving P2A peptide (hCD16A-P2A-eGFP), and Activation Induced Clipping System (AICS) carrying anti- CD19 single-chain fragment-variable and eGFP fused with a self-cleaving P2A peptide (FMC63- AICS-P2A-eGFP).
- eGFP served as an internal fluorescent marker to assess transduction efficiency.
- CD16A and FMC63-AICS Surface expression analysis of human endogenous CD16A and FMC63-AICS on both untransduced and transduced THP-1 cells was conducted via flow cytometry.
- Fluorescent antibodies specific to CD16 PE-conjugated anti-human CD16 mouse monoclonal antibody clone 3G8 from CellSignaling Technology, catalog # 82004S
- FMC63 APC-conjugated REAffinity CD19 CAR FMC63 Idiotype Antibody from Miltenyi Biotec, catalog # 130-127-343 were used for this purpose.
- FIG.44 illustrates the importance of the FC-binding domain of CD16A in ensuring the proper surface expression of the Activation Induced Clipping System (AICS).
- AICS Activation Induced Clipping System
- Jurkat cells were transduced with CD27-AICS (A) or CD27-AICS constructs lacking the F C -binding domain (B and C), all linked to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide.
- the cells were subsequently stained with antibodies specific for the CD27 ectodomain and CD16, and the expression of CD27-AICS constructs was evaluated on eGFP-positive live cells using flow cytometry.
- Activation Induced Clipping System fused to the CD27 ectodomain (amino acids 25-124, using endogenous human CD27 numbering) and enhanced GFP (eGFP), linked with a self-cleaving P2A peptide (CD27-AICS-P2A-eGFP); Activation Induced Clipping System lacking the FC-binding domain (lacking amino acids 1-189, using endogenous human CD16A numbering) with or without the CD8A hinge (CD27-AICS ⁇ Fc-P2A-eGFP and CD27-CD8A hinge-AICS ⁇ Fc- P2A-eGFP).
- eGFP was incorporated as an internal fluorescent marker to facilitate the assessment of transduction efficiency.
- the surface expression of the various CD27- AICS constructs on transduced Jurkat cells was analyzed using flow cytometry. Fluorescent antibodies specific to CD16 (PE-conjugated anti-human CD16 mouse monoclonal antibody clone 3G8 from CellSignaling Technology, catalog # 82004S) and CD27 (APC-conjugated anti-human CD27 mouse monoclonal antibody clone L128 from BD Biosciences, catalog # 337169) were employed for this purpose.
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Abstract
Disclosed are nucleic acids encoding fusion proteins containing a target binding domain, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain.
Description
ACTIVATION INDUCED CLIPPING SYSTEM (AICS) RELATED APPLICATIONS [0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No.63/424,270, filed on November 10, 2022, the entire contents of which are incorporated herein by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] The contents of the electronic sequence listing (M065670537WO00-SEQ-JAV.xml; Size: _______ bytes; and Date of Creation: _______, 2023) are herein incorporated by reference in their entirety. FIELD OF THE INVENTION [0003] Disclosed are nucleic acids, and related compositions and methods, encoding fusion proteins containing an a target binding domain, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain. SUMMARY OF THE DISCLOSURE [0004] Chimeric antigen receptor (CAR)-expressing Natural Killer (NK) cells have shown promising clinical responses, such as during adoptive cell therapy (ACT) in some cancer types (Liu et al., N. Engl. J. Med. 382:545-553 (2020)). However, solid tumors have been difficult to treat with ACT. Unlike dispersed hematologic malignancies (so called liquid tumors), solid tumors, especially late-stage solid tumors, exhibit an inhibitory tumor microenvironment (TME) due to, for example, hypoxia, low pH, suppressive cytokines, lactate, and prostaglandins. These inhibitory factors circumvent the ability of CAR-NK cells to infiltrate and kill cancerous cells (Martinez and Moon, Front. Immunol.10:128-21 (2019)). [0005] In addition, immune checkpoint inhibitors are important therapies to prevent cancerous cells from using peripheral tolerance pathways to limit anti-tumor cytotoxic responses. However, systemic delivery of immune checkpoint inhibitors results in toxicities and may cause inflammatory disorders of many different organs, diarrhea, polyneuropathy, inflammatory neuropathies, anemia, neutropenia, thromboyxtopenia, rashes, pulmonary, neurological,
endocrinopathy, hepatic, and renal complications (Martins et al., Nat Rev. Clin. Oncol. 16:563- 580 (2019)). [0006] Also, CAR T cells, such as those directed against the CD19 antigen, have been introduced in therapy of autoimmune disease. Autoreactive B cells play a key role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. B-cell-depleting monoclonal antibodies, such as rituximab, have poor therapeutic efficacy in autoimmune diseases, mainly due to the persistence of autoreactive B cells in lymphatic organs and inflamed tissues. CAR T cells have been found to induce rapid and sustained depletion of circulating B cells. However, despite the tremendous progress in the clinical management of autoimmune diseases, many patients do not respond to currently used treatments. [0007] Provided herein, are methods and compositions, that can enhance cell killing via targeting with fusion proteins that target a first target and that also have a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17A, which allows for the release of the targeting domain. To date, no evidence exists that a cleavable membrane-bound fusion protein with a target binding domain could be produced to provide controllable release of the target binding domain. Cell killing can also be enhanced with the fusion proteins as provided herein that target a first target and that also have a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17A and that allow for ADCC. Preferably, in some embodiments, the fusion proteins provided herein allow for targeting of a CAR as well as ADCC. Cell killing can be useful in the treatment of a number of diseases and disorders, such as cancer, autoimmune disease, transplant rejection and graft versus host disease (GVHD). [0008] Thus, a first aspect of the present disclosure is directed to a nucleic acid or set of nucleic acids that together comprise a sequence that encodes a fusion protein, wherein the fusion protein comprises a target binding domain that binds a first target, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain, wherein the cleavage domain is located between the target binding domain and the transmembrane domain. In one embodiment, the cleavage domain is of any one of the proteins provided herein cleavable by ADAM 17, such as CD16A. In one embodiment, the cleavage domain comprises the extracellular domain of of any one of the proteins provided herein cleavable by ADAM 17, such as CD16A. In one embodiment of any one of the compositions or methods provided herein, the cleavage domain also comprises an Fc-binding domain.
[0009] In one embodiment of any one of the compositions or methods provided herein, the cleavage domain comprises any one of the relevant specific sequences provided herein. In one embodiment of any one of the compositions or methods provided herein, the extracellular domain of the protein cleavable by ADAM 17 comprises any one of the relevant specific sequences provided herein. [0010] In one embodiment, a composition comprising any one of the nucleic acids or sets of nucleic acids provided herein is provided. [0011] In one embodiment of any one of the compositions or methods provided herein the transmembrane domain interacts with signaling adaptor proteins CD3ζ and Fc Fragment of IgE Receptor Ig (FCER1G). In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain comprises a transmembrane domain of a protein cleavable by ADAM 17. In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain is of any one of the relevant proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain comprises any one of the relevant specific sequences provided herein. [0012] In one embodiment of any one of the compositions or methods provided herein, the fusion protein further comprises an intracellular domain connected to the transmembrane domain, and the nucleic acid or set of nucleic acids encodes the intracellular domain. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain is of a protein cleavable by ADAM 17. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain is of any one of the relevant proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain comprises any one of the relevant specific sequences provided herein. [0013] In one embodiment of any one of the compositions or methods provided herein, the first target is a receptor or ligand on a cell, the killing of which cell is desirable. In one embodiment of any one of the compositions or methods provided herein, the first target is an antigen or other ligand on a cancer cell. In one embodiment of any one of the compositions or methods provided herein, the first target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell. In one embodiment of any one of the compositions or methods provided herein, the first target is a receptor or other ligand on an immune cell. In one embodiment of any one of the compositions or methods provided herein, the immune cell is a B cell or T cell.
[0014] In one embodiment of any one of the compositions or methods provided herein, the killing of the cell by CAR targeting and/or antibody-dependent cellular cytotoxicity (ADCC) is desirable. [0015] In one embodiment of any one of the compositions or methods provided herein, the cancer antigen is any one of the cancer antigens provided herein. [0016] In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds a receptor or other ligand on a B cell. In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds any one of the receptors or other ligands on a B cell provided herein. [0017] In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds a receptor or other ligand on a T cell. In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds any one of the receptors or other ligands on a T cell provided herein. [0018] In one embodiment of any one of the compositions or methods provided herein, the target binding domain comprises an antibody fragment. In one embodiment of any one of the compositions or methods provided herein, the target binding domain comprises a single-chain variable antibody fragment (scFv). [0019] In one embodiment of any one of the compositions or methods provided herein, the target binding domain binds CD19, PDL1 or CD70. In one embodiment of any one of the compositions or methods provided herein, the target binding domain comprises an anti-CD19 antibody fragment, PD1 or an anti-PDL1 antibody fragment, or CD27. [0020] In one embodiment of any one of the compositions or methods provided herein, the nucleic acid or set of nucleic acids further comprise a sequence that encodes a CAR polypeptide, wherein the CAR polypeptide comprises a second target binding domain that binds a second target and a transmembrane domain. In one embodiment of any one of the compositions or methods provided herein, the first target and second target are different. In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain is of a protein cleavable by ADAM 17. In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain is of any one of the relevant proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the transmembrane domain comprises any one of the relevant specific sequences provided herein.
[0021] In one embodiment of any one of the compositions or methods provided herein, the CAR polypeptide further comprises an intracellular domain. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain is of a protein cleavable by ADAM 17. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain is of any one of the relevant proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the intracellular domain comprises any one of the relevant specific sequences provided herein. [0022] In one embodiment of any one of the compositions or methods provided herein, the second target is an antigen or other ligand on a cancer cell. In one embodiment of any one of the compositions or methods provided herein, the second target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell. In one embodiment of any one of the compositions or methods provided herein, the second target is a receptor or other ligand on an immune cell. In one embodiment of any one of the compositions or methods provided herein, the immune cell is a B cell or T cell. [0023] In one embodiment of any one of the compositions or methods provided herein, the cancer antigen is any one of the cancer antigens provided herein. [0024] In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds a receptor or other ligand on a B cell. In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds any one of the receptors or other ligands on a B cell provided herein. [0025] In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds a receptor or other ligand on a T cell. In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds any one of the receptors or other ligands on a T cell provided herein. [0026] In one embodiment of any one of the compositions or methods provided herein, the second target binding domain comprises an antibody fragment. In one embodiment of any one of the compositions or methods provided herein, the second target binding domain comprises a single-chain variable antibody fragment (scFv). [0027] In one embodiment of any one of the compositions or methods provided herein, the second target binding domain binds CD19 or PDL1. In one embodiment of any one of the
compositions or methods provided herein, the second target binding domain comprises an anti- CD19 antibody fragment, PD1 or an anti-PDL1 antibody fragment. [0028] In one embodiment of any one of the compositions or methods provided herein, the fusion protein is any one of the fusion proteins provided herein. In one embodiment of any one of the compositions or methods provided herein, the sequence of the fusion protein is any one of the specific relevant sequences provided herein. [0029] In one embodiment of any one of the compositions or methods provided herein, the fusion protein and CAR polypeptide is any one of the combinations of fusion proteins and CAR polypeptides provided herein. In one embodiment of any one of the compositions or methods provided herein, the sequences of the fusion protein and CAR polypeptide is any one of the combinations of specific relevant sequences provided herein. [0030] In one embodiment of any one of the compositions or methods provided herein, the first and second sequences are operatively linked to the same or different promoters. [0031] In one aspect, a vector or set of vectors comprising any one of the nucleic acids or sets of nucleic acids provided herein is provided. In one embodiment of any one of the compositions or methods provided herein, the vector is a viral vector. In one embodiment of any one of the compositions or methods provided herein, the vector is a non-viral vector. In one embodiment of any one of the compositions or methods provided herein, the non-viral vector is a plasmid. [0032] In one aspect, an immune cell or population of immune cells comprising any one of the nucleic acids or sets of nucleic acids or any one of the vectors or sets of vectors provided herein is provided. In another aspect, an immune cell that expresses any one or any one combination of the fusion proteins and CAR polypeptides provided herein is provided. [0033] In one embodiment of any one of the compositions or methods provided herein, the immune cell(s) express signaling adaptor proteins CD3ζ and Fc Fragment of IgE Receptor Ig (FCER1G). In one embodiment of any one of the compositions or methods provided herein, the cell(s) is/are NK cell(s). In one embodiment of any one of the compositions or methods provided herein, the cell(s) is/are T cell(s). In one embodiment of any one of the compositions or methods provided herein, the cell(s) is a/are monocyte(s) or macrophage(s). [0034] In one aspect, a composition comprises any one of the immune cells or populations of immune cells provided herein is provided. In one embodiment of any one of such embodiments, the immune cell(s) are transformed with any one of the vector or sets of vectors encoding a fusion
protein provided herein. In some embodiments of any one of such compositions, the immune cell(s) is transformed with a second vector containing a nucleic acid or set of nucleic acids that encode a CAR polypeptide. [0035] In one embodiment of any one of the compositions or methods provided herein, the composition further comprises a pharmaceutically effective carrier. In one embodiment of any one of the compositions or methods provided herein, the cell(s) is/are in a therapeutically effective amount. [0036] In one aspect, a method of killing cells, comprising contacting cells with any one of the compositions provided herein is provided. In one embodiment of any one of such methods provided herein, the contacting may be in vitro or may be in vivo by administering any one of the compositions provided herein. [0037] In one aspect, a method of treating a subject with cancer, comprising administering to the subject any one of the compositions provided herein is provided. [0038] In one aspect, a method of treating a subject with autoimmune disease, comprising administering to the subject any one of the compositions provided herein is provided. [0039] In one aspect, a method of treating a subject with a transplant, comprising administering to the subject any one of the compositions provided herein is provided. [0040] In one aspect, a method of treating a subject with GVHD, comprising administering to the subject any one of the compositions provided herein is provided. [0041] In one embodiment of any one of the methods provided herein, the subject is one that has received or is to receive an antibody therapy. [0042] In one embodiment of any one of the methods provided herein, the method further comprises administering to the subject an antibody therapy. [0043] In one embodiment of any one of compositions or methods provided herein, the immune cells are allogeneic but have a complete or partial HLA-match with the subject. [0044] In one embodiment of any one of compositions or methods provided herein, the immune cells are autologous. [0045] In one embodiment of any one of the methods provided herein, the method further comprises isolating immune cells from a tissue or body fluid sample of the subject prior to the contacting or administering of any one of the compositions provided herein. In one embodiment of any one of such methods, the immune cells are isolated based on CD56 expression.
[0046] Working Examples and Figures herein demonstrate ADAM 17-mediated cleavage of the target binding domain of the fusion proteins, which can enable the transformed immune cells to exert effects, such as cell killing, via numerous therapeutic avenues. BRIEF DESCRIPTION OF THE DRAWINGS [0047] FIG. 1 is a schematic illustration that shows a therapeutic avenue of an activation induced clipping system (AICS) in which NK cell activation via CAR engagement causes PL1- CD16 fusion protein shedding, release of PD1-containing target binding domain, binding of PDL1 on cancer cells, and subsequent blockade of PDL1-mediated inhibitory signals. [0048] FIGs.2A – 2C are a set of flow cytometry and bar plots showing that NK92 MI cells express and shed the PD1-CD16 fusion protein. FIG.2A is set of flow cytometer plots that show PD1 and CD16 expression in cells with and without the PD1-CD16 fusion protein. FIG. 2B is a set of bar plots showing CD16 expression in NK92 MI cells expressing either CD19-CAR alone, PD1-CD16 fusion protein alone, PD1-CD16 fusion protein in combination with CD19-CAR, or PDL1scFv-CD16 fusion protein in combination with CD19-CAR exposed to different stimulations. FIG.2C is a set of bar plots showing PD1 expression in the same cells and conditions in FIG.2B. [0049] FIG.3 is a set of line plots showing that PD1-fusion protein expressing cells kill target cells. FIG. 3 is a set of line plots showing NK92 MI cells with and without expression of anti- CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16, or a combination thereof kill K562 target cells. [0050] FIG. 4 is a set of line plots of the same cells and conditions presented in FIG. 3 but graphed as a comparison between the different NK cell preparations. [0051] FIG.5 is a set of line plots showing that PD1-fusion protein expressing cells kill target cells. FIG.5 is a set of line plots showing primary NK (pNK) cells with and without expression of anti-CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16 fusion protein, or a combination thereof kill Raji target cells. [0052] FIGs. 6A – 6B are a set of bar plots showing the functional aspects of PD1-fusion protein expressing cells after co-incubation with target cells. FIG.6A is a set of bar plots showing expression of CD107a by pNK cells with and without expression of anti-CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16 fusion protein, or a combination thereof after co-incubation with
K562 or Raji target cells. FIG. 6B is a set of bar plots showing IFNγ expression under the same conditions as FIG.6A. [0053] FIG.7 is a set of bar plots showing the kinetics of CD16 shedding on NK92 MI cells. FIG.7 is a set of bar plots showing CD16 expression on NK92 MI cells expressing the PDL1scFv- CD16 fusion protein after co-incubation with K562 or Raji target cells with and without exogenous expression of PDL1. [0054] FIG. 8 is a set of bar plots showing the kinetics of PDL1 engagement on cancer cells after treatment with NK92 MI cells. FIG.8 is a set of bar plots showing PDL1 expression on K562 and Raji target cells with and without exogenous expression of PDL1 for different time points after co-culture with NK92 MI cells expressing the PDL1scFv-CD16 fusion protein and the CD19- CAR. [0055] FIG. 9 is a schematic illustration of embodiments comparing cell cytotoxicity in cells expressing a traditional CAR to those using a second therapeutic avenue in which cells express the CD19scFv-CD16 fusion protein without a CAR protein, where NK cell activation causes CD19scFv-CD16 fusion protein cleavage and direct killing of cancer cells. [0056] FIG. 10 is a set of line plots comparing the cytotoxicity ability of NK92 MI cells expressing either the CD19scFv-CD16 fusion protein or anti-CD19 CAR. FIG.10 is a set of line plots that show the cells tested against three cancer targets, cells from the Raji, Daudi, and Ramos cell lines. [0057] FIGs.11A – 11B are a set of bar plots showing the functional aspects of cells expressing the CD19scFv-CD16 fusion protein or anti-CD19 CAR after co-incubation with target cells. FIG. 11A is a set of bar plots showing CD107a expression on NK92 MI cells with and without expression of anti-CD19-CAR, PD1-CD16 fusion protein, PDL1scFv-CD16 fusion protein, or a combination thereof after co-incubation with K562 or Raji target cells with and without exogenous expression of PDL1. FIG.11B is bar plots showing IFNγ expression under the same conditions as FIG.11A. [0058] FIG.12 is a set of flow cytometry plots showing T cells with and without transduction of CD19scFv-CD16 fusion protein or anti-CD19 CAR containing virus. FIG.12 is flow cytometry plots showing T cells transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and assayed for cell scatter, alive/dead, GFP (exogenous expression), CD16, and 4-1BBL.
[0059] FIG. 13 is a set of line plots showing T cell killing of target cells. FIG. 13 is flow cytometry plots showing T cells transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and co-incubated with cells from the cancer cell lines Raji, Daudi, and Ramos. [0060] FIG.14 is a set of bar plots showing the functional aspects of T cells after co-incubation with target cells. FIG. 14 is a set of bar plots showing CD107a expressing T cells (all cells and gated on GFP+ cells) transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and co-incubated with cells from the cancer cell lines Raji, Daubi, and Ramos, or no target cell control. [0061] FIG.15 is a set of bar plots showing the functional aspects of T cells after co-incubation with target cells. FIG.15 is a set of bar plots showing IFNγ expressing T cells (all cells and gated on GFP+ cells) transduced with no virus (negative control), CD19-CAR, CD19scFv-CD16 fusion protein, or CD19scFv-CD16-4-1BBL fusion protein and co-incubated with cells from the cancer cell lines Raji, Daubi, and Ramos, or no target cell control. [0062] FIG.16 is a schematic illustration of embodiments comparing cell cytotoxicity in cells expressing a traditional PDL1-CAR to those using a third therapeutic avenue in which cells express either a PDL1scFv-CD16 fusion protein or a PD1-CD16 fusion protein without a CAR protein, where NK cell activation causes fusion protein cleavage and direct killing of cancer cells. [0063] FIG.17 is a set of line plots showing cells expressing fusion proteins killing of target cells. NK92 MI cells expressing a traditional PDL1-CAR, PDL1scFv-CD16 fusion protein, or PD1-CD16 fusion protein were co-incubated with K562 with and without exogenous PDL1 target cells and assayed for target cell killing. [0064] FIG.18 is a set of line plots showing the cells and conditions in FIG.17 but comparing each cell condition separately. [0065] FIGs.19A – 19B are a set of bar plots showing the functional aspects of T cells after co-incubation with target cells. NK92 MI NK cells transduced with no virus (negative control), or virus containing PDL1-CAR, PDL1scFv-CD16 fusion protein, or PDl-CD16 fusion protein were co-incubated with cells from the cancer cell line K562 with and without exogenous PDL1 expression and assayed for CD107a expression (FIG.19A) or IFNγ expression (FIG.19B). [0066] FIG. 20 is an illustration of how NK cells can kill cells, such as cancer cells, through various mechanisms.
[0067] FIGs. 21A – 21C are a set of flow cytometry plots and line plots that show memory- like NK cells in the peripheral blood lymphocyte compartment after ACT. FIG. 20A is a set of flow cytometry plots that show memory-like NK cells in the peripheral blood after ACT. FIG.20B is a plot that shows the percentage of NKG2A positive NK cell subsets. FIG.20C is line plot that shows the percentage of PD1 positive NK cell subsets. [0068] FIG.22 illustrates the hinge region of CD16 carries the ADAM17 cleavage site. [0069] FIG.23 illustrates the function of a regular CAR (for example, targeting CD19 antigen on cancer cells) versus a CD16-based fusion protein (for example, CD19scFv-CD16 fusion protein also targeting CD19 antigen on cancer cells). [0070] FIG.24 provides examples of construct designs. [0071] FIG. 25 shows that T cells expressing CAR or CD16-based receptor fusion proteins provided herein were comparable in their function. [0072] FIG.26 shows that T cells expressing CD16-based receptors fusion proteins provided herein were less activated than CAR T cells upon engagement of Raji cells. [0073] FIG.27 provides results that demonstrate that CAR T cells and T cells expressing fusion proteins provided herein were able to eliminate Raji cells more efficiently than untransduced T cells, however cells expressing fusion proteins provided herein were more potent than CAR T cells at later timepoints (day 12 and day 16) and lead to better clearance of Raji cells. [0074] FIG. 28 show the proportion and number of transduced T cells in the culture. The results demonstrate that T cells expressing fusion proteins provided herein expand more than CAR T cells. [0075] FIG. 29 show the proportion and number of transduced CD8+ T cells in the culture. The results demonstrate that the expansion of the CD8+ subset of T cells expressing fusion proteins provided herein is more robust than the CD8+ subset of CAR T cells. [0076] FIG. 30 show the proportion and number of transduced CD4+ T cells in the culture. The results demonstrate that the expansion of the CD4+ subset of T cells expressing fusion proteins provided herein is more robust than that of CAR T cells. [0077] FIG.31 illustrates tumor evasion via antigen downregulation. [0078] FIG.32 illustrates regular CAR versus expressed CD16 fusion proteins provided herein. [0079] FIG.33 illustrates regular CAR versus expressed CD16 fusion proteins provided herein with CD19 antigen loss.
[0080] FIG. 34 demonstrates the generation of CD19-deficient Raji cells as model of tumor escape. [0081] FIG. 35 shows results from co-culturing control antibody (IgG) or rituximab treated Raji and CD19-deficient Raji (CD19KO-Raji) with T cells expressing the indicated constructs for 24h. The results show that CD19-deficient Raji cells are resistant to CAR and SAR-mediated killing through CD19 targeting. However, T cells expressing CD19scFv-haCD16-GFP (high affinity CD16-based SAR) were able to kill CD19-deficient Raji cells treated with rituximab which targets CD20, thus inducing antibody-dependent cell-mediated cytotoxicity (ADCC). [0082] FIG.36 illustrates an experiment where T cells were co-cultured with CD19-deficient Raji cells (CD19KO-Raji cells) and re-challenged with CD19KO-Raji cells at day 8 and day 16 to study ADCC capability in a model of tumor escape where CD19 antigen was absent. The cells were cultured in the presence of IL-2 cytokine to promote T cell survival and proliferation. [0083] FIG.37 show the number of CD19KO-Raji cells remaining in the culture in the presence of control antibody (IgG) or rituximab (anti-CD20). The results demonstrate that T cells expressing fusions proteins provided herein were more potent at eliminating CD19KO-Raji cells in the presence of rituximab as compared to untransduced T cells or CAR T cells, suggesting that the CD16 moiety of the fusion protein is capable of inducing ADCC. [0084] FIG. 38 show the viability of CD19KO-Raji cells (proportion of live cells and proportion of dead cells). [0085] FIG. 39 show the proportion and number of transduced T cells in the culture. The results demonstrate that T cells expressing fusion proteins provided herein expand more than CAR T cells when co-cultured with CD19KO-Raji cells in the presence of rituximab. [0086] FIG. 40 show the proportion and number of transduced CD4+ T cells in the culture. The results demonstrate that the expansion of the CD4+ subset of T cells expressing fusion proteins provided herein is more robust than the CD4+ subset of CAR T cells when co-cultured with CD19KO-Raji cells in the presence of rituximab. [0087] FIG. 41 show the proportion and number of transduced CD8+ T cells in the culture. The results demonstrate that the expansion of the CD8+ subset of T cells expressing fusion proteins provided herein is similar or lower than the CD8+ subset of CAR T cells when co-cultured with CD19KO-Raji cells in the presence of control IgG or rituximab, although the expansion of CD8+ T cells was overall weaker than that of CD4+ T cells.
[0088] FIG. 42 shows the mRNA expression levels of CD3ζ (A) and FcεRIγ (B) in various human immune cells isolated from peripheral blood samples of healthy donors. CD3ζ and FcεRIγ are essential plasma membrane signaling adaptor proteins that play a crucial role in the interaction with CD16A and the subsequent expression of CD16A on the cell surface. [0089] FIG.43 demonstrates the surface expression of the Activation Induced Clipping System (AICS) carrying the anti-CD19 single-chain fragment-variable (humanized FMC63 clone) in THP- 1, a monocyte cell line derived from an acute monocytic leukemia patient. Untransduced THP-1 cells (A), THP-1 cells expressing human CD16A (B), and FMC63-AICS (C), connected to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide, were subjected to staining with an isotype control, as well as antibodies specific for human CD16 and FMC63 scFv. Surface expression of the constructs was subsequently analyzed using flow cytometry. [0090] FIG. 44 illustrates the importance of the FC-binding domain of CD16A in the proper surface expression of the Activation Induced Clipping System (AICS). Jurkat cells were transduced with CD27-AICS (A) or CD27-AICS constructs lacking the FC-binding domain (B and C), all linked to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide. The cells were subsequently stained with antibodies specific for the CD27 ectodomain and CD16, and the expression of CD27-AICS constructs was evaluated on eGFP-positive live cells using flow cytometry. [0091] FIG.45 illustrates wild-type CD16A as well as an examplary fusion protein. DETAILED DESCRIPTION OF THE DISCLOSURE [0092] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated to facilitate the understanding of the present disclosure. [0093] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” mean “one or more” and therefore include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an antibody therapy” includes mixtures of two or more such antibody therapies, and the like.
[0094] Unless stated otherwise, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.” [0095] The term “approximately” as used herein refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [0096] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of” is associated). The transitional phrase “consisting essentially of” limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure. Nucleic Acids [0097] In one aspect, the disclosure provides a nucleic acid or set of nucleic acids that encode(s) a fusion protein comprising a target binding domain that binds a first target, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17, and a transmembrane domain. The term “nucleic acid” as used herein 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. The term nucleotide, unless specifically stated or obvious from context, 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). 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. As provided herein, the sequences for the fusion proteins or combinations of fusion proteins and CAR polypeptides may be encoded on a single nucleic acid or may be encoded by more than one nucleic acid (a set of nucleic acids). [0098] The first target may be an antigen. In some embodiments, the target binding domain is an antibody fragment. In some embodiments, 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. 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. Thus, for example, 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. [0099] The term “antigen” as used herein 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. [0100] In some embodiments of any one of the compositions or methods provided herein the antigen is on a cancer (e.g., tumor) cell or a cognate receptor or cognate ligand of the antigen on an immune cell. In some embodiments, the cancer antigen may be “tumor-associated” or “tumor- specific” antigen. “Tumor-associated antigen” (TAA), as used herein, 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. The term "tumor-specific antigen”, as used herein, refers to antigens that are present on a cancer, tumor or neoplastic cell but is not detectable on a normal cell derived from the same tissue or lineage as the cancer, tumor or neoplastic cell.
[0101] Cancer and tumor antigens include, without limitation, EGFR, CD19, CD20, CD22, NKG2D ligands, CS1, GD2, 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, PD-L1, BCMA, 41BB, CTAG1B, and CD33. Other examples include CD44v7/8, CD138, CD244, CEA, Csl, EBNA3C, EGP-2, EGP-40, E CAM, erb-B2, erb-B 2,3,4, FBP, GD2, GD3, GPA7, Her2, Her2/neu, IL-13R-a2, KDR, k-light chain, LeY, L 1 cell adhesion molecule, MAGE-A 1, Mesothelin, oncofetal antigen hST4, PSCA, TAG-72, etc. [0102] In some embodiments, 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. In some embodiments, 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 β (TGFβ), EGF, NKG2A (CD159), and NKG2D. [0103] In some embodiments, the target binding domain binds AFP, ALPP, AXL, B7-H3, B- cell maturation antigen (BCMA), 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, FRα, GD2 ganglioside, GFRα4, GLV, GP100, GPC3, GUCY2C, ERB-B2 receptor tyrosine kinase 2 (HER2), ICAM-1, IL13Rα2, KLK2, KNG2DL, LeY, LMP1, mesothelin, MG7, major histocompatibility complex, class I, E (HLA-E), MHC Class I polypeptide-related sequence A (MICA), MHC Class I polypeptide-related sequence B (MICB), MSLN, MUC16, mucin 1 (MUC1), Nectin4, NY-ESO-1, PSCA, PSMA, ROR2, or VEGFR2. [0104] In some embodiments, 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 anticancer 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. In some embodiments, the target binding domain is a scFv that binds CD19. In some embodiments, the target binding domain is derived from the sequence of a commercially available anti-CD19 antibody, antibody fragment, or derivative thereof. Amino acid sequences of representative anti-CD19 antibody heavy and light chains of which are set forth in Table 1. 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
301 nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsre 361 emtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr 421 wqqgnvfscs vmhealhnhy tqkslslspg k
nts, the target binding domain is a scFv that binds to CD20. In some embodiments, 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. Table 2: Amino acid sequences of representative anti-CD20 antibody fragments Polypeptide Sequence ofatumumab 1 evqlvesggg lvqpgrslrl scaasgftfn dyamhwvrqa pgkglewvst iswnsgsigy
heavy chain 181 slssvvtvps sslgtqtyic nvnhkpsntk vdkkaepksc dkthtcppcp apellggpsv (SEQ ID NO: 241 flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty 15) 301 rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt lppsrdeltk
nts, 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. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0107] In some embodiments, the target binding domain binds mesothelin. In some embodiments, 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 8,481,703 9,023,351 9,416,190 9,719,996, and 10,851,175 and U.S. Patent Application Publications 2019/0218294 and 2022/0056147, and such sequences are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0108] In some embodiments, the target binding domain binds PD-L1. When targeting a checkpoint molecule, 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. In some embodiments, the target binding domain is derived from at least a portion of the PD1 extracellular domain. In some embodiments, 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 3. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 3: Amino acid sequences of representative anti-PD-L1 antibody fragments Polypeptide Sequence atezolizumab 1 evqlvesggg lvqpggslrl scaasgftfs dswihwvrqa pgkglewvaw ispyggstyy h h i 61 adsvkgrfti sadtskntay lqmnslraed tavyycarrh wpggfdywgq gtlvtvssas [
] n some em o ments, t e us on prote n targets t e pat way y n ing EGFR. In some embodiments, the target binding domain is a scFv that binds EGFR. In some embodiments, the target binding domain is derived from the sequence of a commercially available anti-EGFR antibody, antibody fragment, or variant thereof, for example, cetuximab (Erbitux®), panitumumab (Vectibix®), necitumumab (Portrazza®), and amivantamab (Rybrevant®), the amino acid sequences of the heavy and light chains of which are set forth 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 representative anti-EGFR antibody fragments
Polypeptide Sequence cetuximab 1 qvqlkqsgpg lvqpsqslsi tctvsgfslt nygvhwvrqs pgkglewlgv iwsggntdyn [
0110] In some embodiments, the fusion protein blocks the NKG2A/HLA-E pathway. In some embodiments, target binding domain is a scFv that binds NKG2A or HLA-E. In some embodiments, the target binding domain is derived from the sequence of a commercially available anti-NKG2A antibody, antibody fragment, or variant thereof, for example, monalizumab (formerly IPH2201) and humanized Z199; the amino acid sequences of the heavy and light chains of which
are set forth in Table 5. Any one of the nucleic acids or sets of nucleiac acids provided herein may encode any one of such sequences. Table 5: Amino acid sequences of representative anti-NKG2A antibody fragments Polypeptide Sequence monalizumab 1 evqlvqsgae vkkpgeslki sckgsgysft sywmnwvrqm pgkglewmgr idpydsethy
uch as a B cell or T cell. Immune cells expressing fusion proteins can be used in, for example, B cell or T cell depletion therapy. As an example, 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. As another example, the target binding domain binds a receptor or other ligand on a T cell where 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. [0112] In some embodiments, the target is a B cell maturation antigen, wherein the B cell maturation antigen is, for example, (BCMA), CD19, CD20, CD27, CD70, or CD117, or mesothelin. [0113] ADAM 17, originally referred to as tumor necrosis factor (TNF)-α-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-α, TNF receptor I, and TNF receptor II. ADAM 17 is
expressed on NK cells generally, as well as the CD3-CD56bright and CD3-CD56dim NK cell subsets. ADAM 17 is also expressed on CD3+CD56+ NKT cells, but ADAM 17 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. 6:153 (2018)). Immune cell activation results in increased ADAM 17 activity and therefore target shedding, for example, stimulation with phorbol myristate acetate, IL-12, and IL- 18. Activation of immune cells expressing a fusion protein as disclosed herein results in ADAM 17-mediated fusion protein cleavage within the cleavage domain, releasing the target binding domain into the extracellular space as a soluble protein. [0114] In some embodiments of any one of the compositions or methods provided herein, the cleavage domain comprises any domain of a protein cleavable by ADAM 17, which includes variants thereof. Such proteins include, but are not limited to, CD16A, CD62L, TNF-α, TNF receptor I, and TNF receptor II. In some embodiments, the cleavage domain contains the amino acid sequence AVSTI (SEQ ID NO: 37), or a variant thereof. [0115] As used herein, a “variant” is any molecule with the same desired activity (such as cleavable by ADAM 17) 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. In any one of the compositions or methods provided herein, any one of the domains or other molecules or entities may be a variant of any one of the relevant sequences provided herein. [0116] In some embodiments, the variant contains a serine at position 3, when numbered according to SEQ ID NO: 37, and one or more variants of amino acids at positions 1, 2, 4, and/or 5. In some embodiments, the cleavage domain contains the amino acid sequence KLDKSFSMIKEGDYN (SEQ ID NO: 38), or a variant thereof. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0117] In some embodiments of any one of the compositions or methods provided herein, the cleavage domain is derived from the extracellular (EC) domain of a protein cleavable by ADAM 17, which includes variants thereof. As used here, fusion proteins that in some embodiments comprise such an extracellular domain are also referred to herein as SAR, and cells, such as T cells, expressing such fusion proteins are referred to as SAR cells, such as SAR T cells. In some embodiments of any one of the compositions or methods provided herein, the cleavage domain is derived from the EC domain of CD16A and, thus, the fusion protein can comprise such an extracellular domain. In some embodiments, the cleavage domain comprises the extracellular
domain of CD16A. In some embodiments, the cleavage domain is derived from the EC domain of CD62L. In some embodiments, the ADAM 17 cleavage domain is embodied in a domain native to or derived from a CD16A isoform or a CD62L isoform. In some embodiments, this cleavage domain contains the extracellular domain of CD16A having the amino acid sequence set forth below (SEQ ID NO: 39), or a variant thereof. 1 mwqlllptal lllvsagmrt edlpkavvfl epqwyrvlek dsvtlkcqga yspednstqw 61 fhneslissq assyfidaat vddsgeyrcq tnlstlsdpv qlevhigwll lqaprwvfke 121 edpihlrchs wkntalhkvt ylqngkgrky fhhnsdfyip katlkdsgsy fcrglfgskn 181 setvniti tqglavsti [0118] In some embodiments of any one of the compositions or methods provided herein, the cleavage domain comprises a variant of SEQ ID NO: 39 such that it has a higher affinity for IgG as compared to the wild-type sequence. In some embodiments of this, for example, the cleavage domain has a F176V substitution (i.e., a valine at position 176 in place of the phenylalanine, shown as a boxed amino acid in SEQ ID NO: 39). In other embodiments of this, for example, the cleavage domain has a Y158V substitution (i.e., a valine at position 158 of SEQ ID NO:39). [0119] In some embodiments, the cleavage domain contains the extracellular domain of CD62L having the amino acid sequence set forth below (SEQ ID NO: 40), or a variant thereof. 1 mgcrrtregp skamifpwkc qstqrdlwni fklwgwtmlc cdflahhgtd cwtyhysekp 61 mnwqrarrfc rdnytdlvai qnkaeieyle ktlpfsrsyy wigirkiggi wtwvgtnksl 121 teeaenwgdg epnnkknked cveiyikrnk dagkwnddac hklkaalcyt ascqpwscsg 181 hgecveiinn ytcncdvgyy gpqcqfviqc epleapelgt mdcthplgnf sfssqcafsc 241 segtnltgie ettcgpfgnw sspeptcqvi qceplsapdl gimncshpla sfsftsactf 301 icsegtelig kkkticessg iwsnpspicq kldksfsmik egdyn [0120] Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of the cleavage domains, including any one of the extracellular domains, provided herein, such as of, for example, CD16A. [0121] In some embodiments of any one of the compositions or methods provided herein, a signal peptide is included in the fusion protein, such as N-terminal to the target binding domain. The term “signal peptide” as used herein 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. Fusion proteins containing a signal peptide and transmembrane domain can be trafficked to the plasma membrane. [0122] In some embodiments, the signal peptide is derived from albumin, CD8α, CD33, erythropoietin (EPO), IL-2, human or mouse Ig-kappa chain V-III (IgK VIII), tissue plasminogen
activator (tPA), or secreted alkaline phosphatase (SEAP). Signal peptides may also be synthetic (i.e., non-naturally occurring). Amino acid sequences of representative signal peptides are listed in [0123] Table 6. 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 signal peptides Signal peptide Sequence Albumin (SEQ ID NO: 41) MKWVTFISLLFLFSSAYS [
y p p in, the fusion protein comprises one or more linkers. In some embodiments, a linker is present between sequences of the target binding domain (e.g., a linker disposed between the variable heavy (VH) and variable light (VL) domains of a scFv target binding domain). In some embodiments, a linker is present between the target binding domain and the cleavage domain. In some embodiments, a linker is present between a target binding domain and the extracellular domain of CD16A, CD62L, TNF-α, TNF receptor I, and TNF receptor II, such as when the fusion protein comprises a target binding domain and an extracellular domain of CD16A, CD62L, TNF-α, TNF receptor I, and TNF receptor II, which includes variants thereof. [0125] In some embodiments, the linker comprises an amino acid having the sequence GGGX, GGGGX (SEQ ID NO: 54), or GSSGSX (SEQ ID NO: 55), where X is either cysteine (C) or serine
(S), or a repeating sequence thereof. In some embodiments, the linker has the amino acid sequence GGGGS (SEQ ID NO: 56), GGGGSGGGGS (SEQ ID NO: 57), GGGGSGGGGSGGGGS (SEQ ID NO: 58), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 59), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 60), KESGSVSSEQLAQFRSLD (SEQ ID NO: 61), EGKSSGSGSESKST (SEQ ID NO: 62), or GSAGSAAGSGEF (SEQ ID NO: 63). [0126] In the fusion proteins provided herein, the transmembrane domain can be connected to the cleavage domain, which is generally between the transmembrane domain and the target binding 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 cleavage domain of the fusion protein from the cell surface after ADAM 17-mediated cleavage. The transmembrane domain generally localizes the fusion protein to the endoplasmic reticulum during translation and delivery to the cell surface. [0127] In some embodiments, the transmembrane domain interacts with signaling adaptor protein CD3ζ and Fc Fragment of IgE Receptor Ig (FCER1G). The transmembrane may be derived from CD3α, CD3β, CD3γ, CD3ζ, CD3ε, CD4, CD5, CD8α, 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)), FcεRIα, FcεRIβ, FcεRIγ, ICOS, KIR2DS2, MHC class I, MHC class II, or NKG2D, which includes variants thereof. In some embodiments, the transmembrane domain is derived from CD16A or CD62L. In some embodiments, the transmembrane domain is derived from CD3ζ, CD4, CD8α, CD28, or CD137 (4-1BB). Amino acid sequences of representative transmembrane domains are listed in Table 7. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 7: Amino acid sequences of transmembrane domains Transmembrane domain Sequence
[0128] In some embodiments of any one of the compositions or methods provided herein, the fusion protein further comprises an intracellular domain (IC) of any one of the proteins cleavable by ADAM 17 provided herein, which includes variants thereof, which can be connected to the transmembrane domain. The IC domain can provide signaling capacities to the fusion protein. In some embodiments, the IC domain is derived from CD16A, which includes variants thereof. The CD16A IC domain can interact with the adaptor proteins CD3ζ 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 CD62L IC domain is known to interact with α- actinin, calmodulin, ezrin, meosin, protein kinase C (PKC) isozymes and AP-1. See, Ivetic et al., Front. Immunol.10:1068 (2019). In some embodiments, the intracellular domain is derived from CD16A, but lacks the CD16 signal peptide. In some embodiments, the intracellular domain is derived from CD62L. [0129] In some embodiments, the nucleic acid or set of nucleic acids encode(s) a fusion protein containing the amino acid sequence set forth below (SEQ ID NO: 69), and which contains the features, from N-terminus to C-terminus, a PD1 single peptide (SP), a PD1 EC domain, a G4S linker, a CD16 EC domain, a CD16 transmembrane (TM) domain, and a CD16 intracellular (IC) domain (which lacks a CD16 signal peptide) set forth in Table 8. 1 mqipqapwpv vwavlqlgwr pgwfldspdr pwnpptfspa llvvtegdna tftcsfsnts 61 esfvlnwyrm spsnqtdkla afpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgt 121 ylcgaislap kaqikeslra elrvterrae vptahpspsp rpagqfqtlv ggggsgmrte 181 dlpkavvfle pqwyrvlekd svtlkcqgay spednstqwf hneslissqa ssyfidaatv 241 ddsgeyrcqt nlstlsdpvq levhigwlll qaprwvfkee dpihlrchsw kntalhkvty 301 lqngkgrkyf hhnsdfyipk atlkdsgsyf crglfgsknv ssetvnitit qglavstiss 361 ffppgyqvsf clvmvllfav dtglyfsvkt nirsstrdwk dhkfkwrkdp qdk Table 8: Amino acid sequences of PD1-CD16 (pHIV-PD1-CD16) Polypeptide Amino acid sequence V E E S N C
Human CD16 TM domain (SEQ ID VSFCLVMVLLFAVDTGLYFSV NO: 73) H D1 I i E ID KT IR TRD KDHKFK RKDP DK
ein containing the amino acid sequence set forth below (SEQ ID NO: 75), and which contains the features, from N-terminus to C-terminus, a CD8 SP, an anti-PDL1 ScFv, a G4S linker, a CD16 EC domain, a CD16 TM domain, and a CD16 IC domain (which lacks a CD16 signal peptide) set forth in Table 9. 1 malpvtalll plalllhaar pdiqmtqsps slsasvgdrv titcrasqdv stavawyqqk 61 pgkapklliy sasflysgvp srfsgsgsgt dftltisslq pedfatyycq qylyhpatfg 121 qgtkveikgg ggsggggsgg ggsevqlves ggglvqpggs lrlscaasgf tfsdswihwv 181 rqapgkglew vawispyggs tyyadsvkgr ftisadtskn taylqmnslr aedtavyyca 241 rrhwpggfdy wgqgtlvtvs sggggsgmrt edlpkavvfl epqwyrvlek dsvtlkcqga 301 yspednstqw fhneslissq assyfidaat vddsgeyrcq tnlstlsdpv qlevhigwll 361 lqaprwvfke edpihlrchs wkntalhkvt ylqngkgrky fhhnsdfyip katlkdsgsy 421 fcrglfgskn vssetvniti tqglavstis sffppgyqvs fclvmvllfa vdtglyfsvk 481 tnirsstrdw kdhkfkwrkd pqdk Table 9: Amino acid sequences of PDL1scFv-CD16 (pHIV-PDL1scFv-CD16) Polypeptide Amino acid sequence CD8 SP (SEQ ID NO: 76) MALPVTALLLPLALLLHAARP K E Q T T E S N C
[0131] In some embodiments, the nucleic acid or set of nucleic acids encode(s) a fusion protein containing the amino acid sequence set forth below (SEQ ID NO: 79), and which contains the features, from N-terminus to C-terminus, a CD8 SP, an anti-CD19 ScFv, a G4S linker, a CD16 EC
domain, a CD16 TM domain, and a CD16 IC domain (which lacks a CD16 signal peptide) set forth in Table 10. 1 malpvtalll plalllhaar peivmtqspa tlslspgera tlscrasqdi skylnwyqqk 61 pgqaprlliy htsrlhsgip arfsgsgsgt dytltisslq pedfavyfcq qgntlpytfg 121 qgtkleikgg ggsggggsgg ggsqvqlqes gpglvkpset lsltctvsgv slpdygvswi 181 rqppgkglew igviwgsett yyssslksrv tiskdnsknq vslklssvta adtavyycak 241 hyyyggsyam dywgqgtlvt vssggggsgm rtedlpkavv flepqwyrvl ekdsvtlkcq 301 gayspednst qwfhneslis sqassyfida atvddsgeyr cqtnlstlsd pvqlevhigw 361 lllqaprwvf keedpihlrc hswkntalhk vtylqngkgr kyfhhnsdfy ipkatlkdsg 421 syfcrglfgs knvssetvni titqglavst issffppgyq vsfclvmvll favdtglyfs 481 vktnirsstr dwkdhkfkwr kdpqdk Table 10: Amino acid sequences of CD19scFv-CD16 (pHIV-CD19scFv-CD16) Polypeptide Amino acid sequence Human CD8 SP (SEQ ID NO: 80) MALPVTALLLPLALLLHAARP P P L T E S N C [
0 3 ] n some embod ments, t e nuc e c ac d or set o nuc e c ac ds encode(s) a arger usion protein that includes the CD19-binding fusion protein shown above (SEQ ID NO: 83), which is connected to a 4-1BBL protein (which includes the EC, TM and IC domains thereof) via a P2A self-cleaving peptide. This latter portion of the larger fusion protein has the amino acid sequence SEQ ID NO: 74; the amino acid sequences of its components are set forth in Table 11. 1 malpvtalll plalllhaar peivmtqspa tlslspgera tlscrasqdi skylnwyqqk 61 pgqaprlliy htsrlhsgip arfsgsgsgt dytltisslq pedfavyfcq qgntlpytfg 121 qgtkleikgg ggsggggsgg ggsqvqlqes gpglvkpset lsltctvsgv slpdygvswi 181 rqppgkglew igviwgsett yyssslksrv tiskdnsknq vslklssvta adtavyycak 241 hyyyggsyam dywgqgtlvt vssggggsgm rtedlpkavv flepqwyrvl ekdsvtlkcq
301 gayspednst qwfhneslis sqassyfida atvddsgeyr cqtnlstlsd pvqlevhigw 361 lllqaprwvf keedpihlrc hswkntalhk vtylqngkgr kyfhhnsdfy ipkatlkdsg 421 syfcrglfgs knvssetvni titqglavst issffppgyq vsfclvmvll favdtglyfs 481 vktnirsstr dwkdhkfkwr kdpqdkgsga tnfsllkqag dveenpgpme yasdasldpe 541 apwppaprar acrvlpwalv aacavflacp wavsgarasp gsaasprlre 601 gpelspddpa glldlrqgmf aqlvaqnvll idgplswysd pglagvsltg glsykedtke 661 lvvakagvyy vffqlelrrv vagegsgsvs lalhlqplrs aagaaalalt vdlppassea 721 rnsafgfqgr llhlsagqrl gvhlhteara rhawqltqga tvlglfrvtp eipaglpspr 781 se Table 11: Amino Acid Sequences of the components of the 4-1BBL protein Polypeptide Amino acid sequence P2A self-cleaving peptide (SEQ ID GSGATNFSLLKQAGDVEENPGP F V A A
Chimeric Antigen Receptor Polypeptides [0133] In some embodiments of any one of the compositions or methods provided herein, the nucleic acid or set of nucleic acids contains an additional sequence that encodes a CAR polypeptide. The CAR polypeptide is made up of a second target binding domain that binds to a second target and a transmembrane domain. The CAR polypeptide may also comprise an intracellular domain. In some embodiments, the intracellular domain may comprise a signaling domain. In some embodiments, this second target is different from the first target (of the fusion protein). In some embodiments, the second target binding domain is an antibody fragment (e.g., a scFv). [0134] The second target may be any one of the targets provided herein, such as any one of the first targets provided herein. In some embodiments, the second target is a cancer antigen as provided herein. In other embodiments, the second target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell. In other embodiments, the second target is a receptor or other ligand on an immune cell. In some embodiments, the immune cell is a B cell or T cell.
[0135] In other embodiments, the second target is BCMA, CD19, CD70, or PD-L1. In some embodiments, the CAR target binding domain is a scFv that binds BCMA. In some embodiments, the CAR target binding domain is derived from the sequence of a commercially available anti- BCMA antibody, antibody fragment, or derivative thereof. In some embodiments, the CAR target binding domain is derived from belantamab (Blenrep®) heavy and light chains. [0136] The amino acid sequence of belantamab heavy chain is set forth below (SEQ ID NO: 88). 1 qvqlvqsgae vkkpgssvkv sckasggtfs nywmhwvrqa pgqglewmga tyrghsdtyy 61 nqkfkgrvti tadkststay melsslrsed tavyycarga iydgydvldn wgqgtlvtvs 121 sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vhtfpavlqs 181 sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkkve pkscdkthtc ppcpapellg 241 gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy 301 nstyrvvsvl tvlhqdwlng keykckvsnk alpapiekti skakgqprep qvytlppsrd 361 eltknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr 421 wqqgnvfscs vmhealhnhy tqkslslspg k [0137] The amino acid sequence of belantamab light chain is set forth below (SEQ ID NO: 89). 1 diqmtqspss lsasvgdrvt itcsasqdis nylnwyqqkp gkapklliyy tsnlhsgvps 61 rfsgsgsgtd ftltisslqp edfatyycqq yrklpwtfgq gtkleikrtv aapsvfifpp 121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 181 lskadyekhk vyacevthqg lsspvtksfn rgec [0138] Additional anti-BCMA antibodies and BCMA-binding fragments thereof are known in the art. See, e.g., U.S. Patents 10,072,088 and 11,084,880 and U.S. Patent Application Publications 2016/0131655, 2017/0226216, 2018/0133296, 2019/0151365, 2019/0381171, 2020/0339699, 2020/0055948, and 2022/0064316, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0139] In some embodiments, the CAR target binding domain is a scFv that binds CD70. Anti- CD70 antibodies and CD70-binding fragments thereof are known in the art. See, e.g., U.S. Patents 7,641,903, 8,337,838, 9,051,372, 7,641,903, and 9,765,149 and U.S. Patent Application Publications 2019/0106498 and 2021/0380707, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0140] In some embodiments, the CAR target binding domain binds CD38. In some embodiments, the CAR target binding domain is derived from a commercially available anti-CD38 antibody, CD38-binding fragments thereof, or derivative thereof, e.g., daratumumab (Darzalex®),
isatuximab (Sarclisa®), and mezagitamab (TAK-079), the amino acid sequences of the heavy and light chains of which are set forth in Table 12. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 12: Amino Acid Sequences of anti-CD38 antibody fragments Polypeptide Sequence daratumumab 1 evqllesggg lvqpggslrl scavsgftfn sfamswvrqa pgkglewvsa isgsgggtyy 61 adsvkgrfti srdnskntly lqmnslraed tavyfcakdk ilwfgepvfd ywgqgtlvtv [
0141] In some embodiments, the CAR target binding domain, e.g., a scFv, binds CD138. Anti- CD138 antibodies and CD138-binding fragments thereof are known in the art. See, e.g., U.S. Patents 9,221,914, 9,387,261, 9,446,146, and 10,975,158 and U.S. Patent Application Publications 2007/0183971, 2009/0232810, 2018/0312561, 2019/0100588, 2020/0384024, and 2020/0392241, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.
[0142] In some embodiments, the CAR target binding domain, e.g., a scFv, binds FCRH5. Anti-FCRH5 antibodies and FCRH5-binding fragments thereof are known in the art, e.g., cevostamab, and U.S. Patents 8,466,260, 9,017,951, 10,323,094, 10,435,471, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0143] The amino acid sequence of a representative anti-FCRH5 heavy chain is set forth below (SEQ ID NO: 96). 1 diqmtqspss lsasvgdrvt itckasqdvr nlvvwfqqkp gkapklliys gsyrysgvps 61 rfsgsgsgtd ftltisslqp edfatyycqq hysppytfgq gtkveikrtv aapsvfifpp 121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 181 lskadyekhk vyacevthqg lsspvtksfn rgec [0144] The amino acid sequence of a representative anti-FCRH5 light chain is set forth below (SEQ ID NO: 97). 1 evqlvesgpg lvkpsetlsl tctvsgfslt rfgvhwvrqp pgkglewlgv iwrggstdyn 61 aafvsrltis kdnsknqvsl klssvtaadt avyycsnhyy gssdyaldnw gqgtlvtvss 121 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181 glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep kscdkthtcp pcpapellgg 241 psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyg 301 styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361 mtknqvslwc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421 qqgnvfscsv mhealhnhyt qkslslspgk [0145] In some embodiments, the CAR target binding domain, e.g., a scFv, binds GPRC5D. Anti-GPRC5D antibodies and GPRC5D-binding fragments thereof are known in the art, e.g., talquetamab, U.S. Patents 10,562,968 and 10,590,196, and U.S. Patent Application Publications 2019/0367612, 2020/0123250, 2020/0190205, 2020/0270326, and 2021/0054094, the sequences of which are incorporated herein by reference. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. [0146] The amino acid sequence of a representative anti-FCRH5 antibody scFv fragment is set forth below (SEQ ID NO: 98). 1 divmtqtpls spvtlgqpas iscrssqslv hsdgntylsw lqqrpgqppr lliykisnrf 61 fgvpdrfsgs gagtdftlki srveaedvgv yycmqatqfp htfgqgtkle ikggsegkss 121 gsgseskstg gsqvtlkesg pvlvkptetl tltctvsgfs ltnirmsvsw irqppgkale 181 wlahifsnde ksyssslksr ltisrdtsks qvvltltnvd pvdtatyyca rmrlpygmdv 241 wgqgttvtvs s [0147] In some embodiments, the CAR target binding domain, e.g., a scFv, binds SLAMF7. In some embodiments, the antigen binding domain is derived from a commercially available anti-
SLAMF7 antibody, SLAMF7-binding fragment, or derivative thereof, e.g., elotuzumab (Empliciti®). [0148] The amino acid sequence of an elotuzumab heavy chain is set forth below (SEQ ID NO: 99). 1 evqlvesggg lvqpggslrl scaasgfdfs rywmswvrqa pgkglewige inpdsstiny 61 apslkdkfii srdnaknsly lqmnslraed tavyycarpd gnywyfdvwg qgtlvtvssa 121 stkgpsvfpl apsskstsgg taalgclvkd yfpepvtvsw nsgaltsgvh tfpavlqssg 181 lyslssvvtv pssslgtqty icnvnhkpsn tkvdkkvepk scdkthtcpp cpapellggp 241 svflfppkpk dtlmisrtpe vtcvvvdvsh edpevkfnwy vdgvevhnak tkpreeqyns 301 tyrvvsvltv lhqdwlngke ykckvsnkal papiektisk akgqprepqv ytlppsrdel 361 tknqvsltcl vkgfypsdia vewesngqpe nnykttppvl dsdgsfflys kltvdksrwq 421 qgnvfscsvm healhnhytq kslslspgk [0149] The amino acid sequence of an elotuzumab light chain is set forth below (SEQ ID NO: 100). 1 diqmtqspss lsasvgdrvt itckasqdvg iavawyqqkp gkvpklliyw astrhtgvpd 61 rfsgsgsgtd ftltisslqp edvatyycqq yssypytfgq gtkveikrtv aapsvfifpp 121 sdeqlksgta svvcllnnfy preakvqwkv dnalqsgnsq esvteqdskd styslsstlt 181 kadyekhk vyacevthqg lsspvtksfn rgec [0150] The transmembrane domain of the CAR polypeptide can connect the CAR target binding domain to an intracellular signaling domain. In some embodiments of any one of the compositions or methods provided herein, the transmembrane domain is directly connected to the CAR target binding domain. The transmembrane domain of the CAR may be any one of the transmembrane domains provided herein, such as described for the fusion proteins. Amino acid sequences of representative transmembrane domains are listed in Table 7. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences.Error! Reference source not found. [0151] The amino acid sequence of a naturally occurring transmembrane domain may be modified by an amino acid substitution to avoid binding of such regions to the transmembrane domain of the same or different surface membrane proteins to minimize interactions with other members of a receptor complex. See, e.g., U.S. Patent Application Publication 2021/0101954; Soudais et al., Nat. Genet. 3:77-81 (1993); Muller et al., Front. Immunol.12:639818-13 (2021); Elazar et al., elife 11:e75660-29 (2022). [0152] In some embodiments of any one of the compositions or methods provided herein, the CAR polypeptide includes a hinge domain disposed between the CAR target binding domain and the transmembrane domain. A hinge domain may provide flexibility in terms of allowing the CAR
target binding domain to obtain an optimal orientation for target binding, enhancing cell killing activities, etc. [0153] In some embodiments, the hinge domain is derived from IgA, IgD, IgE, IgG, or IgM. In some embodiments, the hinge domain is derived from CD3ζ, CD4, CD8α, CD28, IgG1, IgG2, or IgG4. Amino acid sequences of representative hinge domains of which are listed in Table 13. Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 13: Amino acid sequences of representative hinge domains Hinge domain Sequence CD3ζ (SEQ ID NO: 101) QSFGLLDPK Y
ellular signaling and immune cell function. The signaling domain may include a primary signaling domain and/or a co-stimulatory signaling domain. In some embodiments, the intracellular domain is capable of delivering a signal approximating that of natural ligation of an ITAM-containing molecule or receptor complex such as a TCR receptor complex. [0155] In some embodiments of any one of the compositions or methods provided herein, the intracellular signaling domain includes a plurality, e.g., 2 or 3, costimulatory signaling domains described herein, e.g., selected from 4-1BB, CD3ζ, CD28, CD27, ICOS, and OX40. In some embodiments, the intracellular signaling domain may include a CD3ζ domain as a primary signaling domain, and 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-CD3ζ; CD3ζ-4-1BB; CD28-CD3ζ; CD3ζ-CD28; CD28- 4-1BB and 4-1BB-CD28. In some embodiments, the primary signaling domain is derived from CD3ζ, CD27, CD28, CD40, KIR2DS2, MyD88, or OX40. In some embodiments, the co- stimulatory signaling domain is derived from one or more of CD3γ, CD3δ, CD3ε, CD3ζ, CD4,
CD5, CD8α, 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, FcαRI, FcγRI, FcγRII, FcγRIII, IL-2RB, ICOS, KIR2DS2, MyD88, OX40, and ZAP70. Amino acid sequences of representative signaling domains are listed in Table . Any one of the nucleic acids or sets of nucleic acids provided herein may encode any one of such sequences. Table 14: Amino acid sequences of representative signaling domains Signaling domain Sequence CD3ζ (SEQ ID NO: 108) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR R L T Y L V
LLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPL GHMPERFDAFICYCPSDIQFVQEMIRQLEQTNYRLKLCVSDRDVLPGTCVW K N G N P R E E S In
some embodiments, the nucleic acid encodes a CAR containing the amino acid sequence set forth below (SEQ ID NO: 126), and which contains the features, from N-terminus to C-terminus, a CD8 SP, an anti-CD19 scFv, a CD8 hinge, a CD8 TM domain, a 4-1BB IC domain, and a CD3ζ IC domain set forth in Table 15. 1 malpvtalll plalllhaar peivmtqspa tlslspgera tlscrasqdi skylnwyqqk 61 pgqaprlliy htsrlhsgip arfsgsgsgt dytltisslq pedfavyfcq qgntlpytfg 121 qgtkleikgg ggsggggsgg ggsqvqlqes gpglvkpset lsltctvsgv slpdygvswi 181 rqppgkglew igviwgsett yyssslksrv tiskdnsknq vslklssvta adtavyycak 241 hyyyggsyam dywgqgtlvt vsstttpapr pptpaptias qplslrpeac rpaaggavht 301 rgldfacdiy iwaplagtcg vlllslvitl yckrgrkkll yifkqpfmrp vqttqeedgc 361 scrfpeeeeg gcelrvkfsr sadapaykqg qnqlynelnl grreeydvld krrgrdpemg 421 gkprrknpqe glynelqkdk maeayseigm kgerrrgkgh dglyqglsta tkdtydalhm 481 qalppr Table 15: Amino acid sequences of anti-CD19-CAR (pHIV-CD19-CAR-GFP) Polypeptide Amino acid sequence P
anti-CD19 ScFv VH (SEQ ID NO: 77) QVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKG LEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAA D
wn above (SEQ ID NO: 64), which is connected to an anti-CD19-CAR via a P2A self-cleaving peptide. This latter portion has the amino acid sequence SEQ ID NO: 130; the amino acid sequences of its components are set forth above. 1 malpvtalll plalllhaar pdiqmtqsps slsasvgdrv titcrasqdv stavawyqqk 61 pgkapklliy sasflysgvp srfsgsgsgt dftltisslq pedfatyycq qylyhpatfg 121 qgtkveikgg ggsggggsgg ggsevqlves ggglvqpggs lrlscaasgf tfsdswihwv 181 rqapgkglew vawispyggs tyyadsvkgr ftisadtskn taylqmnslr aedtavyyca 241 rrhwpggfdy wgqgtlvtvs sggggsgmrt edlpkavvfl epqwyrvlek dsvtlkcqga 301 yspednstqw fhneslissq assyfidaat vddsgeyrcq tnlstlsdpv qlevhigwll 361 lqaprwvfke edpihlrchs wkntalhkvt ylqngkgrky fhhnsdfyip katlkdsgsy 421 fcrglfgskn vssetvniti tqglavstis sffppgyqvs fclvmvllfa vdtglyfsvk 481 tnirsstrdw kdhkfkwrkd pqdkgsgatn fsllkqagdv eenpgpmalp vtalllplal 541 llhaarpeiv mtqspatlsl spgeratlsc rasqdiskyl nwyqqkpgqa prlliyhtsr 601 lhsgiparfs gsgsgtdytl tisslqpedf avyfcqqgnt lpytfgqgtk leikggggsg 661 gggsggggsq vqlqesgpgl vkpsetlslt ctvsgvslpd ygvswirqpp gkglewigvi 721 wgsettyyss slksrvtisk dnsknqvslk lssvtaadta vyycakhyyy ggsyamdywg 781 qgtlvtvsst ttpaprpptp aptiasqpls lrpeacrpaa ggavhtrgld facdiyiwap 841 lagtcgvlll slvitlyckr grkkllyifk qpfmrpvqtt qeedgcscrf peeeeggcel 901 rvkfsrsada paykqgqnql ynelnlgrre eydvldkrrg rdpemggkpr rknpqeglyn 961 elqkdkmaea yseigmkger rrgkghdgly qglstatkdt ydalhmqalp pr [0158] In some embodiments, the nucleic acid encodes a CAR containing the amino acid sequence set forth below (SEQ ID NO: 131), and which contains the features, from N-terminus to C-terminus, a CD8 SP, anti-PD-L1 scFv, CD8 hinge, CD8 TM domain, 4-1BB IC domain, and CD3ζ IC domain set forth in Table 16. 1 malpvtalll plalllhaar pdiqmtqsps slsasvgdrv titcrasqdv stavawyqqk 61 pgkapklliy sasflysgvp srfsgsgsgt dftltisslq pedfatyycq qylyhpatfg 121 qgtkveikgg ggsggggsgg ggsevqlves ggglvqpggs lrlscaasgf tfsdswihwv 181 rqapgkglew vawispyggs tyyadsvkgr ftisadtskn taylqmnslr aedtavyyca 241 rrhwpggfdy wgqgtlvtvs stttpaprpp tpaptiasqp lslrpeacrp aaggavhtrg 301 ldfacdiyiw aplagtcgvl llslvitlyc krgrkkllyi fkqpfmrpvq ttqeedgcsc 361 rfpeeeeggc elrvkfsrsa dapaykqgqn qlynelnlgr reeydvldkr rgrdpemggk 421 prrknpqegl ynelqkdkma eayseigmkg errrgkghdg lyqglstatk dtydalhmqa 481 lppr
Table 16: Amino acid sequences of components of the anti-PD-L1 CAR Domain Amino acid sequence Human CD8 SP (SEQ ID NO: 75) MALPVTALLLPLALLLHAARP A Q K S D
, a fusion protein-encoding nucleic acid is connected to a CAR-encoding nucleic acid by a nucleic acid encoding a self-cleaving peptide (e.g., P2A). In some embodiments, the sequences encoding the fusion protein and the CAR polypeptide are under the control of the same regulatory elements. In some embodiments, the sequences encoding the fusion protein and the CAR polypeptide are under the control of the different regulatory elements. In some embodiments of any one of the compositions or methods provided herein, the fusion protein-encoding nucleic acid(s) and the CAR-encoding nucleic acid(s) are separate nucleic acid molecules. Vectors [0160] The fusion protein-encoding nucleic acid(s) may be introduced to an immune cell by a suitable vector or set of vectors. In embodiments that include a CAR, the CAR-encoding nucleic acid(s) may be introduced into an immune cell by the same vector or set of vectors or by a separate 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. [0161] The term "promoter" as used herein 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 fusion protein-encoding sequence, a CAR polypeptide-encoding sequence, or a sequence encoding a fusion protein linked to a CAR polypeptide by a nucleic acid encoding a self-cleaving peptide. 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. [0162] The term "operatively linked" as used herein is to be understood that 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 fusion protein-encoding nucleic acid sequence). [0163] In some embodiments, a vector contains a single promoter operatively linked to a fusion- protein encoding nucleic acid and/or a CAR-encoding nucleic acid. In some embodiments, the fusion-protein encoding nucleic acid and the CAR-encoding nucleic acid are separated by a nucleic acid encoding a self-cleaving peptide. In other embodiments, the fusion-protein encoding nucleic acid and the CAR-encoding nucleic acid are separated by nucleic acid encoding an internal ribosome entry site (IRES). [0164] In some embodiments, a vector contains a first promoter operatively liked to a fusion- protein encoding nucleic acid and a second promoter operatively liked to a CAR-encoding nucleic acid. In some embodiments, two vectors are provided, a first vector containing a promoter operatively linked to a fusion-protein encoding nucleic acid and a second vector containing a promoter operatively linked to a CAR-encoding nucleic acid. [0165] In some embodiments, 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 β-Actin, and splice acceptor of rabbit β -Globin)) or promoters derived from the β-actin,
phosphoglycerate kinase (PGK), or factor EF1α genes. In some embodiments, the promoter may have a core region located close to the nucleic acid coding sequence. In some embodiments, 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. In some embodiments, 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. [0166] In some embodiments, 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. As used herein, the term “lentiviral vector” is intended to mean an infectious lentiviral particle. Lentivirinae (lentiviruses) is a subfamily of enveloped retrovirinae (retroviruses), that are distinguishable from other viruses by virion structure, host range, and pathological effects. An infectious lentiviral particle will be capable of invading a target host cell, including infecting, and transducing non-dividing cells and immune cells. [0167] In some embodiments, 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. [0168] A lentiviral vector can exhibit functions additional to, or different from, a naturally occurring lentivirus. For example, 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. [0169] The terms “pseudotyping”, “pseudotyped”, “pseudotyped vector”, and “pseudotyped vector particle” are used herein to refer to 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 thusly be expanded or altered depending on the type of cell surface receptor bound by the glycoprotein derived from the second virus. [0170] In some embodiments, the lentiviral vector is pseudotyped with a baboon endogenous retroviral (BaEV) envelope glycoprotein (BaEV-gp). The nucleic acid sequence of a representative BaEV-gp is set forth below (SEQ ID NO: 132). 1 atgggtttca ctacgaaaat tatctttctg tataatctgg tactcgtata tgcgggtttc 61 gacgatccca ggaaagcgat cgaacttgtc cagaagagat acgggaggcc ctgtgactgc 121 agcggagggc aagtatcaga acccccctct gatcgggtca gccaagttac ttgcagcggc 181 aaaacagctt acctgatgcc ggatcagaga tggaaatgca aatccatacc caaggacacc 241 agtccgagtg gaccattgca ggaatgtccg tgtaatagtt accaatcaag cgtccattca 301 agttgctaca cgtcatacca gcaatgtcgc tcaggaaata aaacctatta tacggcgaca 361 ctgcttaaaa cccaaacggg tggcacctct gatgttcagg ttctcggaag tacgaataag 421 ttgattcaga gtccctgcaa cggtatcaaa ggccagtcaa tttgttggtc tacgacagcg 481 cctatccatg tgagtgacgg cggtgggccg ttggatacaa cacgaataaa aagtgtacag 541 cggaaacttg aggagataca caaagccctc taccccgagc ttcagtacca tcccctggcc 601 atccctaagg tcagggacaa tctcatggta gacgctcaaa ccctcaacat cctcaatgcc 661 acctacaatc tcttgttgat gtctaacaca agcttggtag atgactgctg gctctgtctt 721 aaattgggcc ctccgactcc cctcgctata cccaacttcc ttctgtcata cgtaacgcgc 781 agctccgaca acatatcatg tctgataatc ccgccgttgc ttgtgcagcc catgcagttc 841 tctaacagct cctgcttgtt cagtccatct tataattcaa cagaagaaat tgatttgggc 901 catgtagctt tcagtaactg tacatcaata actaacgtca ctggccccat ctgcgccgtg 961 aacggttctg tcttcctctg cggcaacaat atggcttata catacttgcc aactaactgg 1021 accggtctgt gtgtattggc cacgctgttg cctgacatag atataatccc tggcgacgaa 1081 cccgtcccta tcccagccat cgaccatttt atttatcgcc ccaagcgcgc gattcagttt 1141 atccctctgc tcgctgggtt gggcattacg gctgctttta ctacgggggc taccggcctt 1201 ggagtgtccg ttacccaata tacgaaactg tccaatcaat tgatttcaga cgtgcaaatc 1261 ttgagctcta ctatccagga tctgcaggac caggtagact ctctggcgga agtcgtcttg 1321 caaaatcggc gggggttgga tctgctgacc gccgagcagg gcggcatctg tcttgctctt 1381 caagaaaaat gctgttttta cgtgaacaaa tcaggtattg taagagataa aataaaaact 1441 ttgcaagaag agctcgaaag gaggcggaaa gacctggcgt ctaatcctct gtggactggc 1501 ctgcaggggc tcctccccta tttgctgccc tttcttggtc cgctcctgac tttgttgctg 1561 ctcctgacta ttgggccatg catcttcaat cgactcaccg cgttcatcaa tgataaactc 1621 aacataatcc acgctatgtg a [0171] The amino acid sequence of a representative BaEV-gp is set forth below (SEQ ID NO: 133). 1 mgfttkiifl ynlvlvyagf ddprkaielv qkrygrpcdc sggqvsepps drvsqvtcsg 61 ktaylmpdqr wkcksipkdt spsgplqecp cnsyqssvhs scytsyqqcr sgnktyytat 121 llktqtggts dvqvlgstnk liqspcngik gqsicwstta pihvsdgggp ldttriksvq 181 rkleeihkal ypelqyhpla ipkvrdnlmv daqtlnilna tynlllmsnt slvddcwlcl 241 klgpptplai pnfllsyvtr ssdnisclii ppllvqpmqf snssclfsps ynsteeidlg 301 hvafsnctsi tnvtgpicav ngsvflcgnn maytylptnw tglcvlatll pdidiipgde 361 pvpipaidhf iyrpkraiqf ipllaglgit aafttgatgl gvsvtqytkl snqlisdvqi 421 lsstiqdlqd qvdslaevvl qnrrgldllt aeqggiclal qekccfyvnk sgivrdkikt 481 lqeelerrrk dlasnplwtg lqgllpyllp flgplltlll lltigpcifn rltafindkl 541 niiham
[0172] BaEV is an endogenous gammaretrovirus that had a recombination event between a Papio cynocephalus endogenous retrovirus and a simian betaretrovirus. 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. In some embodiments, the lentiviral vector is pseudotyped with the feline endogenous retrovirus RD114 glycoprotein. [0173] In some embodiments, 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. IL-18 pretreatment further enables transduction. CD56 bright (CD56bright; CD56br) NK cells express higher levels of ASCT2 compared to CD56 low expressing cells (CD56dim) 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 fusion protein that achieves 40-60% transduction efficiency. In some embodiments, cytokine pretreatment of the immune cells followed by transduction with BaEV-LV results in selective expansion of NK cells without the expansion of T regulatory cells, and the expanded memory-like NK cells dominate the peripheral blood lymphocyte compartment in vivo (Shapiro et al., J. Clin. Invest. 132(11):e154334-17 (2022)). [0174] The term “bright” as used herein in the context of marker expression (e.g., CD56bright) 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.
[0175] In other embodiments, the vector is a non-viral vector, representative examples of which include plasmids, mRNA, linear single stranded (ss) DNA or linear double stranded (ds) DNA, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac(PB)-based vectors. In yet other embodiments, the vector may include both viral and non-viral elements. [0176] In some embodiments, the vector is a plasmid. In addition to a promoter operatively linked to the nucleic acids, 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. In some embodiments, the vector multiple plasmids, a first plasmid encoding the fusion protein and a second plasmid encoding the CAR polypeptide. [0177] In some embodiments, a carrier encapsulates the vector. The carrier may be lipid-based, e.g., lipid nanoparticles (LNPs), liposomes, lipid vesicles, or lipoplexes. In some embodiments, the carrier is an LNP. In certain embodiments, 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. [0178] 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 a pegylated lipids, which are lipids conjugated to polyethylene glycol. 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. LNPs, their method of preparation, formulation, and delivery are disclosed in, e.g., U.S. Patent Application Publication Nos.2004/0142025, 2007/0042031, and 2020/0237679 and U.S. Patents 9,364,435, 9,518,272, 10,022,435, and 11,191,849. [0179] 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). Cells [0180] 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. As used 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. Representative examples of immune cells include those as shown in FIG. 45. Combination of different immune cells may be used. Representative examples of T cells include cytotoxic lymphocytes, cytotoxic T cells (CD8+ T cells), T helper cells (CD4+ T cells), αβ T cells and/or γδ T cells NK T (NKT) cells, and Th17 T-cells. In some embodiments, the immune cells are CD8+ T cells. In some embodiments, the immune cells are CD4+ T cells. In some embodiments, 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 fusion protein and, optionally, a CAR polypeptide. [0181] In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are a NK cell line, primary NK cells, memory-like NK cells, or induced memory like NK cells. [0182] In some embodiments, the immune cells are monocytes or macrophages. [0183] 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. In some embodiments, 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. See, for example, Brown et al., PloS One 5:e11373-9 (2010), Rivera et al., Curr. Protoc. Stem Cell Biol.
54:e117-21 (2020), Seki et al., Cell Stem Cell 7:11-4 (2010), Takahashi et al., Cell 126:663-76 (2006), Fusaki et al., Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 85:348-62 (2009), Park et al., Nature 451:141-6 (2008), and U.S. Patents 10,214,722, 10,370,452, 10,428,309, 10,844,356, 11,141,471, 11,162,076, and 11,193,108 and U.S. Patent Application Publications 2012/0121544, 2018/0362927, 2019/0112577, and 2021/0015859. [0184] NK cells are produced in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56brightCD16- to CD56dimCD16+ cells that represents the most abundant population in peripheral blood. In some embodiments, 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). In some embodiments, 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. Immunol. 7:504-9 (2016), Shah et al., PLoS One 8:e76781-9 (2013); Zhao et al., Front Immunol 11:584099-8 (2020)). In some embodiments, 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)). [0185] In some embodiments, the cells are primary NK cells, also known as “conventional NK cells” (cNK). Typically, 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. In some embodiments, 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. [0186] In some embodiments, 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-γ, a prototype NK cell functional readout, in response to leukemia target cells or after stimulation with IL-12, IL-15, and IL-18. Upon restimulation with cytokines or target tumor cells, a larger fraction of CIML NK cells produce higher levels of IFN-γ as compared with cNsK cells. CIML NK cells adoptively transferred into
leukemia-bearing mice inhibit tumor growth to a greater degree as compared to conventional NK cells. See, e.g., Cooper et al., Proc. Natl. Acad. Sci. USA 106:1915-9 (2009); Ni et al., J. Exp. Med. 209:2351-65 (2012); Keppel et al., J. Immunol. 190:4754-62 (2013); Romee et al., Sci. Transl. Med.8(357):357ra123-26 (2016). [0187] NK cells may be effectively used in ACT. As illustrated in FIG.20A, NK cells grouped by visualization of t-Distributed Stochastic Neighbor Embedding (viSNE) represented the largest cell population after ACT, at the expense of CD3+ cells, CD8+ cells, and ungated cells before ACT (labeled ‘Day Pre’). Different NK cell subsets positive for NKG2A and PD1 were further tracked after NK ACT infusion (FIG. 20B – FIG. 20C). These data demonstrate this treatment regimen could be adapted to deliver AICS NK cells expressing a fusion protein. [0188] In some embodiments, the cells are allogeneic to the subject receiving the cells, that is, the cells have a complete or at least partial HLA-match with the subject. In some embodiments, the cells are autologous. The term “autologous” as used herein refers to any material (e.g., NK cells or T cells) derived from the same subject to whom it is later re-introduced. The term “allogeneic” as used herein 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). [0189] In some embodiments, 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). Furthermore, the use of 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). [0190] Methods of introducing the vectors containing the fusion protein-encoding nucleic acids and/or CAR polypeptide-encoding nucleic acids into immune cells are known in the art. See, e.g., U.S. Patents 7,399,633, 7,575,925, 10,072,062, 10,370,452, and 10,829,735 and U.S. Patent Publications 2019/0000880 and 2021/0407639. [0191] In some embodiments, a lentiviral vector is transduced into immune cells. In other embodiments, 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. In some embodiments, 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. See, e.g., U.S. Patent 10,563,226, and U.S. Patent Application Publications 2019/0225991, 2020/0080108, and 2022/0186263. [0192] In some embodiments, the method entails ex vivo or in vivo delivery of linear, circular, or self-amplifying mRNAs. See, e.g., U.S. Patents 7,442,381, 7,332,322, 9,822,378, 9,254,265, 10,532,067, and 11,291,682. In some embodiments, 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. [0193] In some embodiments, a plasmid containing a fusion protein-encoding nucleic acid and/or CAR polypeptide-encoding nucleic acid is/are transfected into immune cells. In some embodiments, 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 [0194] Pharmaceutical compositions of the disclosure include compositions comprising therapeutically effective numbers of genetically modified immune cells and a pharmaceutically acceptable carrier. The term “therapeutically effective number of immune cells” (which indirectly includes a corresponding amount of the fusion protein and, optionally, CAR polypeptide) as used herein refers to a sufficient number of the immune cells that contain the fusion protein-encoding nucleic acid(s) and, optionally, CAR polypeptide-encoding nucleic acid(s) to provide a desired effect. [0195] 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, and 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 1×105 to approximately 1×1010 cells per subject. In some embodiments,
the effective number of the genetically modified immune cells is between approximately 1×105 to approximately 6×108 cells per kg of subject body weight. [0196] 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. In some embodiments, 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. 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®) may be used to achieve the desired isotonicity. Depending on the carrier and the immune cells, 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. Diseases and Disorders [0197] 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. Such diseases or disorders include cancer as well as diseases and disorders where B cell depletion or T cell depletion may be beneficial. [0198] In some aspects, 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 fusion proteins and, optionally, a CAR polypeptide as described herein. The term “cancer” as used herein 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. [0199] In some embodiments, 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 (AML)
is an MDS and a cancer of the blood and bone marrow. AML (also known as myelogenous leukemia and acute nonlymphocytic leukemia) is the most common type of acute leukemia in adults that usually progress quickly if left untreated. In some embodiments, a subject may be suffering from relapse after haploidentical hematopoietic cell transplantation (haplo-HCT) (Shapiro et al., J. Clin. Invest.132(11):e154334-17 (2022)). [0200] In some embodiments, 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, or blastic plasmacytoid dendritic cell neoplasm. [0201] In some embodiments, the cancer is characterized by the presence of a solid tumor. In some embodiments, 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), multiple myeloma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, epithelial carcinomas, fallopian tube cancer, and primary peritoneal cancer), pancreatic cancer, gastrointestinal cancer (e.g., adenocarcinoma, primary gastric lymphoma, gastrointestinal cancers (e.g., gastrointestinal stromal tumor (GIST)), and neuroendocrine (carcinoid) cancers), or blastic plasmacytoid DC neoplasm. [0202] In some aspects, 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. [0203] 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. Examples of autoimmune diseases for which the compositions and methods provided herein may be useful 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 (CD), Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan’s syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn’s disease, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressler’s syndrome, Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert- Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lyme disease chronic, Meniere’s disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonage-Turner syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia
rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Raynaud’s phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjögren’s syndrome, Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sympathetic ophthalmia (SO), Takayasu’s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, and Vogt-Koyanagi-Harada Disease. [0204] In some aspects, the compositions or methods provided herein may be used for a subject that has received a transplant. As used herein, “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. [0205] In some aspects, the compositions or methods provided herein may be used for a subject that has graft versus host disease (GVHD). “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. [0206] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from the indicated disease or disorder. In some embodiments, 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). [0207] 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. [0208] In some embodiments, the genetically modified immune cells are T cells, NK cells or monocytes or macrophages. In some embodiments, 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. Administration [0209] 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).
[0210] Administration can be autologous or allogeneic. For example, 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). Combination Therapy [0211] In some embodiments, the present methods may include co-administration of another agent, such as an anti-cancer agent, antibody therapy, immunotherapy, etc. The term “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. Thus, if given sequentially, at the onset of administration of the second therapy, 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). For example, 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. Thus, the terms are not limited to the administration of the active agents at exactly the same time. [0212] 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. This may be achieved by contacting the cancer cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cancer cells with two distinct compositions or formulations, at
the same time, wherein one composition includes recipient cells and the other includes the second agent(s). [0213] In some embodiments, 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. In some embodiments, the immune cells of the present disclosure are administered after the subject receives lymphodepletion chemotherapy. In some embodiments, the lymphodepletion chemotherapy includes melphalan. In some embodiments, the subject receives a stem cell transplant after the lymphodepletion chemotherapy. [0214] Additional ACT potentiating treatments that may be used with the aspects of the present disclosure 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. In some cases, 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. [0215] 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 thereof. [0216] 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. [0217] 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. Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. [0218] 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®). Clinically available examples of 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. [0219] 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, Blinatumomab, Brentuximab vedotin, Briakinumab, Canakinumab, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, Cedelizumab, Certolizumab pegol,
Cetuximab, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Daclizumab, Daratumumab, Denosumab, Detumomab, Dorlimomab aritox, Dorlixizumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enlimomab pegol, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab , Foravirumab, Fresolimumab, Galiximab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, GC1008, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan, Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab-CD3, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab, Oportuzumab monatox, Oregovomab, Otelixizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Pascolizumab, Pemtumomab, Pertuzumab, Pexelizumab, Pintumomab, Priliximab, Pritumumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab Reslizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab, Ruplizumab, Satumomab pendetide, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Ticilimumab (tremelimumab), Tigatuzumab, Tocilizumab (atlizumab), Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, and Zolimomab aritox. [0220] These and other aspects of the present application will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain embodiments of the application but are not intended to limit its scope, as defined by the claims.
EXAMPLES EXAMPLE 1: Materials and Methods [0221] CAR vector design. The pHIV-CD19-CAR-GFP plasmid was obtained. The CD19- CAR consists of the CD8 signal peptide, an CD19 scFv, CD8 hinge, CD8 transmembrane domain, 4-1BB intracellular domain and CD3ζ intracellular domain. [0222] Gene fragments encoding PD1-CD16 and PDL1scFv-CD16 fusion proteins were synthesized (Integrated DNA Technologies) and cloned into pHIV-CD19-CAR-GFP plasmid upstream of the CD19-CAR to generate pHIV-PD1-CD16-CD19-CAR-GFP and pHIV- PDL1scFv-CD16-CD19-CAR-GFP. The fusion protein (PD1-CD16 or PDL1scFv-CD16), CAR, and GFP are separated by P2A (SEQ ID NO: 79). The PD1-CD16 fusion protein consists of the PD1 signal peptide, the PD1 extracellular domain, a short linker of Gly-Gly-Gly-Gly-Ser (G4S) (SEQ ID NO: 56) and CD16 gene without the CD16 signal peptide. The PDL1scFv-CD16 fusion protein consists of the CD8 signal peptide, an anti-PDL1 scFv, a short linker (G4S) (SEQ ID NO: 56) and CD16 gene without the CD16 signal peptide. The anti-PDL1 scFv was generated using codon-optimized DNA sequences of the VL and VH regions of atezolizumab. The CD19scFv- CD16 fusion protein was generated by combining the CD19scFv from CD19-CAR with CD16 of PDL1scFv-CD16 fusion protein. [0223] Lentivirus production. Lentivirus was produced by co-transfecting HEK293 with the transfer plasmids (pHIV-CD19-CAR-GFP, pHIV-PD1-CD16-CD19-CAR-GFP or pHIV- PDL1scFv-CD16-CD19-CAR-GFP) along with the packaging plasmids pCMV-Δ8.9 and envelope (pCMV-VSVG or pCMV-BaEV) and pAdv plasmids. The pAdv plasmid is commercially available as pAdvAdvantage™ from Promega, and the nucleic acid sequence is provided at NCBI Accession No. U47294, version U47294.2, which sequence is incorporated herein by reference. Culture supernatants were collected at 24h and 36h, and lentivirus particles were pelleted by ultracentrifugation at 25,000 rpm for 2h at 4°C. Lentivirus particles were resuspended in 200μl DMEM medium and stored at -80°C. [0224] Cell Lines. K562, Raji, Daudi, Ramos and NK92 MI cell lines were obtained from ATCC. K562, Raji, Daudi and Ramos cells were cultured in RP-10 medium (RPMI 1640 supplemented with 10% FBS, 1× penicillin/streptomycin, 2mM L-glutamine, and 7.5 mmol
HEPES). NK92 MI cells were cultured in X-VIVOTM 15 Hematopoietic serum-free culture media (Lonza) supplemented with 10% human AB serum (Millipore Sigma), 10% FBS, 1× penicillin/streptomycin, and 2mM L-glutamine. K562 and Raji cells were transduced with VSVG- pseudotyped lentivirus encoding human PDL1 and/or mCherry-luciferase. NK92 MI cells were transduced using BaEV-pseudotyped lentivirus. [0225] Generation and transduction of human primary NK cells. Human primary NK cells were isolated from leukoreduction system chamber from a healthy donor after incubation with RosetteSep™ Human NK Cell Enrichment Cocktail (StemCell) followed by Ficoll density gradient centrifugation. Cells were stimulated for 18 hours with 10ng/mL recombinant human IL- 12 (R&D Systems) and 50ng/mL recombinant human IL-18 (R&D Systems) in RP-10. NK cells were transduced with BaEV-pseudotyped lentivirus on RetroNectin-coated plates (Takara Bio) with the addition of Vectofusin-1 (Miltenyi). After spinfection, cells were rested for 2 days in RP- 10 with 1ng/mL recombinant human IL-15 (Miltenyi). NK cells were then expanded in NK MACS medium (Miltenyi) supplemented with 5% human serum and 100 U/mL recombinant human IL-2 (Miltenyi). Alternatively, freshly isolated NK cells were cultured in the presence of irradiated K562 feeder cells expressing mbIL-21, 4-1BBL and OX40L in RP-10 with 100U/mL recombinant human IL-2 (Miltenyi) for 5 days, then transduced as described above and cultured for another 10 days with the addition of feeders one day after transduction. [0226] Generation and transduction of human primary T cells. Human primary NK cells were isolated from leukoreduction system chamber from a healthy donor after incubation with RosetteSep™ Human NK Cell Enrichment Cocktail (StemCell) followed by Ficoll density gradient centrifugation. Cells were stimulated for 18 hours with 10ng/mL recombinant human IL- 12 (R&D Systems) and 50ng/mL recombinant human IL-18 (R&D Systems) in RP-10. NK cells were transduced with BaEV-pseudotyped lentivirus and rested for 2 days in RP-10 with 1ng/mL recombinant human IL-15 (Miltenyi). NK cells were then expanded in NK MACS medium (Miltenyi) supplemented with 5% human serum and 100 U/mL recombinant human IL-2 (Miltenyi). [0227] Cytotoxicity of NK cells in vitro and intracellular cytokine staining. Target cells were cultured in RP-10 medium and labelled with 5uM of CellTrace Violet (Thermo Fisher Scientific) in PBS for 20 min at 37°C. Target cells and effector cells were washed twice with RP-10 and co- cultured at the indicated E:T ratios. To measure NK cell cytotoxicity, cells were co-cultured for
4h, then stained with 2 µL of PE-Annexin V (Biolegend) and 2 µL of 7-AAD (BD Biosciences) in 50uL Annexin V binding buffer (Biolegend) for 15 min at RT. To measure intracellular IFNγ and degranulation, cells were co-cultured for 1h, followed by the addition of 0.2uL BD GolgiPlug™ (BD Biosciences), 0.13uL BD GolgiStop™ (BD Biosciences) and 1uL of APC- CD107a (Biolegend). After an additional 5h of co-culture, cells were stained for intracellular IFNγ using BD Cytofix/Cytoperm™ (BD Biosciences). Cells were acquired using BD LSRFortessa™ and analyzed using FlowJo (Tree Star). [0228] Cytotoxicity of NK cells in vivo. Six to eight week-old NSG mice were obtained from The Jackson Laboratories. NSG mice were injected intravenously with 1 million luciferase- expressing K562, Raji or Ramos cells. Three days after tumor inoculation, primary NK cells were injected intravenously. Mice were injected intraperitoneally with IL-2 (up to 75,000 IU/mouse) every 3 days. In vivo bioluminescent imaging (BLI) was performed once or twice a week to monitor tumor burden. Blood samples were collected from the tail vein. After 4-8 weeks, mice were euthanized for organ harvest for flow cytometric analysis. Mice were sacrificed when they became moribund and exhibited significant weight loss. [0229] Kinetics of AICS Shedding. NK cells were co-cultured with target cells and stained for surface expression of CD16 at different timepoints. CD16 shedding was analyzed by measuring soluble CD16 (or PDL1scFv-CD16 fusion protein) in the supernatant by ELISA or cytometric bead array (CBA). [0230] Testing AICS in blocking PD1-PDL1 inhibition. To determine if shed fusion protein can interfere with PD1-PDL1 inhibition on neighboring cells, NK cells were transduced to overexpress PD1 and co-cultured with PDL1-expressing target cells. PDL1-expressing target cells were pre-treated with conditioned media containing released PDL1scFv-CD16 (from NK cell and target cell co-culture) before co-culture with PD1-expressing NK cells. Target cell death was then measured by Annexin V and 7AAD staining. To determine if PDL1scFv-CD16 fusion protein can prevent PD1-PDL1 inhibition when expressed on the same cell, NK cells were transduced to express CD19-CAR as well as both native PD1 protein and the PDL1scFv-CD16 fusion protein. NK cells were then co-cultured with PDL1-expressing target cells, and target cell death was measured by Annexin V and 7AAD staining. [0231] Flow Cytometry. Cells were stained with the following antibodies: anti-CD16 (3G8), anti-CD56 (HCD56), anti-PD1 (NAT105), anti-PDL1 (29E.2A3), anti-CD3 (OKT3), anti-CD4
(RPA-T4), anti-CD8 (SK1), CD19 (HIB19), anti-CD20 (2H7), and anti-CD107a (H4A3) from Biolegend; anti-IFNg (B27), and anti-CD3 (HIT3a) from BD biosciences. CD19-CAR was stained using PE-labeled human CD19 (20-291) protein (ACROBiosystems). EXAMPLE 2: Activation Induced Clipping System (AICS) NK Cells Link Tumor Antigen Recognition to Regulatory Payload Delivery [0232] NK cells expressing an anti-PD1 scFV fusion protein and CD19 CAR enable the targetable delivery of the anti-PD1 payload after CD19 tumor antigen engagement (FIG.1). The inhibitory PD1/PDL1 pathway in the TME is the target in this example, which will allow these enhanced memory-like NK CAR cell’s effect on the TME to be examined in vitro. The NK cell line NK92 MI does not normally express CD16 or PD1 as measured by Flow cytometry (FIG.2A, top panel), but transfection of the anti-PD1-CD16 fusion protein results in surface expression of both antigens (FIG.2A, bottom panel). [0233] NK cell activation can be measured by co-culture with cancer cell lines, with and without exogenous CD19 expression. Cancer cell lines include K562 leukemia cells, which do not express CD19, but can still activate NK cells by the NK activating receptor NKG2D and Raji B cell lymphoma cells, which do express CD19, and which will activate CD19 CAR expressing cells. [0234] NK92 MI cells expressing CD19 CAR, PD1-CD16 fusion protein, PD1-CD16 fusion protein and CD19 CAR, and CD19 CAR and PDL1scFv-CD16 fusion proteins were tested for CD16 expression with negative control (no stimulation), or positive control (PMA/Iono), and co- cultured with K562 (±exogenous CD19) and Raji (±exogenous CD19) cells (FIG. 2B). The co- culture of NK cells with Raji cells induced the shedding of PD1-CD16 fusion protein (based on downregulation of CD16 expression on the surface of NK cells), which was induced by CAR engagement (absence of CAR did not induce CD16 shedding upon co-culture with Raji cells). Cancer cells expressing PDL1 induced more downregulation of cell-associated CD16, which can be due to increased shedding or binding of the PD1-CD16 fusion protein with PDL1. The co- culture of NK cells with K562 cells also induced the shedding of PD1-CD16 fusion protein, and shedding is more pronounced when K562 express PDL1. Shedding of the entire fusion protein is confirmed when the same cells are stained for cell-surface PD1 (FIG.2C). [0235] NK cells kill cancer cells through antibody-dependent cell-mediated cytotoxicity (ADCC) when tumor specific antibodies are present. The inventive fusion protein-expressing NK
cells were tested for their ability to kill target cells in the absence of antibodies (FIG.3). NK cells were co-cultured with K562 cells (±exogenous CD19) and the killing of target cells by NK cells was measured. Compared to the CD19- parental K562 cells, PDL1+ K562 cells strongly inhibited the cytotoxic functions of control NK cells or CD19-CAR expressing NK cells (FIG.3, top left two panels). However, NK cells that express the PD1-CD16 fusion protein showed enhanced killing of the PDL1+ K562 cells, indicating that indeed, the PD1-CD16 fusion protein can induce the killing of the target cells by NK cells in the absence of antibody. FIG. 4 shows a direct comparison of the different NK cell conditions as assayed in FIG. 3. Cells expressing the PDL1scFv-CD16 fusion protein induced the highest level of cytotoxicity. Similar results were obtained when the same NK cells were tested against Raji cells (±exogenous CD19), as illustrated in FIG.5 and FIG.6. [0236] Next, the activation of the NK92 MI cells were measured based on IFNγ expression. Cells expressing the PDL1scFv-CD16 fusion protein along with CD19-CAR induced strong IFNγ expression when stimulated with K562 (±exogenous CD19) or Raji (±exogenous CD19) cells (FIG. 7). To assess the kinetics of PDL1scFv-CD16 fusion protein shedding, NK92 MI cells expressing PDL1scFv-CD16 fusion protein and CD19-CAR (PDL1scFv-CD16-CD19-CAR) were co-cultured with K562 (±exogenous CD19) or Raji (±exogenous CD19) cells. The PDL1+ cell lines were compared with the parental cell lines (PDL1-). PMA/ionomycin resulted in robust CD16 shedding and was used as a positive control. Co-culture of NK cells with K562 or Raji cells induced the shedding of PDL1scFv-CD16 fusion protein (shedding is based on downregulation of CD16 expression on the surface of NK cells) within 1 hour. Cancer cells expressing PDL1 decreased surface CD16, due to increased shedding or binding of the PDL1scFv-CD16 fusion protein with PDL1 (FIG.7). [0237] The kinetics of PDL1 engagement on cancer cells was assessed as illustrated in FIG.8. NK cells were engineered to express both PDL1scFv-CD16 fusion protein and the CD19-CAR (PDL1scFv-CD16-CD19-CAR). These NK cells were co-cultured with K562 or Raji engineered to express exogenous PDL1. The PDL1+ cell lines (FIG. 8, right two panels) were compared with the parental cell lines (PDL1-, FIG.8, left two panels) for PDL1 expression over time. NK cells expressing PDL1scFv-CD16 fusion protein and CD19-CAR cocultured with either K562 or Raji cells expressing PDL1 induced the downregulation of PDL1 on cancer cells within 1 hour and
continued to decrease over time, suggesting that PDL1 was quickly engaged by the PDL1scFv- CD16 fusion protein (FIG.8). EXAMPLE 3: CD19-CD16 Fusion Protein Killing [0238] A different approach is a CD16-based fusion protein with an ability to activate and result in CAR-independent cytotoxicity. Described herein are CD19-CD16 fusion protein-expressing cells, which are compared to CD19-CAR expressing cells (FIG.9). The CD19 portion of the fusion protein is an CD19 antibody fragment (e.g., scFv) targeting the CD19 antigen on cancer cells. To test whether the CD19scFv-CD16 fusion protein can induce the killing of the target cells by NK cells, NK92 MI cells were engineered to express no CAR (negative control) (FIG. 10, circles), CD19-CAR (regular CAR, positive control) (FIG. 10, squares), and CD19scFv-CD16 fusion protein (FIG. 10, triangles). The NK92 cells were sorted to obtain a pure population (>95% positive for CD19-CAR or CD19scFv-CD16 fusion protein), based on GFP expression. NK cells were co-cultured with the B cell lymphoma cell lines Raji, Daudi, and Ramos, and the killing of target cells were measured by AnnexinV+ and 7AAD+ stained cells. [0239] Negative control, untransduced NK cells, were effective at killing Raji cells (based on % cell death on y axis), but less so at killing Daudi and Ramos cells. NK cells expressing CD19scFv-CD16 fusion protein showed enhanced killing of the target cells as compared to negative control NK cells, and at a level similar to that of NK cells expressing the regular CD19- CAR as illustrated in FIG.10. [0240] The activation of NK cells based on the expression of CD107a (a degranulation marker indicating the release of cytotoxic molecules by NK cells) and IFNγ was next examined (FIG.12). When co-cultured with B cell lymphoma cell lines (Raji, Daudi and Ramos), NK cells expressing CD19scFv-CD16 fusion protein showed increased activation based on CD107a expression as compared to control NK cells and was similar to NK cells expressing the regular CD19-CAR. [0241] NK cells expressing CD19scFv-CD16 fusion protein also showed increased production of IFNg cytokine as compared to control NK cells, but less than NK cells expressing the regular CD19-CAR, suggesting that the regular CAR may be better than the CD16-based fusion protein at inducing cytokine production. [0242] Comparatively, CD19-CD16 fusion protein are also functional in T cells. T cells from healthy donor peripheral blood was engineered to express 1) No CAR (control), 2) CD19-CAR,
3) CD19scFv-CD16 fusion protein, 4) CD19scFv-CD16 fusion protein + 4-1BBL. To generate CD19scFv-CD16, the scFv of CD19-CAR was fused with the complete CD16 protein (excluding the signal peptide). The regular CD19-CAR contains the cytoplasmic domain of 4-1BB, a co- receptor that activates NK cells and T cells function and helps these cells persist in vivo. [0243] Since the CD19scFv-CD16 fusion protein does not contain the cytoplasmic domain of 4-1BB, CD19scFv-CD16 fusion protein and 4-1BBL, the ligand of 4-1BB, was co-expressed, these additional proteins may also help the cells persist in vivo. [0244] The results of transduction were analyzed by flow cytometry (FIG.12). The third rows of plots indicate the efficiency of transduction based on GFP expression after 9 days of culture. Control T cells (top row) were untransduced, hence they showed 0.03% of GFP+ cells. T cells transduced with regular CD19-CAR or CD19scFv-CD16 fusion protein were 18.8% and 17.7% GFP+, indicating that they are similar in the levels of transduction, and therefore a similar number of transduced T cells co-cultured with target cancer cells are in the assays, and thus a fair comparison in regard to the number of target cells killed can be made (the ratios of cancer cells versus T cells will be similar). T cells transduced with CD19scFv-CD16 fusion protein plus 4- 1BBL only showed 2.68% GFP+, which is a very low level of transduction. [0245] To test cytotoxicity, T cells were co-cultured with B cell lymphoma cell lines (Raji, Daudi and Ramos) and the killing of target cells by T cells was measured (FIG. 13). Negative control, untransduced T cells were effective at killing Raji cells (based on % cell death on y axis), but less so at killing Daudi and Ramos cells. T cells expressing CD19scFv-CD16 fusion protein showed enhanced killing of the target cells as compared to control T cells, but at a lower level than that of T cells expressing the regular CD19-CAR. T cells expressing CD19scFv-CD16 fusion protein + 4-1BBL were less efficient at killing the target cells, however, only 2.68% of these cells were GFP+, and therefore transduced. [0246] Next, the activation of T cells based on the expression of CD107a (a degranulation marker indicating the release of cytotoxic molecules by T cells) was measured. The top graphs show the expression of CD107a on all T cells (untransduced and transduced, GFP- and GFP+). The bottom graphs show the expression of CD107a on transduced T cells only (GFP+ only). When co-cultured with B cell lymphoma cell lines (Raji, Daudi and Ramos), T cells expressing the CD19scFv-CD16 fusion protein had increased activation based on CD107a expression as compared to control T cells, but less than T cells expressing the regular CD19-CAR (FIG.13).
[0247] The activation of T cells based on the production of the IFNγ cytokine (FIG.15) was similar to CD107a (FIG. 13). The top graphs show the production of IFNγ on total T cells (untransduced and transduced, GFP- and GFP+), and the bottom graphs show the production of IFNγ on transduced T cells only (GFP+ only). T cells expressing CD19scFv-CD16 fusion protein also showed increased activation based on their production of IFNγ cytokine as compared to control T cells, but less than T cells expressing the regular CD19-CAR. [0248] Next, the shedding of CD19scFv-CD16 fusion protein was measured. The right two columns of plots indicate the expression of CD16 on T cells (FIG. 17). Primary human T cells genetically engineered to express the CD19scFv-CD16 fusion protein were co-cultured with B cell lymphoma cell lines (Raji, Daudi and Ramos), and the expression of CD16 was measured. The co- culture of T cells with all cancer cells induced the downregulation of CD16 expression on the surface of T cells, which may indicate that the CD19scFv-CD16 fusion protein is being engaged by CD19 antigen on the cancer cells, or that the CD19scFv-CD16 fusion protein is being cleaved by the protease ADAM 17 and shed (released) from the cell surface. [0249] Primary human T cells genetically engineered to express the CD19scFv-CD16 fusion protein in combination with 4-1BBL (FIG.18) resulted in similar results of loss of CD16 on the cell surface. EXAMPLE 4: NK Expressing PDL1-CD16 Fusion Protein [0250] NK cells were engineered to express a PDL1-CD16 fusion protein, using the PDL1 repressive pathway ligand instead of the target antigen of CD19 (FIG. 16). The function of a regular CAR (targeting PDL1 antigen on cancer cells) versus the CD16-based fusion protein (PDL1scFv-CD16 or PD1-CD16 fusion proteins) was compared. To test whether the PDL1scFv- CD16 or PD1-CD16 fusion protein-expressing cells can induce target cell killing, NK cells were co-cultured with leukemia cell line cells with and without exogenous PDL1. NK92 MI cells were engineered to express 1) No CAR (negative control), 2) PDL1-CAR (standard CAR, positive control), 3) PDL1scFv-CD16 fusion protein, and 4) PD1-CD16 fusion protein (FIG.17). [0251] To generate PD1-CD16 fusion protein, the scFv targeting PDL1 was replaced with the extracellular domain of PD1. It was expected that the PDL1scFv will bind with stronger affinity to PDL1 as compared with PD1. These cells were co-cultured with either K562 cells, a leukemia cell line that activates NK cells via the activating receptor NKG2D on NK cells or K562 engineered
to express PDL1 (PDL1+ K562). Control nontransduced NK cells were effective at killing K562 cells (based on % cell death on y axis), but less so at killing PDL1+ K562 cells. This result was expected as the PDL1 pathway represses NK cell killing. [0252] NK cells expressing PDL1scFv-CD16 fusion protein showed enhanced killing of the PDL1+ K562 cells as compared to control NK cells, and at a level similar to that of NK cells expressing the regular PDL1-CAR. NK cells expressing PD1-CD16 fusion protein also showed enhanced killing of the PDL1+ K562 cells as compared to control NK cells, but at a lower level than that of T cells expressing the regular CD19-CAR or PDL1scFv-CD16 fusion protein. FIG. 18 illustrates the data shown in FIG.17 but compares each NK cell line separately as a function of target cell killing (K562 with and without PDL1 expression). [0253] When co-cultured with PDL1+ K562 cells, NK cells expressing PDL1scFv-CD16 fusion protein showed increased activation based on CD107a expression as compared to control NK cells and was similar to NK cells expressing the regular CD19-CAR. NK cells expressing PDL1scFv-CD16 fusion protein also showed decreased production of IFNγ cytokine as compared to NK cells expressing the regular CD19-CAR. EXAMPLE 5: Comparing CD16-based Antigen Receptor (SAR) with Conventional CAR in Human Primary T Cells [0254] NK cells can kill cancer cells through various mechanisms. They can target cancer cells directly when engineered to express CAR, but they also have numerous intrinsic receptors that allow them to kill target cells. For example, NK cells express the activating receptor NKG2D that can recognize stress ligands on the surface of the cancer cells (e.g., MICA/B, ULBP1-6). NK cells can also recognize tumor antigens indirectly via a mechanism called ADCC where CD16A can bind the Fc portion of an antibody. [0255] Chimeric antigen receptors (CARs) are synthetic receptors that allow cells, such as NK cells and T cells, to recognize a tumor antigen on the surface of the cancer cell and trigger the release of granules to kill the cancer cell. A subset of NK cells naturally express CD16A. In the presence of a tumor antigen specific antibody, NK cells can kill the cancer cell through CD16A by ADCC. Primary NK cells from the peripheral blood express high levels of CD16A. When NK cells are activated, with various stimuli, such as using PMA/ionomycin (activator of intracellular signaling pathways), IL-12 and IL-18 (cytokines), or through engagement with cancer cells (K562
leukemia cell line), CD16A can be cleaved and released from the cell surface. This phenomenon allows the NK cells to disengage the target cell and move on to 2nd and 3rd, etc. target cells allowing serial killing. CD16A shedding may also prevent NK cell exhaustion. The shedding of CD16A is mediated by the metalloprotease ADAM 17. [0256] The function of a regular CAR (targeting CD19 antigen on cancer cells) versus a CD16- based fusion protein (CD19scFv-CD16 fusion protein) also targeting CD19 antigen on cancer cells was compared to see whether the CD19scFv-CD16 fusion protein can induce the killing of target cells by T cells. The cytotoxicity of T cells against Raji lymphoma cells was evaluated. It was found (FIGS.25-30) that T cells expressing CAR or CD16-based receptor (SAR) were comparable in their function. All showed enhanced killing of target cells in contrast to untransduced T cells. IFNγ cytokine production and degranulation (CD107a) of T cells upon co-culture with Raji lymphoma cells was also compared. CAR-T cells were co-cultured with Raji (B cell lymphoma cell line) for up to 24 days. The CAR-T cells were re-challenged with Raji cells at day 8 and day 16 to induce chronic stimulation of T cells and to study long-term persistence and expansion. T cells expressing the CD16-based receptors (SAR) were less activated than CAR-T cells upon engagement of Raji cells. The cells were cultured in the presence of IL-2 cytokine to promote T cell survival and proliferation. CAR-T cells and SAR-T cells were able to eliminate Raji cells more efficiently than untransduced T cells, however SAR-T cells were more potent than CAR-T cells at later timepoints (day 12 and day 16) and lead to better clearance of Raji cells. It was also found that SAR-T cells expanded more than CAR-T cells, as the numbers of SAR-T cells were about 5-fold higher than the number of CAR-T cells at day 12 and day 16, and about 10-fold higher at day 24. The results also demonstrate that the expansion of the CD4+ subset of SAR-T cells and the CD8+ subset of SAR-T cells is more robust than the CAR-T cells, respectively. Overall, SAR- T cells kill as good as CAR-T cells, but seem to be less activated (produce less IFNγ and CD107a). EXAMPLE 6: Tumor Escape [0257] When lymphoma patients are treated with CAR-T cells (for example, CAR targeting CD19 antigen on cancer cell), in some cases, the cancer cells develop a resistance mechanism by downregulating the expression of the CAR-specific antigen (CD19) and become resistant to CAR- T cell therapy. Whether the CD16-based antigen receptor (SAR) could also mediate antibody- dependent cell-mediated cytotoxicity (ADCC), which could limit tumor escape, by allowing the
SAR to engage both CD19 (via scFv) and CD20 (via using a therapeutic antibody targeting CD20 called rituximab), was examined. In the presence of a tumor antigen specific antibody, it was found that cancer cells can be killed through CD16A through ADCC. [0258] In addition, a model of tumor escape was established using CRISPR-Cas9 technology to knockout (delete) the CD19 gene in Raji cells. These cells still express other markers such as CD20 and CD22 which can be targeted. In this experiment, two guide RNAs (gRNAs) targeting CD19 were mixed with Cas9-RFP enzyme to form RNPs (ribonucleoproteins). Raji cells were electroporated with RNPs. In the flow cytometry plots (FIG.34), CD19 antigen was deleted in about 33% of Raji cells. The CD19 knockout Raji cells were sorted to obtain a pure population (>99%). Control antibody (IgG) or rituximab treated Raji and CD19-deficient Raji (CD19KO- Raji) were co-cultured with T cells expressing the indicated constructs for 24h. The results (FIG. 35) show that CD19-deficient Raji cells are resistant to CAR and SAR-mediated killing through CD19 targeting. However, T cells expressing CD19scFv-haCD16-GFP (high affinity CD16-based SAR) were able to kill CD19-deficient Raji cells treated with rituximab which targets CD20, thus inducing antibody-dependent cell-mediated cytotoxicity (ADCC). [0259] CAR-T cells were co-cultured with CD19KO-Raji cells for up to 24 days. The CAR-T cells were re-challenged with CD19KO-Raji cells at day 8 and day 16 to study the ADCC capability of SAR in a model of tumor escape where CD19 antigen was absent. The cells were cultured in the presence of IL-2 cytokine to promote T cell survival and proliferation. The cells were also treated with either a non-targeting negative control antibody (IgG) or rituximab (anti- CD20) to induce ADCC. The results are shown in FIGS.37-39. The figures show the number of CD19KO-Raji cells remaining in the culture in the presence of control antibody (IgG) or rituximab (anti-CD20). The results demonstrate that SAR-T cells were more potent at eliminating CD19KO- Raji cells in the presence of rituximab as compared to untransduced T cells or CAR-T cells, suggesting that the CD16 moiety of the SAR in capable of inducing ADCC. The high-affinity CD16-based SAR (CD19scFv-haCD16, variant V158) was slightly better than the low affinity version of SAR (CD19scFv-CD16, variant F158) at inducing killing of CD19KO-Raji cells in the presence of rituximab. The results demonstrate that SAR-T cells expand more than CAR-T cells when co-cultured with CD19KO-Raji cells in the presence of rituximab, as the numbers of SAR- T cells were about 3-fold higher than the number of CAR-T cells at day 20.
[0260] FIG.40 show the proportion and number of transduced CD4+ T cells in the culture. The results demonstrate that the expansion of the CD4+ subset of SAR-T cells is more robust than the CD4+ subset of CAR-T cells when co-cultured with CD19KO-Raji cells in the presence of rituximab, as the numbers of CD4+ SAR-T cells were about 3-fold higher than the number of CD4+ CAR-T cells at day 20. FIG.41 show the proportion and number of transduced CD8+ T cells in the culture. The results also demonstrate that the expansion of the CD8+ subset of SAR-T cells is similar or lower than the CD8+ subset of CAR-T cells when co-cultured with CD19KO- Raji cells in the presence of control IgG or rituximab, although the expansion of CD8+ T cells was overall much weaker than that of CD4+ T cells. EXAMPLE 7: Rationale for AICS Compatibility Across Diverse Immune Cell Types. [0261] The Activation Induced Clipping System (AICS) provided herein can leverage the innate characteristics of CD16A, including its transmembrane domain, to engage with crucial signaling adaptor proteins CD3ζ and FCER1G. The current understanding is that CD16A interacts with adaptor signaling proteins CD3ζ and FCER1G exclusively via transmembrane domains. Notably, both CD3ζ and FCER1G adaptor signaling proteins are transmembrane entities characterized by extremely short extracellular domains, whereas CD16A is characterized by an extremely short intracellular domain, which has no known signaling function. The interaction of CD16A with these adaptor proteins is not only necessary but also sufficient for its surface expression and signaling, devoid of a predilection for selective interactions between the two entities. Furthermore, the functional capacity of CD16A to activate diverse immune cell types, such as NK cells, T cells, monocytes, and macrophages has been established. [0262] FIG.42 shows the full spectrum of immune cell types that express CD3ζ and FCER1G, and expression of fusion proteins as provided herein in human NK cells, T cells, and monocytes has been demonstrated. FIG.42 (A) CD3ζ mRNA and (B) FCER1G mRNA show expression in human peripheral blood-derived immune cells. Population-averaged gene expression data for purified human immune cells was acquired from the ImmGen Human Cell Atlas dataset. This dataset was generated through ultra-low-input RNA-sequencing using peripheral blood mononuclear cells from two healthy donors. The data is publicly accessible and can be found at immgen.org. [0263] It has also been demonstrated that the extracellular domain of CD16A, specifically its Fc binding domain, is required for surface expression in addition to ADCC function. This finding
is demonstrated with constructs that incorporate the CD16A hinge, transmembrane, and cytoplasmic domains but omitting the Fc binding domain not being expressed on the cell surface. FIG.43 demonstrates the surface expression of the Activation Induced Clipping System (AICS) carrying the anti-CD19 single-chain fragment-variable (humanized FMC63 clone) in THP-1, a monocyte cell line derived from an acute monocytic leukemia patient. Untransduced THP-1 cells (A), THP-1 cells expressing human CD16A (B), and FMC63-AICS (C), connected to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide, were subjected to staining with an isotype control, as well as antibodies specific for human CD16 and FMC63 scFv. Surface expression of the constructs was subsequently analyzed using flow cytometry. The THP-1 cell line (TIB-202) was procured from the American Type Culture Collection (ATCC) and maintained in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum, 5 mM L-glutamine, and 1% penicillin/streptomycin. Genetic engineering of THP-1 cells involved the use of VSV-G pseudotyped lentiviral particles to introduce two distinct genetic constructs: a biscistronic cDNA encoding human endogenous CD16A and enhanced GFP (eGFP) fused with a self-cleaving P2A peptide (hCD16A-P2A-eGFP), and Activation Induced Clipping System (AICS) carrying anti- CD19 single-chain fragment-variable and eGFP fused with a self-cleaving P2A peptide (FMC63- AICS-P2A-eGFP). In both cDNA constructs, eGFP served as an internal fluorescent marker to assess transduction efficiency. Surface expression analysis of human endogenous CD16A and FMC63-AICS on both untransduced and transduced THP-1 cells was conducted via flow cytometry. Fluorescent antibodies specific to CD16 (PE-conjugated anti-human CD16 mouse monoclonal antibody clone 3G8 from CellSignaling Technology, catalog # 82004S) and FMC63 (APC-conjugated REAffinity CD19 CAR FMC63 Idiotype Antibody from Miltenyi Biotec, catalog # 130-127-343) were used for this purpose. Prior to antibody staining, the cells were pre- incubated with Human TruStain FcX (BioLegend, catalog # 422301) to block non-specific binding of fluorescent antibodies to endogenous Fc receptors on THP-1 cells. As an isotype control, PE- conjugated mouse IgG1 kappa clone MOPC-21 (BioLegend, catalog # 400111) was employed. [0264] FIG.44 illustrates the importance of the FC-binding domain of CD16A in ensuring the proper surface expression of the Activation Induced Clipping System (AICS). Jurkat cells were transduced with CD27-AICS (A) or CD27-AICS constructs lacking the FC-binding domain (B and C), all linked to enhanced green fluorescent protein (eGFP) via the P2A self-cleaving peptide. The cells were subsequently stained with antibodies specific for the CD27 ectodomain and CD16, and
the expression of CD27-AICS constructs was evaluated on eGFP-positive live cells using flow cytometry. The Jurkat cell line, clone E6-1 (TIB-152), was procured from the American Type Culture Collection (ATCC) and cultured in RPMI medium supplemented with 10% heat- inactivated fetal bovine serum, 5 mM L-glutamine, and 1% penicillin/streptomycin. To enable the expression of various genetic constructs, Jurkat cells underwent genetic engineering using VSV- G pseudotyped lentiviral particles. Three distinct genetic constructs were introduced: Biscistronic cDNA encoding Activation Induced Clipping System fused to the CD27 ectodomain (amino acids 25-124, using endogenous human CD27 numbering) and enhanced GFP (eGFP), linked with a self-cleaving P2A peptide (CD27-AICS-P2A-eGFP); Activation Induced Clipping System lacking the FC-binding domain (lacking amino acids 1-189, using endogenous human CD16A numbering) with or without the CD8A hinge (CD27-AICSΔFc-P2A-eGFP and CD27-CD8A hinge-AICSΔFc- P2A-eGFP). [0265] In all cDNA constructs, eGFP was incorporated as an internal fluorescent marker to facilitate the assessment of transduction efficiency. The surface expression of the various CD27- AICS constructs on transduced Jurkat cells was analyzed using flow cytometry. Fluorescent antibodies specific to CD16 (PE-conjugated anti-human CD16 mouse monoclonal antibody clone 3G8 from CellSignaling Technology, catalog # 82004S) and CD27 (APC-conjugated anti-human CD27 mouse monoclonal antibody clone L128 from BD Biosciences, catalog # 337169) were employed for this purpose. Prior to antibody staining, the cells were pre-incubated with Human TruStain FcX (BioLegend, catalog # 422301) to prevent non-specific binding of fluorescent antibodies. [0266] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [0267] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be
devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.
Claims
CLAIMS What is claimed is: 1. A nucleic acid or set of nucleic acids comprising a first sequence that encodes a fusion protein, wherein the fusion protein comprises: a target binding domain that binds a first target, a cleavage domain cleavable by A Disintegrin and Metalloproteinase (ADAM) 17 and which comprises the extracellular domain of CD16A, and a transmembrane domain; wherein the cleavage domain is located between the target binding domain and the transmembrane domain.
2. The nucleic acid or set of nucleic acids of claim 1, wherein the transmembrane domain interacts with signaling adaptor proteins CD3ζ and Fc Fragment of IgE Receptor Ig (FCER1G).
3. The nucleic acid or set of nucleic acids of claim 1 or 2, wherein the first target is a receptor or ligand on a cell, the killing of which cell is desirable.
4. The nucleic acid or set of nucleic acids of claim 3, wherein the killing of the cell by antibody-dependent cellular cytotoxicity (ADCC) is desirable.
5. The nucleic acid or set of nucleic acids of any one of claims 1-4, wherein the first target is an antigen or other ligand on a cancer cell.
6. The nucleic acid or set of nucleic acids of any one of claims 1-4, wherein the first target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell.
7. The nucleic acid or set of nucleic acids of any one of claims 1-4, wherein the first target is a receptor or other ligand on an immune cell.
8. The nucleic acid or set of nucleic acids of claim 7, wherein the immune cell is a B cell or T cell.
9. The nucleic acid or set of nucleic acids of any one of claims 1-8, wherein the target binding domain binds a cancer antigen, and the cancer antigen is EGFR, CD19, CD20, a NKG2D ligand, CS1, GD2, 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, PD-L1, BCMA, CD33, 41BB, CTAG1B or CD33.
10. The nucleic acid or set of nucleic acids of any one of claims 1-8, wherein the target binding domain binds a receptor or other ligand on a B cell, and wherein the receptor or other ligand is 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.
11. The nucleic acid or set of nucleic acids of any one of claims 1-8, wherein the target binding domain binds a B cell maturation antigen, wherein the B cell maturation antigen is (BCMA), CD19, CD20, CD27, CD70, or CD117, or mesothelin.
12. The nucleic acid or set of nucleic acids of any one of claims 1-9, wherein the target binding domain binds a receptor or other ligand on a T cell, and wherein the receptor or other ligand is αβ T cell receptor, γδ T cell receptor, 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.
13. The nucleic acid or set of nucleic acids of any one of claims 1-12, wherein the target binding domain comprises an antibody fragment.
14. The nucleic acid or set of nucleic acids of claim 13, wherein the target binding domain comprises a single-chain variable antibody fragment (scFv).
15. The nucleic acid or set of nucleic acids of any one of claims 1-4, 13 and 14, wherein the target binding domain binds CD19, PDL1 or CD70.
16. The nucleic acid or set of nucleic acids of claim 15, wherein the target binding domain comprises an anti-CD19 antibody fragment, PD1 or an anti-PDL1 antibody fragment, or CD27, respectively.
17. The nucleic acid or set of nucleic acids of any one of claims 1-16, wherein the target binding domain and the extracellular domain of CD16A are connected via a hinge or linker and the nucleic acid also encodes the hinge or linker.
18. The nucleic acid or set of nucleic acids of any one of claims 1-17, wherein the transmembrane domain comprises a transmembrane domain of a protein cleavable by ADAM 17.
19. The nucleic acid or set of nucleic acids of claim 18, wherein the transmembrane domain is of CD16A, CD62L, TNF-α, TNF receptor I or TNF receptor II.
20. The nucleic acid or set of nucleic acids of any one of claims 1-19, wherein the cleavage domain comprises the amino acid sequence AVSTI set forth as SEQ ID NO: 37.
21. The nucleic acid or set of nucleic acids of any one of claims 1-19, wherein the cleavage domain comprises the amino acid sequence set forth as SEQ ID NO: 39.
22. The nucleic acid or set of nucleic acids of any one of claims 1-19, wherein the cleavage domain comprises the amino acid sequence set forth as SEQ ID NO: 39 and the transmembrane domain comprises the amino acid sequence set forth as SEQ ID NO: 68.
23. The nucleic acid or set of nucleic acids of any one of claims 1-22, wherein the fusion protein further comprises an intracellular domain connected to the transmembrane domain and the nucleic acid encodes the intracellular domain.
24. The nucleic acid or set of nucleic acids of claim 23, wherein the intracellular domain is of a protein cleavable by ADAM 17.
25. The nucleic acid or set of nucleic acids of claim 24, wherein the intracellular domain is of CD16A, CD62L, TNF-α, TNF receptor I or TNF receptor II.
26. The nucleic acid or set of nucleic acids of any one of claims 1-25, further comprising a second sequence that encodes a chimeric antigen receptor (CAR) polypeptide, wherein the CAR polypeptide comprises a second target binding domain that binds a second target, wherein the first target and second target are different, and a transmembrane domain.
27. The nucleic acid or set of nucleic acids of claim 26, wherein the CAR polypeptide further comprises an intracellular domain.
28. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target is an antigen or other ligand on a cancer cell.
29. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target is a cognate receptor or cognate ligand of a cancer antigen on an immune cell.
30. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target is a receptor or other ligand on an immune cell.
31. The nucleic acid or set of nucleic acids of claim 30, wherein the immune cell is a B cell or T cell. 4880-6768-7197.2
32. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target binding domain binds a cancer antigen, and the cancer antigen is EGFR, CD19, CD20, a NKG2D ligand, CS1, GD2, 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, PD-L1, BCMA, CD33, 41BB, CTAG1B or CD33.
33. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target binding domain binds a receptor or other ligand on a B cell, and wherein the receptor or other ligand is 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.
34. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target binding domain binds a B cell maturation antigen, wherein the B cell maturation antigen is (BCMA), CD19, CD20, CD27, CD70, or CD117, or mesothelin.
35. The nucleic acid or set of nucleic acids of claim 26 or 27, wherein the second target binding domain binds a receptor or other ligand on a T cell, and wherein the receptor or other ligand is αβ T cell receptor, γδ T cell receptor, 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.
36. The nucleic acid or set of nucleic acids of any one of claims 26-35, wherein the second target binding domain comprises an antibody fragment.
37. The nucleic acid or set of nucleic acids of claim 36, wherein the second target binding domain comprises a single-chain variable antibody fragment (scFv).
38. The nucleic acid or set of nucleic acids of any one of claims 26, 27, 36 and 37, wherein the second target binding domain binds CD19 or PDL1.
39. The nucleic acid or set of nucleic acids of claim 38, wherein the second target binding domain comprises an anti-CD19 antibody fragment, PD1 or an anti-PDL1 antibody fragment.
40. The nucleic acid or set of nucleic acids of any one of claims 26-39, wherein the first and second sequences are operatively linked to the same or different promoters.
41. A vector or set of vectors comprising the nucleic acid or set of nucleic acids of any one of claims 1-40.
42. The vector or set of vectors of claim 41, wherein the vector is a viral vector.
43. The vector or set of vectors of claim 41, wherein the vector is a non-viral vector.
44. The vector or set of vectors of claim 43, wherein the non-viral vector is a plasmid.
45. An immune cell or population of immune cells comprising the nucleic acid or set of nucleic acids of any one of claims 1-40 or the vector or set of vectors of any one of claims 41-44.
46. The immune cell or population of immune cells of claim 45, wherein the immune cell(s) express signaling adaptor proteins CD3ζ and Fc Fragment of IgE Receptor Ig (FCER1G).
47. The immune cell or population of immune cells of claim 46, wherein the cell(s) is/are NK cell(s).
48. The immune cell or population of immune cells of claim 46, wherein the cell(s) is/are T cell(s).
49. The immune cell or population of immune cells of claim 46, wherein the cell(s) is a/are monocyte(s).
50. A composition comprising the immune cell(s) of any one of claims 45-49.
51. The composition of claim 50, further comprising a pharmaceutically effective carrier.
52. The composition of claim 50 or 51, wherein the cell(s) is/are in a therapeutically effective amount.
53. A method of treating a subject with cancer, comprising: administering to the subject the immune cell(s) of any one of claims 45-49 or the composition of any one of claims 50-52.
54. A method of treating a subject with autoimmune disease, comprising: administering to the subject the immune cell(s) of any one of claims 45-49 or the composition of any one of claims 50-52.
55. A method of treating a subject with a transplant, comprising: administering to the subject the immune cell(s) of any one of claims 45-49 or the composition of any one of claims 50-52.
56. A method of treating a subject with graft versus host disease (GVHD), comprising: administering to the subject the immune cell(s) of any one of claims 45-49 or the composition of any one of claims 50-52.
57. The method of any one of claims 53-56, wherein the immune cells are allogeneic but have a complete or partial HLA-match with the subject.
58. The method of any one of claims 53-56, wherein the immune cells are autologous.
59. The method of any one of claims 53-56 and 58, further comprising isolating immune cells from a tissue or body fluid sample of the subject prior to the administering of the immune cell(s).
60. The method of claim 59, wherein the immune cells are isolated based on CD56 expression.
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