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WO2025076473A1 - Proteins bispecific to cd3 and nkg2dl and associated uses thereof - Google Patents

Proteins bispecific to cd3 and nkg2dl and associated uses thereof Download PDF

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Publication number
WO2025076473A1
WO2025076473A1 PCT/US2024/050126 US2024050126W WO2025076473A1 WO 2025076473 A1 WO2025076473 A1 WO 2025076473A1 US 2024050126 W US2024050126 W US 2024050126W WO 2025076473 A1 WO2025076473 A1 WO 2025076473A1
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Prior art keywords
peptide
seq
protein
nkg2d
amino acid
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French (fr)
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Thomas M. Schmitt
Roland K. STRONG
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Fred Hutchinson Cancer Center
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Fred Hutchinson Cancer Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • Natural killer group 2, member D is an activating receptor expressed by natural killer (NK) cells and subsets of T cells which bind to several related families of stressinducible ligands.
  • NKG2D is known to be upregulated in response to malignant transformation. Tumor cells identify themselves as “altered self” by inducing expression of NKG2D ligands (NKG2DL) and elicit targeted destruction by the immune system.
  • NKG2DLS are broadly expressed by most tumors, and as such represent promising targets for immunotherapy.
  • senescent cells In addition to transformed cells, senescent cells also express NKG2DLs. Senescent cells accumulate in tissues as individuals age and have a deleterious effect on tissue function. Efforts to eliminate senescent cells using senolytic drugs may provide a promising strategy for preventing or treating many age-related conditions in humans. At present, options for clinically eliminating senescent cells are limited.
  • the present technology comprises engineered proteins a CD3 binding domain and a single peptide chain comprising a first Natural killer group 2, member D (NKG2D) peptide, and a second NKG2D peptide.
  • NKG2D Natural killer group 2, member D
  • the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
  • the first NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
  • the first NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
  • the first NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
  • the first NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
  • the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an N-terminal domain of an NKG2D protein.
  • one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
  • the mammalian peptide is a human peptide or a murine peptide.
  • the first NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
  • the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
  • the NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
  • the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
  • the CD3 binding domain comprises a T-cell activating sequence.
  • the engineered protein further comprises one or more of an immunoglobulin leader peptide, a purification tag, a protease scission site, or a second linking peptide sequence.
  • the immunoglobulin leader peptide is a Ig K leader peptide.
  • the purification tag is selected from the group consisting of a hexa-histidine tag, a FLAG tag, a Myc tag, and a Human influenza hemagglutinin (HA) tag.
  • the protease scission site is a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-C scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, or an endopeptidase Arg-C scission site.
  • TSV Tobacco Etch Virus
  • trypsin scission site a trypsin scission site
  • a chymotrypsin scission site a pepsin scission site
  • a papain scission site a thermolysin scission
  • the present technology comprises a vector comprising a nucleotide sequence encoding an engineered protein of the present technology or a peptide thereof.
  • the present technology comprises vectors comprising a nucleotide sequence encoding the CD3 binding domain of the engineered proteins of the present technology.
  • the present technology comprises vectors comprising a nucleotide sequence encoding the single chain peptide comprising first NKG2D peptide and the second NKG2D peptide of the engineered proteins of the present technology.
  • the present technology comprises methods of generating an engineered protein comprising a CD3 binding domain, and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide, comprising the steps of (i) synthesizing the CD3 binding domain from a first vector; (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector; and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
  • the present technology comprises methods of activating senolysis of senescent cells in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
  • the one or more biomarkers comprises a protein, a transcript, a DNA damage marker, or a reactive oxygen species (ROS) marker.
  • ROS reactive oxygen species
  • the protein is selected from the group consisting of a senescence-associated beta-galactosidase (SA-p-gal) protein, a cyclin-dependent kinase inhibitor 1 or cyclin-dependent kinase-interacting protein 1 (p21WAF1/CIP1 ) protein, a p16INK4a protein, a tumor protein p53 (p53) protein, a retinoblastoma protein (pRB) protein, a deleted in esophageal cancer 1 (DEC1 ) protein, a phosphorylated histone H2AX (y-H2AX) protein, a p53-binding protein 1 (53BP1 ) protein, an Ataxia-telangiectasia mutated (ATM) protein, a meiotic recombination 11 (MRE11 ) protein, a superoxide dismutase (SOD) protein, superoxide dismutase (SOD)
  • SA-p-gal
  • the transcript is selected from the group consisting of a lysosomal p-galactosidase 1 (GLB1 ) transcript, a cyclin-dependent kinase inhibitor 2A (CDKN2A) transcript, a p21 WAF1/CIP1 transcript, a Tumor Protein P53 (TP53) transcript, a DEC1 transcript, a cyclin-dependent kinase (CDK) protein, a CDK transcript, a tumor protein p53 binding protein 1 (TP53BP1 ) transcript, an ATM transcript, a MRE11 transcript, a SOD transcript, and a CAT transcript.
  • the DNA damage marker is selected from the group consisting of a y-H2AX phosphorylation level, a thymine dimer, a phosphorylation of an ATM kinase target, a UV-induced DNA photoproduct, a guanine oxidation, and a double-strand break.
  • the ROS marker is selected from the group consisting of a malondialdehyde (MDA) level, a hydrogen peroxide (H2O2) level, oxidized glutathione (GSSG) protein, a protein carbonylation, oxidized thiol group, a tyrosine nitration, and a GSSG to reduced glutathione (GSH) ratio.
  • MDA malondialdehyde
  • H2O2O2 hydrogen peroxide
  • GSSG oxidized glutathione
  • GSH reduced glutathione
  • the present technology comprises methods for lymphatic cell activation in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
  • the one or more lymphatic cell activation markers is a protein or a transcript.
  • the protein is selected from the group consisting of a CD25 protein, a OD69 protein, a CD71 protein, a CD44 protein, a CD154 protein, a CD28 protein, a CD86 protein, a CD19 protein, a CD3 protein, a CD8 protein, a CD4 protein, a CD56 protein, a CD62L protein, a HLA-DR protein, a granzyme B protein (GZMB) protein, an interferon-gamma (IFN-y) protein, an interleukin-2 (IL-2) protein, a CD38 protein, a CD45RO protein, and a CD20 protein.
  • GZMB granzyme B protein
  • he transcript is selected from the group consisting of a CDC25A transcript, a CDC25B transcript, a CDC25C transcript, a CD69 transcript, a TFRC gene, a CD44 transcript, a CD154 transcript, a CD28 transcript, a CD86 transcript, a CD19 transcript, a CD3 transcript, a CD8 transcript, a CD4 transcript, CD56 transcript, a CD62L transcript, a HLA-DR transcript, a GZMB transcript, an interferon-gamma (IFNG) transcript, an interleukin-2 (IL2) transcript, a CD38 gene, a CD45RO transcript, and a CD20 transcript.
  • IFNG interferon-gamma
  • IL2 interleukin-2
  • the present technology comprises methods of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the lymphatic cell is a T cell, a B cell, or an NK cell.
  • the subject has a cancer or an age-related disease.
  • the age-related disease comprises one or more of osteoporosis, arthritis, obesity, cardiovascular disease, or a neurodegenerative disorder.
  • the present technology comprises methods of treating cancer in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
  • the cancer is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
  • the first NKG2D peptide and/or the second NKG2D peptide comprises a NKG2DL binding domain.
  • the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
  • one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
  • the mammalian peptide is a human or murine peptide.
  • the first NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
  • the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
  • the NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
  • the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
  • the CD3 binding domain comprises a T-cell activating sequence.
  • the engineered protein is present in a pharmaceutical composition.
  • FIG. 1 A illustrates a native form of an NKG2D dimer (left) compared to a singlechain NKG2D dimer (right) in accordance with embodiments of the present technology.
  • FIG. 1 B illustrates an engineered protein comprising the single chain NKG2D single-chain dimer of FIG. 1 A and a CD3 binding domain in accordance with embodiments of the present technology.
  • FIG. 1 C illustrates a structural comparison between the native NKG2D dimer (NKG2D2) (RCSB Protein Data Bank (PDB) accession code I MPU.pdb) and the singlechain NKG2D dimer (NKG2D scd ) (RCSB PDB accession code 8TM2.pdb) of FIG. 1 A.
  • FIG. 1 D illustrates an engineered protein comprising two NKG2D single-chain dimers of FIG. 1 A each fused to a fragment crystallizable (Fc) region of an anti-CD3 antibody in accordance with embodiment of the present technology.
  • Fc fragment crystallizable
  • FIG. 1 E illustrates the engineered protein of FIG. 1 D further comprising singlechain variable fragment (scFv) domains in accordance with embodiments of the present technology.
  • scFv singlechain variable fragment
  • FIG. 2 illustrates NKG2DL transcript levels among cell lines expressing major histocompatibility complex (MHC) class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), LIL16 binding protein 2 (LILBP2), or UL16 binding protein 4 (ULBP4).
  • MHC major histocompatibility complex
  • MICA major histocompatibility complex
  • MIMB MHC class I chain-related protein B
  • LIL16 binding protein 2 LIL16 binding protein 2
  • ULBP4 UL16 binding protein 4
  • FIG. 4 illustrates levels of T cell activation in response to titrated NKG2D-SCD- TCE for T cells in the presence of MDA-MB-231 breast cancer cells (squares) relative to T cells alone (circles).
  • FIG. 6 shows qualitative amounts of the engineered protein of FIGS. 5A-5C in the supernatant of HEK293F cells 10 days after transduction of a lentiviral vector comprising a nucleotide sequence encoding the engineered protein.
  • FIGS. 7A-7C illustrate dose-response results for MDA-MB-231 (FIG. 7A), H1299 (FIG. 7B), and HeLa (FIG. 7C) cells transduced with the lentiviral vector of FIGS. 5A-5C (“ADI”) or a lentiviral vector comprising a nucleotide sequence encoding an engineered protein having the amino acid sequence SEQ ID NO: 93 (“OKT3”).
  • the present technology provides engineered proteins comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide for treating a variety of diseases, including cancer and age-related disease. Also provided are vectors for synthesizing the engineered proteins of the present technology.
  • a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents of the present technology are merely exemplary and that equivalents of such are known in the art. Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
  • ranges is intended as a continuous range, including every value between the minimum and maximum values recited, as well as any ranges that may be formed by such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that may be formed by dividing a disclosed numeric value into any other disclosed numeric value. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios may be unambiguously derived from the numerical values presented herein and in all instances, such ratios, ranges, and ranges of ratios represent various embodiments of the present technology.
  • the term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
  • Any one or more of engineered proteins of the present technology may be present in a pharmaceutical composition, whereby therapeutically effective amounts of the engineered protein are combined in admixture with a pharmaceutically acceptable carrier or excipient. Salts and/or prodrugs of the engineered proteins may also be used.
  • excipients include water, saline, dextrose, glycerol, or the like and combinations thereof.
  • An “excipient” of the pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • Salts as used herein comprises pharmaceutically acceptable salts.
  • a pharmaceutically acceptable salt includes any salt that retains the activity of the engineered protein is acceptable for pharmaceutical use.
  • a pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.
  • the terms “treat,” “treatment,” and “treating” refer to a manner of providing a pharmaceutical composition and/or engineered protein to alleviate disease outcomes. This includes utilizing administration techniques of the present technology. Efficacy of treatment may be determined by various assessment methods of the present technology (e.g., assessment of senolysis, lymphatic cell activation, and measurement of biomarkers).
  • biomarker refers to a biological output that is used as a measure of cellular response, whether that be to assess response to therapeutics, disease status, such as cancer or an age-related disease, or as a predictor of clinical outcomes. Biomarkers evaluated in the context of cells, tissue, or whole organisms.
  • Disease herein refers to any disorder adversely affecting biological status. Disease also may be in the context of human and animal health.
  • treat may also refer to the reduction or inhibition of the progression and/or duration of a disease (e.g., cancer or an age-related disease), the reduction or amelioration of the severity of the disease, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies.
  • a disease e.g., cancer or an age-related disease
  • the terms “effective amount” or “therapeutically effective amount”, refer to that amount of the engineered protein being administered which will relieve to some extent one or more of the symptoms of the disease being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • An appropriate “effective amount” may differ from one individual to another.
  • An appropriate “effective amount” in any individual case may be determined using techniques, such as a dose escalation study.
  • subject refers to anyone being evaluated for disease or condition or being administered a therapeutic or pharmaceutical composition. This includes people without diagnosed or confirmed disease or condition. This also includes people with diagnosed or confirmed disease or condition, such as cancer or an age-related disease.
  • control may refer to any subject used as a basis for comparison to the subject of the present technology (e.g., a subject having cancer or an age-related disease).
  • a control includes, but is not limited to, any subject who has not been administered the therapeutic (e.g., an engineered protein) or pharmaceutical composition or administered a placebo, and the subject of the present technology at baseline or prior to administration of a pharmaceutical composition.
  • binding refers to all types of physical and chemical binding, reactions, complexing, attraction, chelating and the like.
  • the “proteins” and “peptides” of the present technology may be (a) naturally- occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.
  • peptide as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids.
  • the amino acids forming all or a part of a peptide may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post- translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, and the like, so that the term “peptide” includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone.
  • peptide also includes dimers or multimers of peptides.
  • a “manufactured” peptide includes a peptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method.
  • the term “peptide” includes peptides containing a variable number of amino acid residues, optionally with nonamino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.
  • amino acids are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid.
  • the key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N — CHR — COOH, wherein R represents a side chain group.
  • the various a-amino acids differ in the side-chain moiety that is attached to the a-carbon.
  • the “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User’s Guide, G. A. Grant, editor, W.H.
  • amino acid also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generally in Synthetic Peptides: A User’s Guide, supra; Hruby et al., Biochem. J. 268:249-262 (1990); and Toniolo, Int. J. Peptide Protein Res. 35:287-300 (1990); the teachings of all of which are incorporated herein by reference.
  • Percent (%) amino acid sequence “identity” with respect to the sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence for each of the peptides and/or engineered proteins after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity may be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared may be determined.
  • percent amino acid sequence identity values generated using the WU-BLAST-2 computer program uses several search parameters, most of which are set to the default values.
  • variants of the proteins and peptides of the present technology may be used. “Variants” include protein sequences having one or more amino acid additions, deletions, stop positions, or substitutions, as compared to a protein sequence disclosed elsewhere herein.
  • An amino acid substitution may be a conservative or a non-conservative substitution.
  • Variants of the proteins and peptides of the present technology include those having one or more conservative amino acid substitutions.
  • a “conservative substitution” or “conservative amino acid substitution” involves a substitution found in one of the following conservative substitutions groups: Group 1 : Ala, Gly, Ser, Thr; Group 2: Glu, Asp; Group 3: Asn, Glu; Group 4: Arg, Lys, His; Group 5: lie, Leu, Met, Vai; and Group 6: Phe, Tyr, Trp.
  • amino acids may be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and lie.
  • Non-conservative substitutions include those that significantly affect: the structure of the peptide backbone in the area of the alteration (e.g., the alpha-helical or beta-sheet structure); the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
  • Non-conservative substitutions which in general are expected to produce the greatest changes in the protein's properties are those in which (i) a hydrophilic residue (e.g. Ser or Thr) may be substituted for (or by) a hydrophobic residue (e.g.
  • a Cys or Phe may be substituted for (or by) any other residue;
  • a residue having an electropositive side chain e.g. Lys, Arg, or His
  • an electronegative residue e.g. Gin or Asp;
  • a residue having a bulky side chain e.g. Phe
  • Creighton (1984) Proteins, W.H. Freeman and Company incorporated herein by reference in its entirety.
  • the present technology provides engineered proteins formed of a binding domain, peptides, and a linker, that are bispecific to both CD3 and NKG2DL.
  • the engineered proteins provide for binding to CD3 and NKG2DL expressed by one or more cells or present in free form.
  • the engineered proteins may bind CD3 on a first cell and NKG2DL on a second cell. In some embodiments, binding of CD3 and NKG2DL occurs simultaneously or sequentially.
  • the engineered proteins may bind CD3 and/or NKG2DL expressed by one or more cell types.
  • an engineered protein of the present technology may be capable of binding CD3 expressed at the surface a first cell type and CD3 expressed at the surface of a second cell type.
  • the engineered protein may be capable of binding NKG2DL expressed at the surface of a first cell type and NKG2DL expressed at the surface of a second cell type.
  • the first cell type and the second cell type are of different lineages.
  • the first cell type and the second cell type are present in different tissues.
  • the engineered protein comprises a CD3 binding domain, a first NKG2D peptide, and a second NKG2D peptide.
  • the first NKG2D peptide and the second NKG2D peptide may be present on a single peptide chain and in some embodiments, the first NKG2D peptide and the second NKG2D peptide are present on the single peptide chain.
  • the CD3 binding domain may be present on the single peptide chain, or on a second single peptide chain that is fused to, operably connected to, or otherwise linked the first single peptide chain.
  • the engineered proteins of the present technology comprise one or more NKG2D peptides designed to bind NKG2DL (e.g., MICA, MICB, RAET1 E/ULBP4, RAET1 G/ULBP5, RAET1 H/ULBP2, RAET1/ULBP1 , RAET1 L/ULBP6, and RAET1 N/ULBP3).
  • NKG2DL e.g., MICA, MICB, RAET1 E/ULBP4, RAET1 G/ULBP5, RAET1 H/ULBP2, RAET1/ULBP1 , RAET1 L/ULBP6, and RAET1 N/ULBP3
  • the one or more NKG2D peptides comprise a first NKG2D peptide and a second NKG2D peptide.
  • the first NKG2D peptide and/or the second NKG2D peptide may be a peptide having fewer amino acids than the wild-type NKG2D protein.
  • the wild-type NKG2D protein is a human wild-type NKG2D protein (NCBI Accession: CAA04925). In some embodiments, the wild-type protein is a murine wild-type protein (NCBI Accession: AAC28245).
  • the peptide having fewer amino acids than the wildtype NKG2D protein may comprise a peptide having one or more amino acids less than the wild-type NKG2D protein. In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein comprises at least about 110 to about 130 residues of the wildtype NKG2D protein.
  • the peptide having fewer amino acids than the wild-type NKG2D protein comprises one or more C-type lectin (CTL)-like or carbohydrate- recognition domain (CRD) (CLECT) domains.
  • CTL C-type lectin
  • CCD carbohydrate- recognition domain
  • the peptide having fewer amino acids than the wild-type NKG2D protein is lacking a transmembrane domain.
  • the transmembrane domain comprises or consist of the amino acid sequence PFFFCCFIAVAMGIRFIIMVAIW (SEQ ID NO: 1 ).
  • the peptide having fewer amino acids than the wild-type NKG2D protein comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
  • the peptide having fewer amino acids than the wild-type NKG2D protein comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
  • the peptide having fewer amino acids than the wild-type NKG2D protein comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
  • the first NKG2D peptide and/or the second NKG2D peptide may comprise a NKG2DL binding domain.
  • the first NKG2D peptide and the second NKG2D peptide comprise dimerization domains, wherein the first NKG2D peptide and the second NKG2D peptide form a dimer which binds a NKG2DL.
  • the first NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence. In some embodiments, the first NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence. In some embodiments, the first NKG2D peptide and the second NKG2D peptide are different.
  • the first NKG2D peptide and/or the second NKG2D peptide comprise or consist of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 1 .
  • the first NKG2D peptide and/or the second NKG2D peptide comprise or consist of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 1 .
  • the first NKG2D peptide and/or the second NKG2D peptide comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 1 .
  • the first NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the first NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the second NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the second NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the first NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the first NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the second NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the second NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the first NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99.
  • the first NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99.
  • the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99.
  • the first NKG2D peptide and the second NKG2D peptide may be linked on the single chain peptide by one or more linking peptides.
  • the one or more linking peptides are used to join the first NKG2D peptide to the second NKG2D peptide and/or to the CD3 binding domain or to another peptide or another type of molecule.
  • the one or more linking peptides comprises a glycineserine (Gly/Ser) linker, a proline-threonine-glycine linker, an alanine linker, a lysine linker, a threonine linker, a valine-glycine-serine-threonine linker, an elastin-like peptide linker, or a hexahistidine linker.
  • Gly/Ser glycineserine
  • the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by one or more linking peptides comprising an N- terminal domain of an NKG2D protein.
  • the one or more linking peptides comprises or consists of an amino acid sequence listed in Table 2.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 2.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 2.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 2.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5. [0129] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8. [0138] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 97.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 97.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 97.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence of GS. [0147] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence of GS.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence of GS.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 98.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 98.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 98.
  • the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 102.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 102.
  • the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 102.
  • the engineered protein of the present technology further comprises a CD3 binding domain, which selectively binds a CD3 protein.
  • the CD3 binding domain may bind at least a portion of one or more CD3 subunits.
  • the one or more CD3 subunits is selected from the group consisting of CD3s, CD3y, CD35, and CD3 .
  • the CD3 protein is a wild-type human CD3 protein.
  • the CD3 binding domain may recognize one or more of wild-type human CD3s (NCBI Accession: NP 000723), wild-type human CD3y (NCBI Accession: NP 000064), wild-type human CD35 (NCBI Accession: ACA05962), and wild-type human CD3 ⁇ (isoform 1 NCBI Accession: NP_932170; isoform 2 NCBI Accession: NP_000725; isoform 3 NCBI Accession: NP_001365444; isoform 4 NCBI Accession: NP_00136544).
  • the CD3 protein is a wild-type murine CD3 protein.
  • the CD3 binding domain may recognize one or more of wild-type murine CD3s (NCBI Accession: NP_031674), wild-type murine CD3y (NCBI Accession: NP_033980); wild-type murine CD35 (NCBI Accession: NP_038515), and wild-type murine CD3 (NCBI Accession: P24161 ).
  • the CD3 binding domain comprises one or more T-cell activating sequences.
  • CD3 is expressed on the surface of cells such as lymphocytes.
  • Nonlimiting examples of CD3-expressing lymphocytes include T cells, NK cells, and B cells. Binding of CD3 through the CD3 binding domain provides a stimulatory signal which engages immune responses, including but not limited to, B cell stimulation, antibody production, antigen recognition, apoptotic signaling, cytotoxic signaling, cytokine storm, and target cell destruction or neutralization.
  • the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
  • the antigen binding fragment thereof may comprise one or more of a Fragment antigen-binding (Fab) or at least a portion thereof of an anti-CD3 antibody, a fragment crystallizable (Fc) region or at least a portion thereof of an anti-CD3 antibody, a single-chain variable fragment (scFv) or at least a portion thereof of an anti-CD3 antibody, a variable light (VL) domain and/or a variable heavy (VH) domain or at least a portion thereof of an anti-CD3 antibody, or one or more complementarity-determining regions (CDRs) or at least a portion thereof of an anti-CD3 antibody.
  • Fab Fragment antigen-binding
  • Fc fragment crystallizable
  • scFv single-chain variable fragment
  • VL variable light
  • VH variable heavy domain
  • CDRs complementarity-determining regions
  • the CD3 binding domain is fused to one or more NKG2D single-chain dimers. In some embodiments, the CD3 binding domain is fused to two or more NKG2D single chain dimers. In some embodiments, when the CD3 binding domain is fused to two or more NKG2D single chain dimers, the two or more NKG2D single chain dimers are each independently fused to different polypeptide chains of the CD3 binding domain.
  • the anti-CD3 antibody or the antigen fragment thereof may comprise a VL domain and/or a VH domain listed in Table 3
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 1 1 .
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 1 1 .
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 1 1 .
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 12.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 12.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 12. [0171] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 103.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 103.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 103.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 104.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 104.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 104.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 105. [0178] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 105.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 105.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 106.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 106.
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 106.
  • the engineered protein of the present technology may further comprise a leader peptide.
  • the leader peptide comprises a signal sequence (e.g., a targeting sequence or a localization sequence).
  • the leader peptide comprises or consist of an Igk or an IgA leader peptide.
  • the Igk leader peptide is a murine peptide sequence.
  • the Igk leader peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical METDTLLLWVLLLWVPGSTG (SEQ ID NO: 73).
  • the Igk leader peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73. [0186] In some embodiments, the Igk leader peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73.
  • the leader peptides comprise or consist of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 4.
  • the leader peptides comprise or consist of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 4.
  • the leader peptides comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 4.
  • the engineered protein of the present technology may further comprise one or more purification tags.
  • the purification tag may provide an epitope for selective binding of the engineered protein.
  • the epitope may comprise a binding sequence for use in purification or pull-down using methods commonly used in the art.
  • the epitope tag may be placed at the amino- or carboxyl-terminus of the engineered protein.
  • an epitope tag is be placed within the amino acid sequence of the engineered protein amino acid sequence.
  • the epitope tag enables the engineered proteins to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag; this also may be useful for binding the engineered protein to a support for heterogeneous screening methods.
  • Nonlimiting examples of purification tags include a hexahistidine tag (HHHHHH; SEQ ID NO: 10), a FLAG tag (DYKDDDDK; SEQ ID NO: 90), a Myc tag, and a Human influenza hemagglutinin (HA) tag (YPYDVPDYA; SEQ ID NO: 91 ).
  • the engineered protein comprises one or more protease scission sites.
  • Nonlimiting examples of protease scission sites include a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-0 scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, and an endopeptidase Arg-C scission site.
  • TSV Tobacco Etch Virus
  • the TEV protease scission site comprises or consist of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to ENLYFQ (SEQ ID NO: 92).
  • the TEV protease scission site comprises or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92.
  • the TEV protease scission site comprises or consist of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to ENLYFQGS (SEQ ID NO: 96)
  • the TEV protease scission site comprises or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 96.
  • the TEV protease scission site comprises or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 96.
  • the engineered protein comprises a CD3 binding domain, a single-chain peptide comprising a first NKG2D peptide and a second NKG2D peptide, a leader peptide, a linker sequence, a purification tag, and a protease scission site, together comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
  • the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 107.
  • the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
  • the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
  • the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 108.
  • the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
  • the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
  • the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
  • the engineered proteins comprise or consist of an anti- CD3 antibody fused to one or more NKG2D single-chain dimers of the present technology. In some embodiments, the engineered proteins comprise or consist of an anti-CD3 antibody fused to two or more NKG2D single-chain dimers of the present technology.
  • the engineered proteins comprise or consist of an Fc region or at least a portion thereof of an anti-CD3 antibody fused to one or more NKG2D single-chain dimers of the present technology. In some embodiments, the engineered proteins comprise or consist of an Fc region or at least a portion thereof of an anti-CD3 antibody fused to two or more NKG2D single-chain dimers of the present technology. In some embodiments, when the engineered proteins comprise two or more NKG2D singlechain dimers, the two or more NKG2D single-chain dimers are each ultimately fused to different chains of the Fc region or the portion thereof.
  • any one of the domains, peptides, or engineered proteins of the present technology may comprise a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
  • one or more of the CD3 binding domain, the first NKG2D peptide, or the second NKG2D peptide comprise a peptide selected from the group consisting of a mammalian peptide, a non- naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
  • the mammalian peptide is a human peptide or a murine peptide.
  • the first NKG2D peptide, the second NKG2D peptide, or any one of the linker sequences comprises or consists of at least a portion of the wild-type human NKG2D protein (NCBI Accession: CAA04925; SEQ ID NO: 94) or the wild-type mouse NKG2D protein (NCBI Accession: AAC28245; SEQ ID NO: 95) shown in Table 6.
  • the portion may comprise any peptide having the full sequence of SEQ ID NO:94 or SEQ ID NO: 95 or less.
  • Covalent modifications of the engineered proteins of the present technology are included within the scope of the present technology.
  • One type of covalent modification includes reacting targeted amino acid residues of the engineered proteins with an organic derivatizing agent that is retractable with selected side chains or the N- or C-terminal residues of the engineered proteins.
  • Derivatization with bifunctional agents may be useful, for instance, for crosslinking the engineered proteins to a water-insoluble support matrix or surface for use in the methods described below, or for in vivo stability.
  • crosslinking agents include 1 ,1 -bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homobifunctional imidoesters, including disuccinimidyl esters (e.g., 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides (e.g., bis-N-maleimido-1 ,8-octane) and agents such as methyl-3- ((p-azidophenyl)dithio) propioimidate, and 1 -ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride.
  • disuccinimidyl esters e.g., 3,3'-dithiobis(succinimidylpropionate
  • bifunctional maleimides e.
  • the engineered proteins of the present technology are glycosylated.
  • Glycosylation is a type of post-translational modification that refers to the covalent attachment of sugars to proteins. Functions of glycosylation include contributing to proper protein folding and facilitating cell-to-cell and/or protein-protein interactions.
  • the engineered proteins of the present technology are N- linked glycosylated.
  • N-linked glycans attach to the nitrogen atom of certain amino acids including N and R.
  • N-linked glycosylation occurs during translation in the endoplasmic reticulum. Therefore, proteins that encode a signal peptide that signals for translation within the endoplasmic reticulum may become N-linked glycosylated.
  • An N-linked glycosylation site may include N-X-(S), where X is any amino acid.
  • the engineered proteins of the present technology are O- linked glycosylated.
  • O-linked glycans attach to the hydroxyl (OH) of amino acids including S, T, Y, hydroxyl-K, and hydroxyl-P.
  • O-linked glycosylation occurs in the Golgi apparatus.
  • An O-linked glycosylation site may include (S or T)-X-X-P, where X is any amino acid.
  • the engineered proteins of the present technology are glycosylated due to protein production using a eukaryotic cell expression system. All domains of life (eukaryote, bacteria, and archaea) are capable of protein glycosylation. However, glycosylation machinery may differ across domains and even across species. Therefore, a protein that is glycosylated when expressed in an autologous system may not become glycosylated upon expression in a heterologous system. One specific reason is if the glycosylation site of the protein is not recognized by the cell type used for expression
  • vectors which comprise nucleotide sequences encoding one or more peptides in accordance with the present technology.
  • the vectors may comprise one or more of a nucleotide sequence encoding a peptide or an engineered protein of the present technology in addition to vector and/or cloning sequences commonly used in the art.
  • the vectors may comprise one or more of an origin of replication (Ori), a selectable marker, a promoter, a transcription start site, a ribosome binding site, a terminator sequence, a polyadenylation signal, a multiple cloning site, an origin of transfer, a regulatory element, or an inducible system.
  • the Ori is selected from the group consisting of a ColE1 Ori, a pUC Ori, a p15A Ori, a pSC101 Ori, a SV40 Ori, a M13 Ori, an OriC.
  • the Ori may comprise an autonomously replicating sequence or a eukaryotic chromosomal origin.
  • the selectable marker may be an antibiotic resistance marker, a nutritional marker, a reporter gene, an herbicide resistance gene, a phage resistance gene, a temperature-sensitive marker, a hygromycin resistance gene, a purine or pyrimidine analogue resistance gene, or an antibiotic-sensitivity marker.
  • the vector comprises one or more selectable markers or two or more selectable markers.
  • Nonlimiting examples of promoters include a T7 Promoter, a TAC (Trp-lac Hybrid Promoter), a IPTG-lnducible Promoters, a IRES (Internal Ribosome Entry Site), a CMV Promoter (Cytomegalovirus Promoter), a SV40 Promoter (Simian Virus 40 Promoter), a EF1 a (Elongation Factor 1 Alpha) Promoter, a PGK Promoter (Phosphoglycerate Kinase Promoter), an Actin Promoter (e.g., CMV-p-Actin Promoter), a GAL1 Promoter, and a CaMV 35S Promoter (Cauliflower Mosaic Virus 35S Promoter).
  • a T7 Promoter e.g., a TAC (Trp-lac Hybrid Promoter), a IPTG-lnducible Promoters, a IRES (Internal Ribosome Entry Site), a CMV Promoter (Cyt
  • the inducible system comprises a Lac operon (lac promoter with IPTG induction), a Tet-On and Tet-Off system, an AraC-Arabinose system (pBAD Promoter), a T7 Promoter with T7 RNA Polymerase, an IPTG-inducible promoters, an AraC-PBAD promoter system, a chemically inducible system (e.g., Shield-1 ), a heat shock promoter (e.g., hsp70 promoter), a pBAD Promoter with L-Arabinose, and a nitrogenresponsive systems (e.g., nif Promoter).
  • lac promoter with IPTG induction a Tet-On and Tet-Off system
  • an AraC-Arabinose system pBAD Promoter
  • T7 Promoter with T7 RNA Polymerase an IPTG-inducible promoters
  • an AraC-PBAD promoter system e.g., Shield-1
  • a heat shock promoter
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:93.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • a vector comprising a nucleotide sequence that encodes the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the vector comprises a nucleotide sequence that encodes at least two peptides, each comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the vector comprises a nucleotide sequence that encodes at least two peptides, each comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • the vector comprises a nucleotide sequence that encodes at least two peptides, each comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • a vector comprising a nucleotide sequence that encodes the CD3 binding domain.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 1 1 or SEQ ID NO: 12.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 11 or SEQ ID NO: 12.
  • the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to identical to a sequence listed in Table 3.
  • the sequence is SEQ ID NO: 1 1 or SEQ ID NO: 12.
  • the engineered protein of the present technology is generated by a method comprising synthesizing the full-length peptide using a single vector.
  • the engineered protein of the present technology is generated by a method comprising (i) synthesizing the CD3 binding domain from a first vector, (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector, and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
  • compositions comprising one or more engineered proteins or vectors of the present technology.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or excipient (e.g., water, saline, dextrose, glycerol).
  • the pharmaceutically acceptable carrier and/or excipient comprises water.
  • the pharmaceutically acceptable carrier comprises one or more of lactose, microcrystalline cellulose (MCC), starch, glycerin, polyethylene glycol (PEG), sorbitol, silicon dioxide (SiO2), magnesium stearate, propylene glycol, polysorbate 80, hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), lecithin, ethanol, water, mineral oil, talc, gelatin, sucrose, or povidone-iodine.
  • MMC microcrystalline cellulose
  • HPMC hydroxypropyl methylcellulose
  • PVP polyvinylpyrrolidone
  • lecithin lecithin
  • ethanol water, mineral oil, talc, gelatin, sucrose, or povidone-iodine.
  • Nonlimiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • the pharmaceutically acceptable carrier may comprise one or more of an absorption delaying agent, an antioxidant, a binder, a buffering agent, a bulking agent or filler, a chelating agent, a coating, a disintegration agent, a dispersion media, a gel, an isotonic agent, a lubricant, a preservative, a salt, a solvent or co-solvent, a stabilizer, a surfactant, and/or a delivery vehicle.
  • an absorption delaying agent an antioxidant, a binder, a buffering agent, a bulking agent or filler, a chelating agent, a coating, a disintegration agent, a dispersion media, a gel, an isotonic agent, a lubricant, a preservative, a salt, a solvent or co-solvent, a stabilizer, a surfactant, and/or a delivery vehicle.
  • Nonlimiting examples of an antioxidant includes ascorbic acid, methionine, and vitamin E.
  • Nonlimiting examples of a buffering agent includes citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and trimethylamine salts.
  • the chelating agent is EDTA.
  • Nonlimiting examples of an isotonic agent includes polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.
  • Nonlimiting examples of a preservative includes phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanoL
  • Stabilizers comprise a broad category of excipients which may range in function from a bulking agent to an additive which solubilizes the engineered protein or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers may include polyhydric sugar alcohols; amino acids, ornithine, L-leucine, 2-phenylalanine, E, and T; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosul
  • the pharmaceutical composition comprises a pharmaceutically acceptable salt that retains the activity of the engineered protein and is acceptable for pharmaceutical use.
  • the salt may comprise a pharmaceutically acceptable acid addition salt.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.
  • the pharmaceutical composition comprises a pharmaceutically acceptable base addition salt.
  • Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, procaine, and the amino acids Lys or Arg.
  • the pharmaceutical composition may be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline.
  • the aqueous solutions may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • the formulation may be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • the pharmaceutical compositions may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like.
  • suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g.
  • lactose sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents may be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms may be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as caramel, peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. may also be used.
  • the pharmaceutical compositions may be formulated as an aerosol.
  • the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers may also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated containing a powder mix (including flavored mixes) of engineered proteins and a suitable powder base such as lactose or starch.
  • the pharmaceutical compositions may also be formulated as suppositories and/or depot preparations. Depot preparations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts. [0256] Additionally, the pharmaceutical compositions may be formulated as sustained- release systems utilizing semipermeable matrices of solid polymers comprising at least one engineered protein. Sustained-release systems may release the engineered proteins following administration for a few weeks up to over 100 days.
  • Depot formulations may comprise one or more bioerodible polymers including, but not limited to, poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)- co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades may result in characteristics that borrow from each of the contributing polymers.
  • bioerodible polymers including, but not limited to, poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)- co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades may result in characteristics that borrow from each of the contributing polymers.
  • solvents e.g., dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof
  • Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.
  • Exemplary release modifiers may comprise surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Del.), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Del.), sucrose acetate isobutyrate (SAIB), salts, and buffers.
  • surfactants e.g., hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Del.), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington,
  • Excipients that partition into the external phase boundary of microparticles may also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.
  • Additional processing of the sustained release depot formulations may utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine.
  • a freeze-dry cycle may also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.
  • the pharmaceutical composition comprises the engineered proteins of the present technology of at least about 0.1% w/v or w/w of the composition, at least about1% w/v or w/w of the composition; at least about 10% w/v or w/w of the composition; at least about 20% w/v or w/w of the composition; at least about 30% w/v or w/w of the composition; at least about 40% w/v or w/w of the composition; at least about 50% w/v or w/w of the composition; at least about 60% w/v or w/w of the composition; at least about 70% w/v or w/w of the composition; at least about 80% w/v or w/w of the composition; at least about 90% w/v or w/w of the composition; at least about 95% w/v or w/w of the composition; or at least about 99% w/v or w/w of the composition.
  • the pharmaceutical compositions of the present technology may be administered to a subject in need thereof.
  • the pharmaceutical composition may be formulated for administration by, for example, injection, inhalation, infusion, insertion, perfusion, lavage, or ingestion.
  • the pharmaceutical compositions may further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous ingestion, injection, or insertion.
  • the pharmaceutical composition is administered by implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through
  • doses therapeutically effective amounts for all methods of the present technology may be initially estimated based on results from in vitro assays and/or animal model studies. Such information may be used to modify the doses administered to the subject.
  • doses therapeutically effective amounts
  • the actual dose amount administered to a particular subject may be determined by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
  • administered doses of pharmaceutical compositions of the present technology may range from about 0.1 to about 5 pg/kg or from about 0.5 to about 1 pg/kg.
  • a dose may comprise about 1 pg/kg, about 5 pg/kg, about 10 pg/kg, about 15 pg/kg, about 20 pg/kg, about 25 pg/kg, about 30 pg/kg, about 35 pg/kg, about 40 pg/kg, about 45 pg/kg, about 50 pg/kg, about 55 pg/kg, about 60 pg/kg, about 65 pg/kg, about 70 pg/kg, about 75 pg/kg, about 80 pg/kg, about 85 pg/kg, about 90 pg/kg, about 95 pg/kg, about 100 pg/kg, about 150 pg/kg, about 200 pg/kg, about 250 pg/kg, about 350
  • a dose may comprise about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg, about 250 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450 mg/kg, about 500 mg/kg, about 550 mg/kg, about 600 mg/kg, about 650 mg/kg, about 700 mg/kg, about 750 mg/kg, about 800 mg/kg, about 850 mg/kg, about 900 mg/kg, about 950 mg/kg, or about 1000 mg/kg or more of a pharmaceutical composition of
  • administered doses of pharmaceutical compositions of the present technology may range from at least 0.1 to at least 5 pg/kg or from at least 0.5 to at least 1 pg/kg.
  • a dose may comprise at least 1 pg/kg, at least 5 pg/kg, at least 10 pg/kg, at least 15 pg/kg, at least 20 pg/kg, at least 25 pg/kg, at least 30 pg/kg, at least 35 pg/kg, at least 40 pg/kg, at least 45 pg/kg, at least 50 pg/kg, at least 55 pg/kg, at least 60 pg/kg, at least 65 pg/kg, at least 70 pg/kg, at least 75 pg/kg, at least 80 pg/kg, at least 85 pg/kg, at least 90 pg/kg, at least 95 pg/kg, at least 100 pg/kg, at least
  • a dose may comprise at least 1 mg/kg, at least 5 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, at least 100 mg/kg, at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 350 mg/kg, at least 400 mg/kg, at least 450 mg/kg, at least 500 mg/kg, at least 550 mg/kg, at least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least 750 mg/kg, at least 800 mg/kg, at least
  • administered doses of pharmaceutical compositions of the present technology may range from at least about 0.1 to at least about 5 pg/kg or from at least about 0.5 to at least about 1 pg/kg.
  • a dose may comprise at least about 1 pg/kg, at least about 5 pg/kg, at least about 10 pg/kg, at least about 15 pg/kg, at least about 20 pg/kg, at least about 25 pg/kg, at least about 30 pg/kg, at least about 35 pg/kg, at least about 40 pg/kg, at least about 45 pg/kg, at least about 50 pg/kg, at least about 55 pg/kg, at least about 60 pg/kg, at least about 65 pg/kg, at least about 70 pg/kg, at least about 75 pg/kg, at least about 80 pg/kg, at least about 85 pg/kg, at least about 90 pg
  • a dose may comprise at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 15 mg/kg, at least about 20 mg/kg, at least about 25 mg/kg, at least about 30 mg/kg, at least about 35 mg/kg, at least about 40 mg/kg, at least about 45 mg/kg, at least about 50 mg/kg, at least about 55 mg/kg, at least about 60 mg/kg, at least about 65 mg/kg, at least about 70 mg/kg, at least about 75 mg/kg, at least about 80 mg/kg, at least about 85 mg/kg, at least about 90 mg/kg, at least about 95 mg/kg, at least about 100 mg/kg, at least about 150 mg/kg, at least about 200 mg/kg, at least about 250 mg/kg, at least about 350 mg/kg, at least about 400 mg/kg, at least about 450 mg/kg, at least about 500 mg/kg, at least about 550 mg/kg, at least about 600 mg/kg, at
  • Therapeutically effective amounts may be achieved by administering single or multiple doses during a treatment regimen.
  • the dose is administered about daily, about every other day, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about weekly, about every 2 weeks, about every 3 weeks, about monthly, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, or about yearly.
  • the dose is administered at least daily, at least every other day, at least every 3 days, at least every 4 days, at least every 5 days, at least every 6 days, at least weekly, at least every 2 weeks, at least every 3 weeks, at least monthly, at least every 2 months, at least every 3 months, at least every 4 months, at least every 5 months, at least every 6 months, at least every 7 months, at least every 8 months, at least every 9 months, at least every 10 months, at least every 11 months, or at least yearly.
  • the dose is administered at least about daily, at least about every other day, at least about every 3 days, at least about every 4 days, at least about every 5 days, at least about every 6 days, at least about weekly, at least about every 2 weeks, at least about every 3 weeks, at least about monthly, at least about every 2 months, at least about every 3 months, at least about every 4 months, at least about every 5 months, at least about every 6 months, at least about every 7 months, at least about every 8 months, at least about every 9 months, at least about every 10 months, at least about every 11 months, or at least about yearly.
  • the subject of the present technology has a disease.
  • the subject has one or more of a cancer, an age-related disease, an immune condition or disease, or graft-versus-host disease or transplant rejection.
  • Nonlimiting examples of age-related diseases include osteoporosis, arthritis, obesity, cardiovascular disease, and neurodegenerative disorders.
  • Nonlimiting examples of cancer includes a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
  • the immune condition or disease comprises an infection (e.g., bacterial infections, fungal infections, or viral infections).
  • the immune condition is an inflammatory response to a vaccine.
  • the immune condition or disease is an autoimmune disease.
  • Nonlimiting examples of autoimmune disease includes rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, Hashimoto's thyroiditis, Graves' disease, celiac disease, psoriasis, inflammatory bowel disease (IBD), Sjogren's syndrome, myasthenia gravis, vitiligo, pernicious anemia, ankylosing spondylitis, Addison's disease, and Goodpasture syndrome.
  • the autoimmune condition is an NKG2D- driven autoimmune condition.
  • the transplant comprises at least a partial organ or tissue or a cell.
  • the transplant comprises an epithelial tissue, a connective tissue, a muscular tissue, or a nervous tissue.
  • the methods of the present technology comprise administering one or more engineered proteins of the present technology to a subject in need thereof, wherein the one or more engineered proteins binds a NKG2DL and a CD3, each expressed on the surface of one or more cells.
  • the NKG2DL may be a cell-surface protein expressed by a cell including, but not limited to a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
  • transformed cells include cancer cells, immortalized cells, virally infected cells, and cells having abnormal growth or behavior driven by a genetic alteration.
  • Nonlimiting examples of cells subjected to genotoxic stress include cells exposed to one or more of ultraviolet (UV) radiation, chemotherapy, radiation, smoke, chemical carcinogens, metabolic stress, infection, or pharmaceuticals.
  • UV ultraviolet
  • CD3 is expressed on the surface of one or more lymphatic cells.
  • the one or more lymphatic cells is a T cell, a B cell, or a NK cell.
  • T cells include CD4 + T cells, CD8 + T cells, regulatory T cells (Tregs), memory T cells, natural killer T (NKT) cells, gamma delta (y6) T Cells, mucosal- associated invariant T (MAIT) cells, innate-like T Cells, follicular helper T (Tfh) cells, and senescent T cells.
  • Tregs regulatory T cells
  • NKT natural killer T
  • y6 gamma delta
  • MAIT mucosal- associated invariant T
  • Tfh follicular helper T
  • senescent T cells include CD4 + T cells, CD8 + T cells, regulatory T cells (Tregs), memory T cells, natural killer T (NKT) cells, gamma delta (y6) T Cells, mucosal- associated invariant T (MAIT) cells, innate-like T Cells, follicular helper T (Tfh) cells, and senescent T cells.
  • Nonlimiting examples of B cells include naive B cells, memory B cells, plasma cells, regulatory B (Bregs) cells, B-1 cells, follicular B cells, marginal zone B cells, B-2 cells, IgM memory B cells, IgA-producing B cells.
  • Bregs regulatory B
  • Nonlimiting examples of NK cells include CD56 dim NK cells, CD56 bri9ht NK cells, CD16 + NK cells, CD57 + NK cells, adaptive NK cells, trained NK cells, hyporesponsive NK cells, tissue-resident NK cells, and decidual NK Cells.
  • the engineered proteins of the present technology are useful in treating, reducing, or preventing a plurality of conditions.
  • Nonlimiting examples of conditions that one or more engineered proteins of the present technology are useful in treating, preventing, or reducing includes cancer, age-associated disease, autoimmune disease, infections, GVHD, and transplant rejection.
  • biomarkers such as proteins or transcripts
  • levels of biomarkers, such as proteins or transcripts, associated with a disease may be assessed before, during, or after administration of one or more engineered proteins to determine efficacy of treatment.
  • biomarkers associated with disease include proteins, transcripts, chemicals (e.g., peroxide), or chemical alterations such as reactive oxygen species (ROS) markers or DNA damage markers (e.g., oxidative damage, UV photoproducts, and double-stranded breaks) correlated with a condition being treated (e.g., cancer, age-associated disease, autoimmune disease, infections, GVHD, and transplant rejection).
  • ROS reactive oxygen species
  • DNA damage markers e.g., oxidative damage, UV photoproducts, and double-stranded breaks
  • the subject administered a dose of one or more engineered proteins exhibits one or more of:
  • SA-p-gal beta-galactosidase
  • CDKN2A cyclin-dependent kinase inhibitor 2A
  • TP53BP1 tumor protein p53 binding protein 1
  • MDA malondialdehyde
  • (gg) a reduction in a GSSG to reduced glutathione (GSH) ratio by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
  • (hh) an increase in mitochondrial membrane potential ( M ⁇ m) level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
  • an increase in an oxygen consumption rate (OCR) by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control
  • OCR oxygen consumption rate
  • a reduction in an extracellular acidification rate (ECAR) by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control
  • mm a reduction in a proton production rate (PPR) by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
  • PPR proton production rate
  • (zz) an increase in a CD4 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
  • (ccc) an increase in a HLA-DR protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
  • (ddd) an increase in a granzyme B protein (GZMB) protein or transcript level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
  • (ggg) an increase in a CD38 protein or transcript level by about 1%, 2%, 3%, 4%,
  • NKG2D scd single-chain dimer forms of NKG2D
  • FIG. 1A NKG2D scd proteins comprising a CD3 binding domain (FIG. 1 B) having a VH (SEQ ID NO: 11 ) and VL (SEO ID NO: 12) sequence.
  • VH SEQ ID NO: 11
  • VL SEO ID NO: 12
  • the C-terminus of the VL sequence was linked to the first NKG2D peptide (SEQ ID NO: 3) by the NKG2D N-terminal arm (SEQ ID NO: 6), forming an engineered protein bispecific to CD3 and NKG2DL (SEQ ID NO: 93) (FIG. 1 B). Because this engineered protein was expected to activate CD3- expressing lymphocytes, it was labeled NKG2D-single chain dimer-T cell engager (NKG2D- SCD-TCE). The engineered NKG2D single chain dimer was structurally similar to a native NKG2D dimer when the two structures were superimposed (FIG. 1C). These single chain structures may be engineered to an Fc region of an anti-CD3 antibody, which may comprise an scFv domain (FIGS. 1 D and 1 E).
  • NKG2D-SCD-TCE were expressed using the Daedalus platform in HEK293F cells (Invitrogen) as fusions with Siderocalin (Sen) to stabilize expression, adapted from the methods disclosed in Garrity, D., Call, M. E., Feng, J., & Wucherpfennig, K. W. (2005).
  • the activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure. Proceedings of the National Academy of Sciences of the United States of America, 102(21 ), 7641-7646 and 9. Bannesayake AD, Correnti C, Ryu BY, Brault M, Strong RK, and Rawlings DJ (201 1 ).
  • Daedalus a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors.
  • a TEV protease scission site (SEQ ID NO: 92) was inserted between the Sen and NKG2D moieties, and N-terminal hexahistidine (SEQ ID NO: 10) and FLAG (SEQ ID NO: 90) tags were incorporated to facilitate purification.
  • Additional TEV protease features may comprise those disclosed in Finton KA, Larimore K, Larman HB, Friend D, Correnti C, Rupert PB, Elledge SJ, Greenberg PD, and Strong RK (2013).
  • Autoreactivity and exceptional CDR plasticity hinder elicitation of the anti-HIV antibody 4E10.
  • PLoS Pathog 9(9), e1003639 incorporated herein by reference in its entirety.
  • Recombinant proteins were purified from culture supernatants by immobilized metal chelate affinity, treated with TEV protease to release the Sen fusion partner, and polished by size exclusion chromatography (SEC).
  • Example 2 Engineered protein NKG2D-SCD-TCE promotes lymphocyte activation in the presence of cells expressing NKG2DL
  • NKG2D-SCD-TCE activates T cells specifically in the presence of NKG2DL
  • primary CD8 + T cells were purified from donor PBMCs. Titrated NKG2D-SCD-TCE was added to samples comprising CD8 + T cells alone or to samples comprising CD8 + T cells mixed 1 :1 with a population of NKG2DL-expressing MDA-MB-231 cells. See Kim Y, Bigelow L, Borovilos M, Dementieva I, Duggan E, Eschenfeldt W, Hatzos C, Joachimiak G, Li H, and Maltseva N, et al (2008). Chapter 3. High-throughput protein purification for x-ray crystallography and NMR.
  • NKG2D-SCD-TCE was added at a concentration of 2600 ng/ml, 260 ng/ml, 26 ng/ml, 2.6 ng/ml, 0.26 ng/ml, 0.026 ng/ml, or 0.0026 ng/ml and cultured with samples for 16 hours.
  • 0.0 ng/ml NKG2D-SCD-TCE was used as an additional control.
  • the samples comprising both CD8 + T cells and MDA-MB-231 cells demonstrated increased T cell activation with increased NKG2D-SCD-TCE dosage, as measured by flow CD137 protein levels using flow cytometry.
  • NKG2D-SCD-TCE-mediated T cell activation dose-response using was also examined using a nonlinear regression model (Graphpad Prism). Percent T cell activation (measured by CD137 protein level, y-axis) was then plotted against NKG2D-SCD-TCE concentration (x-axis) for both samples comprising primary CD8 + T cells only (circles) compared to samples comprising primary CD8+ T cells plus MDA-MB-231 cells (squares) (FIG. 4). The resulting EC50 for NKG2D-SCE-TCE was about 3.2 ng/ml (0.056 nM) in samples comprising MDA-MB-231.
  • MDA-MB- 231 (FIG. 5A), HeLa (FIG. 5B), H1299 (FIG. 5C), and HEK233F (FIG. 6) cells were transduced with lentivirus comprising a nucleotide sequence encoding SEQ ID NO: 107 (“ADI”), and supernatant was collected from confluent cultures of untransduced (“Parental”) or ADI-transduced cells for HEK293T cells. Supernatants were collected and added to cultures containing the indicated tumor cell line and primary CD8 T cells that had previously been non-specifically activated and expanded for 10 days prior to the assay at a 3:1 ratio.
  • ADI nucleotide sequence encoding SEQ ID NO: 107
  • MDA-MB-231 (FIG. 7A), H1299 (FIG. 7B), and HeLa (FIG. 5C) cells were transduced with lentivirus comprising nucleotide sequences encoding the engineered proteins of Examples 1 -3 (SEQ ID NO: 93: “OKT3” and SEQ ID NO: 107: “ADI) at increasing concentrations.
  • Both the OKT3 and the ADI engineered proteins resulted in increased T cell activation relative to untransduced controls (“Parental”), as measured by the percent of CD137+ T cells.
  • An engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first Natural killer group 2, member D (NKG2D) peptide, and a second NKG2D peptide.
  • NKG2D Natural killer group 2, member D
  • the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a variable heavy (VH) domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11.
  • VH variable heavy
  • VL variable light domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12.
  • the engineered protein of embodiment 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105.
  • 1 The engineered protein of embodiment 3 or 10, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106.
  • NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
  • NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
  • NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
  • NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
  • NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an N-terminal domain of an NKG2D protein.
  • linker peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 10, or SEQ ID NO: 98.
  • 21 The engineered protein of embodiment 18, wherein the linker peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 10, or SEQ ID NO: 98.
  • An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 1 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least
  • An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about
  • An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at
  • An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at
  • NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
  • NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
  • IgK leader peptide comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73.
  • protease scission site is a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-C scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, or an endopeptidase Arg-C scission site.
  • TSV Tobacco Etch Virus
  • TEV protease scission site comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92 or SEQ ID NO: 96.
  • a pharmaceutical composition comprising the engineered protein of any one of embodiments 1 -39, and one or more pharmaceutically acceptable carriers.
  • a vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 1 , (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • a vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 , (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • a vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103, (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • a vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105, (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
  • a vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
  • a vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 107.
  • a vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 108.
  • a vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
  • a method of generating an engineered protein comprising a CD3 binding domain, and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide comprising the steps of (i) synthesizing the CD3 binding domain from a first vector; (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector; and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
  • a method of activating senolysis of senescent cells in a subject in need thereof comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
  • biomarkers comprises a protein, a transcript, a DNA damage marker, or a reactive oxygen species (ROS) marker.
  • ROS reactive oxygen species
  • a senescence-associated beta-galactosidase SA-[3-gal) protein, a cyclin-dependent kinase inhibitor 1 or cyclin-dependent kinase-interacting protein 1 (p21 WAF1/CIP1 ) protein, a p16INK4a protein, a tumor protein p53 (p53) protein, a retinoblastoma protein (pRB) protein, a deleted in esophageal cancer 1 (DEC1 ) protein, a phosphorylated histone H2AX (y-H2AX) protein, a p53-binding protein 1 (53BP1 ) protein, an Ataxia-telangiectasia mutated (ATM) protein, a meiotic recombination 11 (MRE11 ) protein, a superoxide dismutase (SOD) protein, superoxide diss
  • SA-[3-gal) protein a cyclin-dependent kinase inhibitor 1 or
  • transcript is selected from the group consisting of a lysosomal p-galactosidase 1 (GLB1 ) transcript, a cyclin- dependent kinase inhibitor 2A (CDKN2A) transcript, a p21WAF1/CIP1 transcript, a Tumor Protein P53 (TP53) transcript, a DEC1 transcript, a cyclin-dependent kinase (CDK) protein, a CDK transcript, a tumor protein p53 binding protein 1 (TP53BP1 ) transcript, an ATM transcript, a MRE11 transcript, a SOD transcript, and a CAT transcript.
  • a lysosomal p-galactosidase 1 (GLB1 ) transcript
  • CDKN2A) transcript a cyclin- dependent kinase inhibitor 2A
  • TP53 Tumor Protein P53
  • DEC1 transcript a cyclin-dependent kinase
  • CDK cyclin-dependent kinase
  • DNA damage marker is selected from the group consisting of a y-H2AX phosphorylation level, a thymine dimer, a phosphorylation of an ATM kinase target, a UV-induced DNA photoproduct, a guanine oxidation, and a double-strand break.
  • ROS marker is selected from the group consisting of a malondialdehyde (MDA) level, a hydrogen peroxide (H2O2) level, oxidized glutathione (GSSG) protein, a protein carbonylation, oxidized thiol group, a tyrosine nitration, and a GSSG to reduced glutathione (GSH) ratio.
  • MDA malondialdehyde
  • H2O2O2 hydrogen peroxide
  • GSSG oxidized glutathione
  • GSH reduced glutathione
  • a method for lymphatic cell activation in a subject in need thereof comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
  • the one or more lymphatic cell activation markers is a protein or a transcript.
  • the protein is selected from the group consisting of a CD25 protein, a CD69 protein, a CD71 protein, a CD44 protein, a CD154 protein, a CD28 protein, a CD86 protein, a CD19 protein, a CD3 protein, a CD8 protein, a CD4 protein, a CD56 protein, a CD62L protein, a HLA-DR protein, a granzyme B protein (GZMB) protein, an interferon-gamma (IFN-y) protein, an interleukin-2 (IL-2) protein, a CD38 protein, a CD45RO protein, and a CD20 protein.
  • GZMB granzyme B protein
  • transcript is selected from the group consisting of a CDC25A transcript, a CDC25B transcript, a CDC25C transcript, a CD69 transcript, a TFRC gene, a CD44 transcript, a CD154 transcript, a CD28 transcript, a CD86 transcript, a CD19 transcript, a CD3 transcript, a CD8 transcript, a CD4 transcript, CD56 transcript, a CD62L transcript, a HLA-DR transcript, a GZMB transcript, an interferon-gamma (IFNG) transcript, an interleukin-2 (IL2) transcript, a CD38 gene, a CD45RO transcript, and a CD20 transcript.
  • IFNG interferon-gamma
  • IL2 interleukin-2
  • a method of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
  • ITAM immunoreceptor tyrosine-based activation motif
  • an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
  • ITAM immunoreceptor tyrosine-based activation motif
  • lymphatic cell is a T cell, a B cell, or an NK cell.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
  • 71 The method or use of embodiment 67, 69, or 70, wherein the cancer is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
  • the cancer is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer
  • NKG2D peptide and/or the second NKG2D peptide comprises a NKG2DL binding domain.
  • CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
  • anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 1.
  • NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. [0442] 83. The method or use of any one of embodiments 52-82, wherein the second
  • NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:3.
  • NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
  • NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
  • NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
  • NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
  • NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a peptide comprising an N-terminal domain of an NKG2D protein.
  • one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
  • NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
  • 94 The method or use of any one of embodiments 52-93, wherein the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
  • NKG2DL is a cellsurface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.

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Abstract

The present technology provides engineered proteins bispecific to CD3 and NKG2DL, and method of using the engineered proteins for modulating lymphocyte function and treating a variety of diseases, including age-associated diseases and cancer.

Description

PROTEINS BISPECIFIC TO CD3 AND NKG2DL AND ASSOCIATED USES THEREOF
STATEMENT OF GOVERNMENT LICENSING RIGHTS
[0001] This invention was made with government support under AH 76563, AI121242, and AH 2332 awarded by the National Institutes of Health. The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATION
[0002] This application claims the benefit of U.S. Provisional Application No. 63/588,433, filed October 6, 2023, the contents of which are incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0003] This application contains a ST.26 compliant Sequence Listing, which was submitted in XML format via Patent Center, and is hereby incorporated by reference in its entirety. The XML copy, created on September 20, 2024, is named SequenceListingXML0703548010WO00.XML and is 101 KB in size.
BACKGROUND
[0004] Natural killer group 2, member D (NKG2D) is an activating receptor expressed by natural killer (NK) cells and subsets of T cells which bind to several related families of stressinducible ligands. NKG2D is known to be upregulated in response to malignant transformation. Tumor cells identify themselves as “altered self” by inducing expression of NKG2D ligands (NKG2DL) and elicit targeted destruction by the immune system. NKG2DLS are broadly expressed by most tumors, and as such represent promising targets for immunotherapy.
[0005] In addition to transformed cells, senescent cells also express NKG2DLs. Senescent cells accumulate in tissues as individuals age and have a deleterious effect on tissue function. Efforts to eliminate senescent cells using senolytic drugs may provide a promising strategy for preventing or treating many age-related conditions in humans. At present, options for clinically eliminating senescent cells are limited.
[0006] Current therapeutic efforts targeting the NKG2D-L-expressing cells often enhance protective NKG2D-mediated effector functions which may be counterproductive. Therefore, therapeutics which target NKG2DL-expressing cells for elimination without stimulating dysregulation of the NKG2D/NKG2DL axis are needed.
SUMMARY
[0007] It is one object of the present technology to provide novel engineered proteins and uses thereof, including disease treatment and modulation of lymphocyte function.
[0008] In some embodiments, the present technology comprises engineered proteins a CD3 binding domain and a single peptide chain comprising a first Natural killer group 2, member D (NKG2D) peptide, and a second NKG2D peptide.
[0009] In some embodiments, the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
[0010] In some embodiments, the first NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
[0011] In some embodiments, the first NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
[0012] In some embodiments, the first NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
[0013] In some embodiments, the first NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
[0014] In some embodiments, the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an N-terminal domain of an NKG2D protein.
[0015] In some embodiments, one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
[0016] In some embodiments, the mammalian peptide is a human peptide or a murine peptide.
[0017] In some embodiments, the first NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
[0018] In some embodiments, the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
[0019] In some embodiments, the NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
[0020] In some embodiments, the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
[0021] In some embodiments, the CD3 binding domain comprises a T-cell activating sequence.
[0022] In some embodiments, the engineered protein further comprises one or more of an immunoglobulin leader peptide, a purification tag, a protease scission site, or a second linking peptide sequence.
[0023] In some embodiments, the immunoglobulin leader peptide is a Ig K leader peptide.
[0024] In some embodiments, the purification tag is selected from the group consisting of a hexa-histidine tag, a FLAG tag, a Myc tag, and a Human influenza hemagglutinin (HA) tag.
[0025] In some embodiments, the protease scission site is a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-C scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, or an endopeptidase Arg-C scission site. [0026] In some embodiments, the present technology comprises pharmaceutical compositions comprising an engineered protein of the present technology and one or more pharmaceutically acceptable carriers.
[0027] In some embodiments, the present technology comprises a vector comprising a nucleotide sequence encoding an engineered protein of the present technology or a peptide thereof.
[0028] In some embodiments, the present technology comprises vectors comprising a nucleotide sequence encoding the CD3 binding domain of the engineered proteins of the present technology.
[0029] In some embodiments, the present technology comprises vectors comprising a nucleotide sequence encoding the single chain peptide comprising first NKG2D peptide and the second NKG2D peptide of the engineered proteins of the present technology.
[0030] In some embodiments, the present technology comprises methods of generating an engineered protein comprising a CD3 binding domain, and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide, comprising the steps of (i) synthesizing the CD3 binding domain from a first vector; (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector; and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
[0031] In some embodiments, the present technology comprises methods of activating senolysis of senescent cells in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
[0032] In some embodiments, the one or more biomarkers comprises a protein, a transcript, a DNA damage marker, or a reactive oxygen species (ROS) marker.
[0033] In some embodiments, the protein is selected from the group consisting of a senescence-associated beta-galactosidase (SA-p-gal) protein, a cyclin-dependent kinase inhibitor 1 or cyclin-dependent kinase-interacting protein 1 (p21WAF1/CIP1 ) protein, a p16INK4a protein, a tumor protein p53 (p53) protein, a retinoblastoma protein (pRB) protein, a deleted in esophageal cancer 1 (DEC1 ) protein, a phosphorylated histone H2AX (y-H2AX) protein, a p53-binding protein 1 (53BP1 ) protein, an Ataxia-telangiectasia mutated (ATM) protein, a meiotic recombination 11 (MRE11 ) protein, a superoxide dismutase (SOD) protein, superoxide dismutase (SOD) protein, F2-lsoprostane protein, 8-Hydroxy-2'- deoxyguanosine (8-OHdG) protein.
[0034] In some embodiments, the transcript is selected from the group consisting of a lysosomal p-galactosidase 1 (GLB1 ) transcript, a cyclin-dependent kinase inhibitor 2A (CDKN2A) transcript, a p21 WAF1/CIP1 transcript, a Tumor Protein P53 (TP53) transcript, a DEC1 transcript, a cyclin-dependent kinase (CDK) protein, a CDK transcript, a tumor protein p53 binding protein 1 (TP53BP1 ) transcript, an ATM transcript, a MRE11 transcript, a SOD transcript, and a CAT transcript.
[0035] In some embodiments, the DNA damage marker is selected from the group consisting of a y-H2AX phosphorylation level, a thymine dimer, a phosphorylation of an ATM kinase target, a UV-induced DNA photoproduct, a guanine oxidation, and a double-strand break.
[0036] In some embodiments, the ROS marker is selected from the group consisting of a malondialdehyde (MDA) level, a hydrogen peroxide (H2O2) level, oxidized glutathione (GSSG) protein, a protein carbonylation, oxidized thiol group, a tyrosine nitration, and a GSSG to reduced glutathione (GSH) ratio.
[0037] In some embodiments, the present technology comprises methods for lymphatic cell activation in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
[0038] In some embodiments, the one or more lymphatic cell activation markers is a protein or a transcript. [0039] In some embodiments, the protein is selected from the group consisting of a CD25 protein, a OD69 protein, a CD71 protein, a CD44 protein, a CD154 protein, a CD28 protein, a CD86 protein, a CD19 protein, a CD3 protein, a CD8 protein, a CD4 protein, a CD56 protein, a CD62L protein, a HLA-DR protein, a granzyme B protein (GZMB) protein, an interferon-gamma (IFN-y) protein, an interleukin-2 (IL-2) protein, a CD38 protein, a CD45RO protein, and a CD20 protein.
[0040] In some embodiments, he transcript is selected from the group consisting of a CDC25A transcript, a CDC25B transcript, a CDC25C transcript, a CD69 transcript, a TFRC gene, a CD44 transcript, a CD154 transcript, a CD28 transcript, a CD86 transcript, a CD19 transcript, a CD3 transcript, a CD8 transcript, a CD4 transcript, CD56 transcript, a CD62L transcript, a HLA-DR transcript, a GZMB transcript, an interferon-gamma (IFNG) transcript, an interleukin-2 (IL2) transcript, a CD38 gene, a CD45RO transcript, and a CD20 transcript.
[0041] In some embodiments, the present technology comprises methods of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
[0042] In some embodiments, the lymphatic cell is a T cell, a B cell, or an NK cell.
[0043] In some embodiments, the subject has a cancer or an age-related disease.
[0044] In some embodiments, the age-related disease comprises one or more of osteoporosis, arthritis, obesity, cardiovascular disease, or a neurodegenerative disorder.
[0045] In some embodiments, the present technology comprises methods of treating cancer in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
[0046] In some embodiments, the cancer is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
[0047] In some embodiments, the first NKG2D peptide and/or the second NKG2D peptide comprises a NKG2DL binding domain.
[0048] In some embodiments, the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
[0049] In some embodiments, one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
[0050] In some embodiments, the mammalian peptide is a human or murine peptide.
[0051] In some embodiments, the first NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
[0052] In some embodiments, the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
[0053] In some embodiments, the NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
[0054] In some embodiments, the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
[0055] In some embodiments, the CD3 binding domain comprises a T-cell activating sequence.
[0056] In some embodiments, the engineered protein is present in a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 A illustrates a native form of an NKG2D dimer (left) compared to a singlechain NKG2D dimer (right) in accordance with embodiments of the present technology.
[0058] FIG. 1 B illustrates an engineered protein comprising the single chain NKG2D single-chain dimer of FIG. 1 A and a CD3 binding domain in accordance with embodiments of the present technology.
[0059] FIG. 1 C illustrates a structural comparison between the native NKG2D dimer (NKG2D2) (RCSB Protein Data Bank (PDB) accession code I MPU.pdb) and the singlechain NKG2D dimer (NKG2Dscd) (RCSB PDB accession code 8TM2.pdb) of FIG. 1 A.
[0060] FIG. 1 D illustrates an engineered protein comprising two NKG2D single-chain dimers of FIG. 1 A each fused to a fragment crystallizable (Fc) region of an anti-CD3 antibody in accordance with embodiment of the present technology.
[0061] FIG. 1 E illustrates the engineered protein of FIG. 1 D further comprising singlechain variable fragment (scFv) domains in accordance with embodiments of the present technology.
[0062] FIG. 2 illustrates NKG2DL transcript levels among cell lines expressing major histocompatibility complex (MHC) class I chain-related protein A (MICA), MHC class I chain- related protein B (MICB), LIL16 binding protein 2 (LILBP2), or UL16 binding protein 4 (ULBP4).
[0063] FIG. 3 illustrates T cell activation levels in response to NKG2D-SCD-TCE- mediated target cell recognition in samples comprising CD8+ T cells and NKG2DL- expressing cells (top row) relative to samples comprising CD8+ T cells and no NKG2DL- expressing cells (bottom row).
[0064] FIG. 4 illustrates levels of T cell activation in response to titrated NKG2D-SCD- TCE for T cells in the presence of MDA-MB-231 breast cancer cells (squares) relative to T cells alone (circles).
[0065] FIGS. 5A-5C illustrate live cell cytotoxicity analysis in MDA-MB-231 (FIG. 5A), HeLa (FIG. 5B) and H1299 (FIG. 5C) cells following transduction of a lentiviral vector comprising a nucleotide sequence encoding an engineered protein having the amino acid sequence SEQ ID NO: 107, in accordance with the embodiments of the present technology.
[0066] FIG. 6 shows qualitative amounts of the engineered protein of FIGS. 5A-5C in the supernatant of HEK293F cells 10 days after transduction of a lentiviral vector comprising a nucleotide sequence encoding the engineered protein.
[0067] FIGS. 7A-7C illustrate dose-response results for MDA-MB-231 (FIG. 7A), H1299 (FIG. 7B), and HeLa (FIG. 7C) cells transduced with the lentiviral vector of FIGS. 5A-5C (“ADI”) or a lentiviral vector comprising a nucleotide sequence encoding an engineered protein having the amino acid sequence SEQ ID NO: 93 (“OKT3”).
DETAILED DESCRIPTION
[0068] The present technology provides engineered proteins comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide for treating a variety of diseases, including cancer and age-related disease. Also provided are vectors for synthesizing the engineered proteins of the present technology.
[0069] While the present technology is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the present technology is to be considered as an exemplification of the present technology and is not intended to limit the present technology to the specific embodiments illustrated. Headings are provided for convenience only and are not to be construed to limit the present technology in any manner. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.
[0070] The use of numerical values in the various quantitative values specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about.” It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about.” It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub- range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. It also is to be understood, although not always explicitly stated, that the reagents of the present technology are merely exemplary and that equivalents of such are known in the art. Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
[0071] Also, the disclosure of ranges is intended as a continuous range, including every value between the minimum and maximum values recited, as well as any ranges that may be formed by such values. Also disclosed herein are any and all ratios (and ranges of any such ratios) that may be formed by dividing a disclosed numeric value into any other disclosed numeric value. Accordingly, the skilled person will appreciate that many such ratios, ranges, and ranges of ratios may be unambiguously derived from the numerical values presented herein and in all instances, such ratios, ranges, and ranges of ratios represent various embodiments of the present technology.
[0072] To the extent any materials incorporated by reference herein conflict with the present disclosure, the present disclosure controls.
Definitions
[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present technology belongs. For the purposes of the present technology, the following terms are defined below.
[0074] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
[0075] The term “about” means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by acceptable levels in the art. Typically, such variation may be as much 10% above and below a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length and such variation may be influenced by standard applicable measurement practices. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.
[0076] Any one or more of engineered proteins of the present technology may be present in a pharmaceutical composition, whereby therapeutically effective amounts of the engineered protein are combined in admixture with a pharmaceutically acceptable carrier or excipient. Salts and/or prodrugs of the engineered proteins may also be used.
[0077] A “pharmaceutically acceptable carrier or excipient” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier or excipient may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier or excipient must be “pharmaceutically acceptable,” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. An “excipient” of the pharmaceutical composition refers to an inert substance added to a composition to further facilitate administration of a compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
[0078] “Salts” as used herein comprises pharmaceutically acceptable salts. A pharmaceutically acceptable salt includes any salt that retains the activity of the engineered protein is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. [0079] The terms “treat,” “treatment,” and “treating” refer to a manner of providing a pharmaceutical composition and/or engineered protein to alleviate disease outcomes. This includes utilizing administration techniques of the present technology. Efficacy of treatment may be determined by various assessment methods of the present technology (e.g., assessment of senolysis, lymphatic cell activation, and measurement of biomarkers). The term “biomarker” refers to a biological output that is used as a measure of cellular response, whether that be to assess response to therapeutics, disease status, such as cancer or an age-related disease, or as a predictor of clinical outcomes. Biomarkers evaluated in the context of cells, tissue, or whole organisms. The term “disease” herein refers to any disorder adversely affecting biological status. Disease also may be in the context of human and animal health.
[0080] The terms “treat”, “treatment”, and “treating” may also refer to the reduction or inhibition of the progression and/or duration of a disease (e.g., cancer or an age-related disease), the reduction or amelioration of the severity of the disease, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies.
[0081] As used herein, the terms “effective amount” or “therapeutically effective amount”, refer to that amount of the engineered protein being administered which will relieve to some extent one or more of the symptoms of the disease being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate “effective amount” may differ from one individual to another. An appropriate “effective amount” in any individual case may be determined using techniques, such as a dose escalation study.
[0082] The term “subject”” refers to anyone being evaluated for disease or condition or being administered a therapeutic or pharmaceutical composition. This includes people without diagnosed or confirmed disease or condition. This also includes people with diagnosed or confirmed disease or condition, such as cancer or an age-related disease.
[0083] The term “control,” as used herein, may refer to any subject used as a basis for comparison to the subject of the present technology (e.g., a subject having cancer or an age-related disease). A control includes, but is not limited to, any subject who has not been administered the therapeutic (e.g., an engineered protein) or pharmaceutical composition or administered a placebo, and the subject of the present technology at baseline or prior to administration of a pharmaceutical composition.
[0084] The terms “bind” and “binding” refer to all types of physical and chemical binding, reactions, complexing, attraction, chelating and the like.
[0085] The “proteins” and “peptides” of the present technology may be (a) naturally- occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides.
[0086] The term “peptide” as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids. The amino acids forming all or a part of a peptide may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post- translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, and the like, so that the term “peptide” includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone. The term “peptide” also includes dimers or multimers of peptides. A “manufactured” peptide includes a peptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method. The term “peptide” includes peptides containing a variable number of amino acid residues, optionally with nonamino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.
[0087] By employing chemical synthesis, a useful means of production, it is possible to introduce various amino acids which do not naturally occur along the chain, modify the N- or C-terminus, and the like, thereby providing for improved stability and formulation, resistance to protease degradation, and the like.
[0088] “Amino acids” are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen and have the generic formula H2N — CHR — COOH, wherein R represents a side chain group. The various a-amino acids differ in the side-chain moiety that is attached to the a-carbon. The “amino acids” of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User’s Guide, G. A. Grant, editor, W.H. Freeman & Co., New York (1992), the teachings of which are incorporated herein by reference, including the text and table set forth at pages 11 through 24. As set forth above, the term “amino acid” also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generally in Synthetic Peptides: A User’s Guide, supra; Hruby et al., Biochem. J. 268:249-262 (1990); and Toniolo, Int. J. Peptide Protein Res. 35:287-300 (1990); the teachings of all of which are incorporated herein by reference.
[0089] In the peptides of the present technology, conventional amino acid residues have their conventional meaning as given in Chapter 2400, of the Manual of Patent Examining Procedure, 8th Ed. Thus, “Ala” is alanine; “Arg” is arginine; “Asn” is asparagine; “Asp” is aspartic acid; “Cys” is cysteine; “Gin” is glutamine; “Glu” is glutamic acid; “Gly” is Glycine; “His” is histidine; “He” is isoleucine; “Leu” is leucine; “Lys” is lysine; “Met” is methionine; “Phe” is phenylalanine; “Pro” is proline; “Ser” is serine; Thr is threonine; “Trp” is tryptophan; “Tyr” is tryosine; and “Vai” is valine. Unless otherwise indicated, all amino acids abbreviations represent either isomer, i.e., the L-isomer, the D-isomer, or combinations thereof may be used.
[0090] Percent (%) amino acid sequence “identity” with respect to the sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence for each of the peptides and/or engineered proteins after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity may be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared may be determined. For example, percent amino acid sequence identity values generated using the WU-BLAST-2 computer program uses several search parameters, most of which are set to the default values. WU-BLAST-2 computer program was implemented using the methods disclosed in Altschul, S. F., & Gish, W. (1996). Local alignment statistics. Methods in enzymology, 266, 460-480, the entire contents of which are incorporated herein by reference. Those that are not set to default values (i.e., the adjustable parameters) are set with the following values: overlap span=1 , overlap fraction=0.125, word threshold (T)=11 and scoring matrix BLOSUM62.
[0091] In some embodiments, variants of the proteins and peptides of the present technology may be used. “Variants” include protein sequences having one or more amino acid additions, deletions, stop positions, or substitutions, as compared to a protein sequence disclosed elsewhere herein.
[0092] An amino acid substitution may be a conservative or a non-conservative substitution. Variants of the proteins and peptides of the present technology include those having one or more conservative amino acid substitutions. A “conservative substitution” or “conservative amino acid substitution” involves a substitution found in one of the following conservative substitutions groups: Group 1 : Ala, Gly, Ser, Thr; Group 2: Glu, Asp; Group 3: Asn, Glu; Group 4: Arg, Lys, His; Group 5: lie, Leu, Met, Vai; and Group 6: Phe, Tyr, Trp.
[0093] Additionally, amino acids may be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and lie. Other groups including amino acids that are considered conservative substitutions for one another include: sulfur-containing: Met and Cys; acidic: Asp, Glu, Asn, Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp.
[0094] Non-conservative substitutions include those that significantly affect: the structure of the peptide backbone in the area of the alteration (e.g., the alpha-helical or beta-sheet structure); the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. Non-conservative substitutions which in general are expected to produce the greatest changes in the protein's properties are those in which (i) a hydrophilic residue (e.g. Ser or Thr) may be substituted for (or by) a hydrophobic residue (e.g. Leu, lie, Phe, Vai, or Ala); (ii) a Cys or Phe may be substituted for (or by) any other residue; (iii) a residue having an electropositive side chain (e.g. Lys, Arg, or His) may be substituted for (or by) an electronegative residue (e.g. Gin or Asp); or (iv) a residue having a bulky side chain (e.g. Phe), may be substituted for (or by) one not having a bulky side chain, (e.g. Gly). Additional information is found in Creighton (1984) Proteins, W.H. Freeman and Company, incorporated herein by reference in its entirety.
[0095] The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference, including but not limited to U.S. Patent No. 10,815,290.
Engineered Proteins
[0096] The present technology provides engineered proteins formed of a binding domain, peptides, and a linker, that are bispecific to both CD3 and NKG2DL. The engineered proteins provide for binding to CD3 and NKG2DL expressed by one or more cells or present in free form. The engineered proteins may bind CD3 on a first cell and NKG2DL on a second cell. In some embodiments, binding of CD3 and NKG2DL occurs simultaneously or sequentially. The engineered proteins may bind CD3 and/or NKG2DL expressed by one or more cell types. For example, an engineered protein of the present technology may be capable of binding CD3 expressed at the surface a first cell type and CD3 expressed at the surface of a second cell type. Similarly, the engineered protein may be capable of binding NKG2DL expressed at the surface of a first cell type and NKG2DL expressed at the surface of a second cell type. In some embodiments, the first cell type and the second cell type are of different lineages. In some embodiments, the first cell type and the second cell type are present in different tissues.
[0097] In some embodiments, the engineered protein comprises a CD3 binding domain, a first NKG2D peptide, and a second NKG2D peptide. The first NKG2D peptide and the second NKG2D peptide may be present on a single peptide chain and in some embodiments, the first NKG2D peptide and the second NKG2D peptide are present on the single peptide chain. The CD3 binding domain may be present on the single peptide chain, or on a second single peptide chain that is fused to, operably connected to, or otherwise linked the first single peptide chain.
NKG2D Peptides
[0098] The engineered proteins of the present technology comprise one or more NKG2D peptides designed to bind NKG2DL (e.g., MICA, MICB, RAET1 E/ULBP4, RAET1 G/ULBP5, RAET1 H/ULBP2, RAET1/ULBP1 , RAET1 L/ULBP6, and RAET1 N/ULBP3). In some embodiments, the one or more NKG2D peptides comprise a first NKG2D peptide and a second NKG2D peptide. The first NKG2D peptide and/or the second NKG2D peptide may be a peptide having fewer amino acids than the wild-type NKG2D protein. In some embodiments, the wild-type NKG2D protein is a human wild-type NKG2D protein (NCBI Accession: CAA04925). In some embodiments, the wild-type protein is a murine wild-type protein (NCBI Accession: AAC28245). The peptide having fewer amino acids than the wildtype NKG2D protein may comprise a peptide having one or more amino acids less than the wild-type NKG2D protein. In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein comprises at least about 110 to about 130 residues of the wildtype NKG2D protein. In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein comprises one or more C-type lectin (CTL)-like or carbohydrate- recognition domain (CRD) (CLECT) domains. In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein is lacking a transmembrane domain. In some embodiments, the transmembrane domain comprises or consist of the amino acid sequence PFFFCCFIAVAMGIRFIIMVAIW (SEQ ID NO: 1 ). [0099] In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
[0100] In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
[0101] In some embodiments, the peptide having fewer amino acids than the wild-type NKG2D protein comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 2.
[0102] In some embodiments, the first NKG2D peptide and/or the second NKG2D peptide may comprise a NKG2DL binding domain. In some embodiments, the first NKG2D peptide and the second NKG2D peptide comprise dimerization domains, wherein the first NKG2D peptide and the second NKG2D peptide form a dimer which binds a NKG2DL.
[0103] In some embodiments, the first NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence. In some embodiments, the first NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence. In some embodiments, the first NKG2D peptide and the second NKG2D peptide are different.
[0104] In some embodiments, the first NKG2D peptide and/or the second NKG2D peptide comprise or consist of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 1 .
[0105] In some embodiments, the first NKG2D peptide and/or the second NKG2D peptide comprise or consist of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 1 .
[0106] In some embodiments, the first NKG2D peptide and/or the second NKG2D peptide comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 1 .
[0107] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. [0108] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0109] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0110] In some embodiments, the second NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0111] In some embodiments, the second NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0112] In some embodiments, the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0113] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0114] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0115] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0116] In some embodiments, the second NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0117] In some embodiments, the second NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. [0118] In some embodiments, the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0119] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99.
[0120] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99.
[0121] In some embodiments, the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99.
Table 1 : Exemplary NKG2D Peptide Sequences
Figure imgf000021_0001
Figure imgf000022_0001
[0122] The first NKG2D peptide and the second NKG2D peptide may be linked on the single chain peptide by one or more linking peptides. In some embodiments, the one or more linking peptides are used to join the first NKG2D peptide to the second NKG2D peptide and/or to the CD3 binding domain or to another peptide or another type of molecule.
[0123] In some embodiments, the one or more linking peptides comprises a glycineserine (Gly/Ser) linker, a proline-threonine-glycine linker, an alanine linker, a lysine linker, a threonine linker, a valine-glycine-serine-threonine linker, an elastin-like peptide linker, or a hexahistidine linker.
[0124] In some embodiments, the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by one or more linking peptides comprising an N- terminal domain of an NKG2D protein. In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence listed in Table 2.
[0125] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 2.
[0126] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 2.
[0127] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 2.
[0128] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5. [0129] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5.
[0130] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5.
[0131] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
[0132] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
[0133] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.
[0134] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7.
[0135] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7.
[0136] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 7.
[0137] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8. [0138] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.
[0139] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.
[0140] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9.
[0141] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9.
[0142] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 9.
[0143] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 97.
[0144] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 97.
[0145] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 97.
[0146] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence of GS. [0147] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence of GS.
[0148] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence of GS.
[0149] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
[0150] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
[0151] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
[0152] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 98.
[0153] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 98.
[0154] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 98.
[0155] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 102. [0156] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 102.
[0157] In some embodiments, the one or more linking peptides comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 102.
Table 2: Exemplary NKG2D Linking Peptide Sequences
Figure imgf000026_0001
CD3 Binding Domains
[0158] The engineered protein of the present technology further comprises a CD3 binding domain, which selectively binds a CD3 protein. The CD3 binding domain may bind at least a portion of one or more CD3 subunits. In some embodiments, the one or more CD3 subunits is selected from the group consisting of CD3s, CD3y, CD35, and CD3 . [0159] In some embodiments, the CD3 protein is a wild-type human CD3 protein. The CD3 binding domain may recognize one or more of wild-type human CD3s (NCBI Accession: NP 000723), wild-type human CD3y (NCBI Accession: NP 000064), wild-type human CD35 (NCBI Accession: ACA05962), and wild-type human CD3^ (isoform 1 NCBI Accession: NP_932170; isoform 2 NCBI Accession: NP_000725; isoform 3 NCBI Accession: NP_001365444; isoform 4 NCBI Accession: NP_00136544).
[0160] In some embodiments, the CD3 protein is a wild-type murine CD3 protein. The CD3 binding domain may recognize one or more of wild-type murine CD3s (NCBI Accession: NP_031674), wild-type murine CD3y (NCBI Accession: NP_033980); wild-type murine CD35 (NCBI Accession: NP_038515), and wild-type murine CD3 (NCBI Accession: P24161 ).
[0161] In some embodiments, the CD3 binding domain comprises one or more T-cell activating sequences.
[0162] In some embodiments, CD3 is expressed on the surface of cells such as lymphocytes. Nonlimiting examples of CD3-expressing lymphocytes include T cells, NK cells, and B cells. Binding of CD3 through the CD3 binding domain provides a stimulatory signal which engages immune responses, including but not limited to, B cell stimulation, antibody production, antigen recognition, apoptotic signaling, cytotoxic signaling, cytokine storm, and target cell destruction or neutralization.
[0163] In some embodiments, the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof. The antigen binding fragment thereof may comprise one or more of a Fragment antigen-binding (Fab) or at least a portion thereof of an anti-CD3 antibody, a fragment crystallizable (Fc) region or at least a portion thereof of an anti-CD3 antibody, a single-chain variable fragment (scFv) or at least a portion thereof of an anti-CD3 antibody, a variable light (VL) domain and/or a variable heavy (VH) domain or at least a portion thereof of an anti-CD3 antibody, or one or more complementarity-determining regions (CDRs) or at least a portion thereof of an anti-CD3 antibody. In some embodiments, the CD3 binding domain is fused to one or more NKG2D single-chain dimers. In some embodiments, the CD3 binding domain is fused to two or more NKG2D single chain dimers. In some embodiments, when the CD3 binding domain is fused to two or more NKG2D single chain dimers, the two or more NKG2D single chain dimers are each independently fused to different polypeptide chains of the CD3 binding domain.
[0164] The anti-CD3 antibody or the antigen fragment thereof may comprise a VL domain and/or a VH domain listed in Table 3
[0165] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 1 1 .
[0166] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 1 1 .
[0167] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 1 1 .
[0168] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 12.
[0169] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 12.
[0170] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 12. [0171] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 103.
[0172] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 103.
[0173] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 103.
[0174] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 104.
[0175] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 104.
[0176] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 104.
[0177] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 105. [0178] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 105.
[0179] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 105.
[0180] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 106.
[0181] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 106.
[0182] In some embodiments, the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 106.
Table 3: Exemplary CD3 binding domain sequences
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Leader Peptides
[0183] The engineered protein of the present technology may further comprise a leader peptide. In some embodiments, the leader peptide comprises a signal sequence (e.g., a targeting sequence or a localization sequence). In some embodiments, the leader peptide comprises or consist of an Igk or an IgA leader peptide. In some embodiments, the Igk leader peptide is a murine peptide sequence.
[0184] In some embodiments, the Igk leader peptide comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical METDTLLLWVLLLWVPGSTG (SEQ ID NO: 73).
[0185] In some embodiments, the Igk leader peptide comprises or consists of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73. [0186] In some embodiments, the Igk leader peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73.
[0187] In some embodiments, the leader peptides comprise or consist of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 4.
[0188] In some embodiments, the leader peptides comprise or consist of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 4.
[0189] In some embodiments, the leader peptides comprise or consist of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 4.
Table 4: Exemplary Leader Peptide Sequences
Figure imgf000038_0001
Figure imgf000039_0001
Additional Embodiments of Engineered Proteins
[0190] The engineered protein of the present technology may further comprise one or more purification tags. The purification tag may provide an epitope for selective binding of the engineered protein. For example, the epitope may comprise a binding sequence for use in purification or pull-down using methods commonly used in the art. The epitope tag may be placed at the amino- or carboxyl-terminus of the engineered protein. However, in some embodiments, an epitope tag is be placed within the amino acid sequence of the engineered protein amino acid sequence.
[0191] In some embodiments, the epitope tag enables the engineered proteins to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag; this also may be useful for binding the engineered protein to a support for heterogeneous screening methods.
[0192] Various tag polypeptides and their respective antibodies are well known in the art. Nonlimiting examples of purification tags include a hexahistidine tag (HHHHHH; SEQ ID NO: 10), a FLAG tag (DYKDDDDK; SEQ ID NO: 90), a Myc tag, and a Human influenza hemagglutinin (HA) tag (YPYDVPDYA; SEQ ID NO: 91 ). [0193] In some embodiments, the engineered protein comprises one or more protease scission sites. Nonlimiting examples of protease scission sites include a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-0 scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, and an endopeptidase Arg-C scission site.
[0194] In some embodiments, the TEV protease scission site comprises or consist of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to ENLYFQ (SEQ ID NO: 92).
[0195] In some embodiments, the TEV protease scission site comprises or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92.
[0196] In some embodiments, the TEV protease scission site comprises or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92.
[0197] In some embodiments, the TEV protease scission site comprises or consist of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to ENLYFQGS (SEQ ID NO: 96)
[0198] In some embodiments, the TEV protease scission site comprises or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 96.
[0199] In some embodiments, the TEV protease scission site comprises or consist of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 96.
[0200] In some embodiments, the engineered protein comprises a CD3 binding domain, a single-chain peptide comprising a first NKG2D peptide and a second NKG2D peptide, a leader peptide, a linker sequence, a purification tag, and a protease scission site, together comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0201] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0202] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0203] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0204] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
107.
[0205] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
107.
[0206] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 107.
[0207] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
108.
[0208] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
108. [0209] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 108.
[0210] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
[0211] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
[0212] In some embodiments, the engineered protein comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
Table 5: Exemplary Engineered Protein Sequence
Figure imgf000042_0001
Figure imgf000043_0001
[0213] In some embodiments, the engineered proteins comprise or consist of an anti- CD3 antibody fused to one or more NKG2D single-chain dimers of the present technology. In some embodiments, the engineered proteins comprise or consist of an anti-CD3 antibody fused to two or more NKG2D single-chain dimers of the present technology.
[0214] In some embodiments, the engineered proteins comprise or consist of an Fc region or at least a portion thereof of an anti-CD3 antibody fused to one or more NKG2D single-chain dimers of the present technology. In some embodiments, the engineered proteins comprise or consist of an Fc region or at least a portion thereof of an anti-CD3 antibody fused to two or more NKG2D single-chain dimers of the present technology. In some embodiments, when the engineered proteins comprise two or more NKG2D singlechain dimers, the two or more NKG2D single-chain dimers are each ultimately fused to different chains of the Fc region or the portion thereof.
[0215] Any one of the domains, peptides, or engineered proteins of the present technology may comprise a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences. In some embodiments one or more of the CD3 binding domain, the first NKG2D peptide, or the second NKG2D peptide comprise a peptide selected from the group consisting of a mammalian peptide, a non- naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences. In some embodiments, the mammalian peptide is a human peptide or a murine peptide. In some embodiments, the first NKG2D peptide, the second NKG2D peptide, or any one of the linker sequences comprises or consists of at least a portion of the wild-type human NKG2D protein (NCBI Accession: CAA04925; SEQ ID NO: 94) or the wild-type mouse NKG2D protein (NCBI Accession: AAC28245; SEQ ID NO: 95) shown in Table 6. The portion may comprise any peptide having the full sequence of SEQ ID NO:94 or SEQ ID NO: 95 or less.
Modifications of the Engineered Proteins
[0216] Covalent modifications of the engineered proteins of the present technology are included within the scope of the present technology. One type of covalent modification includes reacting targeted amino acid residues of the engineered proteins with an organic derivatizing agent that is retractable with selected side chains or the N- or C-terminal residues of the engineered proteins. Derivatization with bifunctional agents may be useful, for instance, for crosslinking the engineered proteins to a water-insoluble support matrix or surface for use in the methods described below, or for in vivo stability. Commonly used crosslinking agents include 1 ,1 -bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homobifunctional imidoesters, including disuccinimidyl esters (e.g., 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides (e.g., bis-N-maleimido-1 ,8-octane) and agents such as methyl-3- ((p-azidophenyl)dithio) propioimidate, and 1 -ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride.
[0217] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of P and L, phosphorylation of hydroxyl groups of S or T residues, methylation of the amino groups of L, R, and H side chains (see, e.g., T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N- terminal amine, and/or amidation of any C-terminal carboxyl group. In addition, modifications such as derivitization with polyethylene glycols (and other glycols) to increase the in vivo stability half-life are also included.
[0218] In some embodiments, the engineered proteins of the present technology are glycosylated. Glycosylation is a type of post-translational modification that refers to the covalent attachment of sugars to proteins. Functions of glycosylation include contributing to proper protein folding and facilitating cell-to-cell and/or protein-protein interactions.
[0219] Sugars that may be linked to proteins through glycosylation include glycans and monosaccharides, such glucose and fucose. Glycans are polysaccharides with sugar subunits linked by glycosidic bonds. Sugars that may be present in glycans include mannose, sialic acid, GIcNAc, galactose, and fucose. Glycans are a diverse class of molecules and may vary in the type of sugar moieties present, in the length of the chains of sugars, and in structure (branched or unbranched sugar chains). There are three major classes of glycans involved in protein glycosylation; N-linked glycans, O-linked glycans, and phospho-linked glycans.
[0220] In some embodiments, the engineered proteins of the present technology are N- linked glycosylated. N-linked glycans attach to the nitrogen atom of certain amino acids including N and R. In eukaryotic cells, N-linked glycosylation occurs during translation in the endoplasmic reticulum. Therefore, proteins that encode a signal peptide that signals for translation within the endoplasmic reticulum may become N-linked glycosylated. An N-linked glycosylation site may include N-X-(S), where X is any amino acid. [0221] In some embodiments, the engineered proteins of the present technology are O- linked glycosylated. O-linked glycans attach to the hydroxyl (OH) of amino acids including S, T, Y, hydroxyl-K, and hydroxyl-P. In eukaryotic cells, O-linked glycosylation occurs in the Golgi apparatus. An O-linked glycosylation site may include (S or T)-X-X-P, where X is any amino acid.
[0222] In some embodiments, the engineered proteins of the present technology are glycosylated due to protein production using a eukaryotic cell expression system. All domains of life (eukaryote, bacteria, and archaea) are capable of protein glycosylation. However, glycosylation machinery may differ across domains and even across species. Therefore, a protein that is glycosylated when expressed in an autologous system may not become glycosylated upon expression in a heterologous system. One specific reason is if the glycosylation site of the protein is not recognized by the cell type used for expression
Table 6: Human NKG2D protein
Figure imgf000046_0001
Vectors
[0223] Also provided herein are vectors which comprise nucleotide sequences encoding one or more peptides in accordance with the present technology. The vectors may comprise one or more of a nucleotide sequence encoding a peptide or an engineered protein of the present technology in addition to vector and/or cloning sequences commonly used in the art. For example, the vectors may comprise one or more of an origin of replication (Ori), a selectable marker, a promoter, a transcription start site, a ribosome binding site, a terminator sequence, a polyadenylation signal, a multiple cloning site, an origin of transfer, a regulatory element, or an inducible system.
[0224] In some embodiments, the Ori is selected from the group consisting of a ColE1 Ori, a pUC Ori, a p15A Ori, a pSC101 Ori, a SV40 Ori, a M13 Ori, an OriC. The Ori may comprise an autonomously replicating sequence or a eukaryotic chromosomal origin.
[0225] The selectable marker may be an antibiotic resistance marker, a nutritional marker, a reporter gene, an herbicide resistance gene, a phage resistance gene, a temperature-sensitive marker, a hygromycin resistance gene, a purine or pyrimidine analogue resistance gene, or an antibiotic-sensitivity marker. In some embodiments, the vector comprises one or more selectable markers or two or more selectable markers.
[0226] Nonlimiting examples of promoters include a T7 Promoter, a TAC (Trp-lac Hybrid Promoter), a IPTG-lnducible Promoters, a IRES (Internal Ribosome Entry Site), a CMV Promoter (Cytomegalovirus Promoter), a SV40 Promoter (Simian Virus 40 Promoter), a EF1 a (Elongation Factor 1 Alpha) Promoter, a PGK Promoter (Phosphoglycerate Kinase Promoter), an Actin Promoter (e.g., CMV-p-Actin Promoter), a GAL1 Promoter, and a CaMV 35S Promoter (Cauliflower Mosaic Virus 35S Promoter).
[0227] In some embodiments, the inducible system comprises a Lac operon (lac promoter with IPTG induction), a Tet-On and Tet-Off system, an AraC-Arabinose system (pBAD Promoter), a T7 Promoter with T7 RNA Polymerase, an IPTG-inducible promoters, an AraC-PBAD promoter system, a chemically inducible system (e.g., Shield-1 ), a heat shock promoter (e.g., hsp70 promoter), a pBAD Promoter with L-Arabinose, and a nitrogenresponsive systems (e.g., nif Promoter). [0228] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:93.
[0229] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0230] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0231] In some embodiments, provided is a vector comprising a nucleotide sequence that encodes the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide. In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0232] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0233] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0234] In some embodiments, the vector comprises a nucleotide sequence that encodes at least two peptides, each comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0235] In some embodiments, the vector comprises a nucleotide sequence that encodes at least two peptides, each comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. [0236] In some embodiments, the vector comprises a nucleotide sequence that encodes at least two peptides, each comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0237] In some embodiments, provided is a vector comprising a nucleotide sequence that encodes the CD3 binding domain. In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 1 1 or SEQ ID NO: 12.
[0238] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is about 80%, 85%, 90%, 95%, 99%, or 100% identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 11 or SEQ ID NO: 12.
[0239] In some embodiments, the vector comprises a nucleotide sequence that encodes a peptide comprising or consisting of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to identical to a sequence listed in Table 3. In some embodiments, the sequence is SEQ ID NO: 1 1 or SEQ ID NO: 12.
[0240] In some embodiments, the engineered protein of the present technology is generated by a method comprising synthesizing the full-length peptide using a single vector. In some embodiments, the engineered protein of the present technology is generated by a method comprising (i) synthesizing the CD3 binding domain from a first vector, (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector, and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
Pharmaceutical Compositions
[0241] Some embodiments of the present technology include a pharmaceutical composition comprising one or more engineered proteins or vectors of the present technology. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or excipient (e.g., water, saline, dextrose, glycerol). In some embodiments, the pharmaceutically acceptable carrier and/or excipient comprises water. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of lactose, microcrystalline cellulose (MCC), starch, glycerin, polyethylene glycol (PEG), sorbitol, silicon dioxide (SiO2), magnesium stearate, propylene glycol, polysorbate 80, hydroxypropyl methylcellulose (HPMC), polyvinylpyrrolidone (PVP), lecithin, ethanol, water, mineral oil, talc, gelatin, sucrose, or povidone-iodine.
[0242] Nonlimiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
[0243] The pharmaceutically acceptable carrier may comprise one or more of an absorption delaying agent, an antioxidant, a binder, a buffering agent, a bulking agent or filler, a chelating agent, a coating, a disintegration agent, a dispersion media, a gel, an isotonic agent, a lubricant, a preservative, a salt, a solvent or co-solvent, a stabilizer, a surfactant, and/or a delivery vehicle.
[0244] Nonlimiting examples of an antioxidant includes ascorbic acid, methionine, and vitamin E.
[0245] Nonlimiting examples of a buffering agent includes citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and trimethylamine salts.
[0246] In some embodiments, the chelating agent is EDTA.
[0247] Nonlimiting examples of an isotonic agent includes polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.
[0248] Nonlimiting examples of a preservative includes phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanoL
[0249] Stabilizers comprise a broad category of excipients which may range in function from a bulking agent to an additive which solubilizes the engineered protein or helps to prevent denaturation or adherence to the container wall. Typical stabilizers may include polyhydric sugar alcohols; amino acids, ornithine, L-leucine, 2-phenylalanine, E, and T; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on the engineered protein weight.
[0250] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable salt that retains the activity of the engineered protein and is acceptable for pharmaceutical use. The salt may comprise a pharmaceutically acceptable acid addition salt. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.
[0251] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable base addition salt. Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, procaine, and the amino acids Lys or Arg.
[0252] In some embodiments the pharmaceutical composition may be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the formulation may be in lyophilized and/or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
[0253] In some embodiments, the pharmaceutical compositions may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid formulations such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g. lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents may be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as caramel, peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. may also be used.
[0254] In some embodiments, the pharmaceutical compositions may be formulated as an aerosol. In some embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers may also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated containing a powder mix (including flavored mixes) of engineered proteins and a suitable powder base such as lactose or starch.
[0255] The pharmaceutical compositions may also be formulated as suppositories and/or depot preparations. Depot preparations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salts. [0256] Additionally, the pharmaceutical compositions may be formulated as sustained- release systems utilizing semipermeable matrices of solid polymers comprising at least one engineered protein. Sustained-release systems may release the engineered proteins following administration for a few weeks up to over 100 days.
[0257] Depot formulations may comprise one or more bioerodible polymers including, but not limited to, poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)- co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades may result in characteristics that borrow from each of the contributing polymers.
[0258] The use of different solvents (e.g., dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) may alter microparticle size and structure to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.
[0259] Exemplary release modifiers may comprise surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127), polysorbates, Span® (Croda Americas, Wilmington, Del.), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Del.), sucrose acetate isobutyrate (SAIB), salts, and buffers.
[0260] Excipients that partition into the external phase boundary of microparticles (e.g., surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, or PVA,) may also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.
[0261] Additional processing of the sustained release depot formulations may utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle may also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.
[0262] In some embodiments, the pharmaceutical composition comprises the engineered proteins of the present technology of at least about 0.1% w/v or w/w of the composition, at least about1% w/v or w/w of the composition; at least about 10% w/v or w/w of the composition; at least about 20% w/v or w/w of the composition; at least about 30% w/v or w/w of the composition; at least about 40% w/v or w/w of the composition; at least about 50% w/v or w/w of the composition; at least about 60% w/v or w/w of the composition; at least about 70% w/v or w/w of the composition; at least about 80% w/v or w/w of the composition; at least about 90% w/v or w/w of the composition; at least about 95% w/v or w/w of the composition; or at least about 99% w/v or w/w of the composition.
Associated Methods
Administration of Pharmaceutical Compositions
[0263] The pharmaceutical compositions of the present technology may be administered to a subject in need thereof. The pharmaceutical composition may be formulated for administration by, for example, injection, inhalation, infusion, insertion, perfusion, lavage, or ingestion. The pharmaceutical compositions may further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and/or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and/or subcutaneous ingestion, injection, or insertion. In some embodiments, the pharmaceutical composition is administered by implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.
[0264] For administration, therapeutically effective amounts (i.e. “doses”) for all methods of the present technology may be initially estimated based on results from in vitro assays and/or animal model studies. Such information may be used to modify the doses administered to the subject. [0265] The actual dose amount administered to a particular subject may be determined by a physician, veterinarian, or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.
[0266] In some embodiments, administered doses of pharmaceutical compositions of the present technology may range from about 0.1 to about 5 pg/kg or from about 0.5 to about 1 pg/kg. In other non-limiting examples, a dose may comprise about 1 pg/kg, about 5 pg/kg, about 10 pg/kg, about 15 pg/kg, about 20 pg/kg, about 25 pg/kg, about 30 pg/kg, about 35 pg/kg, about 40 pg/kg, about 45 pg/kg, about 50 pg/kg, about 55 pg/kg, about 60 pg/kg, about 65 pg/kg, about 70 pg/kg, about 75 pg/kg, about 80 pg/kg, about 85 pg/kg, about 90 pg/kg, about 95 pg/kg, about 100 pg/kg, about 150 pg/kg, about 200 pg/kg, about 250 pg/kg, about 350 pg/kg, about 400 pg/kg, about 450 pg/kg, about 500 pg/kg, about 550 pg/kg, about 600 pg/kg, about 650 pg/kg, about 700 pg/kg, about 750 pg/kg, about 800 pg/kg, about 850 pg/kg, about 900 pg/kg, about 950 pg/kg, about 1000 pg/kg, about 0.1 to about 5 mg/kg or from about 0.5 to about 1 mg/kg, a pharmaceutical composition of the present technology. In other non-limiting examples, a dose may comprise about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, about 150 mg/kg, about 200 mg/kg, about 250 mg/kg, about 350 mg/kg, about 400 mg/kg, about 450 mg/kg, about 500 mg/kg, about 550 mg/kg, about 600 mg/kg, about 650 mg/kg, about 700 mg/kg, about 750 mg/kg, about 800 mg/kg, about 850 mg/kg, about 900 mg/kg, about 950 mg/kg, or about 1000 mg/kg or more of a pharmaceutical composition of the present technology.
[0267] In some embodiments, administered doses of pharmaceutical compositions of the present technology may range from at least 0.1 to at least 5 pg/kg or from at least 0.5 to at least 1 pg/kg. In other non-limiting examples, a dose may comprise at least 1 pg/kg, at least 5 pg/kg, at least 10 pg/kg, at least 15 pg/kg, at least 20 pg/kg, at least 25 pg/kg, at least 30 pg/kg, at least 35 pg/kg, at least 40 pg/kg, at least 45 pg/kg, at least 50 pg/kg, at least 55 pg/kg, at least 60 pg/kg, at least 65 pg/kg, at least 70 pg/kg, at least 75 pg/kg, at least 80 pg/kg, at least 85 pg/kg, at least 90 pg/kg, at least 95 pg/kg, at least 100 pg/kg, at least 150 pg/kg, at least 200 pg/kg, at least 250 pg/kg, at least 350 pg/kg, at least 400 pg/kg, at least 450 pg/kg, at least 500 pg/kg, at least 550 pg/kg, at least 600 pg/kg, at least 650 pg/kg, at least 700 pg/kg, at least 750 pg/kg, at least 800 pg/kg, at least 850 pg/kg, at least 900 pg/kg, at least 950 pg/kg, at least 1000 pg/kg, at least 0.1 to at least 5 mg/kg or from at least 0.5 to at least 1 mg/kg, a pharmaceutical composition of the present technology. In other nonlimiting examples, a dose may comprise at least 1 mg/kg, at least 5 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 55 mg/kg, at least 60 mg/kg, at least 65 mg/kg, at least 70 mg/kg, at least 75 mg/kg, at least 80 mg/kg, at least 85 mg/kg, at least 90 mg/kg, at least 95 mg/kg, at least 100 mg/kg, at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 350 mg/kg, at least 400 mg/kg, at least 450 mg/kg, at least 500 mg/kg, at least 550 mg/kg, at least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least 750 mg/kg, at least 800 mg/kg, at least 850 mg/kg, at least 900 mg/kg, at least 950 mg/kg, or at least 1000 mg/kg or more of a pharmaceutical composition of the present technology.
[0268] In some embodiments, administered doses of pharmaceutical compositions of the present technology may range from at least about 0.1 to at least about 5 pg/kg or from at least about 0.5 to at least about 1 pg/kg. In other non-limiting examples, a dose may comprise at least about 1 pg/kg, at least about 5 pg/kg, at least about 10 pg/kg, at least about 15 pg/kg, at least about 20 pg/kg, at least about 25 pg/kg, at least about 30 pg/kg, at least about 35 pg/kg, at least about 40 pg/kg, at least about 45 pg/kg, at least about 50 pg/kg, at least about 55 pg/kg, at least about 60 pg/kg, at least about 65 pg/kg, at least about 70 pg/kg, at least about 75 pg/kg, at least about 80 pg/kg, at least about 85 pg/kg, at least about 90 pg/kg, at least about 95 pg/kg, at least about 100 pg/kg, at least about 150 pg/kg, at least about 200 pg/kg, at least about 250 pg/kg, at least about 350 pg/kg, at least about 400 pg/kg, at least about 450 pg/kg, at least about 500 pg/kg, at least about 550 pg/kg, at least about 600 pg/kg, at least about 650 pg/kg, at least about 700 pg/kg, at least about 750 pg/kg, at least about 800 pg/kg, at least about 850 pg/kg, at least about 900 pg/kg, at least about 950 pg/kg, at least about 1000 pg/kg, at least about 0.1 to at least about 5 mg/kg or from at least about 0.5 to at least about 1 mg/kg, a pharmaceutical composition of the present technology. In other non-limiting examples, a dose may comprise at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 15 mg/kg, at least about 20 mg/kg, at least about 25 mg/kg, at least about 30 mg/kg, at least about 35 mg/kg, at least about 40 mg/kg, at least about 45 mg/kg, at least about 50 mg/kg, at least about 55 mg/kg, at least about 60 mg/kg, at least about 65 mg/kg, at least about 70 mg/kg, at least about 75 mg/kg, at least about 80 mg/kg, at least about 85 mg/kg, at least about 90 mg/kg, at least about 95 mg/kg, at least about 100 mg/kg, at least about 150 mg/kg, at least about 200 mg/kg, at least about 250 mg/kg, at least about 350 mg/kg, at least about 400 mg/kg, at least about 450 mg/kg, at least about 500 mg/kg, at least about 550 mg/kg, at least about 600 mg/kg, at least about 650 mg/kg, at least about 700 mg/kg, at least about 750 mg/kg, at least about 800 mg/kg, at least about 850 mg/kg, at least about 900 mg/kg, at least about 950 mg/kg, or at least about 1000 mg/kg or more of a pharmaceutical composition of the present technology.
[0269] Therapeutically effective amounts may be achieved by administering single or multiple doses during a treatment regimen. In some embodiments, the dose is administered about daily, about every other day, about every 3 days, about every 4 days, about every 5 days, about every 6 days, about weekly, about every 2 weeks, about every 3 weeks, about monthly, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, or about yearly.
[0270] In some embodiments, the dose is administered at least daily, at least every other day, at least every 3 days, at least every 4 days, at least every 5 days, at least every 6 days, at least weekly, at least every 2 weeks, at least every 3 weeks, at least monthly, at least every 2 months, at least every 3 months, at least every 4 months, at least every 5 months, at least every 6 months, at least every 7 months, at least every 8 months, at least every 9 months, at least every 10 months, at least every 11 months, or at least yearly.
[0271] In some embodiments, the dose is administered at least about daily, at least about every other day, at least about every 3 days, at least about every 4 days, at least about every 5 days, at least about every 6 days, at least about weekly, at least about every 2 weeks, at least about every 3 weeks, at least about monthly, at least about every 2 months, at least about every 3 months, at least about every 4 months, at least about every 5 months, at least about every 6 months, at least about every 7 months, at least about every 8 months, at least about every 9 months, at least about every 10 months, at least about every 11 months, or at least about yearly.
Subjects
[0272] In some embodiments, the subject of the present technology has a disease. In some embodiments, the subject has one or more of a cancer, an age-related disease, an immune condition or disease, or graft-versus-host disease or transplant rejection.
[0273] Nonlimiting examples of age-related diseases include osteoporosis, arthritis, obesity, cardiovascular disease, and neurodegenerative disorders.
[0274] Nonlimiting examples of cancer includes a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
[0275] In some embodiments, the immune condition or disease comprises an infection (e.g., bacterial infections, fungal infections, or viral infections). In some embodiments, the immune condition is an inflammatory response to a vaccine. In some embodiments, the immune condition or disease is an autoimmune disease. Nonlimiting examples of autoimmune disease includes rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes, Hashimoto's thyroiditis, Graves' disease, celiac disease, psoriasis, inflammatory bowel disease (IBD), Sjogren's syndrome, myasthenia gravis, vitiligo, pernicious anemia, ankylosing spondylitis, Addison's disease, and Goodpasture syndrome. In some embodiments, the autoimmune condition is an NKG2D- driven autoimmune condition.
[0276] In some embodiments, the transplant comprises at least a partial organ or tissue or a cell. In some embodiments, the transplant comprises an epithelial tissue, a connective tissue, a muscular tissue, or a nervous tissue. Target Cells
[0277] The methods of the present technology comprise administering one or more engineered proteins of the present technology to a subject in need thereof, wherein the one or more engineered proteins binds a NKG2DL and a CD3, each expressed on the surface of one or more cells.
[0278] The NKG2DL may be a cell-surface protein expressed by a cell including, but not limited to a transformed cell, a cell subjected to genotoxic stress, or a senescent cell. Nonlimiting examples of transformed cells include cancer cells, immortalized cells, virally infected cells, and cells having abnormal growth or behavior driven by a genetic alteration. Nonlimiting examples of cells subjected to genotoxic stress include cells exposed to one or more of ultraviolet (UV) radiation, chemotherapy, radiation, smoke, chemical carcinogens, metabolic stress, infection, or pharmaceuticals.
[0279] In some embodiments, CD3 is expressed on the surface of one or more lymphatic cells. In some embodiments, the one or more lymphatic cells is a T cell, a B cell, or a NK cell.
[0280] Nonlimiting examples of T cells include CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), memory T cells, natural killer T (NKT) cells, gamma delta (y6) T Cells, mucosal- associated invariant T (MAIT) cells, innate-like T Cells, follicular helper T (Tfh) cells, and senescent T cells.
[0281] Nonlimiting examples of B cells include naive B cells, memory B cells, plasma cells, regulatory B (Bregs) cells, B-1 cells, follicular B cells, marginal zone B cells, B-2 cells, IgM memory B cells, IgA-producing B cells.
[0282] Nonlimiting examples of NK cells include CD56dim NK cells, CD56bri9ht NK cells, CD16+ NK cells, CD57+ NK cells, adaptive NK cells, trained NK cells, hyporesponsive NK cells, tissue-resident NK cells, and decidual NK Cells.
Uses of the Engineered Proteins
[0283] The engineered proteins of the present technology are useful in treating, reducing, or preventing a plurality of conditions. Nonlimiting examples of conditions that one or more engineered proteins of the present technology are useful in treating, preventing, or reducing includes cancer, age-associated disease, autoimmune disease, infections, GVHD, and transplant rejection.
[0284] Levels of biomarkers, such as proteins or transcripts, associated with a disease may be assessed before, during, or after administration of one or more engineered proteins to determine efficacy of treatment. Nonlimiting examples of biomarkers associated with disease include proteins, transcripts, chemicals (e.g., peroxide), or chemical alterations such as reactive oxygen species (ROS) markers or DNA damage markers (e.g., oxidative damage, UV photoproducts, and double-stranded breaks) correlated with a condition being treated (e.g., cancer, age-associated disease, autoimmune disease, infections, GVHD, and transplant rejection).
[0285] In some embodiments, the subject administered a dose of one or more engineered proteins (e.g., an engineered protein present in a pharmaceutical composition) exhibits one or more of:
[0286] (a) a reduction in senescence-associated beta-galactosidase (SA-p-gal) protein level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0287] (b) a reduction in a cyclin-dependent kinase inhibitor 1 or cyclin-dependent kinase-interacting protein 1 (p21 WAF1/CIP1 ) protein level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0288] (c) a reduction in a p16INK4a protein level by about 1 %, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0289] (d) a reduction in a tumor protein p53 (p53) protein level by about 1 %, 2%, 3%,
4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0290] (e) a reduction in a retinoblastoma protein (pRB) protein level by about 1 %, 2%,
3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0291] (f) a reduction in a deleted in esophageal cancer 1 (DEC1 ) protein level by about
1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0292] (g) a reduction in a phosphorylated histone H2AX (y-H2AX) protein level by about
1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0293] (h) a reduction in a p53-binding protein 1 (53BP1 ) protein level by about 1 %, 2%,
3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0294] (i) a reduction in an Ataxia-telangiectasia mutated (ATM) protein level by about
1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0295] (j) a reduction in a meiotic recombination 11 (MRE1 1 ) protein level by about 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control
[0296] (k) a reduction in a superoxide dismutase (SOD) protein level by about 1 %, 2%,
3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0297] (I) a reduction in a catalase (CAT) protein level by about 1 %, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0298] (j) a reduction in a F2-lsoprostane protein level by about 1 %, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0299] (k) a reduction in an 8-Hydroxy-2'-deoxyguanosine (8-OHdG) protein level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0300] (I) a reduction in a lysosomal [3-galactosidase 1 (GLB1 ) transcript level by about
1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0301] (m) a reduction in a cyclin-dependent kinase inhibitor 2A (CDKN2A) transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0302] (n) a reduction in a p21 WAF1/CIP1 transcript level by about 1 %, 2%, 3%, 4%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0303] (o) a reduction in a Tumor Protein P53 (TP53) transcript level by about 1 %, 2%,
3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0304] (p) a reduction in a DEC1 transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0305] (q) a reduction in a cyclin-dependent kinase (CDK) protein level by about 1 %, 2%,
3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0306] (r) a reduction in a tumor protein p53 binding protein 1 (TP53BP1 ) transcript level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0307] (s) a reduction in an ATM transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0308] (t) a reduction in a MRE1 1 transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0309] (u) a reduction in a SOD transcript level by about 1%, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control
[0310] (v) a reduction in a CAT transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0311] (w) a reduction in a y-H2AX phosphorylation level by about 1 %, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0312] (x) a reduction in a thymine dimer level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0313] (y) a UV-induced DNA photoproduct level by about 1 %, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0314] (z) a reduction in a guanine oxidation level by about 1 %, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0315] (aa) a reduction in a malondialdehyde (MDA) level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0316] (bb) a reduction in a hydrogen peroxide (H2O2) level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0317] (cc) a reduction in a oxidized glutathione (GSSG) protein level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0318] (dd) a reduction in a protein carbonylation level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0319] (ee) a reduction in an oxidized thiol level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0320] (ff) a reduction in a tyrosine nitration level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0321] (gg) a reduction in a GSSG to reduced glutathione (GSH) ratio by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0322] (hh) an increase in mitochondrial membrane potential ( M^m) level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0323] (ii) a reduction in a mitochondrial ROS level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0324] (jj) an increase in an Adenosine Triphosphate (ATP) level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0325] (kk) an increase in an oxygen consumption rate (OCR) by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0326] (II) a reduction in an extracellular acidification rate (ECAR) by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0327] (mm) a reduction in a proton production rate (PPR) by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0328] (nn) an increase in a citrate synthase protein level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0329] (oo) an increase in a citrate synthase transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0330] (pp) an increase in a CD25 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0331] (qq) an increase in a CD69 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0332] (rr) an increase in a CD71 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0333] (ss) an increase in a CD44 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0334] (tt) an increase in a CD154 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0335] (uu) an increase in a CD28 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0336] (vv) an increase in a CD86 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0337] (ww) an increase in a CD19 protein or transcript level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0338] (xx) an increase in a CD3 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0339] (yy) an increase in a CD8 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0340] (zz) an increase in a CD4 protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0341] (aaa) an increase in a CD56 protein or transcript level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0342] (bbb) an increase in a CD62L protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0343] (ccc) an increase in a HLA-DR protein or transcript level by about 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control; [0344] (ddd) an increase in a granzyme B protein (GZMB) protein or transcript level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0345] (eee) an increase in an interferon-gamma (IFN-y) protein or transcript level by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0346] (fff) an increase in an interleukin-2 (IL-2) protein or transcript level by about 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100% relative to a control;
[0347] (ggg) an increase in a CD38 protein or transcript level by about 1%, 2%, 3%, 4%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%
85%, 90%, 95%, or 100% relative to a control;
[0348] (hhh) an increase in a CD45RO protein or transcript level by about 1 %, 2%, 3%,
4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 100% relative to a control; and
[0349] (iii) an increase in a CD20 protein or transcript level by about 1 %, 2%, 3%, 4%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 100% relative to a control.
EXAMPLES
Example 1 : Synthesis of Engineered Proteins Bispecific to CD3 and NKG2DL
[0350] To engineer proteins bispecific to CD3 and NKG2DL, single-chain dimer forms of NKG2D (NKG2Dscd) were designed using a segment of the N-terminal arm of NKG2D as a linker between domains within the native homodimer (FIG. 1A). NKG2Dscd proteins comprising a CD3 binding domain (FIG. 1 B) having a VH (SEQ ID NO: 11 ) and VL (SEO ID NO: 12) sequence. The C-terminus of the VH sequence and the N-terminus of the VL sequence were linked with a Gly/Ser linker (SEQ ID NO: 9). The C-terminus of the VL sequence was linked to the first NKG2D peptide (SEQ ID NO: 3) by the NKG2D N-terminal arm (SEQ ID NO: 6), forming an engineered protein bispecific to CD3 and NKG2DL (SEQ ID NO: 93) (FIG. 1 B). Because this engineered protein was expected to activate CD3- expressing lymphocytes, it was labeled NKG2D-single chain dimer-T cell engager (NKG2D- SCD-TCE). The engineered NKG2D single chain dimer was structurally similar to a native NKG2D dimer when the two structures were superimposed (FIG. 1C). These single chain structures may be engineered to an Fc region of an anti-CD3 antibody, which may comprise an scFv domain (FIGS. 1 D and 1 E).
[0351] NKG2D-SCD-TCE were expressed using the Daedalus platform in HEK293F cells (Invitrogen) as fusions with Siderocalin (Sen) to stabilize expression, adapted from the methods disclosed in Garrity, D., Call, M. E., Feng, J., & Wucherpfennig, K. W. (2005). The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure. Proceedings of the National Academy of Sciences of the United States of America, 102(21 ), 7641-7646 and 9. Bandaranayake AD, Correnti C, Ryu BY, Brault M, Strong RK, and Rawlings DJ (201 1 ). Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res 39(21 ), e143, each incorporated herein by reference in their entireties. A TEV protease scission site (SEQ ID NO: 92) was inserted between the Sen and NKG2D moieties, and N-terminal hexahistidine (SEQ ID NO: 10) and FLAG (SEQ ID NO: 90) tags were incorporated to facilitate purification. Additional TEV protease features may comprise those disclosed in Finton KA, Larimore K, Larman HB, Friend D, Correnti C, Rupert PB, Elledge SJ, Greenberg PD, and Strong RK (2013). Autoreactivity and exceptional CDR plasticity (but not unusual polyspecificity) hinder elicitation of the anti-HIV antibody 4E10. PLoS Pathog 9(9), e1003639, incorporated herein by reference in its entirety. Recombinant proteins were purified from culture supernatants by immobilized metal chelate affinity, treated with TEV protease to release the Sen fusion partner, and polished by size exclusion chromatography (SEC). Purity and proper folding were confirmed by comparative reduced/nonreduced sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical SEC. Endotoxin levels were checked using the PyroGene rFc assay (Lonza).
[0352] Surface plasmon resonance (SPR) interaction analyses confirming that NKG2Dscd proteins retain proper binding to NKG2DL were performed at 25°C with Biacore T100 instrumentation on a Series S CM5 chip (GE Healthcare). NKG2Dscd proteins were immobilized using standard amine coupling chemistry, yielding surfaces with -310 or -240 SPR response units, respectively. A reference surface was generated by activating and deactivating a flow cell in the absence of protein. Serial two-fold dilutions of soluble, wildtype MICA (240 nM to 0.47 nM, or 100 nM to 0.78 nM), were prepared in a running buffer of HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.05% surfactant P-20; GE Healthcare) with 0.1 mg/ml bovine serum albumin. Duplicate MICA samples, interspersed with multiple buffer blanks, were randomly injected at 50 pl/min, with 3 minutes of association and 5 minutes of dissociation. Surfaces were regenerated with a 10-second injection of 10 mM glycine, pH 2.0, followed by 2 minutes of buffer stabilization. Doublereferenced data were fit with a 1 :1 binding model using BIAevaluation 2.0.4 software (GE Healthcare).
Example 2: Engineered protein NKG2D-SCD-TCE promotes lymphocyte activation in the presence of cells expressing NKG2DL
Identifying NKG2DL-Expressing Cells
[0353] To identify cell lines which express NKG2DL, cell lines expressing MICA, MICB, ULBP2, or LILBP4 were examined using DepMap data explorer (Broad Institute). (FIG. 2). A multitude of cancer cell lines were confirmed to express NKG2DL transcripts. This included the breast cancer cell line MDA-MB-231 , which was utilized in subsequent experimentation.
Assessing NKG2D-SCD-TCE-mediated T cell Activation
[0354] To assess whether NKG2D-SCD-TCE activates T cells specifically in the presence of NKG2DL, primary CD8+ T cells were purified from donor PBMCs. Titrated NKG2D-SCD-TCE was added to samples comprising CD8+ T cells alone or to samples comprising CD8+ T cells mixed 1 :1 with a population of NKG2DL-expressing MDA-MB-231 cells. See Kim Y, Bigelow L, Borovilos M, Dementieva I, Duggan E, Eschenfeldt W, Hatzos C, Joachimiak G, Li H, and Maltseva N, et al (2008). Chapter 3. High-throughput protein purification for x-ray crystallography and NMR. Adv Protein Chem Struct Biol 75, 85-105 and Li P, Morris DL, Willcox BE, Steinle A, Spies T, and Strong RK (2001 ). Complex structure of the activating immunoreceptor NKG2D and its MHC class l-like ligand MICA. Nat Immunol 2(5), 443-451 , the disclosures of which are each incorporated herein by reference in their entireties.
[0355] NKG2D-SCD-TCE was added at a concentration of 2600 ng/ml, 260 ng/ml, 26 ng/ml, 2.6 ng/ml, 0.26 ng/ml, 0.026 ng/ml, or 0.0026 ng/ml and cultured with samples for 16 hours. 0.0 ng/ml NKG2D-SCD-TCE was used as an additional control. As illustrated in FIG. 3, the samples comprising both CD8+T cells and MDA-MB-231 cells (top row) demonstrated increased T cell activation with increased NKG2D-SCD-TCE dosage, as measured by flow CD137 protein levels using flow cytometry. The samples comprising CD8+ T cells alone (bottom row) did not demonstrate any T cell activation, regardless of NKG2D-SCD-TCE dose. This suggests that NKG2D-SCD-TCE selectively recognizes and/or activates T cells in the presence of NKG2DL, but not in the absence of NKG2DL.
NKG2D-SCD-TCE Dose-Response
[0356] NKG2D-SCD-TCE-mediated T cell activation dose-response using was also examined using a nonlinear regression model (Graphpad Prism). Percent T cell activation (measured by CD137 protein level, y-axis) was then plotted against NKG2D-SCD-TCE concentration (x-axis) for both samples comprising primary CD8+ T cells only (circles) compared to samples comprising primary CD8+ T cells plus MDA-MB-231 cells (squares) (FIG. 4). The resulting EC50 for NKG2D-SCE-TCE was about 3.2 ng/ml (0.056 nM) in samples comprising MDA-MB-231. In samples having only CD8+ T cells only and were absent of NKG2DL, T cell activation did not occur at any concentration of NKG2D-SCD-TCE treatment. This further demonstrates that NKG2D-SCE-TCE-mediated T cell activation occurs in the presence, but not the absence, of NKG2DL-expressing cells.
Example 3: Assessing Additional Engineered Proteins
[0357] To assess engineered proteins comprising other CD3 binding domains, MDA-MB- 231 (FIG. 5A), HeLa (FIG. 5B), H1299 (FIG. 5C), and HEK233F (FIG. 6) cells were transduced with lentivirus comprising a nucleotide sequence encoding SEQ ID NO: 107 (“ADI”), and supernatant was collected from confluent cultures of untransduced (“Parental”) or ADI-transduced cells for HEK293T cells. Supernatants were collected and added to cultures containing the indicated tumor cell line and primary CD8 T cells that had previously been non-specifically activated and expanded for 10 days prior to the assay at a 3:1 ratio. Cells were assessed using cytotoxicity assays for live-cell analysis (FIGS. 5A-5C) and engineered protein levels were qualitatively assessed (FIG. 6). The qualitative assessment was also assessed using the engineered protein of Examples 1 and 2 as an additional control. This demonstrated that the engineered proteins of SEQ ID NO: 107 (having a VH domain of SEQ ID NO:101 and a VL domain of SEQ ID NO: 100) were effectively translated in the cell types assessed and were detectable for at least 10 days post-transduction of the viral vectors.
Example 4: Assessing T Cell Activation
[0358] To assess effects of the engineered proteins of the present technology on T cell activation, MDA-MB-231 (FIG. 7A), H1299 (FIG. 7B), and HeLa (FIG. 5C) cells were transduced with lentivirus comprising nucleotide sequences encoding the engineered proteins of Examples 1 -3 (SEQ ID NO: 93: “OKT3” and SEQ ID NO: 107: “ADI) at increasing concentrations. Both the OKT3 and the ADI engineered proteins resulted in increased T cell activation relative to untransduced controls (“Parental”), as measured by the percent of CD137+ T cells.
Additional Examples
[0359] The present technology includes, but is not limited to, the following specific examples set forth herein below in paragraphs [0360]-[0457]:
[0360] 1. An engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first Natural killer group 2, member D (NKG2D) peptide, and a second NKG2D peptide.
[0361] 2. The engineered protein of embodiment 1 , wherein the first NKG2D peptide and/or the second NKG2D peptide comprises a NKG2D ligand (NKG2DL) binding domain.
[0362] 3. The engineered protein of embodiment 1 or 2, wherein the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof. [0363] 4. The engineered protein of embodiment 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a variable heavy (VH) domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11.
[0364] 5. The engineered protein of embodiment 3 or 4, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a variable light (VL) domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12.
[0365] 6. The engineered protein of embodiment 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101.
[0366] 7. The engineered protein of embodiment 3 or 6, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100.
[0367] 8. The engineered protein of embodiment 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103.
[0368] 9. The engineered protein of embodiment 3 or 8, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104.
[0369] 10. The engineered protein of embodiment 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105. [0370] 1 1. The engineered protein of embodiment 3 or 10, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106.
[0371] 12. The engineered protein of any one of the preceding embodiments, wherein the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0372] 13. The engineered protein of any one of the preceding embodiments, wherein the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0373] 14. The engineered protein of any one of embodiments, 1 -13 wherein the first
NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
[0374] 15. The engineered protein of any one of embodiments, 1 -14 wherein the first
NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
[0375] 16. The engineered protein of any one of embodiments, 1 -13 wherein the first
NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
[0376] 17. The engineered protein of any one of embodiments, 1 -13 wherein the first
NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
[0377] 18. The engineered protein of any one of embodiments, 1 -17, wherein the first
NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an N-terminal domain of an NKG2D protein.
[0378] 19. The engineered protein of embodiment 18, wherein the NKG2D protein consists of SEQ ID NO: 94.
[0379] 20. The engineered protein of embodiment 18, wherein the linker peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 10, or SEQ ID NO: 98. [0380] 21 . The engineered protein of any one of embodiments 1 -20, wherein one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
[0381] 22. The engineered protein of embodiment 21 , wherein the mammalian peptide is a human peptide or a murine peptide.
[0382] 23. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 1 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
[0383] 24. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
[0384] 25. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
[0385] 26. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
[0386] 27. The engineered protein of anyone embodiments 1 -26, wherein the first
NKG2D peptide and the second NKG2D peptide comprise domains for dimerization. [0387] 28. The engineered protein of any one of embodiments 1 -27, wherein the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
[0388] 29. The engineered protein of any one of embodiments 2-28, wherein the
NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
[0389] 30. The engineered protein of embodiment 29, wherein the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
[0390] 31 . The engineered protein of any one of embodiments 1 -30, wherein the CD3 binding domain comprises a T-cell activating sequence.
[0391] 32. The engineered protein of any one of embodiments 1 -31 , further comprising one or more of an immunoglobulin leader peptide, a purification tag, a protease scission site, or a second linking peptide sequence.
[0392] 33. The engineered protein of embodiment 32, wherein the immunoglobulin leader peptide is a Igr leader peptide.
[0393] 34. The engineered protein of embodiment 33, wherein the IgK leader peptide comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73.
[0394] 35. The engineered protein of embodiment 32, wherein the purification tag is selected from the group consisting of a hexa-histidine tag, a FLAG tag, a Myc tag, and a Human influenza hemagglutinin (HA) tag.
[0395] 36. The engineered protein of embodiment 32, wherein the hexa-histidine tag comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
[0396] 37. The engineered protein of embodiment 32, wherein the protease scission site is a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-C scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, or an endopeptidase Arg-C scission site.
[0397] 38. The engineered protein of embodiment 37, wherein the TEV protease scission site comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92 or SEQ ID NO: 96.
[0398] 39. The engineered protein of embodiment 32, wherein the second linking peptide sequence comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 10, or SEQ ID NO: 98.
[0399] 40. A pharmaceutical composition comprising the engineered protein of any one of embodiments 1 -39, and one or more pharmaceutically acceptable carriers.
[0400] 41. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 1 , (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0401] 42. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 , (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0402] 43. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103, (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0403] 44. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105, (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
[0404] 45. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
[0405] 46. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 107.
[0406] 47. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 108.
[0407] 48. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
[0408] 49. A vector comprising a nucleotide sequence encoding the CD3 binding domain of the engineered protein of any one of embodiments 1 -39. [0409] 50. A vector comprising a nucleotide sequence encoding the single chain peptide comprising first NKG2D peptide and the second NKG2D peptide of the engineered protein of any one of embodiments 1 -39.
[0410] 51 . A method of generating an engineered protein comprising a CD3 binding domain, and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide, comprising the steps of (i) synthesizing the CD3 binding domain from a first vector; (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector; and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
[0411] 52. A method of activating senolysis of senescent cells in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
[0412] 53. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method of activating senolysis of senescent cells in a subject in need thereof, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
[0413] 54. The method of embodiment 52 or the use of embodiment 53, wherein the one or more biomarkers comprises a protein, a transcript, a DNA damage marker, or a reactive oxygen species (ROS) marker.
[0414] 55. The method or use of embodiment 54, wherein the protein is selected from the group consisting of a senescence-associated beta-galactosidase (SA-[3-gal) protein, a cyclin-dependent kinase inhibitor 1 or cyclin-dependent kinase-interacting protein 1 (p21 WAF1/CIP1 ) protein, a p16INK4a protein, a tumor protein p53 (p53) protein, a retinoblastoma protein (pRB) protein, a deleted in esophageal cancer 1 (DEC1 ) protein, a phosphorylated histone H2AX (y-H2AX) protein, a p53-binding protein 1 (53BP1 ) protein, an Ataxia-telangiectasia mutated (ATM) protein, a meiotic recombination 11 (MRE11 ) protein, a superoxide dismutase (SOD) protein, superoxide dismutase (SOD) protein, F2- Isoprostane protein, 8-Hydroxy-2'-deoxyguanosine (8-OHdG) protein.
[0415] 56. The method or use of embodiment 54, wherein the transcript is selected from the group consisting of a lysosomal p-galactosidase 1 (GLB1 ) transcript, a cyclin- dependent kinase inhibitor 2A (CDKN2A) transcript, a p21WAF1/CIP1 transcript, a Tumor Protein P53 (TP53) transcript, a DEC1 transcript, a cyclin-dependent kinase (CDK) protein, a CDK transcript, a tumor protein p53 binding protein 1 (TP53BP1 ) transcript, an ATM transcript, a MRE11 transcript, a SOD transcript, and a CAT transcript.
[0416] 57. The method or use of embodiment 54, wherein the DNA damage marker is selected from the group consisting of a y-H2AX phosphorylation level, a thymine dimer, a phosphorylation of an ATM kinase target, a UV-induced DNA photoproduct, a guanine oxidation, and a double-strand break.
[0417] 58. The method or use of embodiment 54, wherein the ROS marker is selected from the group consisting of a malondialdehyde (MDA) level, a hydrogen peroxide (H2O2) level, oxidized glutathione (GSSG) protein, a protein carbonylation, oxidized thiol group, a tyrosine nitration, and a GSSG to reduced glutathione (GSH) ratio.
[0418] 59. A method for lymphatic cell activation in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
[0419] 60. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method for lymphatic cell activation in a subject in need thereof, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
[0420] 61 . The method of embodiment 59 or the use of embodiment 580, wherein the one or more lymphatic cell activation markers is a protein or a transcript. [0421] 62. The method or use of embodiment 61 , wherein the protein is selected from the group consisting of a CD25 protein, a CD69 protein, a CD71 protein, a CD44 protein, a CD154 protein, a CD28 protein, a CD86 protein, a CD19 protein, a CD3 protein, a CD8 protein, a CD4 protein, a CD56 protein, a CD62L protein, a HLA-DR protein, a granzyme B protein (GZMB) protein, an interferon-gamma (IFN-y) protein, an interleukin-2 (IL-2) protein, a CD38 protein, a CD45RO protein, and a CD20 protein.
[0422] 63. The method or use of embodiment 61 , wherein the transcript is selected from the group consisting of a CDC25A transcript, a CDC25B transcript, a CDC25C transcript, a CD69 transcript, a TFRC gene, a CD44 transcript, a CD154 transcript, a CD28 transcript, a CD86 transcript, a CD19 transcript, a CD3 transcript, a CD8 transcript, a CD4 transcript, CD56 transcript, a CD62L transcript, a HLA-DR transcript, a GZMB transcript, an interferon-gamma (IFNG) transcript, an interleukin-2 (IL2) transcript, a CD38 gene, a CD45RO transcript, and a CD20 transcript.
[0423] 64. A method of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
[0424] 65. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
[0425] 66. The method or use of any one of embodiments 59-64, wherein the lymphatic cell is a T cell, a B cell, or an NK cell.
[0426] 67. The method or use of any one of embodiments 59-66, wherein the subject has a cancer or an age-related disease. [0427] 68. The method or use of embodiment 67, wherein the age-related disease comprises one or more of osteoporosis, arthritis, obesity, cardiovascular disease, or a neurodegenerative disorder.
[0428] 69. A method of treating cancer in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
[0429] 70. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in method of treating cancer in a subject in need thereof, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
[0430] 71. The method or use of embodiment 67, 69, or 70, wherein the cancer is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
[0431] 72. The method or use of any one of embodiments 52-71 , wherein the first
NKG2D peptide and/or the second NKG2D peptide comprises a NKG2DL binding domain.
[0432] 73. The method or use of any one of embodiments 52-72, wherein the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
[0433] 74. The method or use of embodiment 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1 1.
[0434] 75. The method or use of embodiment 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12.
[0435] 76. The method or use of embodiment 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101.
[0436] 77. The method or use of embodiment 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100.
[0437] 78. The method or use of embodiment 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
103.
[0438] 79. The method or use of embodiment 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
104.
[0439] 80. The method or use of embodiment 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
105.
[0440] 81 . The method or use of embodiment 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:
106.
[0441] 82. The method or use of any one of embodiments 52-75, wherein the first
NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. [0442] 83. The method or use of any one of embodiments 52-82, wherein the second
NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:3.
[0443] 84. The method or use of any one of embodiments 52-83, wherein the first
NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
[0444] 85. The method or use of any one of embodiments 52-83, wherein the first
NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
[0445] 86. The method or use of any one of embodiments 52-83, wherein the first
NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
[0446] 87. The method or use of any one of embodiments 52-83, wherein the first
NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
[0447] 88. The method or use of any one of embodiments 52-87, wherein the first
NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a peptide comprising an N-terminal domain of an NKG2D protein.
[0448] 89. The method or use of embodiment 88, wherein the NKG2D protein consists of SEQ ID NO: 94.
[0449] 90. The method or use of embodiment 88 or 89, wherein the peptide comprising the N-terminal domain is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
[0450] 91 . The method or use of any one of embodiments 52-90, wherein one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
[0451] 92. The method or use of embodiment 91 , wherein the mammalian peptide is a human or murine peptide.
[0452] 93. The method or use of any one of embodiments 52-92, wherein the first
NKG2D peptide and the second NKG2D peptide comprise domains for dimerization. [0453] 94. The method or use of any one of embodiments 52-93, wherein the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
[0454] 95. The method or use of embodiment 64 or 65, wherein the NKG2DL is a cellsurface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
[0455] 96. The method or use of embodiment 95, wherein the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
[0456] 97. The method or use of any one of embodiments 52-96, wherein the CD3 binding domain comprises a T-cell activating sequence.
[0457] 98. The method or use of any one of embodiments 52-97, wherein the engineered protein is present in a pharmaceutical composition.

Claims

CLAIMS l/We claim:
1 . An engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first Natural killer group 2, member D (NKG2D) peptide, and a second NKG2D peptide.
2. The engineered protein of claim 1 , wherein the first NKG2D peptide and/or the second NKG2D peptide comprises a NKG2D ligand (NKG2DL) binding domain.
3. The engineered protein of claim 1 or 2, wherein the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
4. The engineered protein of claim 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a variable heavy (VH) domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11.
5. The engineered protein of claim 3 or 4, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a variable light (VL) domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12.
6. The engineered protein of claim 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 .
7. The engineered protein of claim 3 or 6, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100.
8. The engineered protein of claim 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103.
9. The engineered protein of claim 3 or 8, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104.
10. The engineered protein of claim 3, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105.
1 1. The engineered protein of claim 3 or 10, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106.
12. The engineered protein of any one of the preceding claims, wherein the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
13. The engineered protein of any one of the preceding claims, wherein the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
14. The engineered protein of any one of claims 1 -13 wherein the first NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
15. The engineered protein of any one of claims 1 -14, wherein the first NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
16. The engineered protein of any one of claims 1 -13, wherein the first NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
17. The engineered protein of any one of claims 1 -13, wherein the first NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
18. The engineered protein of any one of claims 1 -17, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an N-terminal domain of an NKG2D protein.
19. The engineered protein of claim 18, wherein the NKG2D protein consists of SEQ ID NO: 94.
20. The engineered protein of claim 18, wherein the linker peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 10, or SEQ ID NO: 98.
21 . The engineered protein of any one of claims 1 -20, wherein one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
22. The engineered protein of claim 21 , wherein the mammalian peptide is a human peptide or a murine peptide.
23. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
24. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
25. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
26. An engineered protein comprising (i) an anti-CD3 antibody or antigen binding fragment thereof comprising or consisting of a first amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105 and a second amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106 and (ii) a single peptide chain comprising a first NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3 and a second NKG2D peptide comprising or consisting of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a linking peptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
27. The engineered protein of anyone claims 1 -26, wherein the first NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
28. The engineered protein of any one of claims 1 -27, wherein the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
29. The engineered protein of any one of claims 2-28, wherein the NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
30. The engineered protein of claim 29, wherein the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
31. The engineered protein of any one of claims 1 -30, wherein the CD3 binding domain comprises a T-cell activating sequence.
32. The engineered protein of any one of claims 1 -31 , further comprising one or more of an immunoglobulin leader peptide, a purification tag, a protease scission site, or a second linking peptide sequence.
33. The engineered protein of claim 32, wherein the immunoglobulin leader peptide is a IgK leader peptide.
34. The engineered protein of claim 33, wherein the IgK leader peptide comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 73.
35. The engineered protein of claim 32, wherein the purification tag is selected from the group consisting of a hexa-histidine tag, a FLAG tag, a Myc tag, and a Human influenza hemagglutinin (HA) tag.
36. The engineered protein of claim 32, wherein the hexa-histidine tag comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 10.
37. The engineered protein of claim 32, wherein the protease scission site is a Tobacco Etch Virus (TEV) protease scission site, a trypsin scission site, a chymotrypsin scission site, a pepsin scission site, a papain scission site, a thermolysin scission site, an endopeptidase Glu-C (V8 protease) scission site, an endopeptidase Lys-C scission site, a cyanogen bromide (CNBr) scission site, an endopeptidase Asp-N scission site, or an endopeptidase Arg-C scission site.
38. The engineered protein of claim 37, wherein the TEV protease scission site comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92 or SEQ ID NO: 96.
39. The engineered protein of claim 32, wherein the second linking peptide sequence comprises or consists of an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NQ:10, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 10, or SEQ ID NO: 98.
40. A pharmaceutical composition comprising the engineered protein of any one of claims 1 -39, and one or more pharmaceutically acceptable carriers.
41 . A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11 , (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
42. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 , (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
43. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103, (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
44. A vector comprising a nucleotide sequence encoding (i) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105, (ii) a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106, (ii) a first NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3, and (iii) a second NKG2D peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
45. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93.
46. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 107.
47. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 108.
48. A vector comprising a nucleotide sequence encoding a peptide comprising or consisting of an amino acid sequence about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 109.
49. A vector comprising a nucleotide sequence encoding the CD3 binding domain of the engineered protein of any one of claims 1 -39.
50. A vector comprising a nucleotide sequence encoding the single chain peptide comprising first NKG2D peptide and the second NKG2D peptide of the engineered protein of any one of claims 1 -39.
51. A method of generating an engineered protein comprising a CD3 binding domain, and a single peptide chain comprising a first NKG2D peptide and a second NKG2D peptide, comprising the steps of (i) synthesizing the CD3 binding domain from a first vector; (ii) synthesizing the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide from a second vector; and (iii) fusing the CD3 binding domain to the single chain peptide comprising the first NKG2D peptide and the second NKG2D peptide.
52. A method of activating senolysis of senescent cells in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
53. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method of activating senolysis of senescent cells in a subject in need thereof, wherein administration of the engineered protein reduces a level of one or more biomarkers of senescence in the subject relative to baseline.
54. The method of claim 52 or the use of claim 53, wherein the one or more biomarkers comprises a protein, a transcript, a DNA damage marker, or a reactive oxygen species (ROS) marker.
55. The method or use of claim 54, wherein the protein is selected from the group consisting of a senescence-associated beta-galactosidase (SA-p-gal) protein, a cyclin- dependent kinase inhibitor 1 or cyclin-dependent kinase-interacting protein 1 (p21 WAF1/CIP1 ) protein, a p16INK4a protein, a tumor protein p53 (p53) protein, a retinoblastoma protein (pRB) protein, a deleted in esophageal cancer 1 (DEC1 ) protein, a phosphorylated histone H2AX (y-H2AX) protein, a p53-binding protein 1 (53BP1 ) protein, an Ataxia-telangiectasia mutated (ATM) protein, a meiotic recombination 11 (MRE11 ) protein, a superoxide dismutase (SOD) protein, superoxide dismutase (SOD) protein, F2- Isoprostane protein, 8-Hydroxy-2'-deoxyguanosine (8-OHdG) protein.
56. The method or use of claim 54, wherein the transcript is selected from the group consisting of a lysosomal [3-galactosidase 1 (GLB1 ) transcript, a cyclin-dependent kinase inhibitor 2A (CDKN2A) transcript, a p21 WAF1/CIP1 transcript, a Tumor Protein P53 (TP53) transcript, a DEC1 transcript, a cyclin-dependent kinase (CDK) protein, a CDK transcript, a tumor protein p53 binding protein 1 (TP53BP1 ) transcript, an ATM transcript, a MRE1 1 transcript, a SOD transcript, and a CAT transcript.
57. The method or use of claim 54, wherein the DNA damage marker is selected from the group consisting of a y-H2AX phosphorylation level, a thymine dimer, a phosphorylation of an ATM kinase target, a UV-induced DNA photoproduct, a guanine oxidation, and a double-strand break.
58. The method or use of claim 54, wherein the ROS marker is selected from the group consisting of a malondialdehyde (MDA) level, a hydrogen peroxide (H2O2) level, oxidized glutathione (GSSG) protein, a protein carbonylation, oxidized thiol group, a tyrosine nitration, and a GSSG to reduced glutathione (GSH) ratio.
59. A method for lymphatic cell activation in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
60. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method for lymphatic cell activation in a subject in need thereof, wherein administration of the engineered protein upregulates a level of one or more lymphatic cell activation markers relative to a control.
61 . The method of claim 59 or the use of claim 580, wherein the one or more lymphatic cell activation markers is a protein or a transcript.
62. The method or use of claim 61 , wherein the protein is selected from the group consisting of a CD25 protein, a CD69 protein, a CD71 protein, a CD44 protein, a CD154 protein, a CD28 protein, a CD86 protein, a CD19 protein, a CD3 protein, a CD8 protein, a CD4 protein, a CD56 protein, a CD62L protein, a HLA-DR protein, a granzyme B protein (GZMB) protein, an interferon-gamma (IFN-y) protein, an interleukin-2 (IL-2) protein, a CD38 protein, a CD45RO protein, and a CD20 protein.
63. The method or use of claim 61 , wherein the transcript is selected from the group consisting of a CDC25A transcript, a CDC25B transcript, a CDC25C transcript, a CD69 transcript, a TFRC gene, a CD44 transcript, a CD154 transcript, a CD28 transcript, a CD86 transcript, a CD19 transcript, a CD3 transcript, a CD8 transcript, a CD4 transcript, CD56 transcript, a CD62L transcript, a HLA-DR transcript, a GZMB transcript, an interferongamma (IFNG) transcript, an interleukin-2 (IL2) transcript, a CD38 gene, a CD45RO transcript, and a CD20 transcript.
64. A method of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
65. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in a method of colocalizing a lymphatic cell and a NKG2DL-expressing cell in a subject in need thereof, wherein administration of the engineered protein increases levels of immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation relative to a control.
66. The method or use of any one of claims 59-64, wherein the lymphatic cell is a T cell, a B cell, or an NK cell.
67. The method or use of any one of claims 59-66, wherein the subject has a cancer or an age-related disease.
68. The method or use of claim 67, wherein the age-related disease comprises one or more of osteoporosis, arthritis, obesity, cardiovascular disease, or a neurodegenerative disorder.
69. A method of treating cancer in a subject in need thereof, comprising administering to the subject an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
70. Use of an engineered protein comprising a CD3 binding domain and a single peptide chain comprising a first NKG2D peptide, and a second NKG2D peptide in method of treating cancer in a subject in need thereof, wherein administration of the engineered protein reduces cancer cell proliferation relative to a control.
71 . The method or use of claim 67, 69, or 70, wherein the cancer is selected from the group consisting of a breast cancer, a lung cancer, a colorectal cancer, a prostate cancer, a skin cancer, a bladder cancer, an ovarian cancer, a pancreatic cancer, a leukemia, a non-Hodgkin lymphoma, a kidney cancer, a liver cancer, a thyroid cancer, a brain cancer, a stomach cancer, an esophageal cancer, a cervical cancer, a uterine cancer, a testicular cancer, and a multiple myeloma.
72. The method or use of any one of claims 52-71 , wherein the first NKG2D peptide and/or the second NKG2D peptide comprises a NKG2DL binding domain.
73. The method or use of any one of claims 52-72, wherein the CD3 binding domain comprises an anti-CD3 antibody or an antigen binding fragment thereof.
74. The method or use of claim 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11.
75. The method or use of claim 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12.
76. The method or use of claim 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 101 .
77. The method or use of claim 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 100.
78. The method or use of claim 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 103.
79. The method or use of claim 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VL domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 104.
80. The method or use of claim 73, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a VH domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 105.
81 . The method or use of claim 73 or 74, wherein the anti-CD3 antibody or antigen binding fragment thereof comprises or consists of a L domain having an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 106.
82. The method or use of any one of claims 52-75, wherein the first NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.
83. The method or use of any one of claims 52-82, wherein the second NKG2D peptide comprises or consists of an amino acid sequence at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:3.
84. The method or use of any one of claims 52-83, wherein the first NKG2D peptide and the second NKG2D peptide comprise the same amino acid sequence.
85. The method or use of any one of claims 52-83, wherein the first NKG2D peptide and the second NKG2D peptide consist of the same amino acid sequence.
86. The method or use of any one of claims 52-83, wherein the first NKG2D peptide and the second NKG2D peptide comprise different amino acid sequences.
87. The method or use of any one of claims 52-83, wherein the first NKG2D peptide and the second NKG2D peptide consist of different amino acid sequences.
88. The method or use of any one of claims 52-87, wherein the first NKG2D peptide and the second NKG2D peptide are linked on the single peptide chain by a peptide comprising an N-terminal domain of an NKG2D protein.
89. The method or use of claim 88, wherein the NKG2D protein consists of SEQ ID NO: 94.
90. The method or use of claim 88 or 89, wherein the peptide comprising the N- terminal domain is at least about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 98.
91 . The method or use of any one of claims 52-90, wherein one or more of the CD3 binding domain, the first NKG2D peptide, the second NKG2D peptide, or the NKG2D protein comprises a peptide selected from the group consisting of a mammalian peptide, a non-naturally occurring peptide, a humanized peptide, and a chimeric peptide comprising a human and non-human amino acid sequences.
92. The method or use of claim 91 , wherein the mammalian peptide is a human or murine peptide.
93. The method or use of any one of claims 52-92, wherein the first NKG2D peptide and the second NKG2D peptide comprise domains for dimerization.
94. The method or use of any one of claims 52-93, wherein the CD3 binding domain, the first NKG2D peptide, and the second NKG2D peptide are each present on a single peptide chain.
95. The method or use of claim 64 or 65, wherein the NKG2DL is a cell-surface protein expressed by a transformed cell, a cell subjected to genotoxic stress, or a senescent cell.
96. The method or use of claim 95, wherein the transformed cell or the cell subjected to genotoxic stress is a cancer cell.
97. The method or use of any one of claims 52-96, wherein the CD3 binding domain comprises a T-cell activating sequence.
98. The method or use of any one of claims 52-97, wherein the engineered protein is present in a pharmaceutical composition.
PCT/US2024/050126 2023-10-06 2024-10-04 Proteins bispecific to cd3 and nkg2dl and associated uses thereof Pending WO2025076473A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150232557A1 (en) * 2012-04-20 2015-08-20 Emergent Product Development Seattle Llc Cd3 binding polypeptides
WO2016166139A1 (en) * 2015-04-14 2016-10-20 Eberhard Karls Universität Tübingen Bispecific fusion proteins for enhancing immune responses of lymphocytes against tumor cells
US20170210819A1 (en) * 2014-07-25 2017-07-27 Memorial Sloan Kettering Cancer Center Bispecific her2 and cd3 binding molecules
US20180371051A1 (en) * 2015-11-10 2018-12-27 Fred Hutchinson Cancer Research Center Nkg2d decoys

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150232557A1 (en) * 2012-04-20 2015-08-20 Emergent Product Development Seattle Llc Cd3 binding polypeptides
US20170210819A1 (en) * 2014-07-25 2017-07-27 Memorial Sloan Kettering Cancer Center Bispecific her2 and cd3 binding molecules
WO2016166139A1 (en) * 2015-04-14 2016-10-20 Eberhard Karls Universität Tübingen Bispecific fusion proteins for enhancing immune responses of lymphocytes against tumor cells
US20180371051A1 (en) * 2015-11-10 2018-12-27 Fred Hutchinson Cancer Research Center Nkg2d decoys

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